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XIV Reunión de Biología Molecular de Plantas
Salamanca, 4 – 6 Julio 2018
Libro de Resúmenes
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ÍNDICE
Comité organizador y científico ……….…pág. 3
Carta de Bienvenida ………………….…….pág. 5
Programa XIV RBMP ……………………….pág. 7
Conferencia Inaugural …………………....pág.13
Conferencia Clausura …………….………pág. 14
Sesión I ……………………………………..pág. 15
Sesión II …………………………………….pág. 31
Sesión III ……………………………….…...pág. 51
Sesión IV …………………………….….…..pág. 59
Sesión V ……………….…………………..pág. 103
Sesión VI ………………….…………….…pág. 121
Sesión VII ………………………………....pág. 143
Sesión VIII ………………………………...pág. 185
Sesión IX ……………………………..……pág. 201
Índice de autores ………….........……….pág. 229
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COMITÉ ORGANIZADOR
Óscar Lorenzo. CIALE-USAL,
Salamanca.
Dolores Rodríguez. CIALE-USAL,
Salamanca
Isabel Mateos. CIALE-USAL,
Salamanca
Mª Inmaculada Sánchez. CIALE-USAL,
Salamanca
Tamara Lechón. CIALE-USAL,
Salamanca
María Guadalupe Fernández. CIALE-
USAL, Salamanca
Isabel Manrique. CIALE-USAL,
Salamanca
Juan Arellano. IRNASA-CSIC,
Salamanca
Mónica Balsera. IRNASA-CSIC,
Salamanca
Rosa Morcuende. IRNASA-CSIC,
Salamanca
COMITÉ CIENTÍFICO
Biología de Sistemas Ana Caño. CRAG, CSIC-IRTA-UAB-
UB, Barcelona Pilar Cubas. CNB-CSIC, Madrid
Biotecnología Vegetal Aplicada
Antonio Granell. IBMCP, CSIC-UPV,
Valencia
Javier Pozueta. IdAB,
CSIC/UPNA/Gobierno de Navarra,
Navarra Herramientas tecnológicas
Antonio Molina. CBGP, UPM-INIA,
Madrid Pilar S. Testillano. CIB-CSIC,
Madrid
Desarrollo
David Posé. IHSM-UMA-CSIC, Málaga
Cristina Ferrándiz. IBMCP, CSIC-UPV,
Valencia Regulación Ambiental del Desarrollo
Miguel A. Blázquez. IBMCP, CSIC-
UPV, Valencia
Myriam Calonje. IBVF-CSIC,
Universidad de Sevilla
Señalización Roberto Solano/Andrea Chini. CNB-
CSIC, Madrid
Salomé Prat. CNB-CSIC, Madrid Estrés Abiótico
Julio Salinas/Rafael Catalá. CIB-CSIC,
Madrid Juan Carlos del Pozo. CBGP, UPM-
INIA, Madrid (representante SEFV) Metabolismo
Francisco Javier Cejudo. IBVF-CSIC,
Universidad de Sevilla (representante
SEBBM) Manuel Rodríguez Concepción. CRAG,
CSIC-IRTA-UAB-UB, Barcelona
Interacción Planta – Otros
organismos
Isabel Díaz. CBGP, UPM-INIA, Madrid
Ainhoa Martínez-Medina. iDiv Halle-
Jena-Leipzig, Alemania
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Carta de bienvenida
Es un placer daros la bienvenida a la XIV Reunión de Biología Molecular de
Plantas que se celebra en Salamanca los días 4, 5 y 6 de julio de 2018, en esta
ocasión adquiriendo un carácter especial en el contexto del VIII centenario de
la Universidad de Salamanca. Tras 800 años trabajando por la educación y la
cultura, hemos de seguir esforzándonos por conseguir una sociedad basada en
la ética y el conocimiento.
La XIV Reunión de Biología Molecular de Plantas (RBMP) es el evento bienal
más importante y con mayor repercusión dentro del ámbito de la investigación
de calidad en plantas, donde Sociedades Científicas e investigadores de gran
prestigio se reúnen para dar a conocer sus últimos resultados y la forma en que
los han llevado a cabo, con el fin de fomentar el enriquecimiento científico
colectivo.
En esta edición, hemos querido dar prioridad a la asistencia de jóvenes
investigadores y de investigadores senior de gran prestigio internacional para
las sesiones plenarias, al acercamiento de la investigación a empresarios de
diferentes sectores relacionados con la agricultura y la biotecnología vegetal
con el apoyo de la plataforma biotecnológica BIOVEGEN, y a la igualdad de
género en el comité científico y organizador.
En nombre del Comité Organizador os damos la bienvenida a Salamanca y
esperamos que disfrutéis de este congreso que hemos preparado con mucha
ilusión, tanto de su excelente calidad científica como de la parte lúdica que nos
brinda esta hermosa ciudad europea de la cultura.
Óscar Lorenzo y Dolores Rodríguez
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Programa
XIV Reunión de Biología Molecular de Plantas Miércoles 4 Julio
9 – 10:30 h: Recepción y recogida de material.
10:30 – 11 h: Acto de apertura.
11 – 11:45 h: Conferencia Inaugural - Arp Schnittger (University of Hamburg,
Germany). “Control of progression through meiosis in Arabidopsis.”
Moderador: José Jarillo (CBGP, UPM-INIA, Madrid)
Sesión I – Biología de Sistemas
Moderadoras: Ana Caño (CRAG, CSIC-IRTA-UAB-UB, Barcelona), Pilar Cubas
(CNB-CSIC, Madrid)
11:45 – 12:05 h: Ponencia 1- Fidel Lozano-Elena (CRAG, CSIC-IRTA-UAB-UB,
Barcelona). “Vascular brassinosteroid receptors confer drought resistance
without penalizing plant growth.”
12:05 – 12:25 h: Ponencia 2- Carlos Tarancón (CNB-CSIC, Madrid). “Axillary
bud development is arrested by BRC1 through several genetic networks in
Arabidopsis.”
12:25 – 12:40 h: Comunicación oral 1- Miguel A. Moreno-Risueño (CBGP,
UPM-INIA, Madrid). “A high-resolution spatiotemporal map of root
postembryonic organogenesis reveals new stem cell types and a regulatory
network of identity transitions.”
12:40 – 12:55 h: Comunicación oral 2- Pedro de los Reyes (IBVF-CSIC,
Universidad de Sevilla, Sevilla). “Interplay between the circadian clock, the
photoperiodic signal and the flowering transition in Arabidopsis thaliana.”
12:55 – 13:10 h: Comunicación oral 3- María-Dolores Rey (Universidad de
Córdoba, Córdoba). “The use of -omics technologies to progress in the Quercus
ilex biology.”
13:10 – 13:25 h: Comunicación oral 4- Ignacio Rubio (CRAG, CSIC-IRTA-UAB-
UB, Barcelona). “A systems biology approach unravels new regulatory
connections between unrelated miRNA-TF pairs.”
13:30 – 15 h: Comida.
Sesión II – Biotecnología Vegetal Aplicada
Moderadores: Javier Pozueta (IdAB, CSIC/UPNA/Gobierno de Navarra,
Pamplona), Antonio Granell (IBMCP, CSIC-UPV, Valencia)
15 - 15:20 h: Ponencia 1- José Luis Rambla (IBMCP, CSIC-UPV, Valencia).
“Breeding and engineering the volatile network in tomato: not just for taste.”
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15:20 – 15:40 h: Ponencia 2- Javier Pozueta (Instituto de Agrobiotecnología
IdAB, CSIC/UPNA/Gobierno de Navarra, Navarra). “The bad little fragrant
critters, beneficial workers concept: a story of serendipity and dirty dishes with
important biotechnological implications.”
15:40 – 15:55 h: Comunicación oral 1- Mª Belén Pascual (Universidad de
Málaga, Málaga). “Biotechnological approaches to increase biomass production
in trees.”
15:55 – 16:10 h: Comunicación oral 2- Raquel Jiménez-Muñoz (Universidad de
Granada, Granada). “Use of Zucchini yellow mosaic virus to express cold
resistance genes in Cucurbita pepo.”
16:10 – 16:25 h: Comunicación oral 3.- Cristina Castillejo (IFAPA, Centro de
Churriana, Málaga). “Multiple origins of white strawberries through independent
mutations in a single transcriptional regulator: FvMYB10.”
16:30 – 17:30 h: Pausa café | Pósteres.
Sesión III – BIOVEGEN. Colaboración Ciencia-Empresa y Financiación I+D+i
Moderadores: Antonio Molina (CBGP, UPM-INIA, Madrid), David Lapuente
(BIOVEGEN)
17:30 – 18 h: Conferencia - Manuel Talón (Director Centro de Genómica del
IVIA, Valencia). “Origen y domesticación de los cítricos a la luz de la
genómica.”
18 – 18:10 h: Ponencia 1- Jesús Agüero (ABIOPEP, Murcia). “ABIOPEP:
experiencia de spin-off en Biotecnología Vegetal.”
18:10 – 18:20 h: Ponencia 2.- Antonio Molina (Director. CBGP, UPM-INIA,
Madrid). “Colaboración Ciencia-Empresa: el caso de PLANT RESPONSE
BIOTECH.”
18:20 – 18:30 h: Ponencia 3- Pedro Prado (Jefe de área de Agrosostenibilidad.
Agencia Estatal Investigación-MEIC). “Agencia Estatal de Investigación: visión
de la colaboración Ciencia-Empresa.”
18:30 – 18:40 h: Ponencia 4- David Lapuente (Técnico de proyectos.
BIOVEGEN). “BIOVEGEN: herramientas y evolución colaboración Ciencia-
Empresa.”
18:40 – 19:15 h: Coloquio y preguntas. “Colaboración público-privada”.
Situación y perspectivas. Todos los ponentes.
Sesión III – Herramientas Tecnológicas
Moderadora: Pilar Testillano (CIB-CSIC, Madrid)
17:30 – 17:45 h: Comunicación oral 1- Sara Selma (IBMCP, CSIC-UPV,
Valencia). “Combinatorial design of modular and programmable transcriptional
regulators in plants.”
17:45 – 18 h: Comunicación oral 2- Samuel Daniel Lup (Instituto de
Bioingeniería, Universidad Miguel Hernández, Elche). “Easymap: a user friendly
analysis tool for mapping-by-sequencing of large insertions and point
mutations.”
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18 – 18:15 h: Comunicación oral 3- Alberto Carbonell (IBMCP, CSIC-UPV,
Valencia). “Fine-tune regulation of artificial small-RNA mediated gene silencing
in plants.”
18:15 – 18:30 h: Comunicación oral 4- Adrian A. Valli (CNB-CSIC, Madrid).
“The viral-derived HAM1 pyrophosphatase is required for the ipomovirus
Ugandan cassava brown streak virus to infect its natural host but not a model
plant.”
18:30 – 18:45 h: Comunicación oral 5- Pilar S. Testillano (CIB-CSIC, Madrid).
“Autophagy and cathepsin-like proteases are activated and play a role in stress-
induced cell death during microspore embryogenesis in barley.”
18:45 – 19 h: Comunicación oral 6- Isabel Betegón-Putze (CRAG, CSIC-IRTA-
UAB-UB, Barcelona). “MyROOT: A novel method and software for the semi-
automatic measurement of plant root length.”
19:30 h: Cocktail de bienvenida. Visita nocturna de la ciudad.
Jueves 5 Julio
Sesión IV – Desarrollo
Moderadores: Cristina Ferrándiz (IBMCP, CSIC-UPV, Valencia), David Posé
(IHSM-UMA-CSIC, Málaga)
9 – 9:20 h: Ponencia 1- David Posé (IHSM-UMA-CSIC, Málaga). “Study of
transcriptional regulatory network controlling strawberry fruit ripening and
quality.”
9:20 – 9:40: Ponencia 2- Vicente Balanzà (IBMCP, CSIC-UPV, Valencia). “Life
span in monocarpic plants: unraveling the basis of Global Proliferative Arrest.”
9:40 – 9:55 h: Comunicación oral 1- Jorge Fung-Uceda (CRAG, CSIC-IRTA-
UAB-UB, Barcelona). “The circadian clock sets the time of DNA replication
licensing to regulate growth in Arabidopsis.”
9:55 – 10:10 h: Comunicación oral 2- Mary Paz González-García (CNB-CSIC,
Madrid). “A transcriptional reprogramming module for stem cell differentiation.”
10:10 – 10:25 h: Comunicación oral 3- Jordi Moreno-Romero (CRAG, CSIC-
IRTA-UAB-UB, Barcelona y Uppsala BioCenter, Sweden). “The effect of
parental-specific epigenetic marks in Arabidopsis endosperm development.”
10:25 – 10:40 h: Comunicación oral 4- Raquel Iglesias-Fernández (CBGP,
UPM-INIA, Madrid). “Regulation of the asparagine synthetase encoding gene
HvASN1 in barley seeds by transcription factors HvbZIP53 and HvBLZ1.”
10:40 – 10:55 h: Comunicación oral 5- Riad Nadi (Universidad Miguel
Hernández, Elche, Alicante). “Arabidopsis INCURVATA11 and
CUPULIFORMIS2 are new components of the epigenetic machinery.”
11 – 12h: Pausa café | Pósteres.
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Sesión V – Regulación Ambiental del Desarrollo
Moderadores: Miguel A. Blázquez (IBMCP, CSIC-UPV, Valencia), Myriam Calonje
(IBVF-CSIC, Universidad de Sevilla)
12 – 12:20 h: Ponencia 1- Ángeles Gómez-Zambrano (IBVF, CSIC-Universidad
de Sevilla, Sevilla). “PRC1-mediated H2A.Z monoubiquitination is crucial to
regulate developmental and environmental responses in Arabidopsis.”
12:20 – 12:40 h: Ponencia 2- Eugenio G. Minguet (IBMCP, CSIC-UPV,
Valencia). “The COP1/SPA complex relays light and temperature information on
DELLA proteins in Arabidopsis.”
12:40 – 12:55 h: Comunicación oral 1- Sandra Fonseca (CNB-CSIC, Madrid).
“DET1 complexes control photomorphogenesis by acting at the interface
between light signaling and epigenome dynamics.”
12:55 – 13:10 h: Comunicación oral 2- Mariano Perales (CBGP, UPM-INIA,
Madrid). “A night hour counter mechanism underlies poplar seasonal growth.”
13:10 – 13:25 h: Comunicación oral 3- Charlotte Gommers (CSIC-IRTA-UAB-
UB, Barcelona). “Blinded by the light- Retrograde signals suppress
photomorphogenesis via GLK1 during high light.”
13:30 – 15 h: Comida.
Sesión VI – Señalización
Moderadores: Andrea Chini (CNB-CSIC, Madrid), Salomé Prat (CNB-CSIC,
Madrid)
15 – 15:20 h: Ponencia 1-Andrea Chini (CNB-CSIC, Madrid). “When Chemistry
meets Biology: identification of a novel OPR3-independent pathway for JA
biosynthesis.”
15:20 – 15:40 h: Ponencia 2- Cristina Martínez (CNB-CSIC, Madrid). “PIF4-
induced BR-synthesis is critical to diurnal and thermomorphogenic growth.”
15:40 – 15:55 h: Comunicación oral 1- Tamara Lechón (CIALE-USAL,
Salamanca). “Nitric oxide regulates maintenance of the root apical meristem by
confining WOX5 expression to the quiescent center.”
15:55 – 16:10 h: Comunicación oral 2- Jorge Hernández-García (IBMCP,
CSIC-UPV, Valencia). “Origin of the gibberellin-dependent transcriptional
regulation by molecular exploitation of a transactivation motif in DELLA
proteins.”
16:10 – 16:25 h: Comunicación oral 3- Irene García (IBVF, CSIC-Universidad
de Sevilla, Sevilla). “Is S-Cyanylation a novel protein post-translational
modification?”
16:30 – 17:30 h: Pausa café | Pósteres.
Sesión VII -Estrés abiótico
Moderadores: Rafael Catalá (CIB-CSIC, Madrid), Juan Carlos del Pozo (CBGP,
UPM-INIA, Madrid)
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17:30 – 17:50 h: Ponencia 1- Rafael Catalá (CIB-CSIC, Madrid). “New insights
on the determination of spliceosome specificity.”
17:50 – 18:10 h: Ponencia 2- Ángela Saez (CBGP, UPM-INIA, Madrid). “Root
responses to phosphate starvation: novel functions of cisZeatin and BiAux.”
18:10 – 18:25 h: Comunicación oral 1- José Julián (IBMCP, CSIC-UPV,
Valencia). “BPM3 and BPM5 subunits of Cullin3-RING E3 ubiquitin ligases
target clade A PP2Cs for degradation.”
18:25 – 18:40 h: Comunicación oral 2- Noemí Ruiz-López (UMA-IHSM, CSIC,
Málaga). “Arabidopsis Synaptotagmins 1 and 3 are involved in lipid
homeostasis at ER-PM contact sites under cold stress.”
18:40 – 18:55 h: Comunicación oral 3- Alejandro Navarro (CNB-CSIC, Madrid).
“Uncoupling growth repressive and abiotic-stress tolerance effects of DELLAs.”
18:55 – 19:10 h: Comunicación oral 4- Laura Lamelas (Universidad de Oviedo,
Oviedo). “Epigenetic regulation of heat stress response is revealed through
subcellular proteomics analysis in Pinus radiata.”
19:10 – 19:25 h: Comunicación oral 5- Reyes Ródenas (CEBAS, CSIC,
Murcia). “Essential regions involved in function and regulation of the
Arabidopsis high-affinity K+ transporter AtHAK5.”
19:30 – 20:30 h: Asamblea ordinaria SEFV.
21 h: Cena del Congreso. Hotel Alameda Palace.
Viernes 6 Julio
Sesión VIII – Metabolismo
Moderadores: Manuel Rodríguez Concepción (CRAG, CSIC-IRTA-UAB-UB,
Barcelona), Francisco J. Cejudo (IBVF-CSIC, Universidad de Sevilla)
9 – 9:20 h: Ponencia 1- Juan Manuel Pérez Ruiz (IBVF-CSIC, Universidad de
Sevilla, Sevilla). “The role of 2-Cys peroxiredoxins in the oxidation of chloroplast
redox-regulated enzymes in the dark.”
9:20 – 9:40 h: Ponencia 2- M. Victoria Barja (CRAG, CSIC-IRTA-UAB- UB,
Barcelona). “Finding the enzymes that feed the carotenoid pathway in different
plant tissues.”
9:40 – 9:55 h: Comunicación oral 1- Alma Burciaga-Monge (CRAG, CSIC-
IRTA-UAB-UB, Barcelona). “Esteryl ester biosynthesis in tomato.”
9:55 – 10:10 h: Comunicación oral 2- Inmaculada Coleto (Universidad del País
Vasco UPV/EHU, Bilbao). “MYB28 and MYB29 genes participate in the
regulation of Arabidopsis response to ammonium stress.”
10:10 – 10:25 h: Comunicación oral 3- M. Luisa Hernández (Instituto de la
Grasa, CSIC, Sevilla). “Insights into the members of the microsomal oleate
desaturase (FAD2) gene family involved in linoleic acid synthesis in olive (Olea
europaea).”
10:30 – 11:30 h: Pausa café | Pósteres.
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Sesión IX – Interacción planta otros organismos
Moderadoras: Isabel Díaz (CBGP, UPM-INIA Madrid), Ainhoa Martínez-Medina
(iDiv Halle-Jena-Leipzig, Alemania)
11:30 – 11:50 h: Ponencia 1- Ainhoa Martinez-Medina (iDiv, Friedrich Schiller
University, Germany). “Root mutualistic symbionts: plant allies in a multitrophic
context.”
11:50 – 12:10 h: Ponencia 2- Ana Arnaiz (CBGP, UPM-INIA, Madrid). “ROS
homeostasis essential in plant defence responses to pests.”
12:10 – 12:25 h: Comunicación oral 1- Andrea Sanchez-Vallet (ETH-Zurich,
Switzerland). ” Partial resistance in wheat is triggered upon recognition of an
avirulence gene.”
12:25 – 12:40 h: Comunicación oral 2- Javier Cabrera (Universidad de Castilla-
La Mancha, Toledo). “Key genes for root development are also relevant for gall
formation by root- knot nematodes.”
12:40 – 12:55 h: Comunicación oral 3- M. Luisa Domingo-Calap (CRAG, CSIC-
IRTA-UAB-UB,Barcelona). “Unexpected negative effect of Watermelon Mosaic
Virus (WMV) P1 gene product on the silencing suppressor activity of Cucurbit
Yellow Stunting Disorder Virus (CYSDV) P25.”
12:55 – 13:10 h: Comunicación oral 4- Pepe Cana-Quijada (IHSM-UMA-CSIC,
Universidad de Málaga, Málaga). “NAHG increases transient transformation of
Arabidopsis leaves by Agrobacterium.”
13:15 – 14:15 h: Conferencia Clausura- José Alonso (North Carolina State
University, USA). “Plant signal integration: lesson from ethylene and auxin.”
14:15: Comida y despedida.
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CONFERENCIA INAUGURAL
CONTROL OF PROGRESSION THROUGH MEIOSIS IN
ARABIDOPSIS.
Arp Schnittger
Department of Developmental Biology, University of Hamburg, Germany;
The foundation of sexual reproduction is the formation of gametes with half the
genomic DNA content of a somatic cell. This reduction in genomic content is
accomplished through meiosis that, in contrast to mitosis, comprises two
subsequent chromosome segregation steps without an intervening S-phase. In
addition, meiosis generates new allele combinations through the compilation of
new sets of homologous chromosomes and the reciprocal exchange of
chromatid segments between homologs. Hence, understanding of meiosis is
not only of fundamental interest for biology but also for plant breeding and
agriculture. In our team, we study how entry and progression through meiosis is
controlled. Particular focus is on the cell-cycle machinery and our data show
that CYCLIN-DEPENDENT KINASE A;1 (CDKA;1), the major regulator of
mitosis in Arabidopsis, also plays a key role in orchestrating chromosome
behavior in meiosis. Using a series of weak loss-of-function alleles of CDKA;1 in
combination with a newly established live cell imaging system of meiosis, we
find that plants with very little Cdk activity are sterile due to several severe
meiotic defects, including a failure of chromosomes to synapse. Subsequently,
we identify that the chromosome axis protein ASYNAPTIC 1 (ASY1) is a
phospho-target of CDKA;1 action and show that phosphorylation of ASY1 by
CDKA;1 is key for the dynamic assembly of the chromosome axis. Moreover,
these data provide a glimpse at the molecular mechanisms how the complex
steps of meiosis can be put into order.
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CONFERENCIA CLAUSURA
PLANT SIGNAL INTEGRATION: LESSON FROM ETHYLENE
AND AUXIN
Jose M. Alonso
Department of Plant and Microbial Biology, Program in Genetics, North Carolina
State University, Raleigh, NC, 27695, USA
Survival of plants greatly depends on the ability of these sessile organisms to
tune their hardwired developmental programs to the constant environmental
changes. Although it is clear that plant hormones play a central role in this
signal integration process, the exact molecular mechanisms involved are still
largely unknown. Using the regulation of root growth by ethylene and auxin as a
model experimental system, we have started to unveil some of the molecular
mechanisms behind the interaction between these two hormones. This work is
also shedding some light onto two poorly understood aspects of plant biology:
the regulation of auxin production and the control of gene expression at the
translational level. Our recent work indicates that the production of auxin in
specific cells during development or in response to biotic and abiotic factors
plays critical roles, although sometimes (but not always) auxin transport can
bypass the need for a local source of this hormone. On the other hand, our work
on translation regulation has uncovered a novel level of regulation in the plant
response to the hormone ethylene. Specifically, we have found that the
signaling molecule EIN2 and the nonsense-mediated decay proteins UPFs play
a central role in a previously uncharacterized ethylene-triggered translational
response. Currently, we are investigating the role of other plant hormones in
gene-specific translational regulation. Our studies are revealing new nodes of
interaction between hormones, as well as implicating 3’UTRs and 5’uORFs of
specific transcripts in the regulation of plant responses to key growth regulators.
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SESIÓN I
BIOLOGÍA DE SISTEMAS
MODERADORES:
Ana Caño (CRAG, CSIC-IRTA-
UAB-UB, Barcelona)
Pilar Cubas (CNB-CSIC, Madrid)
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S-I. Ponencia 1
VASCULAR BRASSINOSTEROID RECEPTORS CONFER DROUGHT RESISTANCE WITHOUT PENALIZING PLANT
GROWTH
Norma Fàbregas1,*, Fidel Lozano-Elena1,*, David Blasco-Escámez1, Takayuki Tohge2, 8, Cristina Martínez-Andújar3, Alfonso Albacete3, Sonia Osorio4,
Mariana Bustamante1, José Luis Riechmann1, 7, Ana Conesa5, 6, Francisco Pérez Alfocea3, Alisdair R. Fernie2 and Ana I. Caño-Delgado1.
1Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Barcelona E-08193, Spain. 2 Max Planck Institute of Molecular Plant Physiology,
Potsdam-Golm D-14476, Germany. 3Department of Plant Nutrition, CEBAS-CSIC,
Murcia E-30100, Spain.4Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”. University of Malaga-Consejo Superior de Investigaciones Científicas.
Department of Molecular Biology and Biochemistry, Málaga 29071, Spain. 5Microbiology and Cell Science Department, IFAS, Genetics Institute, University of
Florida, Gainesville, USA. 6Genomics of Gene Expression Lab, Centro de
Investigaciones Príncipe Felipe, Valencia,Spain. 7Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, 08010, Spain 8Current Address: NAIST
Graduate school of Biological Sciences, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
* Both authors contributed equally to this work.
Correspondence to: [email protected]
Drought is a major environmental stress causing large crop losses worldwide and the harmful effects of drought are expected to dramatically increase due climate change. One of the toughest challenges in plant biotechnology will be the development of better-adapted crops able to cope with water-limited agricultural scenarios. The exogenous application of brassinosteroid (BR) has been widely used in agriculture to tolerate abiotic stress without growth arrest but how BRs are able to sustain growth in challenging conditions remains largely unknown. Studies with BR signaling and synthesis mutants in responses to stress failed to provide a linear picture for their involvement in drought adaptation, depicting a complex BRs role on abiotic stress. Our study unveils that the BR receptors, in addition to promoting growth, guides the responses of several traits towards drought adaptation phenotypes. Analyses of a quadruple BR loss-of-function mutant revealed extremely drought resistant phenotypes, likely caused by drought avoidance linked to growth arrest. However plants overexpressing BRL3 receptor yielded drought resistance while maintaining wild-type plant size. System analysis of BRL3 overexpressing plants revealed differential dynamics of metabolite mobilization upon drought. We found these plants to have transcriptional and metabolically primed roots that are better prepared to front drought stress. Interestingly upon tissue-enrichment analysis, great part of these responses was revealed to be vascular-driven, especially from the phloem, which is the native expression domain of BRL3. Future cell- type specific engineering of signaling cascades stands as promising strategy to surpass the typical growth arrest caused by drought stress
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S-I. Ponencia 2
AXILLARY BUD DEVELOPMENT IS ARRESTED BY BRC1
THROUGH SEVERAL GENETIC NETWORKS IN ARABIDOPSIS
Carlos Tarancón1, Eduardo González1, Pedro De Los Reyes2, Fran Romero2,
Pilar Cubas1
1Plant Molecular Department, Spanish National Centre for Biotechnology, Madrid,
Spain, 2Computer Science and Artificial Intelligence Department, University of Sevilla,
Sevilla, Spain,
Corresponding author: Carlos Tarancón ([email protected])
In Arabidopsis, axillary meristems develop into axillary buds at the base of cauline and rosette leaves. These buds remain dormant until environmental and/or developmental stimuli promote shoot branching. The dormancy-activation switch is reversible and implies a drastic genetic reprogramming, which is carefully controlled by the TCP transcription factor BRANCHED1 (BRC1) (Aguilar-Martínez, Poza-Carrión, & Cubas, 2007). It has been described that BRC1 represses bud growth in part by promoting ABA biosynthesis and turning on a gene regulatory network related to ABA. So far this is the best-characterized process controlled by BRC1 (Gonzalez-Grandio et al., 2017). Other gene networks controlled by BRC1 (González-Grandío, Poza-Carrión, Sorzano, & Cubas, 2013) are still poorly known and their relationships with this factor and their implication in bud dormancy are still unclear. Using microarray data from axillary buds and other public microarray data, we have obtained several coexpression networks downstream of BRC1 and have inferred their potential global functions during bud dormancy. Currently we are using a Systems Biology approach to study in depth the BRC1 downstream pathways. We have combined ChIP-seq and RNA-seq data obtained from seedlings with microarray data obtained from axillary buds to infer BRC1 transcriptional networks. We are now studying their topology and properties to propose new hypotheses and potential roles during bud dormancy. In addition, we have identified candidate master regulators, which could mediate and amplify transcriptional responses downstream of BRC1. We are analysing their function in the control of shoot branching.
Aguilar-Martínez, J. A., Poza-Carrión, C., & Cubas, P. (2007). Arabidopsis BRANCHED1 acts
as an integrator of branching signals within axillary buds. The Plant Cell, 19(2), 458–72. https://doi.org/10.1105/tpc.106.048934
Gonzalez-Grandio, E., Pajoro, A., Franco-Zorrilla, J. M., Tarancon, C., Immink, R. G. H., &
Cubas, P. (2017). Abscisic acid signaling is controlled by a BRANCHED1/HD-ZIP i cascade in Arabidopsis axillary buds. Proceedings of the National Academy of Sciences of the United States of America, 114(2). https://doi.org/10.1073/pnas.1613199114
González-Grandío, E., Poza-Carrión, C., Sorzano, C. O. S., & Cubas, P. (2013). BRANCHED1
promotes axillary bud dormancy in response to shade in Arabidopsis. The Plant Cell, 25(3), 834–50. https://doi.org/10.1105/tpc.112.108480
Acknowledgements & Funding: This work is supported by MINECO for the grant (BIO2014-
57011-R). Carlos Tarancon is a La Caixa Foundation predoctoral fellow.
18
S-I. Comunicación 1
A HIGH-RESOLUTION SPATIOTEMPORAL MAP OF ROOT
POSTEMBRYONIC ORGANOGENESIS REVEALS NEW STEM
CELL TYPES AND A REGULATORY NETWORK OF IDENTITY
TRANSITIONS
Alvaro Sanchez-Corrionero1, Maria Angels De Luis Balaguer2, Pablo Perez-Garcia1,
Rossangela Sozzani2, Miguel A. Moreno-Risueno1
1Center for Plant Biotechnology and Genomics (CBGP) UPM-INIA, Department of
Biotechnology and Plant Biology, Universidad Politecnica de Madrid, Pozuelo de Alarcon
(Madrid), Spain, 2 Department of Plant and Microbial Biology, College of Agriculture and Natural
Sciences, North Carolina State University, Raleigh, North Carolina, USA.
Corresponding author: Miguel A. Moreno-Risueno ([email protected])
Plants develop continuously through the formation and growth of organs. Organogenesis after embryogenesis starts with the formation of organ founder cells, which have the developmental potential –pluripotency-of giving rise to all distinct tissues which make up an organ. In Arabidopsis thaliana lateral root founder cells divide asymmetrically to generate a large and a daughter cell. Subsequent divisions are not stereotypic but they result in an organized pattern. Although cells with distinctive fates have been identified, little is known about how they are specified during this morphogenic process. We gain further insight into postembryonic organogenesis we have reconstructed a transcriptional map of first stages of lateral root formation in Arabidopsis. We have identified markers expressed at specific times and performed fluorescent activated cell sorting coupled with RNA sequencing. We have profiled expression of 11 markers which recapitulates expression of 8 different cell-types. Analysis of these data recapitulates expression of known regulators of lateral root formation, such as SCARECROW and PLETHORA (Du, Y. et al., 2017; Goh, T. et al., 2016), identifying involvement of specific cellular functions at certain stages of organogenesis. Our data shows that distinctive transcriptomes for founder cells and theirs daughter which leads to formation of a stem cell type which shares certain percentage of identity which the quiescent center of the root apical meristem, based on Spec calculation. We designated this stem cell as organizing stem cell. To further understand identity transitions and cell fate specification between newly formed cell types during organogenesis we performed clustering based on appearance of cell types, which provides developmental time, in combination with dynamic Bayesian network inference, using GENIST method (de Luis Balaguer, M.A. et al., 2017). The inferred network shows characteristic topology, which arranges as a continuous progression with highly connected hubs at the transitions between distinctive cell identities. Involvement of new regulation will be discussed.
References:
de Luis Balaguer MA, et al. (2017) Proc Natl Acad Sci U S A. 114(36):E7632
Du Y, Scheres B. (2017) Proc Natl Acad Sci U S A. 114:11709
Goh T, et al. (2016) Development. 143:3363
Funding: This work was supported by grants from Ministerio de Economía y Competitividad
(MINECO) of Spain and ERDF, BFU2013-41160-P and BFU2016-80315-P.
19
S-I. Comunicación 2
INTERPLAY BETWEEN THE CIRCADIAN CLOCK, THE
PHOTOPERIODIC SIGNAL AND THE FLOWERING TRANSITION
IN Arabidopsis thaliana.
Pedro de los Reyes1, Francisco J. Romero1,2, José M. Romero1, Federico Valverde1.
1Plant Development Unit - Institute for Plant Biochemistry and Photosynthesis, Consejo
Superior de Investigaciones Científicas, Universidad de Sevilla, Seville, Spain,
2Department of Computer Science and Artificial Intelligence, Universidad de Sevilla,
Seville, Spain
Corresponding author: Federico Valverde ([email protected])
Arabidopsis thaliana constitutes a powerful tool to study the molecular systems underpinning physiological and developmental processes such as the circadian clock, the photoperiodic signal and the flowering transition. In this plant, the circadian clock has been shown to act as an input to the photoperiod and flowering pathway allowing CONSTANS (CO) to promote the florigenic gene FT transcription and induce flowering in long day (LD) conditions (1,2). However, up to date, no evidence has been reported regarding a feedback loop from the photoperiod and flowering pathway back to the circadian clock. In this work, we have followed a moleclar systems biology approach to explore this possibility. We have constructed a transcriptional network called CircadianFloralNet integrating Chip-seq and Chip-on-chip data freely available from the GEO database from 33 key transcription factors (TF) in the circadian clock, photoperiod and other flowering pathways using our own RNA-seq data from plants overexpressing CO. Our results, based on identification of TF binding sites, network motifs and differential gene expression analysis, suggest that CO and HY5 regulate and share a common regulome including central genes in the circadian clock at the end of a LD, such as TOC1, PRR5 and PRR7. This is in agreement with recent results that report the interaction between CO and several PRRs (3) and suggests the presence of a bZIP TF, such as HY5, regulating central genes expression in the circadian clock (4,5). In order to experimentally validate this hypothesis, we have shown that CO interacts with HY5 using transient expression in Nicotiana benthamiana. Additionally, we have shown using QPCR that CO overexpression affects in LD conditions the circadian expression patterns of key regulators such as PRR5 and PRR7 and outputs of the clock such as GIGANTEA. Altogether, these results suggest that CO, HY5 and the PRRs could form a complex needed to establish a feedback loop between the circadian clock, the photoperiod and the flowering pathway contributing to the orchestration of these key processes in the life cycle of plants.
References
1- Johansson, M. and Staiger, D. (2015) J. Exp. Bot. 66, 719–30.
2- Valverde, F. (2011) J. Exp. Bot. 62, 2453–63.
3- Hayama, R. et al. (2017) EMBO J 36, 904-918.
4- Liu, T. L. et al. (2016). Plant physiol. 170, 528-539.
5- Zhang, H. et al. (2011) Plant J. 65, 346-358.
Acknowledgements & Funding. The authors would like to thank funding from projects BIO2014-
52425-P and BIO2017-83629-R (Spanish Ministry of Economy and Competitiveness, MINECO)
partially supported by FEDER funding.
29
S-I. Comunicación 3
THE USE OF -OMICS TECHNOLOGIES TO PROGRESS IN THE Quercus ilex BIOLOGY
María-Dolores Rey, Víctor M. Guerrero-Sánchez, Rosa Sánchez-Lucas, Cristina López-Hidalgo, Ana Mª Maldonado-Alconada, Jesús V. Jorrín-Novo
Department Biochemistry and Molecular Biology, Universidad de Córdoba, Córdoba,
Spain.
Corresponding author: María-Dolores Rey ([email protected]) Continuing advances in -omics methodologies are enhancing the understanding of how plants cope with the dynamic nature of their growing environment. - Omics approaches have been only recently extended to cover forest trees, in general, and holm oak (Quercus ilex L. subsp. ballota [Desf] Samp), in particular. The holm oak, despite being a species of regional interest, has received little attention from the point of molecular view. In this context, our research group leaded by Prof. Jesús V. Jorrín has carried out the optimization of several protocols to extract DNA, RNA and protein, protein analysis by mass spectrometry, NIRS analysis, and microscopic analysis to progress in the Q. ilex biology such as natural variation, seed germination and seedling growth, physiology, biotic and abiotic stress-responses and combing classical biochemistry. Moreover, recently, our research group has integrated those multidisciplinary “-omics” analysis (transcriptomics, proteomics and metabolomics) in this forest specie. A reference transcriptome of holm oak (Guerrero-Sánchez et al. 2017) has recently been published, and the first proteome and metabolome will be published very soon. However, the lack of available genomic information for Q. ilex supposes a notable obstacle to successfully carry out these global studies at molecular level. This indicates an urgent need to obtain the first draft of Q. ilex genome. Also, this lack of genomic data has caused a total absence of molecular cytogenetic data in this specie. At present, we are optimizing molecular cytogenetics protocols previously used in other herbaceous species, such as Arabidopsis or wheat, to characterize the genome of holm oak. Once we achieve to sequence the holm oak genome and its characterization by molecular cytogenetics, we will combine them with previous data from transcriptomic, proteomic and metabolomic analysis to obtain a better understanding of molecular mechanisms involved in physiological processes such as seed germination, seedling establishment, response to abiotic and biotic stresses, which are essential for selection of superior phenotypes for restoration and reforestation programs under the impeding climate change in the Mediterranean region.
Guerrero-Sánchez, V.M., Maldonado-Alconada, A.M., Amil-Ruiz, F., and Jorrín-Novo, J.V. (2017) Front. Mol. Biosci. 4:70.
This work is supported by grants from the Spanish Ministry of Economy and Competitiveness
(Project BIO2015-64737-R2).
21
S-I. Comunicación 4
A SYSTEMS BIOLOGY APPROACH UNRAVELS NEW REGULATORY CONNECTIONS BETWEEN UNRELATED miRNA-
TF PAIRS Shubhada Kulkarni2, 3,4, Klaas Vandepoele2, 3,4, Detlef Weigel5, Ignacio Rubio-
Somoza1
1Molecular Reprogramming and Evolution (MoRE) Laboratory, Centre for Research in
Agricultural Genomics (CRAG), Cerdanyola del Vallés, Spain. 2Ghent University, Department of Plant Biotechnology and Bioinformatics, Ghent, Belgium. 3VIB Center for
Plant Systems Biology, Ghent, Belgium. 4Bioinformatics Institute Ghent, Ghent University, Ghent, Belgium 5Department of Molecular Biology, Max Planck Institute for
Developmental Biology, Tübingen, Germany.
Corresponding author: Ignacio Rubio-Somoza ([email protected]) Micro-RNAs (miRNAs) are key regulators embedded in the genetic networks that orchestrate development and stress responses. The relationship between plant miRNAs and transcription factors (TFs) is notably asymmetrical, with 20% of miRNA families in the Arabidopsis thaliana genome regulating TFs, but only 3% of all TFs being under miRNA regulation. Some of the TFs engage in common regulatory complexes and share downstream targets. Thus, miRNAs would be selecting the TFs present in a given protein complex and their combinatorial regulatory outcome. In order to better define the pathways under the control of unrelated miRNA-TF pairs during plant development, we have undertaken a systems biology approach. We have leveraged techniques based on next generation sequencing (ChIP- and RNA-seq), protein-protein interactions and genetic epistasis tests to confirm and better describe formerly known and new miRNA-TF interactions in regulatory circuits. We focus on six different miRNAs and ten of their TF targets that together tailor both leaf and root development. We will present results revealing new connections between unrelated miRNA-TF pairs and the processes under their regulation.
Acknowledgements and funding: We acknowledge the guidance and support from Christa Lanz and Julia Hildebrandt in NGS library preparation and sequencing. We are grateful to Michael Chistie, Jia-Wei Wang, Elliot Meyerowitz, Stephan Wenkel and Yuhai Cui for donating seeds and plasmids used in this work.
The work in the MoRE group is funded by Spanish Ministry of Economy and Competitiveness
(I.R-S is supported by BFU-2014-58361-JIN, RYC-2015-19154; L. V-M is supported by BES- 2016-
076989) and through the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016-
2019 (SEV-2015-0533) and the CERCA programme from the Generalitat de Catalunya. Work at
MPI is funded by the Max Planck Society. S.R.K. is funded by Research Foundation– Flanders
[G001015N].
22
S-I. Póster 1
WBGS ANALYSIS OF m5C-METHYLATION THROUGHOUT THE
RIPENING OF Fragaria vesca FRUITS
Félix Juan Martínez-Rivas, Rosario Blanco Portales, Francisco Javier Molina
Hidalgo, Enriqueta Moyano Cañete, José Luis Caballero, Juan Muñoz Blanco
and Antonio Rodríguez Franco,
Departamento de Bioquímica y Biología Molecular. Universidad de Córdoba. Campus
de Rabanales. 14071 Córdoba
Corresponding author: Antonio Rodríguez Franco ([email protected])
Ripening of fleshy fruits is clearly a process regulated by epigenetic marks, involving the methylation and demethylation of key genes implied in this process. This is very well characterized in climacteric fruits such as tomato, where key genes implied in the ripening of this fruit are regulated by cytosine methylation in the CpG, CHH and CHG contexts (Gallusci et al, 2016). The analysis of this process in non-climacteric fruits is, however, very scarce. In tomato, a treatment with the demethylating agent 5-Azacitidine, lead to and advancement of the ripening of this fruit (Zhong et al, 2013). In strawberries however, the treatment with this agent lead to a substantial delay in the ripening. This means that the methylation status of key genes is also involved in the ripening of this non-climacteric fruit and that the genes controlling this process are substantially different of those of climacteric plants. We are analysing by means of massive WBGS sequencing the cytosine methylation of DNA extracted from diploid strawberries fruits (Fragaria vesca) in the green and red stages. This was done by treating DNA with bisulfite, by sequencing through Illumina, by mapping with bismark, and analysing the data by using bioinformatic approaches. An RNA-Seq analysis has also been done in parallel from these same fruits. Here, we present preliminary data showing the general genomic methylation state of the DNA in these two stages of fruit development, and an analysis of the DMR regions and of several promotors from key genes regulating this process. All these results will be discussed to build a model able to explain how methylation is involved in the ripening of non-climacteric fruits.
References
• Gallusci P, Hodgman C, Teyssier E, Seymour GB (2016) DNA methylation and
chromatin regulation during fleshy fruit development and ripening. Front. Plant Sci.
7:807. doi: 10.3389/fpls.2016.00807
• Zhong S, Fei Z, Chen Y, Zhen Y, Huang M, Vrebalov J, Mqquinn R, Gapper N, Liu B,
Xiang J, Shao Y, Giavannoni (2013). Single base resolution methylomes of tomato fruit
development reveal epigenome modifications associated with ripening. Nat Biotechnol
31: 154-159. doi:10.1038/nbt.2462
Acknowledgements & Funding: This work was funded by grants AGL2014-55784-C2-2-R and
AGL2017-86531-C2-2-R.
RBP was supported by “Ramón y Cajal” programme from the Spanish Ministerio de Ciencia e
Innovación.
23
S-I. Póster 2
DELLA PROTEINS ACTED AS TRANSCRIPTIONAL HUBS IN
THE COMMON ANCESTOR OF ALL LAND PLANTS
Asier1 Briones-Moreno1, Jorge1 Hernández-García1, Carlos2 Vargas-Chávez2,
David1 Alabadí1, Miguel A.1 Blázquez1
1Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de
Investigaciones Científicas – Universidad Politécnica de Valencia, Valencia, Spain,
2Institute for Integrative Systems Biology (I2SysBio), University of Valencia, Valencia,
Spain
Corresponding author: Miguel A. Blázquez ([email protected])
DELLA proteins are land plant-specific transcriptional regulators known to interact through their C-terminal GRAS domain with over 100 transcription factors (TFs) in Arabidopsis, where they undergo proteasome-mediated degradation in response to gibberellins; therefore, they are proposed to be hubs that relay environmental information to pre-wired transcriptional circuits. To understand the distinct molecular features that characterise hubs of gene regulatory networks, we have investigated the origin of DELLA function and the acquisition of their ‘hub' properties. We have found that surface conservation of the GRAS domain of DELLA proteins is much higher than that of other GRAS proteins, suggesting that promiscuity is a trait that can be selected for. Large- scale systematic analysis of DELLA-TF interactions in seven species covering all major taxa in the land plant lineage have indicated that the ancestral DELLA protein, but not other GRAS ancestors, already displayed a remarkable ability to connect multiple transcriptional programs and that this advantageous property has been actively maintained. Finally, we have used comparative network analysis to understand the functional advantage provided by DELLAs to transcriptional regulation, and we have found an increase in the coordination between otherwise independent processes in plants with DELLAs, which is even more apparent in plants displaying GA-dependent DELLA regulation.
24
S-I. Póster 3
METABOLOMIC CHARACTERIZATION OF STRAWBERRY
CULTIVARS DURING POSTHARVEST
Delphine Pott1, Lothar Willmitzer2, Alisdair R. Fernie2, Sonia Osorio1 José G.
Vallarino2
1Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, University of
Malaga-Consejo Superior de Investigaciones Científicas, Department of Molecular
Biology and Biochemistry, Campus de Teatinos, 29071 Málaga, Spain. 2Max-Planck-
Institute für Molekulare Planzenphysiologie, Am Mühlenberg 1, 14476 Golm, Germany.
Corresponding authors: Sonia Osorio ([email protected]) and José G. Vallarino
Strawberry (Fragaria x ananassa) is the berry most consumed worldwide, being
well-known for its delicate flavour and nutritional characteristics. However,
strawberries possess a very short postharvest shelf-life, and its extension is a
major economic goal. Measures are commercially taken to delay senescence,
including low temperature and controlled atmosphere (Pedreschi and Lurie,
2015). To improve our understanding of the mechanisms underlying the
deterioration of fruit quality during senescence, we monitored the metabolomic
profiles of five strawberry cultivars under different postharvest treatments. Ripe
fruits of Fragaria x ananassa were kept at 4ºC during three, six and ten days in
normal, CO2 and O3-enriched atmospheres. We used a combination of gas
chromatography-mass spectrometry (GC-TOF-MS), ultra-performance liquid
chromatography-Orbitrap mass/mass spectrometry (UPLC-Orbitrap-MS/MS)
and headspace solid phase micro extraction (HS-SPME) coupled with GC-MS
to semi-quantify 49 primary metabolites, 132 polar secondary metabolites and
70 volatiles. Multivariate statistical approaches and network-based methods will
allow us to point out the regulatory factors underlying fruit senescence.
Pedreschi R, Lurie S. 2015. Advances and current challenges in understanding postharvest
abiotic stresses in perishables. Postharvest Biology and Technology 107, 77–89.
The work was supported by the MINECO (grant AGL2012-40066-C02-02, Spain). SO
acknowledges the support by Spanish Ministry of Science and Innovation (Ramón and Cajal
contract, RYC2011-09170). DP has been granted with a PhD fellowship from MINECO (grant
BES-2013-062856). The authors also acknowledge the support by the Plan Propio from
University of Malaga, Campus de Excelencia Internacional de Andalucía. This project has
received funding from the European Union’s Horizon 2020 research and innovation programme
under Grant Agreement Number 679303.
25
S-I. Póster 4
METHYLATION STATUS GOBERNS STRAWBERRY (Fragaria x
ananassa) FRUIT RIPENING. Félix Juan Martínez-Rivas1, Rosario Blanco-Portales1, Francisco Javier Molina-Hidalgo1,
Enriqueta Moyano1, José Luis Caballero1, Juan Muñoz Blanco1 and Antonio Rodríguez-Franco1
Department of Biochemistry and Molecular Biology, University of Cordoba, Córdoba, Spain. Corresponding author: Antonio Rodriguez Franco ([email protected])
Fruit ripening is a genetically programmed process having distinctive and irreversible stages. In strawberry, those changes lead to the development of a soft and edible red receptacle. Many changes take place at the level of DNA methylation, which plays a key role in many developmental processes in plants. This process is well known in climacteric fruits such as tomato, but in general, there is a lack of information in non- climacteric fruits such as the strawberry (Gallusci et al., 2016). The demethylating agent 5-azacytidine (5-AZA) has been used in plants mainly in seeds (Xu et al., 2016) and in tomato fruits (Zhong et al., 2013). We analysed the changes taking place in green fruits that were injected with 5-AZA. This inhibitor clearly leads to a delay of the ripening of strawberry, as the injected part remained white, while the control zones turned to red. Total RNA was extracted from both parts. RNA-Seq was performed after mapping the reads using Kallisto. Data were imported and analysed under R with the tximport and DESeq2 packages. A total of 1916 genes were significative (p<0.01) down regulated (log2FoldChange>2) while 3216 genes were significative up regulated in the red region versus the white one. Medina-Puche et al., 2016 describe up to 10 gene clusters involved in ripening of the strawberry. These clusters were used to clarify the role played by methylation patterns in strawberry ripening process. Aroma related genes, as acetyl-transferases, and phenylpropanoids synthesis genes, as well as cell wall related genes, as polygalacturonases and RGlyases were down regulated indicating a general delay on secondary metabolism and ripening process. Among these results, some transcription factors which are described to regulate these pathways were down regulated, as FaMYB10 and FaEOBII (Medina-Puche et al., 2014; Medina-Puche et al., 2015). Also, key genes on ABA biosynthesis pathway, as FaNCED1 (Jia et al., 2013), phytoene synthase and neoxanthin synthase were down regulated. Some other, like those responsible for the degradation of ABA were clearly induced Taken together these results seems to indicate that methylations status could regulate the overall process of ripening in strawberry.
• Gallusci P, Hodgman C, Teyssier E, Seymour GB 2016 DNA methylation and chromatin regulation
during fleshy fruit development and ripening. Front. Plant Sci. 7:807.
• Jia HF, Chai YM, Li CL, Lu D, Luo JJ, Qin L, Shen YY. 2011. Abscisic acid plays an Important Role in the regulation of strawberry fruit ripening. Plant Physiol,157(1):188-99.
• Medina-Puche L, Blanco-Portales R, Molina-Hidalgo FJ, Cumplido-Laso G, García-Caparrós N, Moyano-Cañete E, Caballero-Repullo JL, Muñoz-Blanco J, Rodríguez-Franco A. 2016. Extensive transcriptomic studies on the roles played by abscisic acid and auxins in the development and ripening of strawberry fruits. Funct Integr Genomics. 16:671-692.
• Medina-Puche L Cumplido-Laso G, Amil-Ruiz F, Hoffmann T, Ring L, Rodríguez-Franco A, Caballero
JL, Schwab W, Muñoz-Blanco J, Blanco-Portales R. 2014. MYB10 plays a major role in the regulation
of flavonoid/phenylpropanoid metabolism during ripening of Fragaria × ananassa fruits- J Exp Bot.
65;401-417
• Medina-Puche L, Molina-Hidalgo FJ, Boersma M, Schuurink R, López-Vidriero I, Solano R, Franco Zorrilla JM, Caballero JL, Blanco-Portales R, Muñoz-Blanco J. 2015. An R2R3-MYB transcription factor
regulates eugenol production in ripe strawberry fruit receptacles. Plant Physiol. 168(2) 598-614.
• Xu J, Tanino KK, Horner KN, Robinson SJ. 2016 Quantitative trait variation is revealed in a novel hypomethylated population of woodland strawberry (Fragaria vesca). BMC Plant Biol. 4;16(1):240
• Zhong S, Fei Z, Chen Y, Zhen Y, Huang M, Vrebalov J, Mcquinn R, Gapper N, Liu B, Xiang J, Shao Y, Giovannoni JJ 2013 Single-base resolution methylomes of tomato fruit development reveal epigenome
modifications associated with ripening. Nat. Biotechnol.
This work was supported by AGL2014-55784-C2 from the Spanish Ministerio de Ciencia e Innovación.
RBP was supported by “Ramón y Cajal” programme from the Spanish Ministerio de Ciencia e Innovación.
26
S-I. Póster 5
THE EMBRYONIC PHOTORECEPTOR CONNECTION MEDIATED
BY HSFA9 ENHANCES VERY EARLY RESPONSES TO BLUE-
AND UV B-LIGHT
Pilar Prieto-Dapena1, Concepción Almoguera1, and Juan Jordano1
1Seed Biotechnology Laboratory, IRNAS (C.S.I.C.), Sevilla, Spain.
Corresponding author: Juan Jordano ([email protected])
HSFA9 is a seed-specific transcription factor involved in desiccation tolerance and
seed longevity (Prieto-Dapena et al., 2008). In sunflower plants, HSFA9 disappears a
few days after seed germination (Almoguera et al., 2002). This "expression window"
supports the involvement of HSFA9 in the initial acquisition of photosynthetic
competence, which occurs immediately after seedling emergence. Within this timing,
we have showed that HSFA9 could enhance red-, and far-red, light responses through
different phytochrome photoreceptors (Prieto-Dapena et al., 2017). Here we will
present evidence that broadens the spectrum of photoreceptors functionally connected
to HSFA9. Transcriptomics analyses of HSFA9-overexpessing plants suggested links
of HSFA9 with blue-light receptors (i.e., Cryptochromes and Phototropins), and with
UVR8, the main ultraviolet-B (UV-B) light receptor. In addition, UVR8-independent UV-
B responses might also be affected by HSFA9. Gain-of-function and loss-of-function
evidence supporting some of these links in transgenic tobacco will be presented. We
will discuss different direct, or indirect, effects of HSAF9 that are compatible with our
current results. We propose that HSFA9 may be very useful for the simultaneous
improvement of morphogenic light responses and stress tolerance. Our work could
facilitate the future engineering of crops that resist enhanced UV-B irradiation, and that
also show resistance to high temperatures and frequent episodes of severe drought;
therefore, crops better fitted to the effects of global climate change.
References
Almoguera, et al. (2002) A seed-specific heat-shock transcription factor involved in
developmental regulation during embryogenesis in sunflower. J. Biol. Chem. 277(46): 43866-
43872.
Prieto-Dapena, et al. (2008) The ectopic overexpression of a seed-specific transcription factor,
HaHSFA9, confers tolerance to severe dehydration in vegetative organs. Plant J. 54(6): 1004-
1014.
Prieto-Dapena, et al. (2017). Seed-specific transcription factor HSFA9 links late embryogenesis
and early photomorphogenesis. J. Exp. Bot. 68: 1097-1108
Acknowledgements & Funding. This work has been funded by FEDER (European
Regional Development Fund) and by “Secretaría de Estado de Investigación,
Desarrollo e Innovación” (projects BIO2014-52303-R and BIO2017-82172-R).
Additional funds were obtained from “Junta de Andalucía” (Group BIO148).
27
S-I. Póster 6
THE EMERGING ROLE OF SUBTELOMERIC
HETEROCHROMATIN ON TELOMERES BIOLOGY
Alejandro Vega-Vaquero,1 Giancarlo Bonora,2 Marco Morselli,2
María I. Vaquero-Sedas,3 Liudmilla Rubbi,2 Matteo Pellegrini,2
and Miguel A. Vega-Palas3
1Technical Superior School of Informatics Engineering, University of Seville, Seville,
Spain; 2Department of Molecular, Cell and Developmental Biology, University of
California, Los Angeles, California, USA; 3Institute of Vegetal Biochemistry and
Photosynthesis, CSIC-University of Seville, Seville, Spain
Corresponding author: Miguel Ángel Vega-Palas ([email protected])
Cytosine methylation regulates the length and stability of telomeres, which can affect a wide variety of biological features, including cell differentiation, development, or illness. Although it is well established that subtelomeric regions are heterochromatic and undergo high levels of DNA methylation, the presence of methylated cytosines at telomeres has remained controversial. We have analyzed multiple bisulfite sequencing studies to address the methylation status of Arabidopsis thaliana telomeres. We found that the levels of estimated telomeric DNA methylation varied among studies. Interestingly, we estimated higher levels of telomeric DNA methylation in studies that produced C-rich telomeric strands with lower efficiency. However, these high methylation estimates arose due to experimental limitations of the bisulfite technique. We found a similar phenomenon for mitochondrial DNA: The levels of mitochondrial DNA methylation detected were higher in experiments with lower mitochondrial read production efficiencies. Based on experiments with high telomeric C-rich strand production efficiencies, we concluded that Arabidopsis telomeres are not methylated, which was confirmed by methylation-dependent restriction enzyme analyses. Thus, our studies indicate that telomeres are refractory to de novo DNA methylation by the RNA-directed DNA methylation machinery. This result, together with previously reported data and our unpublished results, reveal that subtelomeric DNA heterochromatin controls the homeostasis of telomere length.
References:
-Vega-Vaquero, A., Bonora, G., Morselli, M., Vaquero-Sedas, M., Rubbi, L., Pellegrini, M. and
Vega-Palas, M. (2016) Novel features of telomere biology revealed by the absence of telomeric
DNA methylation. Genome Research26: 1047-1056.
-Vaquero-Sedas et al. Unpublished results.
28
S-I. Póster 7
OMICS EXPLORATION OF THE UNFOLDED PROTEIN
RESPONSE IN ARABIDOPSIS
Sharareh Ghasemi 1, Miguel Ángel Delgado-Gutiérrez 1, Raquel Iglesias-
Fernández 1, José Domínguez-Figueroa 1, Joaquín Medina 1, Stephan
Pollmann, Jesús Vicente-Carbajosa 1
1Centre for Plant Biotechnology and Genomics, Madrid Technical University, Madrid,
Spain, Corresponding author: Jesús Vicente-Carbajosa ([email protected])
Eukaryotic cells under certain physiological and environmental conditions
produce a massive accumulation of proteins in the endoplasmic reticulum,
leading to the “endoplasmic reticulum stress”. Return to homeostasis is
achieved through the activation of the UPR (unfolded protein response), a
complex pathway that alleviates from toxic misfolded proteins and their effects.
In recent years, a significant advance has taken place in the unravelling of the
ER-stress perception and response pathways in different eukaryotic organisms.
Nevertheless, the current knowledge of these pathways still lags behind in the
case of plants (Howell SH, 2013). We have initiated a search for novel
components in ER-stress signalling and UPR in the model plant Arabidopsis,
using genetic and systems biology approaches (Hossain A, 2016). Here, we
present an integrative strategy based on the analyses of transcriptomic data
together with multiple yeast two-hybrid screenings, using previously known ER-
stress components in Arabidopsis. Our results identified key elements involved
in different abiotic/biotic stresses linked to ER-stress, autophagy and cell death
processes.
References:
Hossain A, et al. Identification of novel components of the Unfolded Protein Response in
Arabidopsis. Front Plant Sci (2016) 7: 650
Howell, SH (2013). Endoplasmic reticulum stress responses in plants. Annu. Rev. Plant Biol. 64,
477-499
Funding: MINECO. BIO2017-82873-R
29
S-I. Póster 8
DISCOVERY OF NOVEL ENVIRONMENTAL TOLERANCE AND
WOOD QUALITY BIOMARKERS IN Pinus pinaster TRHOUGH
INTEGRATION OF NATURAL VARIATION AND SYSTEMS
BIOLOGY
Mónica Meijón1, Isabel Feito2, Juan Majada3, Luis Valledor1
1Plant Physiology, Faculty of Biology, University of Oviedo, Oviedo, Spain.2Regional Institute for
Research and Agro-Food Development in Asturias (SERIDA), Experimental Station “La Mata”,
Grado, Spain. 3CETEMAS, Carbayin Bajo, Spain.
Corresponding author: Luis Valledor ([email protected])
Natural variation of the metabolome and proteome of Pinus pinaster was studied to improve our understanding of phenotypic diversity, and wood quality. The metabolome and proteome of needles and the apical and basal section of buds were analyzed in three provenances of P. pinaster with contrasting growth capacity selected from mountain in the northwest (CDVO) to the coastal region of southeast Spain (ORIA) also considering a provenance from a sandy Moroccan area (TAMR). The three provenances were grown in a common garden for five years and metabolite and protein extraction were performed from the same sample. For metabolite detection two complementary mass spectrometry techniques: GC-MS and LC-Orbitrap-MS were used, while for protein identification GeLC-Orbitrap/MS combined with the development of custom peptide databases was used. Metabolome, proteome and environmental and growth data were integrated employing modelling and statistical tools to provide a comprehensive picture of phenotypic diversity.
A total of 1576 metabolites and 2069 proteins were identified. The characteristic metabolites of each tissue are related to primary metabolism, while provenances were distinguishable when tissues were analysed independently (Meijón et al., 2017) being secondary metabolites, and in particular flavonoid and terpenoid pathways, essential to reach this differential clustering. In relation to proteome, according to the MapMan categories, the proteins that participate in the synthesis of tetrapyrrole, mitochondrial electron transport or in the metabolism of amino acids are overaccumulated in TAMR while in CDVO the metabolism of nitrogen in the apical part of the bud. Integrative studies of metabolome, proteome and physiological parameters showed strong separation of the three population. Key proteins linked by sPLS networks to wood quality traits were identified in this analysis, such as, LIPOXYGENASE related to abscisic and jasmonic acids response according to Melan et al. (1993) and Pauwels et al. (2008). Altogether these results provide a new perspective of how tree metabolism adapt to different environment, and how these adaptions are also reflected in wood quality, providing these results a new set of biomarkers for breeding programs and forest management practices.
References:
Melan MA, et al. (1993) Plant Physiology 101:441-450.
Pauwels L, et al. (2008) Proceedings of National Academy of Sciences of the USA 105: 1380-
1385.
Acknowledgements & Funding: This work is an output of the VAMPIRO project financed by the
Spanish Ministry of Economy, Industry and Competitiveness (AGL2017-83988-R). MM and LV
were generously supported by Ramón y Cajal program (Spanish Ministry of Economy, Industry
and Competitiveness, grant references RYC-2014- 14981 and RYC-2015-17871, respectively).
39
S-I. Póster 9
DECIPHERING THE Quercus ilex TRANSCRIPTOME USING
COMPLEMENTARY SEQUENCING AND ASSEMBLY
STRATEGIES
Víctor M. Guerrero-Sánchez1, Ana M. Maldonado-Alconada1, Francisco Amil-
Ruiz2, María-Dolores Rey1, Jesús V. Jorrín-Novo1.
1Agroforestry and Plant Biochemistry, Proteomics and Systems Biology; University of
Cordoba; Cordoba, Spain
2Servicio Central de Apoyo a la Investigación, University of Córdoba, Córdoba, Spain,
Corresponding author: Víctor Manuel Guerrero Sánchez ([email protected]) Holm oak (Quercus ilex L.) is the dominant tree species in the Mediterranean forest
with great ecological and economic value. Holm oak fruit, the acorn is used for feed
and fatten the exclusive Iberian race pigs. Acorns have also been used for human
nutrition and it was reported to have interesting antioxidant compounds (Vinha et al.
2016), which have been associated with anti-tumoral, and anti-inflamatory activities.
The holm oak genome has not yet been sequenced, which causes an important
limitation in the identification of gene sequences in response to abiotic and biotic
stresses. Recently, our group published a de novo transcriptome assembly of Q. ilex
(Guerrero-Sánchez et al. 2017). Quercus ilex transcriptome does not only provide
interesting information about its biology, but also it can be used to carry out a genetic
study of drought response, being drought stress the main cause of mortality of holm
oak seedlings. Here, we present an improved transcriptome of Q. ilex generated by
using two sequencing platforms reads (Illumina and Ion Torrent) for a new and better
transcriptome assembly, making a comparison with orthologous plant sequences
selecting the best workflow for the annotation. We improved the transcriptome of holm
oak, with less redundancy and a higher completeness of the transcripts. All the genes
related to drought stress responses previously identified in our group (Simova-Stoilova
et al. 2015), were found in the holm oak transcriptome annotations. Moreover, up to 14
new gene groups related with drought stress were identified in holm oak for the first
time. Understanding the molecular mechanics of gene expression in holm oak, we
could tackle the drought stress, improve the production of acorns and select interesting
varieties for human nutrition.
Guerrero-Sanchez VM et al. (2017) Frontiers in Molecular Biosciences. 4:70 Simova-
Stoilova, L. P., et al. (2015) Frontiers in plant science. 6: 627.
Vinha, A. F. et al. (2016) Comprehensive Reviews in Food Science and Food Safety. 15(6),
947-98
31
SESIÓN II
BIOTECNOLOGÍA VEGETAL
APLICADA
MODERADORES:
Javier Pozueta (IdAB,
CSIC/UPNA/Gobierno de
Navarra, Navarra)
Antonio Granell (IBMCP, CSIC-
UPV, Valencia)
32
S-II. Ponencia 1
BREEDING AND ENGINEERING THE VOLATILE NETWORK IN TOMATO: NOT JUST FOR TASTE
Rambla JL1, Medina A1, Fernandez-Munoz R2, Tieman D3, Garcia-Martinez S4, Perez-Hedo M5, Tikunov Y6, Bovy A6, Urbaneja A5, Ruiz JJ4, Klee HJ3, Monforte
AJ1, Granell A1
1Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), Ingeniero Fausto Elio s/n, 46022 Valencia, email: [email protected] 2Univ Malaga, CSIC, Inst Hortofruticultura Subtrop Mediterranea, Malaga 29750, Spain 3Univ Florida, Dept Hort Sci, Gainesville, FL 32611 USA 4EPSO UMH, Dept Biol Aplicada, Ctra,Beniel Km 3,2, Alicante 03312, Spain 5IVIA, Unidad Entomol UJI IVIA, Ctr Protecc Vegetal & Biotecnol, Carretera Moncada Naquera Km 4,5, Valencia 46113, Spain 6Wageningen Univ & Res, Plant Breeding, Wageningen, Netherlands
Volatile compounds are specialized metabolites plants produce and use to convey and exchange information on specific organs and their interaction with the environment. There are two main scenarios where tomato volatiles have a major impact in AgriFood. On the one hand tomatoes produce a define set of volatiles that determine fruit aroma and flavor with consequences in consumer preferences. On the other hand volatiles produced by leaves inform insects/pests/plant about the plant nature/status and its understanding is necessary for a successful integrated pest management strategy. We will describe the volatile network of tomato fruit, the underlying genetic and molecular program and how the ability of fruit volatiles evolved during early domestication and modern breeding. We will illustrate examples on the use of genetic resources in combination with high throughput genotyping and phenotyping and how this is contributing to 1) identifying genomic regions and markers that can be used to guide breeding for the production of specific volatiles and to the identification of the underlying genes, and 2) to designing of strategies for identifying the key genes and how to use this knowledge to engineering tomato plants for improved flavor and modifying the interaction with pests/ beneficial insects for better pest management practice.
33
S-II. Ponencia 2
THE “BAD LITTLE FRAGRANT CRITTERS, BENEFICIAL WORKERS” CONCEPT: A STORY OF SERENDIPITY AND DIRTY
DISHES WITH IMPORTANT BIOTECHNOLOGICAL IMPLICATIONS
Ángela María Sánchez-López1, Goizeder Almagro1, Pablo García-Gómez1,
Marouane Baslam1,2, Toshiaki Mitsui2, Edurne Baroja-Fernández1, Francisco José Muñoz1, Abdellatif Bahaji1, Adriana Ricarte-Bermejo1, Kinia Ameztoy1,
Nuria De Diego3, Lukás Spíchal3, Karel Dolezal3 and Javier Pozueta-Romero1
1Instituto de Agrobiotecnología (CSIC/UPNA/Gobierno de Navarra). Iruñako etorbidea
123, 31192 Mutiloabeti, Nafarroa, Spain, 2Graduate School of Science and Technology and Department of Applied Biological Chemistry, Niigata University, 2-8050 Ikarashi,
Niigata 950-2181, Japan, 3Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science,
Palacký University, Olomouc, CZ-78371, Czech Republic.
Corresponding author: Javier Pozueta-Romero; E-mail: [email protected]
Microbes emit complex mixtures of volatile compounds (VCs) that are produced as part of their metabolisms. It is well known that beneficial bacteria and fungi produce volatiles that promote plant growth as well as developmental and metabolic changes. However, in a case of serendipity, we have recently shown that this capacity is not restricted to beneficial microorganisms, but also extends to plant pathogens. When Arabidopsis plants were exposed to airborne signals released by diverse microbial phytopathogens, growth promotion was accompanied by enhanced photosynthesis, accelerated flowering and leaf starch over-accumulation through non-canonical biosynthetic pathway(s). Short- term exposition to VCs emitted by the fungal phytopathogen A. alternata and plant growth promoting bacteria induced similar transcriptomic changes in Arabidopsis, indicating that plants react to microbial VCs through highly conserved mechanisms. However, proteomic analyses of plants exposed to A. alternata VCs revealed that nearly 20% of the proteins that were differentially regulated by VCs are encoded by genes whose expression also was differentially regulated by fungal VCs (Sánchez-López et al. 2016a). Furthermore, quantitative thiol site-specific redox proteomic analyses showed that the plant´s response to VCs is associated with global reduction of the proteome, indicating that plant´s response to microbial VCs is largely subject to post-transcriptional regulation. Notably, irrigation of plants of agronomic interest with solutions containing microbial volatiles increased yield. These findings extended knowledge of the diversity and complexity of the mechanisms involved in modulation of plant physiology and growth when plants interact with microorganisms, and raised questions regarding their ecological significance. Based on these findings the “bad little fragrant critters, beneficial workers” concept was proposed according to which non-beneficial microorganisms represent both a yet to be explored promising source of environmentally friendly biostimulants with considerable biotechnological potential.
34
S-II. Comunicación 1
BIOTECHNOLOGICAL APPROACHES TO INCREASE BIOMASS
PRODUCTION IN TREES
Mª Belén Pascual, Marina Rueda-López, Francisco M. Cánovas, Concepción
Ávila
1Department of Molecular Biology and Biochemistry, Málaga University, Málaga, Spain.
Corresponding author: Mª Belén Pascual ([email protected])
Nutrient use efficiency is one of the factors influencing growth and therefore of high importance for biomass production in trees. Poplar is a model tree widely used for molecular and functional studies and the characterization of transgenic poplars overexpressing structural and regulatory genes involved in glutamine biosynthesis has provided insights on how glutamine metabolism is involved in N economy and biomass production in woody plant models. Numerous studies have shown the relevance of GS isoenzymes in plant development, biomass production, and yield (Cánovas et al. 2006; Castro-Rodríguez et al. 2015). In this communication two examples of functional analysis of plant genes in poplar, and their potential interest for biotechnological approaches are presented (Pascual et al. 2018; Rueda-López et al. 2017). Overexpression of cytosolic NADP+-isocitrate dehydrogenase (ICDH), one of the major enzymes involved in the production of 2-oxoglutarate for amino acid biosynthesis in plants, yields poplar trees with increased growth and enhanced vascular development in young leaves and apical stems. These plants also show an increased expression of genes associated with vascular differentiation and altered amino acids and organic acids content (Pascual et al. 2018). In other study, we observed that overexpression of Dof5, a transcriptional regulator of lignin production and the carbon-nitrogen balance, produced poplar trees with increased growth and biomass production when N availability in the soil is sufficient (Rueda-López et al. 2017). Taken together, these results suggest a close relationship between carbon and nitrogen metabolism and highlights the relevance of glutamine and glutamate biosynthesis in the control of growth and development.
Research supported by Spanish Ministry of Economy and Competitiveness and Junta de Andalucía (Grants BIO2015-69285-R, BIO2012-0474 and research group BIO-114).
Cánovas et al. (2006). In Tree Transgenic. Recent Developments. Heildelberg: Springer Verlag. Castro-Rodríguez et al. (2015). BMC Plant Biology 15:20 Pascual et al. (2018). Tree Physiology (in Press) Rueda-López et al. (2017). PLoS ONE 12 (4): e0174748
35
S-II. Comunicación 2 USE OF Zucchini yellow mosaic virus TO EXPRESS COLD
RESISTANCE GENES IN Cucurbita pepo
Raquel Jiménez-Muñoz1, Fátima Carvajal1,2, Francisco Palma1, Amada Pulido1,
Manuel Jamilena3, Diana Leibman4, Amit Gal-On4, Dolores Garrido1
1Department of Plant Physiology, University of Granada, Granada, Spain 2 Plant
Research International, 6700 AA Wageningen, The Netherlands 3Department of
Biology y Geology, Agrifood, (ceiA3), University of Almería, Spain, 4Department of
Virology, Agricultural Research Organization, the Volcani Center, Bet Dagan, Israel.
Corresponding author: Dolores Garrido Garrido ([email protected])
Plant RNA viruses are a powerful genetic tool for rapid assessment of plant
gene functions in the post-genomic era. Viruses can be used for silencing and
overexpression of endogenous genes providing new knowledge about the
genes implicated in plant stress, development and defence responses. The use
of this tool facilitates a rapid and useful comparation of over and knockdown
phenotypes of the same gene in different cultivars using the same vector.
Moreover, it is especially useful for genes that cause embryo lethality and for
plants that are recalcitrant for transformation. ZYMV-AGII (Zucchini yellow
mosaic virus-AGII) is a recombinant attenuated potyvirus vector system widely
used in cucurbits, since it does not elicit the severe phenotype or developmental
impairment caused by wild-type virus, and mild or no symptoms are developed
in Cucurbita pepo (Gal-On, 2000). The aim of our work is to use this technology
for overexpression or silencing genes that are implicated in cold resistance in
zucchini fruit during postharvest. From a transcriptomic comparison of zucchini
cultivars that show contrasting behaviour to chilling injury after cold postharvest
we have selected several genes that appear to be important for chilling
resistance (Carvajal et al, 2018). In this work we describe the use of this vector
to express some of the candidate genes.
Carvajal, F., Rosales, R., Palma, F., Manzano, S., Cañizares, J., Jamilena, M., & Garrido, D. (2018). Transcriptomic changes in Cucurbita pepo fruit after cold storage: differential response between two cultivars contrasting in chilling sensitivity. BMC genomics, 19(1), 125.
Gal-On, A. (2000). A point mutation in the FRNK motif of the potyvirus helper component- protease gene alters symptom expression in cucurbits and elicits protection against the severe homologous virus. Phytopathology, 90(5), 467-473.
Acknowledgements & Funding
This research has been funded by FPI Grant (MEC) in the Project AGL2014-54598-C2-R.
36
S-II. Comunicación 3
MULTIPLE ORIGINS OF WHITE STRAWBERRIES THROUGH
INDEPENDENT MUTATIONS IN A SINGLE TRANSCRIPTIONAL
REGULATOR: FvMYB10
Cristina Castillejo1, Julie Caruana2, Nicolás Oiza1, José Vallarino3, Pilar Muñoz-
del Rio1, Juan C. Triviño4, Sonia Osorio3, Zhongchi Liu2, Timo Hytönen5, José
F. Sánchez-Sevilla1, Iraida Amaya1
1Genómica y Biotecnología, IFAPA Centro de Churriana, Málaga, Spain. 2Department of Cell
Biology and Molecular Genetics, University of Maryland, MD 20742, USA. 3Department of
Biochemistry and Molecular Biology, IHSM-University of Málaga-CSIC, Málaga, Spain.
4Sistemas Genómicos, Valencia, Spain. 5Department of Agricultural Sciences, Viikki Plant
Science Centre (ViPS), University of Helsinki, Helsinki, Finland.
Corresponding author: Iraida Amaya ([email protected])
Strawberries are synonymous with the color red. However, there are numerous white strawberries as well. Anthocyanins are the pigment responsible for fruit coloration. The expression of anthocyanin biosynthetic pathway enzymes is controlled at the transcriptional level by a protein complex consisting of R2R3-MYB and bHLH transcription factors associated with a WD40-repeat protein.
In order to map the locus responsible for the white berry phenotype we performed a bulk segregant analysis (BSA) linked with high-throughput genome sequencing using a segregating population derived from a F. vesca accession bearing white fruits (ESP138.596) and ‘Reine des Vallées’, with red fruits. Whole-genome sequencing was performed on two pools of DNA from white- or red-fruited F2 plants. Significant SNPs were only detected in chromosome 1, with the higher ΔSNP-index spanning a region from 11.1 to 18.5 Mb. Non-synonymous mutations or frame-shifts were detected in ~300 genes, including the R2R3-MYB FvMYB10. Previous studies showed that white/yellow fruits of several F. vesca accessions result from a single nucleotide mutation on FvMYB10. This mutation was not present in the white-fruited ESP138.596 accession. Instead, we identified a gypsy-like retrotransposon inserted in the third exon of FvMYB10 which introduces a premature STOP codon. The truncated protein obtained lacks its C-terminal 141 residues. The presence of this retroelement in homozygosis co-segregated with white fruits in the complete F2 mapping population. We further extended this analysis to other white fruited F. vesca genotypes but none of them harbored this retroelement in FvMYB10. Sequence analysis of MYB10 from these accessions allowed us to identify two other independent polymorphisms, (1) a single nucleotide insertion, which generates a truncated protein, and (2) a large deletion of ~100 Kb spanning a genomic region that contains 7 genes, one of them being FvMYB10.
These results show that white strawberry alleles have arisen from at least four independent mutational events in a single transcriptional regulator, FvMYB10. As it has been shown for fruits of other species, an R2R3-MYB, FvMYB10, appears to be the primary gene responsible for anthocyanin synthesis and fruit coloration in strawberry.
This work was supported by grants RFP2015-00011-00-00 (Spanish Ministry of Economy and
Competitivity and FEDER) and EI.AVA.AVA201601.10 (IFAPA, FEDER funds).
37
S-II. Póster 1
FUNCTIONAL ANALYSIS OF MASTER GENES INVOLVED IN
STOMATAL DEVELOPMENT IN TOMATO AND GRAPEVINE
Alfonso Ortega, Alberto de Marcos, Jonatan Illescas, Carmen Fenoll & Montaña
Mena
Departamento de Ciencias Ambientales, Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-la Mancha, Toledo 45071, Spain
Corresponding author: Alfonso Ortega, [email protected]
Stomatal development has become an attractive system to study
initiation, maintenance and terminal differentiation of lineage-specific stem cells
as well as regulation of tissue patterning. The gene networks controlling the
development of stomata from protodermal cells in aerial organs have been
described in Arabidopsis, but the information for crops is still unknown. In
Arabidopsis stomatal lineage cells is specified by sequential activities of three
basic-helix–loop–helix (bHLH) proteins that positively regulate stomata
development: SPCH, MUTE and FAMA. Those are the key to establish stomatal
abundance in the different organs and alleles. Modifyingtheir activity they can
also have a great potential to change stomatal numbers and their plant
performance under different growth conditions.
By phylogenetic analysis we identified the putative orthologues of the
three Arabidopsis bHLH-coding stomatal regulator genes in Solanum
lycopersicum and in Vitisvinífera and cloned the corresponding full length
cDNAs and genomic promoter regions. We have used these coding regions and
their fusions to GFP in functional assays, testing whether they could
complement the corresponding Arabidopsis loss-of-function mutants when
expressed with their respective Arabidopsis promoters, and the effects of their
overexpression with a constitutive, β-estradiol inducible promoter. The promoter
regions (2-3 kb of genomic sequences upstream from the ORFs) were fused to
GFP and to GUS and introduced in Arabidopsis. Through this approach we
have established the functional orthology for some of these genes, both interms
of protein function and of promoter expression domain. At the moment we are
identifying mutants in tomato and grapevine (by TILLING or eco-TILLING), in a
search for altered stomata abundance phenotypes which might correlate with
different physiological traits (such as those related to water status, leaf cooling
or photosynthesis).
Work was supported by grants AGL2015-65053-Rand PPII10-0194-4164. AA and JI
arerecipients of predoctoral grants from JCCM.
38
S-II. Póster 2 A STUDY OF DUCKWEED NATURAL VARIATION FOR SWINE
WASTEWATER TREATMENT
Almudena Molla-Morales, Cristian Mateo, Odette Deen, Yolanda Leo-del
Puerto, Belén Méndez-Vigo, Mercedes Ramiro, Antonio Leyva, Carlos Alonso-
Blanco
Genética Molecular de Plantas, CNB-CSIC, Cantoblanco (Spain)
Corresponding author: Carlos Alonso-Blanco ([email protected])
Duckweeds (Lemnaceae) are a family of aquatic plants with simple morphology
and rapid growth rate. Due to their high biomass production, tolerance to a wide
range of environmental conditions and good biochemical profile, they have been
recently approached for different industrial and environmental applications,
including phytoremediation, bioenergy and nutrition.
The pollution caused by the high amount of swine wastewater production
in pig farms is an important environmental problem that causes eutrophication
and toxicity on the land. As it is illegal to release it to the environment, farms
need to find a clean process for water remediation. In this project, we aim to
exploit the natural variation of duckweeds for reducing the toxicity and nutrient
content of swine wastewater.
To achieve this goal, we are developing a collection of duckweed natural
varieties (accessions), including the species Lemna gibba, L. minor, L. minuta
and Spirodela polyrrhiza, found in the Iberian Peninsula. This collection is being
characterized at different levels in order to select the optimal varieties for
nutrient removal. First, to determine the inter- and intraspecific variation in this
duckweed collection, we are performing a genetic characterization using
chloroplastic and nuclear molecular markers to study polymorphisms, and flow
cytometry to determine genome size and ploidy level. Additionally, we are
beginning a physiological characterization of growth rate, tolerance of the
duckweed accessions for swine wastewater at different concentrations, and
ability for nutrient (phosphate and nitrate) uptake and removal.
Our preliminary results show substantial inter- and intraspecific variation
in all the analyses performed. Thus, the information obtained in this study can
be useful not only to select the best variety for our particular goal, but also for
the selection of optimal accessions in other industrial and environmental
applications.
This work is funded by the European Union (LEMNA-LIFE15 ENV/ES/000382).
39
S-II. Póster 3 STRUCTURAL BASIS OF THE ALLERGENICITY TO
STRAWBERRIES DUE TO Fra a 1.02
Begoña Orozco-Navarrete1, Florine Dupeux2 Delphine Pott1, Ana Casanal3, María Garrido-Arandia4, Araceli Díaz Perales4, Catharina Merchante1, José A.
Márquez2 and Victoriano Valpuesta1.
1Dpto de Biología Molecular y Bioquímica, Instituto de Hortofruticultura Subtropical y Mediterránea (IHSM), Universidad de Málaga-CSIC, Málaga, Spain, 2European Molecular Biology Laboratory, Grenoble Outstation and Unit of Virus Host-Cell
Interactions, UJF-EMBL-CNRS, Grenoble, France, 3MRC-Laboratory of Molecular Biology, Cambridge, UK, 4Unidad de Bioquímica, Departamento de Biotecnología,
E.T.S. Ingenieros Agrónomos, Madrid, Spain. Corresponding author: Victoriano Valpuesta ([email protected])
Strawberry fruits are highly valued due to their flavor, aroma, and benefits for human health. Despite this, 30% of the population with food hypersensitivity also shows adverse reactions to strawberry (Franz-Oberdorf et al, 2016). The FaFra a 1 protein family, homologs of the major birch pollen allergen Bet v 1, is involved in this allergenicity to strawberry. By RNAseq we have identified transcripts for 18 members of the FaFra a 1 family (from 1.01 to 1.18) in strawberry fruits. Although expressed in all tissues analyzed, each family member presents a unique pattern of expression, which suggests functional specialization for each FaFra a 1 protein. FaFra a 1.02 (Fra2 from now on) is the most expressed one in red fruits and is also the most allergenic among the family members tested (Muñoz et al. 2010; Franz- Oberdorf et al, 2016). In order to understand the molecular bases of this allergenicity we crystalized Fra2 and obtained its structure by X-ray diffraction. Fra2 showed a very high structural homology to Bet v 1, and we asked whether the two proteins were recognized by the immune system in a similar way. For this, we generated five different mutant versions of Fra2 in sites described as important for allergenicity in Bet v 1 (Fernandes et al, 2016), and studied their potential allergenicity as well as their crystal structures. Three of the mutants had substitutions in loop 4 (E46R, D48R, E46/48A) and the other two facing the cavity (A141F and Q64W). Compared to Fra2, all the mutants showed a significant reduction in their capacity to be recognized by the serum of patients with allergies to Bet v 1, and their crystal structures revealed conformational changes in the Bet v 1- IgG interaction sites. Together, these results support that Fra2 and Bet v 1 have similar allergenic determinants We hope this research will aid in understanding how human IgGs interact with Fra2 and might help in the development of new cultivars with a lesser allergenic potential.
-Fernandes, et al (2013). The FEBS journal, 280(5), 1169-1199. -Franz-Oberdorf et al. (2016).Journal of agricultural and food chemistry 64.18: 3688-3696. -Muñoz et al. (2010). Molecular Plant, 3: 113–124.
Acknowledgements & Funding. This work was supported by the Grants BIO2013-44199R and BES-2014-068723 (MINECO). The authors also acknowledge the support by the Plan Propio from University of Malaga, Campus de Excelencia Internacional de Andalucía.
40
S-II. Póster 4 Chlamydomonas MITOCHONDRIAL F-ATPASE F0 A SUBUNIT
(ATP6): A NUCLEUS ENCODED ABA RESPONSIVE SUBUNIT
CONFERRING ABIOTIC STRESS RESISTANCE.
María Carbó1, Francisco J Colina1, Mónica Meijón1, María Jesús Cañal1, Luis Valledor1.
1Plant Physiology Area, Organisms and Systems Biology Department, University of Oviedo, Oviedo, Spain.
Corresponding author: María Carbó ([email protected])
Mitochondrial F-ATP-synthase catalyzes ATP synthesis using the electron transfer chain derived electrochemical gradient. In this complex, F0 region is a stator with different subunits and ATP6 (F0 subunit a) between them. This protein manages proton transport across mitochondrial internal membrane. Mutations in this typically mitochondrial gene are behind multiple human congenital diseases. Interestingly, this is a nuclear gene in Chlamydomonas reinhardtii, coding a uniquely structured protein and an efficient mechanism for its transport to the mitochondria. Also, ATP6 overexpression confers abiotic stress resistance in plants and yeast. Previous results found that Chlamydomonas ATP6 is accumulated under osmotic and saline stress and has stress related cis elements into its promoter. Similar allotopic genes in Arabidopsis are under ABA control. Considering all this, ATP6 could be not only a stress tolerance enhancer candidate but a potential ABA-mediated stress signaling/sensing mechanism into the barely known ABA signaling in Chlamydomonas.
Chlamydomonas ATP6 overexpression lines were generated by a genetic engineering approach. Saline stress phenotyping was evaluated during 72 h by growth measurement and accumulation analysis of pigments, starch and lipids. ATP6 ABA response was checked under control and saline stress by expression changes in oxidative stress responsive genes.
Applied stress triggers changes in ATP6 overexpressing Chlamydomonas, like increased growth. Thus, it can be assumed that ATP6 overexpression tolerance is caused by faster ROS damaged A subunits replacement. This may contribute to increased ATP synthesis under stress. ATP surplus can contribute to ATP demanding stress tolerance mechanisms. Moreover, ATP pool can be translated into bioenergetic changes, such as the starch and lipid accumulation. In addition, Chlamydomonas ATP6 nuclear localization and bZIP related promoter features opens an alternatively regulation pathway. According to this, ATP6 might be an ABA regulated element and responsible for a better ABA stress resistance under oxidative stress in Chlamydomonas.
Acknowledgements & Funding This work is an output of the projects financed by the Spanish Ministry of Economy, Industry and Competitiveness (AGL2016-77633-P) and the Government of Principado de Asturias (GRUPIN14-055). FC was supported by a fellowship from the Severo Ochoa Program (BP14- 138; Government of Principado de Asturias, Spain). MM and LV were generously supported by Ramón y Cajal program (Spanish Ministry of Economy, Industry and Competitiveness, grant references RYC-2014- 14981 and RYC-2015-17871, respectively).
41
S-II. Póster 5 DON’T LET YOUR FRIDGE LED YOUR RESEARCH, pSticky, A
NOVEL GLOBAL MOLECULAR EDITION TOOL FOR
Chlamydomonas reinhardtii
Francisco J Colina1, María Carbó1, Luis Valledor1, María Jesús Cañal1. 1Plant Physiology Area, Organisms and Systems Biology Department, University of
Oviedo, Oviedo, Spain. Corresponding author: Francisco J. Colina ([email protected])
Once Chlamydomonas chloroplastic and nuclear genomes were successfully transformed, vectors became a widespread tool into the microalgae research. From protein production/overexpression/tagging to knock out/silenced lines generation, all relies almost completely on these systems. Chlamydomonas contributed early with its own particularities to these systems, as differential codon usage, low protein yield, difficult homologous recombination and inability to endure Cas9 expression between others. Different solutions have been yet proposed, but developed independently resulting into the high amount of available constructions and strategies.
Tuning a Chlamydomonas transgenic system requires multiple genetic elements to be tried before reaching a successful combination. Different elements are usually contained into different vectors which are commonly highly different and incompatible. To this is to be added the nonetheless important plasmids availability, sequence engineering and verification problems. To ease this problem, different approaches have been taken searching for the modularity and compatibility of the vector elements. Besides this, these systems are still somewhat stiffened allowing only gene exchange.
Following this flexibility and simplicity concept, we have designed a construction were exchangeability spreads also to promoters and selectable markers into a fast and easy editing system. This plasmid is based on a pBluescript II KS backbone associated to a double Chlamydomonas expression cassette. Under two independent promoters this cassette directs selectable marker and a second gene expression which in the last case is also under the control of exchangeable promoters.
The pSticky system has been tried successfully for endowing Chlamydomonas with different herbicide/antibiotic resistances, involving AphVIII and PPX1rs3 resistance markers, and for different protein expression under different promoter control as fusion HSP70-RbcS2 and nitrate inducible NIT1. The selectable marker versatility of this modular construction offers the possibility of fast and simple double gene transformations, starting from the same vector, which is usually required in the characterization of multiple gene families.
Acknowledgements & Funding This work is an output of the projects financed by the Spanish Ministry of Economy, Industry and Competitiveness (AGL2016-77633-P) and the Government of Principado de Asturias (GRUPIN14-055). FC was supported by a fellowship from the Severo Ochoa Program (BP14- 138; Government of Principado de Asturias, Spain). The Spanish Ministry of Economy and Competitiveness supported LV through the Ramón y Cajal program (RYC-2015-17871).
42
S-II. Póster 6 DECREASE OF GLOBAL DNA METHYLATION BY 5-
AZACYTIDINE PROMOTES PROLIFERATION OF CORK OAK
SOMATIC EMBRYOS
Elena Carneros1, Yolanda Pérez-Pérez1, Beatriz Pintos2, Aránzazu Gómez- Garay2, María C. Risueño1, Pilar S.Testillano1
1 Pollen Biotechnology of Crop Plants, Biological Research Center, CIB-CSIC, Madrid,
Spain, 2Dep. Plant Biology I, Plant Physiology, UCM, Madrid, Spain
Corresponding author: Pilar S. Testillano ([email protected])
Cell reprogramming, totipotency acquisition and somatic embryogenesis initiation involve changes in the developmental program of the cell, which affect global genome organization. Epigenetic marks are involved in the regulation of global gene expression programs. DNA methylation, accomplished by DNA methyltransferases, is a key epigenetic modification of the chromatin, associated with gene silencing that can change during cell proliferation and differentiation processes. Somatic embryogenesis is a widely used biotechnological tool for in vitro plant regeneration, still with limited efficiency in woody species. Recent studies in rapeseed and barley have reported that microspore embryogenesis initiation is associated with DNA hypomethylation, and its efficiency can be improved by the DNA demethylating agent 5’- azacytidine (AzaC) (Solís et al. 2015). Little is known about DNA methylation dynamics during in vitro embryogenesis in trees (Rodríguez-Sanz et al. 2014; Corredoira et al. 2017). This work analyses the changes in global DNA methylation during somatic embryogenesis progression in cork oak, as well as the effect of AzaC in the process. Results showed low DNA methylation levels at early stages of somatic embryogenesis. AzaC treatments reduced global DNA methylation and promoted the proliferation of somatic embryos, while it prevents embryo differentiation. These findings provide new insights into mechanisms underlying in vitro embryogenesis in woody species where information is still scarce, opening up new intervention pathways to improve somatic embryogenesis yields for forestry breeding programs.
References:
Corredoira et al. (2017) J Plant Physiol. 213: 42-54
Rodríguez-Sanz et al. (2014) BMC Plant Biol. 14:224
Solís et al. (2015) Front. Plant Sci. 6:472
Funding:
Supported by projects (AGL2014-52028-R and AGL2017-82447-R) funded by Spanish MINECO and European Regional Development Fund (ERDF/ FEDER).
43
S-II. Póster 7
CAS-MEDIATED RNA TARGETING IN PLANTS
Albert Bernal1, 2, Luis Villar-Martin2, Ignacio Rubio-Somoza2
1Master program in Plant Biology, Genomics and Biotechnology, Centre for Research
in Agricultural Genomics (CRAG)- University of Barcelona (UB)- Autonomous
University of Barcelona (UAB), 2Molecular Reprogramming and Evolution (MoRE) Laboratory, Centre for Research in Agricultural Genomics (CRAG), Vall Moronta s.n.-
Edifici CRAG. 08193 Cerdanyola del Vallés, Spain.
Corresponding author: Ignacio Rubio-Somoza, [email protected]
RNA interference (RNAi) is widely used to achieve customized inhibition of target genes in eukaryotes. Approaches based on such technology rely on supplying a RNA sequence that specifically targets endogenous factors to interfere with the stability of messenger RNAs in a sequence homology manner. Nevertheless, it has been shown that the activity of the endogenous players, such as proteins from the DICER and ARGONAUTE families, and their efficiency on downregulating their mRNA targets are sensitive to growth conditions and developmental stage (Szittya et al. 2003; von Born, Bernardo- Faura, and Rubio-Somoza 2018). In order to circumvent the possible limitations imposed by those parameters, we are currently developing an exogenous and highly-specific CRISPR/CAS system that allows customized mRNA down- regulation in plants. To that end, we are comparing the ability of different Type II or Type VI CAS proteins to target RNA without affecting DNA sequence. We will discuss our progress on such endeavor.
Born, Patrick von, Marti Bernardo-Faura, and Ignacio Rubio-Somoza. 2018. “An Artificial MiRNA System Reveals That Relative Contribution of Translational Inhibition to MiRNA-Mediated Regulation Depends on Environmental and Developmental Factors in Arabidopsis Thaliana.” Edited by Sebastien Pfeffer. PLOS ONE 13 (2). Public Library of Science: e0192984. https://doi.org/10.1371/journal.pone.0192984.
Szittya, György, Dániel Silhavy, Attila Molnár, Zoltán Havelda, Agnes Lovas, Lóránt Lakatos, Zsófia Bánfalvi, and József Burgyán. 2003. “Low Temperature Inhibits RNA Silencing-Mediated Defence by the Control of SiRNA Generation.” The EMBO Journal 22 (3). EMBO Press: 633– 40. https://doi.org/10.1093/emboj/cdg74.
Acknowledgements and funding: The work in the MoRE group is funded by Spanish Ministry of Economy and Competitiveness
(I.R-S is supported by BFU-2014-58361-JIN, RYC-2015-19154; L. V-M is supported by BES-
2016-076986) and through the “Severo Ochoa Programme for Centres of Excellence in R&D”
2016-2019 (SEV-2015-0533) and the CERCA programme from the Generalitat de Catalunya.
44
S-II. Póster 8
GENETIC DIVERSITY AND POPULATION STRUCTURE AMONG
A COLLECTION OF 175 Fragaria ACCESSIONS
Iraida Amaya1, Pilar Muñoz del Rio1, Eduardo Cruz-Rus1, Gema Martín
Corredoira1, Carmen Soria1, José F. Sánchez-Sevilla1
1Genómica y Biotecnología, Centro de Málaga (Churriana), Instituto Andaluz de
Investigación y Formación Agraria y Pesquera (IFAPA), Málaga, Spain
Corresponding author: José F. Sánchez Sevilla ([email protected])
F. virginiana and F. chiloensis were used during the 19th and 20th centuries to introgress traits of interest into strawberry (Fragaria × ananassa). However, traditional strawberry breeding has since been performed by intraspecific crosses between cultivars adapted to similar agro-climatic areas, which led to a reduction of genetic diversity in modern cultivars. High-throughput genotyping platforms allow in depth analysis of genetic diversity and in combination with phenotypic scoring facilitates genome wide association studies. A total of 175 strawberry (Fragaria ssp.) accessions was selected from the Fragaria germplasm collection at the IFAPA Centre of Málaga, and included F. × ananassa cultivars, F. chiloensis, F. virginiana and hybrids between F. × ananassa and F. chiloensis. In this study we used the strawberry 90 K SNP Affymetrix® Axiom® array to analyse the genetic diversity, structure and linkage disequilibrium (LD) in the selected accessions. A total of 23,696 high quality SNPs was selected among the 33,668 polymorphic SNPs (PolyHighResolution conversion type). Population structure and diversity analyses using the model- based software STRUCTURE and other clustering strategies revealed diversification into two main groups and several (6-8) subpopulations, reflecting a degree of stratification and the wide diversity of the selected accessions. A large subpopulation encompassed all F. chiloensis and F. virginiana accessions together with old F. × ananassa cultivars. The other main subpopulation consisted of recent varieties gathered by shared pedigree or specific genetic contributions characteristic of specific breeding programs. All the analysed hybrids between F. chiloensis and modern F. × ananassa, with only one exception, shared more genetic similarity to the modern strawberry cultivars lines than to F. chiloensis. This result reflects a bias due to the selection of those particular hybrid siblings among the F1 progenies based in the phenotype for superior agronomic and fruit quality traits and agrees with reported phenotypic data suggesting that few generations are needed to restore fruit size and yield to commercial levels.
This work was supported by grants RFP2015-00011-00-00 (Spanish Ministry of Economy and Competitivity and FEDER) and EI.AVA.AVA201601.10 (IFAPA, FEDER funds).
45
S-II. Póster 9
INSIGHTS INTO THE MECHANISMS OF MUTE FUNCTION THROUGH THE DESIGN AND IN VIVO ANALYSIS OF MUTANT
VARIANTS.
Jonatan Illescas, Alfonso Ortega, Marina Moreno, Amanda F. Martín-Forero, Alberto de Marcos, Carmen Fenoll and Montaña Mena
Departamento de Ciencias Ambientales, Facultad de Ciencias Ambientales y
Bioquímica, Universidad de Castilla-la Mancha, Toledo 45071, España. Corresponding author: Jonatan Illescas ([email protected])
In developmental biology, hypomorphic mutants help study genes whose loss- of-function (LOF) determines lethality, premature developmental arrest or aberrant phenotypes. The two related bHLH-type transcription factors MUTE and SPCH regulate cell differentiation through sequential steps of stomatal development in Arabidopsis thaliana and their LOF confers seedling lethality. From LOF mutant phenotypes it was established that SPCH is essential for the asymmetric cell divisions necessary for the initiation of stomatal lineages, and therefore mutants lacking SPCH function do not produce stomatal lineages. MUTE acts later during lineage progression and it is needed to exit the asymmetric division program and commit the last cell product of the asymmetric divisions (a late meristemoid) to become a guard mother cell. This GMC is in turn committed to a final symmetric division that produces the stomata. Loss of MUTE function prevents stomata formation, but allows production of aborted stomatal lineages. We isolated a novel SPCH allele, spch-5, which carries a point mutation in a highly conserved residue of the bHLH domain, the DNA binding domain in this protein family1. Homozygous spch-5 plants, however, displayed normal growth, although their leaves formed an extremely low number of stomata which were sometimes clustered, while the hypocotyls lacks stomata. Two related alleles, spch-2 and SPCHPPP, also behaved as hypomorphic mutants: plants can generate stomata but in lower numbers and with altered distribution patterns. We generated three mutant versions of MUTE that mimic these SPCH alleles, and tested if they can complement the loss-of-function mute-3 stomataless mutant. We also generated lines for the conditional overexpression of the various alleles. We are using these hypomorphic alleles to: 1) explore their potential to modify stomatal abundance –and thence photosynthesis and transpiration, and 2) understand how, despite of mutations in residues necessary for DNA binding, these SPCH and MUTE alleles can partially sustain stomata lineage completion and produce viable and fertile plants.
1Alberto de Marcos, Anaxi Houbaert, Magdalena Triviño, Dolores Delgado, Mar Martín-Trillo, Eugenia Russinova, Carmen Fenoll, and Montaña Mena (2018). A Mutation in the bHLH Domain of the SPCH Transcription Factor Uncovers a BR-Dependent Mechanism for Stomatal Development. Plant Physiol. Vol. 174, pp. 823–842.
Work was funded by grants AGL2015-65053-R and PPII10-0194-4164. AO and JI are
supported by PhD grants from JCCM.
46
S-II. Póster 10
ALTERATION OF KREBS CYCLE IN TRANSGENIC TOMATO
PLANTS SHOWS A CRUCIAL ROLE OF ORGANIC ACIDS IN
CROP DEVELOPMENT AND FRUIT QUALITY
Lidia Jiménez1, Delphine Pott1, Elsa Martínez-Ferri2, Alisdair R. Fernie3, José
G. Vallarino3, Sonia Osorio1.
1Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, University of
Malaga-Consejo Superior de Investigaciones Científicas, Department of Molecular
Biology and Biochemistry, Campus de Teatinos, 29071 Málaga, Spain. 2Centro de
Churriana, Instituto Andaluz de Investigación y Formación Agraria y Pesquera, (IFAPA)
Málaga, Spain. 3Max-Planck-InstitutfürMolekularePflanzenphysiologie, Potsdam-Golm,
Germany.
Corresponding author: José G. Vallarino ([email protected]) and Sonia Osorio
Tomato (Solanum lycopersicum) is the second most consumed vegetable in the world (FAOstat, 2018), but also a model plant for the study of fleshy fruit. Organic acids (OA) play a crucial role in the plant primary metabolism (Araújo et al., 2012) and are one of the most important fruit quality traits (Oms-Oliuet al., 2011). Transgenic tomato plants expressing a bacterial maleate isomerase, which converts maleate to fumarate, were generated in order to improve our knowledge about the role of OA in the crop and fruit metabolism. Growth and reproduction were affected by the unbalance of OA The plants show a dwarf phenotype and a flowering delay. Furthermore, a lower level in chlorophyll content and a decrease in stomata indicated alterations of photosynthesis. Postharvest was also impaired. Transgenic fruits showed increased water loss and deterioration, pointing an implication of the OA in the cell wall metabolism. These changes have been confirmed by the expression analysis of the cell wall genes related. Our results in metabolomics analyses pointed out changes during fruit ripening in flavor-related primary metabolites, such as OA, amino acids, and sugars, revealing the importance of OA in fruit metabolism. Our findings indicate a pivotal role of tricarboxylic cycle intermediates, such as malate or fumarate, as regulatory metabolites. Besides their roles in quality fruit characteristics, they are also involved in functions like growth or photosynthesis.
Nunes-Nesi A, Nikoloski Z, Sweetlove LJ and Ferni AR, (2012) Metabolic control and regulation of the tricarboxylic acid cycle in photosynthetic and heterotrophic plant tissues. Plant, Cell and Enviroment 35: 1-21. Oms-Oliu G, Hertog MLATM, Van de Poel B, Ampofo-Asiama J, Geeraerd AH, Nicolaï GM (2011) Metabolic characterization of tomato fruit during preharvest development, ripening, and postharvest shelf life. Postharvest Biology ad Technology 62: 7-16
Work in was supported in part by grants from the Max-Planck- Gesellschaft (to SO and ARF), and by Ministerio de Ciencia e Innovacion and University of Malaga through the grant Ramon and Cajal program (SO, RYC- 09170).The authors also acknowledge the support by the Plan Propio from University of Malaga, Campus de Excelencia Internacional de Andalucía.
47
S-II. Póster 11
HORMONAL CROSSTALK REGULATES ADVENTITIOUS ROOT
FORMATION IN DIVERSE CARNATION GENOTYPES
María Salud Justamante1, Joan Villanova1, Antonio Cano2, Emilio A. Cano3,
Manuel Acosta2, José Manuel Pérez-Pérez1
1Instituto de Bioingeniería, Universidad Miguel Hernández, Elche, Spain,
2Departamento de Fisiología Vegetal, Universidad de Murcia, Spain, 3Dümmen
Orange, Murcia, Spain.
Corresponding author: José Manuel Pérez-Pérez ([email protected])
Adventitious rooting of stem cuttings is an essential process needed for
vegetative propagation in many ornamental plants. In cultivated carnation,
adventitious roots (ARs) are formed in the base of the cuttings in response to a
localized auxin gradient. Differences in biosynthesis, transport and conjugation
of auxin levels in the stem cutting base during the first hours after excision
accounts for the differences observed in AR formation between cultivars with
contrasting rooting efficiencies (Cano et al. 2018). To better understand the
genetic basis of this complex trait, we studied AR formation in 177 lines derived
from a cross between two hybrid cultivars with contrasting rooting performance
(Birlanga et al. 2015). Several architectural traits were quantified in fourteen of
these lines displaying extreme AR phenotypes. Combining hormonal and
phenotypic results with selective transcriptome profiling will allow us to identify a
collection of candidate SNPs linked to adventitious rooting performance, which
will be used to establish a novel marker-assisted selection approach to improve
adventitious rooting in this species.
Birlanga, V., et al. (2015) PLOS ONE 10: e0133123
Cano et al. (2018). Front Plant Sci doi: 10.3389/fpls.2018.00566
Work in the laboratory of J.M.P.-P. (AGL2012-33610 and BIO2015-64255-R) is funded by the
Ministry of Economy and Competitiveness and by FEDER funds of the EC - "A way to build
Europe“.
48
S-II. Póster 12
TOWARDS THE DESIGN OF A PIPELINE FOR THE RAPID
GENERATION OF SOURCES OF RESISTANCE TO VIRUSES IN
TOMATO
Pascual Rodríguez-Sepúlveda1, Raquel N. Sempere2, Luis Rodríguez-Moreno2,
Carlos García-Almodovar1, Blanca Gonsalvez2, Fabiola Ruiz-Ramón2, Jesús
Agüero1, Pau Bretó1 y Miguel A. Aranda2
1Abiopep S.L., Parque Científico de Murcia. 30100. Murcia. España
2Departamento de Biología del Estrés y Patología Vegetal, CEBAS-CSIC, 30100,
Murcia, España
Corresponding author: Pascual Rodriguez-Sepúlveda ([email protected])
Abstract
Emerging plant diseases caused by viruses are becoming more frequent in the
current scenario of global change. Resistance traits have been identified for a
number of viruses and crop species, and cultivars with varying degrees of
resistance have been released. However, availability of sufficient sources of
resistance and resistance durability are aspects of serious concern to
producers, breeders and pathologists. It is therefore necessary to pursue
alternative approaches to identify new sources of resistance to viruses in crop
species. Taking advantage of the rapid development of genome editing tools,
we are working on the design and implementation of a pipeline for the efficient
identification of genetic targets whose edition may result in loss-of-susceptibility
to plant viruses; our hypothesis proposes that host proteins interacting with viral
proteins are likely used by the virus for its own multiplication or transport, and
modification or loss of one of these host proteins may result in loss-of-
susceptibility, i.e., resistance. Within this conceptual framework, we have
identified tomato proteins which interact with Pepino mosaic virus proteins.
Virus Induced Gene Silencing (VIGS) has shown that at least some of these
host proteins are required for full viral infection. Mutants in the corresponding
genes have been identified within a TILLING platform, with limited success.
Therefore, genome editing using CRISPR/Cas9 has been set up in our team for
tomato, and is under way for melon. Mutants in candidate genes have been
generated and are being characterised phenotypically.
49
S-II. Póster 13 IMPACT OF ENVIRONMENT AND GENOTYPE ON L-ASCORBIC
ACID CONTENT IN STRAWBERRY FRUIT WITHIN THE EU
GOODBERRY PROJECT
Pilar Muñoz del Rio1, Béatrice Denoyes2, Philippe Chartier3, Aurélie Petit3,
Klaus Olbricht4, Silke Lesemann4, Luca Mazzoni5, Bruno Mezzetti5, Agnieszka
Masny6, Edward Zurawicz6, Antonio Arjona7, Annika Haugeneder8, Johanna
Trinkl8, Wilfried Schwab8, José A. Gómez9, Luis Miranda9, Lidia Jiménez10,
Sonia Osorio10, José F. Sánchez Sevilla1, Iraida Amaya1
1Centro de Churriana, Instituto Andaluz de Investigación y Formación Agraria y
Pesquera, (IFAPA) Málaga, Spain; 2UMR 1332 Biologie du Fruit et Pathologie, INRA,
Villenave d’Ornon, France; 3Ciref Création Variétale Fraises Fruits Rouges, Douville,
France; 4 Hansabred GmbH & Co. KG, Dresden, Germany; 5Dipartimento di Scienze
Agrarie, Alimentari e Ambientali, Università Politecnica delle Marche, Ancona, Italy; 6Department of Breeding of Horticultural Crops Research Institute of Horticulture,
Skierniewice, Poland; 7Viveros California, S.L. Sevilla, Spain; 8Technische Universität
München, Freising, Germany; 9Finca el Cebollar, IFAPA Centro las Torres, Huelva,
Spain; 10Departamento de Biología Molecular y Bioquímica, IHSM, Universidad de
Málaga-CSIC, Málaga, Spain
Corresponding author: Iraida Amaya ([email protected])
Strawberry fruits are considered an excellent source of vitamin C, or L-ascorbic acid (AsA), a water-soluble vitamin that is an essential dietary component for humans. However, AsA content varies widely between Fragaria species and also between strawberry cultivars, ranging from about 10 to more than 80 mg/100 g FW. One of the objectives of the EU GoodBerry project is unraveling the genetic architecture of fruit quality traits that interact with the environment and culture management. To achieve this goal, we have developed a pseudo test-cross population between two cultivars, ‘Candonga’ and ‘Senga Sengana’, adapted to contrasting Southern and Northern European areas, respectively. The population has been grown in five locations across Europe (Poland, Germany, France, Italy and Spain) and it is being phenotyped for several agronomic and fruit quality traits during two seasons. Linkage maps have been constructed using the DArT platform from Diversity Arrays Technology Pty Ltd for strawberry and will be used together with phenotypic data for QTL analysis. Data from a subset of 37 lines of the population evaluated for AsA content during the first season show significant genetic and environmental effects. Although AsA content relative to fresh weight was reproducible between different individual genotypes, it was higher in fruits cultivated in Germany and Spain than in the rest of European regions. AsA in fruits of ‘Candonga’ was higher than in ‘Senga Sengana’ in all locations. A large variation in AsA was found within the population, ranging from less than 40 to about 80 mg/100 g FW. These data indicate that this population represents a useful tool for the identification of mQTL for AsA content in strawberry.
This work is funded by the European Union’s Horizon 2020 research and innovation programme (GoodBerry; grant agreement 714 number 679303).
50
51
SESIÓN III: BIOVEGEN
CONFERENCIA
Manuel Talón
Director Centro de Genómica del Instituto Valenciano de Investigaciones
Agrarias (IVIA), Valencia
ORIGEN Y DOMESTICACIÓN DE LOS CÍTRICOS A LA LUZ DE LA GENÓMICA
En esta exposición proponemos una explicación para el origen de los cítricos y otros temas relacionados que han permanecido esquivos durante los últimos siglos y han sido, por tanto, motivo de grandes controversias. Nuestros análisis genómicos, filogenéticos y biogeográficos han proporcionado por primera vez evidencias sólidas que respaldan la propuesta de que el centro de origen de los cítricos se sitúa en las estribaciones del sudeste del Himalaya, en una región que incluye la zona oriental de la provincia de Assam, el norte de Myanmar y el oeste de la provincia de Yunnan. Esta nueva visión también sugiere que los cítricos evolucionaron durante el Mioceno tardío mediante una rápida radiación que correlaciona con un sorprendente debilitamiento de los monzones, asociado a un período de enfriamiento global. Los cítricos australianos y las mandarinas de Japón y otras islas orientales del Pacifico emergieron más tarde a partir de los cítricos del continente asiático durante el Plioceno y Pleistoceno tempranos, respectivamente. El estudio detallado de los genomas también nos ha permitido distinguir entre las especies puras de cítricos, los genotipos híbridos y las mezclas de genomas o mestizajes, un conocimiento con el que hemos definido la genealogía de los cítricos y dilucidado la constitución genética de las especies salvajes y de las variedades cultivadas de interés comercial. Los datos, por ejemplo, permiten recrear los procesos de domesticación de los cítricos, al revelar que tanto las mandarinas como las naranjas en realidad contienen introgresiones de “pummelo” de diversa extensión, un aspecto que probablemente determina los fenotipos que nosotros reconocemos como mandarinas o naranjas. Estos genomas también muestran una inesperada pero extensa red de conexiones “familiares” que sugiere una domesticación compleja a partir de unos pocos especímenes salvajes. En resumen, este trabajo presenta ideas originales y novedosas sobre el origen, la evolución, la dispersión, la domesticación y la genealogía de las variedades silvestres y cultivadas de cítricos. Tomados en su conjunto, los hallazgos dibujan un nuevo marco evolutivo para estos frutales, un escenario que desafía los actuales pensamientos taxonómicos y filogenéticos y por ende apunta hacia una inexcusable reformulación o redefinición del género Citrus.
52
SESIÓN III
HERRAMIENTAS
TECNOLÓGICAS
MODERADORA:
Pilar S. Testillano (CIB-CSIC,
Madrid)
53
S-III. Comunicación 1
COMBINATORIAL DESIGN OF MODULAR AND
PROGRAMMABLE TRANSCRIPTIONAL REGULATORS IN
PLANTS. Sara Selma1, Joan Bernabé1, Maria Ajenjo1, Borja Diego1, Marta Vázquez1, Antonio
Granell1, Diego Orzaez1. 1Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), Valencia, Spain.
Corresponding author: Sara Selma ([email protected])
The RNA-guided DNA binding activity of CRISPR/Cas9 offers unprecedented possibilities in the fields of synthetic biology and metabolic engineering, among others. Modifications of the Cas9 protein or its guide RNA (gRNA) enable the expansion of the range of Cas9 activities, from its original nuclease activity to other DNA binding-related activities, like transcriptional activation/repression. This opens the possibility of modulating the expression of selected genes in plants through CRISPR/Cas9-based programmable transcription factors. The objective of this work is to design modular gRNA modifications that allow the attachment of regulatory domains to the CRISPR/Cas9 complex for plants. To achieve this, the CRISPR/gRNA-directed synergistic activation mediator or SAM strategy was adapted via Goldenbraid cloning system (Vazquez, 2016). SAM strategy consists of the adding of an RNA aptamer that binds the Ms2 phage coat protein, to free positions of the gRNA. This creates a structure comprising the chimeric RNA-guide and Cas9, which binds to Ms2 fused to a domain of interest. To optimize the process, two chimeric RNA-guides were designed containing two copies of the Ms2 aptamer in different positions. One of them adds the aptamer to the 3´end of the RNA-guide, whereas the second one incorporates it in a free loop of the RNA-guide hairpin (Nowak, 2016). These structures were used for the attachment of several combinations of transcriptional activators to the gRNA/Cas9 complex fused to the Ms2 protein. Taking advantage of the modular Goldenbraid cloning system, the construction of a combinatorial array of programmable transcriptional regulation with other proteins that bind aptamer sequences, like PP7 and COM, and several regulatory domains was facilitated. After test different strategies, the conclusion was that SAM/Ms2 strategy with the aptamer in the 3’ of the scaffold was the most efficient in N. benthamiana. Furthermore, the efficiency of using SAM/Ms2 strategy was compared with others where the activator domain was fused directly to the dCas9 testing different promoters, like Nopaline synthase or dihydroflavonol 4-reductase promoter, among others, in transient and stable expression in N. benthamiana. The results show the great potential of the Cas9/SAM system to modulate metabolic pathways and optimize the production of metabolites of interest.
References: Chance M. Nowak, Seth Lawson, Megan Zerez, and Leonidas Bleris. Guide RNA engineering for versatile Cas9 functionality. Nucleic Acis Res. 44:20 (2016).
Vazquez-Vilar. Marta, Bernabé-Orts . Joan M, Fernandez-del-Carmen. Asun, Ziarsolo . Pello, Jose Blanca, Granell. Antonio and Orzáez Diego. A modular toolbox for gRNA–Cas9 genome
engineering in plants based on the GoldenBraid standard. Plants Methods 12:10 (2016).
54
S-III. Comunicación 2
EASYMAP: A USER FRIENDLY ANALYSIS TOOL FOR
MAPPING-BY-SEQUENCING OF LARGE INSERTIONS AND
POINT MUTATIONS
Samuel Daniel Lup, David Wilson-Sánchez, Sergio Andreu-Sánchez, José Luis
Micol
Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202
Elche Alicante, Spain
Corresponding author: J.L. Micol ([email protected])
Forward genetic screens have identified many genes and continue to be
powerful tools for the dissection of gene action and interactions in Arabidopsis
and other plant species. Moreover, next-generation sequencing (NGS) has
revitalized the time-consuming genetic approaches to identify the mutation
causing a phenotype of interest. Mapping-by-sequencing combines NGS with
classical mapping strategies and allows rapid identification of point mutations
(Schneeberger et al., 2009).
Currently, there are programs that analyze whole-genome sequencing
data in order to map the position of phenotype-causing mutations, but either
they are excessively complicated to install or use, require additional software to
perform complete analysis workflows, or require purchasing very expensive
licenses. We developed Easymap, a program that simplifies the data analysis
workflow from raw reads to candidate mutations. Two main workflow types are
available: bulked segregant mapping, for EMS-induced mutations, and tagged-
sequence mapping, for large insertions such as transposons or T-DNAs.
Easymap performs initial checks on the user data, which are then
processed depending on the needs. Large insertions are mapped by a series of
alignment and filtering steps. For mutations induced by EMS, SNPs are
analyzed based on their allelic frequencies in a phenotyped F2 or M2 mapping
population. The variations found are analyzed against the reference genome
annotation to detect their putative effects on gene function. All the information
relevant for the user is finally presented in a unified report by means of a
graphic interface. In our tests, we found Easymap useful for mapping-by-
sequencing raw data obtained from Arabidopsis and other plant and animal
model systems; our software can be easily installed on Windows, Linux and
Mac OS X.
Schneeberger, K., et al. (2009). Nature Methods 6, 550-551.
55
S-III. Comunicación 3 FINE-TUNE REGULATION OF ARTIFICIAL SMALL-RNA
MEDIATED GENE SILENCING IN PLANTS
Alberto Carbonell1, Lucio López1 and José-Antonio Daròs1
1 Instituto de Biología Molecular y Celular de Plantas (CSIC-Universitat Politècnica de València), Valencia, Spain
Corresponding author: Alberto Carbonell ([email protected])
Artificial small RNAs (sRNAs) are 21-nucleotide RNAs designed to
selectively and effectively silence transcripts with complementary sequences. Typically, active artificial sRNAs accumulate in vivo to high levels, and possess a high degree of base-pairing with their cognate target RNA(s). In plants, they have been extensively used to silence genes of interest in gene function studies, and to generate antiviral resistance or other type of crop improvement. The use of this technology has benefited from the recent development of new methods for designing and generating artificial sRNA constructs such as the P- SAMS web tool (Fahlgren et al., 2016) for the automated design of artificial sRNAs, and a new generation of B/c vectors optimized for one-step cloning and high expression of artificial sRNAs in plants (Carbonell et al., 2014 and 2015). These methodologies have been optimized for time and cost effectiveness, high-throughput applicability and gene silencing efficacy. However, methods for fine-tuning the activity of artificial sRNAs to obtain the desired degree of silencing of the genes of interest have not been reported yet.
In this work, we took advantage of the above-mentioned high-throughput methods for artificial sRNA construct production to explore different approaches to modulate the degree of gene silencing induced by synthetic trans-acting small interfering RNAs (syn-tasiRNAs), a particular class of artificial sRNAs produced through a refined multi-step processing of TAS transcripts. In particular, we analysed the effect on syn-tasiRNA-mediated gene silencing of altering (i) syn-tasiRNA accumulation as a result of modifying syn-tasiRNA position in its TAS precursor, and (ii) the degree of base-pairing between syn- tasiRNA and target RNA(s). Our results indicate that syn-tasiRNA-mediated silencing can be modulated, and thus open the possibility of using artificial sRNAs to achieve different degrees of gene suppression. This could be particularly attractive when lethal or toxic genes need to be silenced, or to partially repress a gene or group of genes in an economically important crop to improve a particular trait and increase the market value of the corresponding product.
References Carbonell, A., et al. (2014). New generation of artificial MicroRNA and synthetic trans-acting
small interfering RNA vectors for efficient gene silencing in Arabidopsis. Plant Physiol 165:15-29.
Carbonell, A. et al. (2015). Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors. Plant J 82:1061-1075.
Fahlgren, N. et al. (2016), A. 2016. P-SAMS: a web site for plant artificial microRNA and synthetic trans-acting small interfering RNA design. Bioinformatics 32:157-158.
Acknowledgements & Funding This study was supported by grants BIO2014-54269-R and BIO2017-83184-R from Ministerio de Economía, Industria y Competitividad (Spain; cofounded FEDER funds) to J.-A.D.. A.C. was the recipient of an Individual Fellowship from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska Curie grant agreement No. 655841.
56
S-III. Comunicación 4 THE VIRAL-DERIVED HAM1 PYROPHOSPHATASE IS
REQUIRED FOR THE IPOMOVIRUS Ugandan Cassava BROWN STREAK VIRUS TO INFECT ITS NATURAL HOST BUT NOT A
MODEL PLANT
Adrian A. Valli1, María Ribaya1, Alfonso González1, Samuel F. Brockington2, Beatriz García1, Fabio Pasin3, Juan Antonio García1
1Department of Plant Molecular Genetics, Spanish National Centre for Biotechnology (CNB-CSIC), Madrid, Spain; 2Department of Plant Sciences, University of Cambridge,
United Kingdom; 3Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan. Corresponding author: Adrian Valli ([email protected])
The family Potyviridae is the biggest and most important group of plant RNA viruses, comprising more than 200 members sorted in ten different genera. Intriguingly, only three of them- one in the Potyvirus genus and two in the Ipomovirus genus - encode a HAM1-like pyrophosphatase highly similar to that present in prokaryotes and eukaryotes (Mbanzibwa et al., 2009). The fact that these three viruses infect plants of the Euphorbiaceae family in nature suggests that HAM1, due to unidentified reasons, is required to infect these particular hosts. Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) are two closely related ipomoviruses expressing HAM1 and causing together what is dubbed the “Ebola of plants”: a viral disease that affect cassavas mainly in Africa with catastrophic consequences. An infectious cDNA clone of UCBSV was recently built in our laboratory (Pasin et al., 2017) and used for reverse genetics experiments aiming to know the relevance and role of viral HAM1 during infection. Hence, we found that complete deletion of HAM1 has no appreciable consequences for UCBSV when infecting Nicotiana benthamiana plants, a classic model for studying viral infections. In contrast, and more importantly, this HAM1 deletion mutant was unable to infect cassava plants, the natural host of UCBSV. By using a MYC-tagged version of HAM1 we have discovered that this factor mainly accumulates as a fusion product attached to the viral replicase (NIb), which is encoded just upstream of HAM1 in the viral genome. Based on our results and the proposed role of HAM1-like proteins in other organisms (Simone et al., 2013), we hypothesise that HAM1 works as a functional domain of NIb to avoid the incorporation of non-canonical nucleotides in the viral genome during replication. Finally, we have carried out a comprehensive phylogenetic study and find that HAM1 pyrophosphatases in those three viruses have a common origin, most probably as a result of a horizontal gene transfer event from plants, which occurred in an ancestor virus that existed before Potyvirus and Ipomovirus. Altogether, our results (i) inform about a novel host-specific requirement for an infection to happen, and (ii) shed light on the evolution of this socio-economically important viral family.
- Mbanzibwa, D.R., Tian, Y., Mukasa, S.B., Valkonen, J.P.T. Cassava brown streak virus (Potyviridae) encodes a putative Maf/HAM1 pyrophosphatase implicated in reduction of mutations and a P1 proteinase that suppresses RNA silencing but contains no HC-Pro (2009) Journal of Virology, 83 (13), pp. 6934-6940. - Pasin, F., Bedoya, L.C., Bernabé-Orts, J.M., Gallo, A., Simón-Mateo, C., Orzaez, D., García, J.A. Multiple T-DNA Delivery to Plants Using Novel Mini Binary Vectors with Compatible Replication Origins (2017) ACS Synthetic Biology, 6 (10), pp. 1962-1968. - Simone, P.D., Pavlov, Y.I., Borgstahl, G.E.O. ITPA (inosine triphosphate pyrophosphatase): From surveillance of nucleotide pools to human disease and pharmacogenetics (2013) Mutation Research - Reviews in Mutation Research, 753 (2), pp. 131-146.
This work is supported by grants BIO2016-80572-R to JAG and BIO2015-73900-JIN to AV.
57
S-III. Comunicación 5
AUTOPHAGY AND CATHEPSIN-LIKE PROTEASES ARE ACTIVATED AND PLAY A ROLE IN STRESS-INDUCED CELL
DEATH DURING MICROSPORE EMBRYOGENESIS IN BARLEY
Ivett Bárány1, Eduardo Berenguer1, María-Teresa Solís1,2, Yolanda Pérez-Pérez1,
M. Estrella Santamaría3, José-Luis Crespo4, María C. Risueño1, Isabel Díaz3, Pilar S. Testillano1
1Pollen Biotechnology of Crop Plants, Biological Research Center, CIB-CSIC, Madrid, Spain, 2Dept. Plant Biology I, UCM, Madrid, Spain,3Center of Plant Biotechnology and Genomics, CBGP, UPM, Madrid, Spain, 4Inst. Plant Biochem.Photosynthesis, IBVF
CSIC, Seville, Spain
Corresponding author: Pilar S. Testillano ([email protected])
Autophagy is a universal cell degradation pathway that plays critical roles in stress response and cell death. Plant cathepsins are Papain-like C1A cysteine- proteases involved in many physiological processes, including programmed cell death. In animals, cathepsins have an integral role in autophagy by degrading autophagic material; however less is known in plants. During stress-induced microspore embryogenesis, in vitro biotechnological process with many applications in plant breeding, many cells die after induction, limiting the efficiency of the process. In this work we have analysed the involvement of autophagy in cell death, in relation to cathepsins activation, during stress- induced microspore embryogenesis in Hordeum vulgare. After stress, ROS and cell death increased and autophagy was activated, including HvATG5 and HvATG6 up-regulation and increase of ATG5, ATG8 and autophagosomes. Concomitantly, cathepsin L/F-, B- and H-like activities were induced, cathepsin- like genes HvPap-1 and HvPap-6 were up-regulated, and HvPap-1, HvPap-6 and HvPap-19 proteins increased and localized in cytoplasm resembling autophagy structures. Inhibitors of autophagy and cysteine-proteases reduced cell death and promoted embryogenesis initiation. Findings reveal a role for autophagy in stress-induced cell death during microspore embryogenesis, with the participation of cathepsins. Similar patterns of activation, expression and localization suggest a possible connection between cathepsins and autophagy. Results open up new possibilities to enhance microspore embryogenesis efficiency with autophagy and/or cysteine-protease modulators.
Reference: Bárány I, Berenguer E, Solís MT, Pérez-Pérez Y, Santamaría ME, Crespo JL, Risueño MC, Díaz I, Testillano PS (2018) Autophagy and cathepsins are activated and involved in cell death during stress-induced microspore embryogenesis in barley. Journal of Experimental Botany 69, 1387-1402.
Funding: Supported by projects (AGL2014-52028-R, AGL2017-82447-R) funded by MINECO and ERDF/ FEDER.
58
S-III. Comunicación 6
MyROOT: A NOVEL METHOD AND SOFTWARE FOR THE SEMI-
AUTOMATIC MEASUREMENT OF PLANT ROOT LENGTH
Isabel Betegón-Putze1, Alejandro González2, Xavier Sevillano2, David Blasco-
Escámez1 and Ana I. Caño-Delgado1
1 Department of Molecular Genetics, Center for Research in Agricultural Genomics (CRAG)
CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra (Cerdanyola del Vallès), 08193 Barcelona,
Spain. 2 GTM- Grup de recerca en Tecnologies Mèdia, La Salle, Universitat Ramon Llull,
08022 Barcelona, Spain.
Corresponding author: Ana I. Caño-Delgado ([email protected])
The root is an essential organ for plant growth and development, so the
characterization of different root traits is crucial for plant research and agriculture.
The analysis of the primary root of Arabidopsis thaliana (Arabidopsis) is widely used
for genetic and molecular studies, however the measurement of its length is still
performed in a time consuming manner with the use of conventional softwares that
require the manual identification of each root.
To save human labour and time in this routine task we developed MyROOT, a
software for the semi-automatic quantification of root length of Arabidopsis seedlings
growing in agar plates. MyROOT is able to automatically determine the scale from an
image of the plate and to accurately measure the root length of the individual plants.
The software determines root length through a bottom-up root tracking process and
the detection of the hypocotyls to identify the starting point of each root. The process
is performed in consecutive steps that can be supervised and modified by the user to
adapt to different image conditions or plant phenotypes. We validated MyROOT
measurements and we found no significant differences with the conventional
methods, thus indicating its usefulness for the scientific community.
Overall, we created MyROOT software that permits high-throughput root length
measurement while minimizing user intervention and time.
59
SESIÓN IV
DESARROLLO
MODERADORES:
Cristina Ferrándiz (IBMCP, CSIC-
UPV, Valencia)
David Posé (IHSM-UMA-CSIC,
Málaga)
60
S-IV. Ponencia 1
STUDY OF TRANSCRIPTIONAL REGULATORY NETWORK
CONTROLLING STRAWBERRY FRUIT RIPENING AND QUALITY
Carmen Martín-Pizarro, Eva Lucas-Reina, Almudena Trapero-Mozos,
Victoriano Meco, David Posé
Departamento de Biología Molecular y Bioquímica, IHSM-UMA-CSIC, Málaga, Spain.
Corresponding author: David Posé (dpose@uma)
Ripening is a critical step for the development of flavor quality in fruits. This
character has significantly declined in many fleshy fruits over recent decades.
This is particularly significant in strawberry (Fragaria ´ ananassa), where
current cultivars are derived from a narrow germplasm collection. Improving fruit
quality requires two important breakthroughs: 1) a precise understanding of the
fruit ripening process that will allow the targeting of relevant genes, and 2) the
identification of novel alleles responsible for fruit quality traits.
In our project, we aim at the identification and characterization of key
transcription factors involved in fruit ripening regulation and their target genes,
in order to infer the Gene Regulatory Network controlling this process. Also, we
are using a collection of around two hundred wild strawberry (Fragaria vesca)
accessions to identify loci involved in important traits such as aroma, size or
resistance to pathogens. Finally, we are implementing the use of the genome-
editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect it might
open opportunities for engineering this species to improve traits of economic
importance.
Acknowledgements & Funding. This work is supported by the Grant ERC-2014-StG 638134
(European Research Council) and the Ramón y Cajal program RYC 2013-1269 (MINECO-
Universidad de Málaga, Spain.
61
S-IV. Ponencia 2
LIFE SPAN IN MONOCARPIC PLANTS: UNRAVELING THE
BASIS OF GLOBAL PROLIFERATIVE ARREST.
Vicente Balanzà, Irene Martínez-Fernández, Cristina Ferrándiz.
Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de
Investigaciones Científicas–Universidad Politécnica de Valencia, Valencia 46022,
Spain
Corresponding author: Cristina Ferrándiz ([email protected])
Monocarpic plants have a single reproductive cycle in their lives; the initiation of
this cycle is determined by the flowering transition, and the end, and therefore
life span, is determined by the coordinated arrest of all meristems, known as
global proliferative arrest (GPA). In Arabidopsis and many other monocarpic
plants, we have a wealth of information about the onset flowering, but, in
contrast, very little is known about the cessation of flowering, that is, the
molecular bases and the signalling mechanisms controlling GPA. Our group has
recently uncovered a novel genetic pathway regulating GPA in Arabidopsis that
responds to age dependent factors and is conserved in different species, the
FRUITFULL-APETALA2 (FUL-AP2) pathway, that controls the temporal
maintenance of WUSCHEL expression and SAM activity (Balanzà et al., 2018).
Interestingly, the FUL-AP2 pathway acts in parallel with systemic cues of
unknown nature from developing seeds, but the possible crosstalk between
them has not been uncovered yet. To address these questions, as well as to
find out new players involved the GPA regulation, acting in parallel or
downstream the module FUL-AP2, we are implementing different genetic,
molecular and whole genome approaches. Our latest progress in these
directions will be presented.
-Genetic control of meristem arrest and life span in Arabidopsis by a FRUITFULL-APETALA2
pathway. 2018. Vicente Balanzà, Irene Martínez-Fernández, Shusei Sato, Martin F Yanofsky,
Kerstin Kaufmann, Gerco C Angenent, Marian Bemer, Cristina Ferrándiz. Nature
communications. 9 (1), 565.
62
S-IV. Comunicación 1 THE CIRCADIAN CLOCK SETS THE TIME OF DNA
REPLICATION LICENSING TO REGULATE GROWTH IN
ARABIDOPSIS Jorge Fung-Uceda1, Kyounghee Lee2 Pil Joon Seo2, Stefanie Polyn3,4, Lieven De
Veylder3,4, Paloma Mas1,5*
1Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra, 08193 Barcelona, Spain, 2Department of Biological
Sciences, Sungkyunkwan University, Suwon 16419, Republic of Korea, 3Center for Plant Systems Biology, VIB, Technologiepark 927, 9052 Gent, Belgium, 4Department of Plant Biotechnology and Bioinformatics, Ghent University,
Technologiepark 927, 9052 Gent, Belgium, 5Consejo Superior de Investigaciones Científicas (CSIC), 08028 Barcelona, Spain.
*Corresponding author: Paloma Mas ([email protected]) Plants, as many organisms, adapt their physiology and metabolism to the surrounding environment. To predict the diurnal and seasonal changes, they rely on an internal biochemical oscillator known as the circadian clock. In plants, this endogenous oscillator consists on a set of core genes with a sequential phase of expression during the day and night. Its synchronization with the environment generates rhythmic patterns of activity in nearly all biological processes. Growth and development are two key biological processes controlled by the circadian clock. Plant growth is also tightly controlled by cell division and expansion. Due to their sessile nature and because plants lack of a defined body plan or cell migration within organs, the spatiotemporal regulation of cell division and expansion is essential for proper morphogenesis and differentiation. The circadian clock and the cell cycle have been well studied in plants as separate pathways. However, their possible interaction to sustain plant growth has been not addressed until now. In our studies, we found that indeed the circadian clock controls the timing of the cell cycle to regulate growth in Arabidopsis. Kinematics assays of leaf cell growth revealed that miss-expression of the core clock component TOC1 (TIMING OF CAB EXPRESSION1) modifies the pace of the mitotic cycle, resulting in alteration of plant growth by affecting the number of leaf cells. Flow cytometry analyses showed that at later stages of development, miss-expression of TOC1 also affects somatic ploidy by altering the endocycle (a variant of the cell cycle consisting on successive rounds of G1 and S phases). Moreover, a detailed study of the cell cycle duration showed that TOC1 safeguards the G1 to S phase transition and that over-expression of TOC1 shortens the S-phase. This shortening correlated well with the binding of TOC1 to the CDC6 promoter and the repression of the CDC6 gene, which encodes a DNA replication licensing factor with a key role in the entry and progression of the S phase. Therefore, our results demonstrate that TOC1 sets the time of the DNA pre-replicative machinery to control plant growth in resonance with the environment. Acknowledgements & Funding We thank R. Deeken for the Agrobacterium strains, Prof. T. Nakagawa for the Gateway vector, Montse Amenós for help with the confocal microscope, Manuela Costa for assistance with the ploidy analyses, Holly Kay for help with the cell size measurements, and Jorge Martínez with the hypocotyl assays. This work was supported by research grants from the Spanish Ministry of Economy and Competitiveness (BFU2016-77236-P), Generalitat de Catalunya (AGAUR) (2017 SGR 1211), Global Research Network of the National Research Foundation of Korea, and European Commission Marie Curie Research Training Network (CHIP- ET, FP7-PEOPLE-2013- ITN607880) to P.M. We also acknowledge the CERCA Programme/Generalitat de Catalunya and the financial support from the Spanish Ministry of Economy and Competitiveness, through the ‘‘Severo Ochoa Programme for Centres of Excellence in R&D’’ 2016–2019 (SEV-2015- 0533).
63
S-IV. Comunicación 2
A TRANSCRIPTIONAL REPROGRAMMING MODULE FOR STEM
CELL DIFFERENTIATION
Mary-Paz González-García1, Ramon Contreras1, Alfonso Muñoz1, Jose-
Juan Sánchez-Serrano1, Maite Sanmartín1 and Enrique Rojo1.
1Plant Genetic Department-Laboratory 316, Institution CSIC- National Centre for
Biotechnology (CNB), Madrid, Spain
Corresponding author: Enrique Rojo ([email protected])
Modulation of plant architecture is a major target for breeding strategies.
Architecture is ultimately depends on the generation of organs from reservoirs
of stem cells present in meristems. Knowledge on stem cell functioning,
maintenance and differentiation therefore of utmost importance to understand
how plants are built and how to modify them at will. MINIYO (IYO) is an RNA
Polymerase II (Pol II) interacting factor that is essential and rate limiting for stem
cell differentiation in Arabidopsis. Moreover, nuclear accumulation of IYO
coincides with the onset of differentiation, suggesting that migration of this
factor into the nucleus acts as a molecular switch to initiate stem cell
differentiation in plants (Sanmartin et al. 2011) and, interestingly, also in
animals (Lynch et al. 2018). We have identified the IYO-interactor RIMA as a
key partner for initiating differentiation (Muñoz et al., 2017) and, in particular, for
activating endoreduplication, a process that often correlates positively with cell
and organ growth and hence with plant yield. We will present data supporting
that IYO and RIMA mediate large scale transcriptional reprogramming to initiate
stem cell differentiation. Knowledge gained in this model plant can readily be
transferred to other species and eventually provide breeders with new tools to
modify traits, such as plant architecture and seed longevity, to optimize and
secure future crop yields.
Lynch et al. 2018 Cell Rep. 22(2):396-410
Muñoz et al. 2017 Plant Cell 29(3):575-588
Sanmartin et al. 2011. Curr Biol 21(12): 999-1008.
Acknowledgements & Funding: The Spanish Ministry of Economy and Competitivity and FEDER
funds (BIO2015-69582-P MINECO/FEDER)
64
S-IV. Comunicación 3 THE EFFECT OF PARENTAL-SPECIFIC EPIGENETIC MARKS IN
ARABIDOPSIS ENDOSPERM DEVELOPMENT
Jordi Moreno-Romero1,2, Hua Jiang2, German Martinez2, Juan Santos-
González2, Claudia Köhler2. 1Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra, Barcelona, Spain. 2Department of Plant Biology, Uppsala BioCenter,
Swedish University of Agricultural Sciences and Linnean Center of Plant Biology, Uppsala, Sweden.
Corresponding author: Jordi Moreno-Romero ([email protected])
The seed is a structure composed by the result of a double fertilization event that leads to the formation of two fertilization products, the zygotic embryo and the endosperm, and the maternally derived seed coat that surrounds them. The endosperm is an ephemeral tissue that nourishes the embryo. Maternal and paternal factors regulate the embryo and endosperm development because, despite their genetic resemblance, the parental genomes are epigenetically distinct. Several of these epigenetic-controlled processes occur in the endosperm during the first days of seed development, such the genomic imprinting, a parent-of-origin dependent gene expression caused by maternally or paternally inherited alleles. These epigenetic mechanisms are involved in the interspecies and interploidy hybridization barrier that leads to seed abortion as a consequence of an endosperm development failure.
For deciphering the parental-specific mechanisms that control endosperm development we have performed genome-wide profiles of several histone marks and DNA methylation of the early endosperm (Moreno-Romero et al., 2016). Using maternal and paternal sequence polymorphisms we are able to differentiate the parental origin of the epigenetics marks. The data reveals that paternal expressed genes (PEGs) are regulated by DNA methylation and the Polycomb-mediated H3 lysine 27 trimethylation (H3K27m3). Moreover, we show a parental-specific asymmetry in the heterochromatic marks H3 lysine 9 dimethylation (H3K9m2) and H3 lysine 27 monomethylation (H3K27m1). After the characterization of these marks in the normal developing endosperm, we have studied their role in seed abortion by analysing the epigenome profiles of interploidy cross derived endosperm. In these studies we have further characterized the already known participation of PEGs and DNA methylation in interploidy cross seed failure and found a new role of H3K9me2 (Hua et al., 2017) and sRNA (Martinez et al., 2018) in the creation of hybridization barriers. By modifying these epigenetic components in the endosperm, we are able to bypass the interploidy hibridization barrier and obtain viable seeds.
Moreno-Romero et al. EMBO J (2016) 35(12):1298-311. Hua et al. Genes Dev. (2017) 31(12): 1272–1287. Martinez et al. Nat Genet (2018) 50(2):193-198.
This work has been supported by grants from European Research Council (N280496), Swedish Science Foundation (2014-3820), Knut och Alice Wallenbergs Stiftelse (KAW 2012.0087), Nilsson Ehle foundation and MINECO-FEDER (BIO2017-85316-R) and the “Centro de Excelencia Severo Ochoa 2016-2019” award SEV-2015-0533.
65
S-IV. Comunicación 4 REGULATION OF THE ASPARAGINE SYNTHETASE ENCODING
GENE HvASN1 IN BARLEY SEEDS BY TRANSCRIPTION
FACTORS HvbZIP53 AND HvBLZ1
*Raquel Iglesias-Fernández, Rosario Alonso, Miguel Ángel Delgado-Gutierrez,
Elena Pastor-Mora, Pilar Carbonero, Jesús Vicente-Carbajosa,
Centro de Biotecnología y Genómica de Plantas (UPM-INIA) and E.T.S. Ingeniería
Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid,28223-
Madrid, Spain
*Corresponding author: Raquel Iglesias-Fernández ([email protected])
Asparagine synthetase (ASN) is a key enzyme involved in nitrogen assimilation, recycling, transport and storage in plants. Glutamine-dependent Asparagine Synthetases (ASNs) are encoded by small multigene families in plants and have been associated to nitrogen remobilization events during seed development (Gaufichon et al., 2017). Transcription regulation upon Hordeum vulgare seed development is controlled by several types of Transcription Factors (TFs), of the bZIP, DOF, B3 and MYB families (Alonso et al., 2009; Abraham et al., 2016; Carbonero et al., 2017). We report here that TFs of the bZIP type, HvbZIP53 and HvBLZ1 are transcriptional activators of HvASN1.
In the Hordeum vulgare genome, two HvASN1 paralog genes (HvASN1.1 and HvASN1.2) have been identified, being only HvASN1 expressed upon seed development. To gain insight into the HvASN1 transcriptional regulation, a bioinformatic search for conserved non-coding cis-elements (phylogenetic shadowing) within the Poaceae orthologous ASN1 gene promoters has been done and a conserved G-box element recognised by bZIP TFs has been identified. The HvASN1.1 promoter is transcriptionally activated in transient expression assays by HvbZIP53 and HvBLZ1 TFs. The protein-protein interaction of these TFs has been confirmed in yeast (Y2H assays) and in plant nuclei by Bimolecular Fluorescence Complementation (BiFC). The transcriptional regulation of HvASN1.1 gene expression by HvbZIP53 and HvBLZ1 has been validated by several molecular techniques such as RT-qPCR analyses and mRNA in situ hybridization experiments.
Abraham Z, Iglesias-Fernández R, Martínez M, Rubio-Somoza I, Díaz I, Carbonero P, Vicente- Carbajosa J A. 2016. Developmental Switch of Gene Expression in the Barley Seed Mediated by HvVP1 (Viviparous-1) and HvGAMYB Interactions. Plant Physiol. 170: 2146-2158.
Alonso R, Oñate-Sánchez L, Weltmeier F, Ehlert A, Diaz I, Dietrich K, Vicente-Carbajosa J, Dröge-Laser W. 2009. A pivotal role of the basic leucine zipper transcription factor bZIP53 in the regulation of Arabidopsis seed maturation gene expression based on heterodimerization and protein complex formation. Plant Cell. 21: 1747-1761.
Carbonero P, Iglesias-Fernández R, Vicente-Carbajosa J. 2017. The AFL subfamily of B3 transcription factors: evolution and function in angiosperm sees. J. Exp. Bot. 68: 871-880.
Gaufichon L et al. 2017. ASN1-encoded asparagine synthetase in floral organs contributes to nitrogen filling in Arabidopsis seeds. Plant J. 91: 371-393.
Acknowledgements & Funding: This work was financially supported by MINECO, Spain (Projects BIO2014-53181-R; p.i. J. V-C and BIO2017-82873-R; p.i. J. V-C and co-IP. R. I-F.).
66
S-IV. Comunicación 5
ARABIDOPSIS INCURVATA11 AND CUPULIFORMIS2 ARE NEW
COMPONENTS OF THE EPIGENETIC MACHINERY
Riad Nadi, Eduardo Mateo-Bonmatí, Lucía Juan-Vicente, José Luis Micol.
Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202
Elche, Alicante, Spain.
Corresponding author: José Luis Micol ([email protected])
All critical developmental and physiological events in a plant’s life cycle
depend on the proper activation and repression of specific gene sets; this
regulation can involve epigenetic mechanisms. Some Arabidopsis thaliana
mutants with disorders of the epigenetic machinery exhibit pleiotropic defects,
including incurved leaves and early flowering, due to the ectopic and
heterochronic expression of developmental regulators (Goodrich et al., 1997;
Barrero et al., 2007). We studied one such mutant class, the incurvata11 (icu11)
loss-of-function mutants, and identified ICU11, the founding member of a small
gene family, that we named CUPULIFORMIS (CP), of the 2-oxoglutarate/Fe(II)-
dependent dioxygenase superfamily. ICU11 and its closest paralog CP2 have
unequally redundant functions: although cp2 mutants are phenotypically wild
type, icu11 cp2 double mutants skip vegetative development and flower upon
germination; this lethal phenotype is reminiscent of loss-of-function mutants of
the Polycomb-group genes EMBRYONIC FLOWER1 (EMF1) and EMF2 (Chen
et al., 1997).
Double mutants harboring icu11 alleles and loss-of-function alleles of
genes encoding components of the epigenetic machinery exhibit synergistic,
severe phenotypes, some similar to those of emf mutants. Hundreds of genes
are misexpressed in icu11 plants, including SEPALLATA3 (SEP3; Pelaz et al.,
2000); derepression of SEP3 causes the leaf phenotype of icu11. ICU11 and
CP2 are nucleoplasmic proteins that act as epigenetic repressors through an
unknown mechanism involving histone methylation, but not DNA methylation.
Barrero, J.M., et al. (2007). Plant Cell 19, 2822-2838.
Chen, L., et al. (1997). Plant Cell 9, 2011-2024.
Goodrich, J., et al. (1997). Nature 386, 44-51.
Pelaz, S., et al. (2000). Nature 405, 200-203.
67
S-IV. Póster 1
DOMINANT ALLELES OF PRE-mRNA PROCESSING FACTOR 8 INCREASE SPLICING FIDELITY IN ARABIDOPSIS
Adrián Cabezas-Fuster1, Rosa Micol-Ponce1, and María Rosa Ponce1.
1Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche,
Alicante, Spain.
Corresponding author: María Rosa Ponce ([email protected])
We performed a screen for EMS-induced suppressors of the morphological
phenotype of ago1-52, a hypomorphic allele of ARGONAUTE1 (AGO1), which
encodes a key factor in microRNA (miRNA) pathways in Arabidopsis (Micol-Ponce et
al, 2014). Five of the extragenic suppressors that we identified were dominant alleles
of MORPHOLOGY OF AGO1-52 SUPPRESSED 5 (MAS5), which was found to
encode pre-mRNA PROCESSING FACTOR 8 (PRP8), a key component of the core
spliceosome. mas5-1 is an informational suppressor of ago1-52 and also suppresses
icu13-1 (a CULLIN1 allele); these two mutations cause missplicing due to the
occurrence and preferential use of alternative splicing acceptor and donor signals,
respectively. However, mas5-1 does not suppress the aberrant splicing caused by
mutations that eliminate splicing acceptor or donor signals. We found increased
levels of the wild-type AGO1 and CUL1 splicing variants and their corresponding
translation products in the ago1-52 mas5-1 and icu13-1 mas5-1 double mutants,
compared to the single ago1-52 and icu13-1 mutants.
On the other hand, we observed synergistic phenotypes in double mutants
involving hypomorphic, recessive prp8 (mas5) mutations and ago1 alleles with
normal splicing. Since human PRP8 seems to be a ribosome biogenesis factor, in
addition to its role in pre-mRNA splicing, we obtained double mutants that revealed
genetic interactions of PRP8 with SMALL ORGAN 4 (SMO4) and RIBOSOMAL RNA
PROCESSING 7 (RRP7), two Arabidopsis genes that encode ribosome biogenesis
factors. Our results (1) explain the observed morphological suppression in ago1-52
mas5 plants, (2) suggest that mas5 alleles increase splicing fidelity, promoting the
use of genuine donor and acceptor splicing signals instead of the new signals
created by the ago1-52 and icu13-1 mutations, and (3) suggest that PRP8 has a role
in both miRNA metabolism and ribosome biogenesis in Arabidopsis.
Micol-Ponce et al. (2014). Sci. Rep. 4, 5533.
Research in the laboratory or M.R.P. is supported by grants from the Ministerio de Economía y
Competitividad of Spain (BIO2014-56889-R and BIO2017-89728-R) and the Generalitat Valenciana
(PROMETEOII/2014/006).
68
S-IV. Póster 2 THE ROLE OF DEVIL PEPTIDES IN CONTROLING CELULAR
PROLIFERATION AND PLANT MORPHOGENESIS
Ana Alarcia-García, Elena Perpiñan, Priscilla Rossetto, Amparo Primo, Cristina
Ferrándiz.
Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones
Científicas—Universidad Politécnica de Valencia, 46022 Valencia, Spain.
DEVIL (DVL) peptides have been identified in Arabidopsis. These are small size and unknown function peptides, encoded by a gene family of 24 members and are located in the plasma membrane. The DVL genes were characterized a decade ago by their overexpression phenotypes, which dramatically affect fruit morphology (Wen et al. 2004; Narita et al. 2004; Guo et al. 2015). However, the loss-of-function mutants do not have any obvious morphological phenotype, so the biological function of these peptides is essentially unknown.
In the lab we have carried out different studies to characterize how DVL peptides interact with the plasma membrane, how are they expressed and accumulate, identify other proteins that interact physically with these peptides, and whether the combination of multiple loss-of-function mutants affect development. Our preliminary results suggest that DVL peptides appear to be mobile signals not integrated in the plasma membrane, and to participate in the control of cell cycle and cell division processes. We are currently extending our work in these directions, generating combinations of multiple mutants in different DVL genes with high sequence similarity (dvl8 dvl11 rtfl9, dvl8 dvl11 rtfl11, and dvl8 dvl11 rtfl9 rtfl11). These combinations do not show obvious loss of function phenotypes, thus supporting the idea of high gene redundancy among members of DVL family (Wen et al. 2004), and that higher order combinations must be required to observe a morphological phenotype. Therefore, we have also used CRISPR/Cas9 technology (Wang et al. 2015) to mutate additional DVL genes to include in this study.
In addition, to confirm and better understand the differences in spatial distribution of mRNA and peptides for DVL1 and DVL8, we have generated pDVLxDVLx:GUS or pDVLx::DVLx:GFP and promoter:reporter lines to try to determine whether this differential distribution is due to peptide active transport. In addition, we are starting to functionally validate the possible protein interactions uncovered in a yeast-2-hybrid screening. These and other preliminary results will be presented, along with possible future directions.
Guo P, Yoshimura A, Ishikawa N, Yamaguchi T, Guo Y, and Tsukaya H. (2015) Comparative analysis of the RTFL peptide family on the control of plant organogenesis. J. Plant Res. 128, 497-510. Narita NN, Moore S, Horiguchi G, Kubo M, Demura T, Fukuda H, Goodrich J, and Tsukaya H. (2004) Overexpression of a novel small peptide Rotundifolia4 decreases cell proliferation and alters leaf shape in Arabidopsis thaliana. Plant J. 38, 699-713. Wang Z, Xing H, Dong L, Zhang H, Han C, Wang X and Chen Q. (2015) Egg cell-specific promoter- controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation. Genome Biology 16,144. Wen J, Lease KA and Walker, J.C. (2004) DVL, a novel class of small polypeptides: overexpression alters Arabidopsis development. Plant J, 37, 668-677.
69
S-IV. Póster 3
DIVIDING TO RULE: CDK INHIBITORS MEDIATE DELLA
SEPARABLE ROLES IN SHOOT MERISTEM ACTIVITY AND STEM
ELONGATION
Antonio Serrano-Mislata 1,2, Sherezade Ortíz-Villajos Cano 1, Robert Sablowski 2, David Alabadí 1, Miguel Ángel Blázquez 1
1 Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), Valencia, Spain 2 John Innes Centre, Norwich, United Kingdom
Corresponding author: Antonio Serrano-Mislata ([email protected])
DELLA proteins are central regulators of plant growth in response to the
environment. When plants encounter any type of stress, DELLAs orchestrate the response by favouring the defence mechanism and decreasing growth rates. DELLA activity relies on interaction with a large set of proteins (mostly transcription factors) involved in many different processes like cell cycle, cell expansion or microtubule organization. As a consequence, DELLA loss- and gain-of-function mutants are highly pleiotropic and, from an agronomical point of view, it would be desirable to separate DELLA functions, i.e. to be able to alter a specific trait of interest without secondary effects.
DELLAs control the activity of the shoot apical meristem (SAM) and the elongation of the inflorescence stem through inhibition of cell proliferation rates in the shoot apex. This DELLA function correlated with direct upregulation of the CDK inhibitor KRP2, a negative regulator of cell cycle progression. KRP2 loss-of-function was sufficient to recover SAM size, but not stem growth, and promote flower production in DELLA semi-dwarf backgrounds (Serrano-Mislata et al., 2017). Therefore, DELLA proteins have genetically separable roles in the control of SAM activity and stem elongation.
The expression level of other CDK inhibitors, such as SIM or SMR1, was also increased in genetic backgrounds with enhanced DELLA activity (Achard et al., 2009). Interestingly, we have observed that SMR1 mutation partially restores the defect on stem elongation of the gai-1D mutant, which encodes a DELLA protein resistant to degradation mediated by gibberellins. Furthermore, revisiting ChIP-seq data (Serrano-Mislata et al., 2017) indicated that DELLA binds to genomic regions in the vicinity of the SMR1 gene. Altogether these observations suggest that regulation of distinct CDK inhibitors can explain the separable roles of DELLAs in SAM and stem tissues. To test this hypothesis, we are currently analysing SAM activity in gai-1D smr1-1 plants. We are also studying whether increased inflorescence growth of gai-1D smr1-1 mutants imposes a penalty on the plant defence response mediated by DELLAs or whether it is also possible to separate DELLA functions in growth and stress.
Achard P, Gusti A, Cheminant S, Alioua M, Dhondt S, Coppens F, Beemster GT, Genschik P. Gibberellin signaling controls cell proliferation rate in Arabidopsis. Current Biology. 2009; 19: 1188-93 Serrano-Mislata A, Bencivenga S, Bush M, Schiessl K, Boden S, Sablowski R. DELLA genes restrict inflorescence meristem function independently of plant height. Nature Plants. 2017; 3: 749-754
70
S-IV. Póster 4
PHENOTYPIC SPACE OF ROOT SYSTEM ARCHITECTURE IN
DIVERSE TOMATO GENOTYPES
Aurora Alaguero-Cordovilla1, Francisco Javier Gran-Gómez1, Joan Sánchez-
Pascual1, Antonio J. Monforte2, Jose Manuel Pérez-Pérez1
1Instituto de Bioingeniería, Universidad Miguel Hernández, Elche, Spain, 2Instituto de
Biología Molecular y Celular de Plantas, UPV–CSIC, Valencia, Spain.
Corresponding author: Jose Manuel Pérez-Pérez ([email protected])
The root system is fundamental for plant growth, with basic functions on the selective
absorption of water and nutrients, as a mechanical support and storage organ, and
for the modulation of some stress responses (Atkinson et al. 2014). Cultivated
tomatoes are particularly sensitive to drought and have poor N and P use efficiency;
hence the manipulation of their root system architecture (RSA) may improve water
and nutrient capture (Koevoets el al. 2016). To explore the phenotypic variation in
RSA traits in tomato, we studied nine representative wild tomato species and four
commercial tomato cultivars during early growth in a controlled environment. We
found extensive differences between the studied lines for a number of meaningful
morphological traits, such as lateral root distribution, lateral root length or
adventitious root development, which might represent local adaptation to soil
conditions during speciation and through domestication. Additionally, early RSA has
been characterized in 26 introgression lines derived from a cross between Solanum
lycopersicum and S. pimpinellifolium. Our results provide a general quantitative
framework to initiate the genetic characterization of early RSA in tomato.
Atkinson, J.A., et al. (2014). Plant Physiol 166: 538-550
Hochholdinger, F., et al. (2018). Trends Plant Sci 23: 79-88
Work in the laboratory of J.M.P.-P. (AGL2012-33610 and BIO2015-64255-R) is funded by the Ministry
of Economy and Competitiveness and by FEDER funds of the EC - "A way to build Europe“.
71
S-IV. Póster 5 OIL BODY RELATED PROTEINS INTERACTION WITH bZIP
TRANSCRIPTION FACTORS AND NITRIC OXIDE
Camilla Molinari1, Inmaculada Sánchez-Vicente1, Oscar Lorenzo1
1Department of Botany and Plant Physiology –Spanish-Portuguese Agricultural Research
Institute (CIALE), University of Salamanca, Salamanca, Spain.
Corresponding author: Oscar Lorenzo ([email protected])
Nowadays nitric oxide (NO) is considered to be a significant chemical messenger in plant biology with a diverse range of functions through the plant development and immune defence. Specifically, it has been involved in de-etiolation, root development and seed germination, among others (Sanz et al., 2015). Furthermore, bZIP transcription factors have a fundamental role during plant growth (Jakoby et al., 2002). In this context, we aim to identify a possible signalling link between both.
Our previous results showed how NO promotes germination though ABI5 degradation which
is modified by S-nitrosylation (Albertos et al,. 2015). This NO bioactivity is the most common
way of plant signalling of the molecule. The homology described between ABI5 and bZIP67,
which is related to fatty acid storage (Mendes et al,. 2013), could indicate a similar regulation
for bZIP67. Thus, finding protein interactors of bZIP67 by TAP-Tagging technology was
crucial to identify novel mechanisms of regulation. We found a wide variety of proteins
particularly those related with oil bodies interact with the transcription factor, such as
HYDROXYSTEROID DEHYDROGENASE 1 (HSD1) and OLEOSINS. The first one is
hypothetically related to brassinosteroids signalling (Li et al,. 2007) and reserves mobilisation
(Baud et al,. 2009). The interplay between HSD1 and bZIP67 shows HSD1 protein regulation in the presence of
ABA and proteasome inhibitor MG132 and highlight that HSD1 degradation is regulated by
the ubiquitin-proteasome system and positive regulated by ABA presence.
Albertos, P., Romero-Puertas, M.C., Tatematsu, K., Mateos, I., Sánchez-Vicente, I., Nambara, E. and
Lorenzo, O. (2015). “S-nitrosylation triggers ABI5 degradation to promote seed germination and
seedling growth”. Nature Communications 6:8669
Baud, S., N. R. Dichow, Z. Kelemen, S. D’Andréa, A. To, N. Berger, M. Canonge et al. (2009)
“Regulation of HSD1 in seeds of Arabidopsis thaliana.” Plant Cell Physiol 50, no. 8: 1463-78.
Jakoby , M., B. Weisshaar, W. Dröge-Laser, J. Vicente-Carbajosa, J. Tiedemann, T. Kroj and F.
Parcy. (2002) "bZIP transcription factors in arabidopsis." Trends Plant Sci, 7, no. 3: 106-11.
Mendes, A., A. A. Kelly, H. van Erp, E. Shaw, S. J. Powers, S. Kurup and P. J. Eastmon. (2013)
“bZIP67 regulates the omega-3 fatty acid content of arabidopsis seed oil by activating FATTY ACID
DESATURASE 3.” Plant Cell 25, no. 8:3104-16.
Sanz, L., Fernández-Marcos, M., Modrego, A., Lewis, D.R., Muday, G.K., Pollman, S., Dueñas, M.,
Santos-Buelga, C., Lorenzo, O. (2014). “Nitric oxide plays a role in stem cell niche homeostasis
through its interaction with auxin”. Plant Physiology 166 (4) 1972-1984.
This work is financed by grants: ERC.KBBE.2012.1.1-01 (EcoSeed-311840). MINECO: (BIO2017-
85758-R), CONSOLIDER (CSD2007-00057). Junta de Castilla y León (SA093U16). Fundación
Solórzano (FS/26-2017).
72
S-IV. Póster 6
DEVELOPMENTAL ROLE OF ARABIDOPSIS ABCE2, A PUTATIVE
RIBOSOME-DISSOCIATING FACTOR
Carla Navarro-Quiles, Eduardo Mateo-Bonmatí, and José Luis Micol
Instituto de Bioingeniería, Universidad Miguel Hernández, Elche, 03202, Alicante, Spain.
Corresponding author: José Luis Micol ([email protected])
Most members of the large, evolutionarily conserved family of ATP-Binding
Cassette (ABC) proteins function as membrane transporters. However, all members
of the ABCE subfamily are soluble and were initially dubbed as RNase L inhibitor
(RLI) proteins. ABCE proteins are present in all eukaryotes and archaea and are
encoded by a single gene in most genomes, or by two genes in a few, as is the case
of Arabidopsis. Functional analysis of ABCE genes, primarily in Saccharomyces
cerevisiae, has shown that ABCE proteins have essential functions as part of the
translational apparatus, through their ribosome-dissociating activity (Nürenberg, et
al., 2013). In all studied eukaryotes and archaea, null alleles or RNA interference of
ABCE genes are lethal, and hypomorphic alleles cause slow growth (Coelho, et al.,
2005; Kougioumoutzi, et al., 2013).
In a screen for Arabidopsis leaf morphological mutants induced by EMS, we
isolated the apiculata7-1 (api7-1) mutant, which has small, dentate and pointed
leaves, with pale margins and aberrant venation pattern. These phenotypes occur in
many mutants with defects in components of the translation machinery. The api7-1
mutation is a hypomorphic allele of ABCE2. The ABCE2 gene is expressed through
all Arabidopsis developmental stages, while the expression levels of its paralog
ABCE1 are very low in all studied organs. We conducted a co-immunoprecipitation
assay of the ABCE2:YFP fusion protein and identified its interactors by liquid
chromatography-tandem mass spectrometry. Consistent with the potential role of
ABCE2 in dissociating ribosomes, we found EUKARYOTIC RELEASE FACTOR 1-2
(ERF1-2) and ERF-3 as its interacting partners. In addition, we detected some
subunits of the eukaryotic translation initiation factor 3 (eIF3), supporting the recently
discovered presence of ABCE proteins on 40S subunits during the early steps of
translation reinitiation in Saccharomyces cerevisiae and rabbit reticulocytes
(Mancera-Martínez, et al., 2017). api7-1 is the first viable mutant allele of ABCE2
described; our study of this allele reveals a developmental role for ABCE proteins in
Arabidopsis leaf organogenesis and venation patterning.
Coelho, C.M., et al. (2005). Development 132, 5411-5424.
Kougioumoutzi, E., et al. (2013). Plant J 73, 533-545.
Mancera-Martínez, E., et al. (2017). RNA Biol 0, 1-7.
Nürenberg, E., and Tampé, R. (2013). Trends Biochem Sci 38, 64-74.
73
S-IV. Póster 7
FaRIF TRANSCRIPTION FACTOR PLAYS A KEY ROLE IN THE
REGULATION OF FRUIT RIPENING IN THE CULTIVATED
STRAWBERRY Fragaria ´ ananassa
Carmen Martín-Pizarro1, Victoriano Meco1, José G. Vallarino2, Jeremy Pillet1, Ana
Casañal3, Catharina Merchante1, James Giovannoni4, Sonia Osorio1, Miguel Ángel
Botella1, David Posé1, Victoriano Valpuesta1.
1Laboratorio de Bioquímica y Biotecnología Vegetal, Instituto de Hortofruticultura Subtropical
y Mediterránea (IHSM), Universidad de Málaga-CSIC. Dpto de Biología Molecular y
Bioquímica, Facultad de Ciencias, Universidad de Málaga, Spain 2 Max-Planck-Institute für
Molekulare Planzenphysiologie, Am Mühlenberg 1, 14476 Golm, Germany.3MRC-Laboratory
of Molecular Biology, Francis Crick AvenueCB20QH, Cambridge UK. 4United States
Department of Agriculture, Robert W. Holley Center and Boyce Thompson Institute for Plant
Research, Cornell University, Ithaca, NY 14853
Corresponding authors: David Posé Padilla ([email protected]) and Victoriano Valpuesta
Strawberry is becoming a model for studying the molecular mechanism of ripening in
non-climacteric fruits. However, a limited number of transcriptional regulators of this
process have been identified so far. In this study, we have identified and
characterized a gene encoding for a NAC transcription factor (TF), named as FaRIF
(Ripening Inducing Factor). FaRIF expression presents a fruit-specific pattern being
upregulated during ripening. In order to functionally characterize this TF, we have
generated silencing (35S::RIF-RNAi) and overexpressing (35S::RIF-GFP) stable
transgenic lines. While the RNAi lines showed an apparent delay of fruit ripening, the
overexpressing lines displayed an acceleration of this process. Transcriptomic
analysis by RNA-seq of the silenced lines showed a significantly altered expression
of genes involved in the flavonoids pathway, as well as genes of the metabolism of
the main sugars of the fruit. Metabolomics analysis confirmed these changes in the
transgenic fruits. Both, transcriptomic and metabolomics data, were in agreement
with the general phenotype observed in the fruits of the FaRIF-silenced lines. All
together, our results support a main role of FaRIF in the control of relevant ripening-
associated processes in strawberry fruit.
Acknowledgements & Funding. This work was supported by the Grants BIO2013-44199R (MINECO),
ERC-2014-StG 638134 (European Research Council – Starting Grant) and the Ramón y Cajal
program RYC 2013-1269 (MINECO-Universidad de Málaga, Spain). The authors also acknowledge
the support by the Plan Propio from University of Malaga, Campus de Excelencia Internacional de
Andalucía.
74
S-IV. Póster 8
TARGETED MUTAGENESIS OF FaTM6 IN THE OCTOPLOID
STRAWBERRY (Fragaria x ananassa) USING THE CRISPR/CAS9
SYSTEM
Carmen1 Martín-Pizarro1, Jose Antonio Duarte-Conde1, David Posé.
1Laboratorio de Bioquímica y Biotecnología Vegetal, Instituto de Hortofruticultura Subtropical
y Mediterránea (IHSM), Universidad de Málaga-Consejo Superior de Investigaciones
Científicas. Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias,
Universidad de Málaga, Spain
Corresponding author: David Posé ([email protected])
The B-class of MADS-box transcription factors has been studied in many plant
species, but remain functionally uncharacterized in the Rosaceae family. APETALA3
(AP3), a member of this class, controls the identity of petals and stamens
in Arabidopsis thaliana. In this work, we identified two members of the AP3 lineage in
the cultivated strawberry (Fragaria × ananassa): FaAP3 and FaTM6.
Interestingly, FaTM6, and not FaAP3, shows an expression pattern equivalent to that
of AP3 in Arabidopsis. Genome editing using Cluster Regularly Interspaced Short
Palindromic Repeats (CRISPR)/Cas9 system is becoming a robust tool for targeted
and stable mutagenesis of DNA. However, whether it can be efficiently used in an
octoploid species such as F. × ananassa is not known. In our study, we report the
application of the CRISPR/Cas9 in F. × ananassa to characterize the function
of FaTM6 in flower development. An exhaustive analysis by high-throughput
sequencing of the FaTM6 locus spanning the target sites showed a high efficiency
genome editing already in the T0 generation. The phenotypic characterization of the
mutant lines indicates that FaTM6 plays a key role in petal and especially in anther
development in strawberry. Our results validate the CRISPR/Cas9 strategy for gene
functional analysis in an octoploid species such as F. × ananassa, and offer new
opportunities for engineering strawberry to improve traits of interest in breeding
programs.
Acknowledgements & Funding..This work was supported by the Grants ERC-2014-StG 638134
(European Research Council) and the Ramón y Cajal program RYC 2013-1269 (MINECO-Universidad
de Málaga, Spain. The authors also acknowledge the support by the Plan Propio from University of
Malaga, Campus de Excelencia Internacional de Andalucía.
75
S-IV. Póster 9
GIBBERELLINS NEGATIVELY MODULATE OVULE NUMBER IN PLANTS
Daniela Barro-Trastoy1, María Dolores Gomez1, Ernesto Escoms1, Maite Saura-
Sánchez2, Inés Sánchez1, Asier Briones-Moreno1, Francisco Vera-Sirera1, Esther Carrera1, Juan José Ripoll3, Martin F. Yanofsky3, Isabel Lopez-Diaz1, José M.
Alonso4, Miguel A. Perez-Amador1
1Instituto de Biología Molecular y Celular de Plantas (IBMCP), CSIC-UPV, Valencia, Spain,
2Facultad de Agronomía, UBA-CONICET, Buenos Aires, Argentina,
3Division of Biological Sciences, University of California at San Diego, La Jolla, USA,
4Department of Plant and Microbial Biology, North Carolina State University, Raleigh, USA
Corresponding author: Miguel A. Perez Amador ([email protected])
Ovule formation is a complex developmental process that occurs early during
gynoecium development. Ovule primordia initiation is controlled by a gene network,
including components of the signaling pathways of auxin, brassinosteroids and
cytokinins. In contrast, gibberellins (GAs), growth regulators that control many
aspects of plant development through plant cell cycle, and their signaling negative
regulators DELLA proteins, have never been shown to be involved in ovule initiation.
We previously reported the role of GAs in the regulation of ovule integument
development, which finally affects ovule and seed size and shape. Here we provide
molecular and genetic evidences that point to DELLA proteins as novel players in the
determination of ovule number in Arabidopsis and in species of agronomic interest
like tomato and rapeseed, adding a new layer of complexity to this important
developmental process. Analysis of DELLA mutants as well as GA treatments
indicate that DELLA activity correlates positively with ovule number, acting as a
positive factor for ovule initiation. In addition, ectopic expression of a dominant
DELLA in the placenta is sufficient to increase ovule number. The role of DELLA
proteins in ovule number seems not to be related to auxins transport or signaling in
the ovule primordia. A possible crosstalk of DELLA proteins with the molecular and
hormonal network controlling ovule initiation is currently being studied.
76
S-IV. Póster 10
A REGULATORY INTERPLAY BETWEEN GIBBERELLIN-DELLA AND
BP MODULATES CELL GROWTH IN THE SHOOT APICAL
MERISTEM OF ARABIDOPSIS
Antonio Serrano Mislata 1,2, Amelia Felipo-Benavent 1,3, Noel Blanco-Touriñán 1,
Ivan Lebovka 1, David Esteve-Bruna 1, Nerea Valdebenito 1, Robert Sablowski 2,
Ignacio Rubio-Somoza 4, Miguel Ángel Blázquez 1, David Alabadí 1
1 Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), Valencia, Spain, 2 John Innes Centre, Norwich, United Kingdom, 3 Institut de Biologie Moléculaire des Plantes,
Strasbourg, France, 4 Centre for Research in Agricultural Genomics, Barcelona, Spain
Corresponding author: David Alabadí ([email protected])
The shoot apical meristem (SAM) gives rise to all of the above-ground organs
of the plant (leaves, flowers and fruits). Because of its primary relevance for plant
development, the molecular and cellular basis behind SAM activity have been widely
studied, not only in the model plant Arabidopsis but also in crops of agronomical
interest such as tomato, maize and rice. A set of works in the early 2000s showed
that the activity of KNOX transcription factors and plant hormones is key to maintain
an appropriate balance between SAM indeterminacy and the determinate growth of
lateral organs (Galinha et al., 2009). KNOX proteins activate cytokinin (CK) but
repress gibberellins (GA) biosynthesis within the SAM and, in the SAM boundaries,
inactivate GA that may diffuse from adjacent tissues. As a result, KNOX proteins
establish a high CK/GA regime that is favourable for SAM activity (Jasinski et al.,
2005).
GA promote growth and differentiation through the degradation of DELLA
proteins. We have observed that DELLAs upregulate the expression of the KNOX
gene BREVIPEDICELLUS (BP) in the Arabidopsis SAM. We have also observed that
exogenous GA3 treatments increase cell volumes and SAM size in bp-1 mutants but
not in the wild-type or in plants that over-express BP (35S::BP). Arabidopsis plants
lack the required enzymes to inactivate GA3 in the SAM periphery what suggests a
negative role for BP in the regulation of GA transport to the SAM. We are currently
testing this hypothesis through a combination of molecular genetics and imaging
approaches. Complementarily, we are studying the relevance of the DELLA-BP-GA
regulatory module for SAM activity and inflorescence development.
Galinha C, Bilsborough G, Tsiantis M. Hormonal input in plant meristems: A balancing act. Seminars
in Cell & Developmental Biology. 2009; 20: 1149-1156
Jasinski S, Piazza P, Craft J, Hay A, Woolley L, Rieu I, Phillips A, Hedden P, Tsiantis M. KNOX action
in Arabidopsis is mediated by coordinate regulation of cytokinin and gibberellin activities. Current
Biology, 2005; 15: 1560-1565
77
S-IV. Póster 11
GENOME-WIDE ANALYSIS OF THE NAC TRANSCRIPTION FACTOR
FAMILY AND THEIR EXPRESSION DURING THE DEVELOPMENT
AND RIPENING OF THE Fragaria × ananassa FRUITS * Pablo Ric-Varas2, Félix J. Martínez-Rivas1, Rosario Blanco-Portales1, Francisco
Javier Molina-Hidalgo1, Antonio J. Matas-Arroyo2, José Luis Caballero1, Juan Muñoz-
Blanco1, Antonio Rodríguez-Franco1, Enriqueta Moyano1
1Department of Biochemistry and Molecular Biology, University of Córdoba, Spain, 2Institute
for Mediterranean and Subtropical Horticulture "La Mayora" (IHSM-UMA-CSIC). Deparment
of Vegetal Biology, University of Málaga, Málaga, Spain.
Corresponding author: Enriqueta Moyano ([email protected])
NAC proteins are a family of transcription factors which have a variety of important
regulatory roles in plants. They present a very well conserved group of NAC
subdomains in the N-terminal region and a highly variable domain at the C-terminus.
Currently, knowledge concerning NAC family in the strawberry plant remains very
limited. In this work, we analyzed the NAC family of Fragaria vesca, and a total of
112 NAC proteins were identified after we curated the annotations from the version
4.0.a1 genome. They were placed into the ligation groups (pseudo-chromosomes)
and described its physicochemical and genetic features. A microarray transcriptomic
analysis showed six of them expressed during the development and ripening of the
Fragaria x ananassa fruit. Their expression patterns were studied in fruit (receptacle
and achenes) in different stages of development and in vegetative tissues. Also, the
expression level under different hormonal treatments (auxins, ABA) and drought
stress was investigated. In addition, they were clustered with other NAC transcription
factor with known function related to growth and development, senescence, fruit
ripening, stress response, and secondary cell wall and vascular development. Our
results indicate that these six strawberry NAC proteins could play different important
regulatory roles in the process of development and ripening of the fruit, providing the
bases for further functional studies and the selection for NAC candidates suitable for
biotechnological applications.
This work was supported by AGL2014-55784-C2 from the Spanish Ministerio Economía, Industria y
Competitividad.
78
S-IV. Póster 12 DECIPHERING STRAWBERRY RIPENING BY TISSUE SPECIFIC
GENE REGULATORY NETWORKS
Eva Lucas-Reina1, Almudena Trapero-Mozos1, María Angels de Luis Balaguer2,
Carmen Martín-Pizarro1, Martín Ramos-Alvelo, Rosangela Sozzani2, Miguel Ángel
Botella, Victoriano Valpuesta1 and David Posé1
1Departamento de Biología Molecular y Bioquímica, IHSM-UMA-CSIC, Málaga, Spain.
2Plant and Microbial Biology Department, North Carolina State University, Raleigh, NC, USA.
Corresponding author: David Posé Padilla (dpose@uma)
During ripening, fruits undergo a number of metabolic and physiological changes
leading to softening and improvement of characters such as flavor and palatability.
Insights into transcriptome changes during strawberry fruit ripening have been
reported, but always using either complete fruits in the analysis or separating
achenes and the fleshy part (receptacle). However, the receptacle is composed of
heterogeneous cell types, each of them with different characteristics and functions.
Hence, transcriptomic studies performed so far may have lost important regulatory
elements which expression is low but important in a specific cell-type specific.
In our study, we use Laser Capture Microdissection (LCM) technique for the isolation
of cells from specific tissue types such as the epidermis, vascular bundles, cortex,
and pith. Transcriptome profiling of these tissue types was performed by RNAseq. A
gene co-expression analysis was performed by Weighted Correlation Network
Analysis (WGCNA). Ontology analysis of each module showed wax biosynthesis as
the main biological pathway enriched at the red epidermis specific module. In order
to elucidate the putative regulatory elements that control the synthesis of waxes in
this tissue, a Gene Regulatory Network (GRN) was generated using GENIST (de
Luis Balaguer, 2017). As a result, we have identified a set of transcription factors that
might regulate the expression of eceriferum genes and a fatty acid elongase
necessary for wax biosynthesis in ripe epidermis.
Ultimately, our results open the possibility of implementing novel targeted breeding
approaches. Moreover, this work shows that LCM followed by RNAseq is a powerful
tool that can be used to clarify the regulatory scenario of tissue-specific biological
processes during strawberry ripening.
References
1. de Luis Balaguer et al (2017). PNAS. 5;114 (36)
Acknowledgements & Funding. This work was supported by the Grant ERC-2014-StG 638134 (European Research Council). The authors also acknowledge the support by the Plan Propio from University of Malaga, Campus de Excelencia Internacional de Andalucía.
79
S-IV. Póster 13
BRASSINOSTEROID-INDUCED MODULATION OF SPHINGOLIPID LONG-CHAIN BASE COMPOSITION AND GENE EXPRESSION
DURING EARLY OLIVE-FRUIT DEVELOPMENT
Jorge1 Corbacho1, Carla2 Inês2, Maria1 C. Camarero1, Beatriz1 Briegas1, Miguel1 A. Paredes1, Juana1 Labrador1, Mercedes3 Gallardo3, Antonio2 M. Corderio2, Maria1 C.
Gomez-Jimenez1
1Department of Plant Physiology, University of Extremadura, Badajoz, Spain, 2UEIS
Biotecnologia e Recursos Genéticos, Instituto Nacional de Investigação Agrária e
Veterinária, I.P. Elvas, Portugal, 3 Department of Plant Physiology, University of Vigo, Vigo,
Spain
Corresponding author: Gomez-Jimenez MC (E-mail: [email protected])
Sphingolipids are abundant membrane components and signalling molecules in various aspects of plant development. However, the role of sphingolipids in early fleshy-fruit growth has been rarely investigated. In this study, we first investigated the temporal changes in sphingolipid long-chain base (LCB) content, composition, and gene expression that occurred during flower opening and early fruit development in olive (Olea europaea L. cv Picual). Moreover, the interaction between sphingolipid and the plant hormone, brassinosteroid (BR), during the early fruit development was also explored. For this, BR levels were manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz) and their effects on early fruit development, sphingolipid LCB content, and gene expression were examined in olive fruit at 14 days post-anthesis (DPA). We here show that sphingolipid with C-4 hydroxylation and Δ8 desaturation with a preference for (E)-isomer formation are quantitatively the most important sphingolipids in olive reproductive organs. In this work, the total LCB amount significantly decreased at the anthesis stage, but olive sphingosine-1-phosphate lyase (OeSPL) gene was expressed exclusively in flower and upregulated during the anthesis, revealing an association with the d18:1(8E) accumulation. However, the LCB content increased in parallel with the up-regulation of the expression of genes for key sphingolipid biosynthetic and LCB modification enzymes during early fruit development in olive. Likewise, we found that EBR exogenously applied in olive tree significantly accelerated the fruit growth rate associated with reduced levels of sphingolipid LCB content and gene expression in olive fruit after 7 and 14 days of treatment, whereas Brz slowed the fruit growth rate and boosted the sphingolipid LCB content and gene expression during early growth. Thus, our data indicate that endogenous sphingolipid LCB and gene-expression levels are intricately controlled during early fruit development in olive and also suggest a possible link between BR, the sphingolipid content/gene expression, and early fruit development.
Acknowledgements & Funding: This work was supported by the Ministerio de Economía y Competitividad, Spain (AGL2014-52194R). The assistance to the congress was possible by the research help financed by the Junta of Extremadura (Spain) and the European Regional Development Fund.
80
4
3 3
S-IV. Póster 14
AMMONIUM NUTRITION MODIFIES ROOT SYSTEM ARCHITECTURE
IN TOMATO PLANTS
Ana Isabel González-Hernández, Loredana Scalschi, Emma Fernández-Crespo,
Eugenio Llorens, Begonya Vicedo, Pilar García-Agustín & Gemma Camañes
Biochemistry and Biotechnology Group. Department of Agricultural and Environmental
Sciences. Jaume I University, 12071, Castellón, Spain.
Corresponding author: [email protected]
Plants take up inorganic nitrogen (N) available in the soil or nutrient solution mainly - + - +
as NO3 or NH4 . NO3 +
assimilation requires more energy than NH4 assimilation. It is
known that NH4 changes root system architecture (RSA) in plants due to several
changes that have an impact in plant development, growth and acclimation (Giehl et
al., 2013). RSA is related with the plant roots morphological and topological
distribution (Lynch, 1995), so it determines water and nutrient uptake and in a long-
term with plant productivity.
To study the effect of NH + nutrition on root system architecture in ten-days old
tomato plants (Solanum lycopersicum var. Ailsa craig), plants were growing under - + - + - + NO3 10 mM (control plants), NH4 10 mM, 1:3 (NO3 : NH4 ) or 2:2 (NO3 : NH4 )
treatments under axenic conditions. In order to know plant ability to use and respond
to different nitrogen sources, we have determined primary root elongation, shoot
height, number of secondary roots, shoot/root ratio, root density and fresh weight. +
Under NH4 nutrition, plants have a decrease in some of these parameters, which are
improved when there is a partial or total NO -
supply (1:3, 2:2 and NO -
treatments).
Moreover, we have analysed several genes related with N uptake and assimilation,
stress indicators and root morphology.
References
Giehl, R., Gruber, B. and von Wirén, N. 2013. It’s time to make changes: modulation of root system
architecture by nutrient signals. Journal of Experimental Botany, Vol. 65, No. 3, pp. 769–778, 2014
Lynch J (1995) Root architecture and plant productivity. Plant Physiol 109: 7–13
Acknowledgements & Funding
Work was supported by grants: AGL2013-49023-C03-02-R, UJI-A2016-09 and UJI-PREDOC/2016/27.
81
S-IV. Póster 15
RETHINKING THE ROLE OF CELL WALL COMPOSITION IN SEED
AND FRUIT DEVELOPMENT
Authors: Vívian Ebeling Viana1, Bruno Gugi3, Azeddine Driouich3, Antonio Costa de Oliveira1, Lucia
Colombo2, Ignacio Ezquer2
1Federal University of Pelotas, Plant genomics and breeding center, Technology Development Center,
P.C. 354, Pelotas, RS, Brazil.
2Università degli Studi di Milano, Department of BioScience, P.C.20133, Milan, Italy.
3Laboratoire Glycobiologie et Matrice Extracellulaire Végétale (EA 4358), Université de Rouen, 76821
Mont Saint Aignan Cedex, France
Corresponding author: [email protected]
In the past years, many of the key genes involved in seed coat development have been
identified and characterized in Arabidopsis thaliana. Although much is known about seed
coat development at the molecular level, many gaps remain in our understanding of how cell
wall alterations can impact developmental processes and how biomechanical alterations can
influence seed and fruit growth. Mutants in Arabidopsis constitute an excellent tool to study
the function of gene families devoted to wall biogenesis. We have generated a collection of
Arabidopsis mutants of cell wall-related genes and screened them looking for developmental
defects in seed and silique growth. We have linked these studies with cell wall composition
analysis. Interestingly, we have found that mutating genes involved in pectin maturation and
hemicellulose deposition altered germination dynamics. Disruption of xylosidase activity also
had a negative influence on seed and fruit growth. We have also studied how several cell
wall biosynthetic/modifying enzymes and structural proteins are regulated at molecular level
by critical transcription factors controlling seed and fruit size. We propose a model whereas
changes in seed coat structure by altering the xyloglucan-cellulose matrix deposition and
pectin maturation are determinant for seed size determination and where many of the players
involved in seed development are major regulators for fruit development.
(We acknowledge the financial support from Università degli Studi di Milano (Linea2 2018-2019-DBS)
and European Council (RISE-EXPOSEED project).
82
S-IV. Póster 16 LOCAL AUXIN BIOSYNTHESIS IS A KEY REGULATOR OF PLANT
DEVELOPMENT
Javier Brumos, Jose Alonso and Anna Stepanova Department of Plant and Microbial Biology, Program in Genetics, North Carolina State
University, Raleigh, NC, 27695-7614, USA Corresponding author: Anna Stepanova ([email protected])
The phytohormone auxin is essential for plant development, coordinating growth and
responses to the environment. Auxin gradients regulate cell fate decisions and are
intrinsic to the plant phenotypic plasticity. Auxin transport is a major element in the
auxin gradient generation (Adamowski and Friml, 2015).
The main pool of auxin, indole-3-acetic acid (IAA), is synthesized via the IPyA
pathway that is composed by the TAA1/TARs and YUCs gene families (Mashiguchi
et al., 2011; Stepanova et al., 2011, Brumos et al., 2014) which have been shown to
exhibit exquisite spatio-temporal expression patterns (Cheng et al., 2006; Stepanova
et al., 2008; Robert et al., 2013). These findings suggest that local auxin production
may contribute to shape the auxin gradients.
Herein, we try to define the role of local auxin biosynthesis and its contribution
in the regulation of plant development and responses to environmental cues by
restricting the spatio-temporal patterns of WEI8.
Our results indicate that local auxin production and transport act redundantly
in the establishment and preservation of robust auxin gradientes essential for root
meristem maintenance. Conversely, local auxin biosynthesis is necessary for flower
fertility and root responses to ethylene and cannot be replaced by just the transport
activity in the formation of morphogenic auxin gradients.
References Adamowski, M., and Friml, J. (2015). PIN-dependent auxin transport: action, regulation, and evolution. Plant Cell 1, 20-32. Brumos, J., Alonso, J.M., and Stepanova, A.N. (2014). Genetic aspects of auxin biosynthesis and its regulation. Physiol Plant 1, 3-12. Cheng, Y., Dai, X., and Zhao, Y. (2006). Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis. Genes Dev. 13, 1790-1799. Mashiguchi, K., Tanaka, K., Sakai, T., Sugawara, S., Kawaide, H., Natsume, M., Hanada, A., Yaeno, T., Shirasu, K., Yao, H. et al. (2011). The main auxin biosynthesis pathway in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 45, 18512-18517. Robert, H.S., Grones, P., Stepanova, A.N., Robles, L.M., Lokerse, A.S., Alonso, J.M., Weijers, D., and Friml, J. (2013). Local auxin sources orient the apical-basal axis in Arabidopsis embryos. Curr. Biol. 24, 2506-2512. Stepanova, A.N., Robertson-Hoyt, J., Yun, J., Benavente, L.M., Xie, D., Dolezal, K., Schlereth, A., Jürgens, G., and Alonso, J.M. (2008). TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development. Cell 1, 177-191. Stepanova, A.N., Yun, J., Robles, L.M., Novak, O., He, W., Guo, H., Ljung, K., and Alonso, J.M. (2011). TheArabidopsisYUCCA1 Flavin Monooxygenase Functions in the Indole-3-Pyruvic Acid Branch of Auxin Biosynthesis. The Plant Cell 11, 3961-3973.
Acknowledgements & Funding This work was supported by the NSF grants MCB 0923727, IOS 1444561 and IOS 1650139 to J.M.A. and A.N.S., MCB 1158181 to J.M.A and the Spanish Ministerio de Educacion postdoctoral fellowship to J.B.
83
S-IV. Póster 17
SVP INTEGRATES SEVERAL ENVIRONMENTAL SIGNALS THAT
CONTROLS THE GROWTH CESSATION IN ASPEN TREES.
José Alfredo Zambrano1, Domenique André1 Bo Zhang1 and Ove Nilsson1
1Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish
University of Agricultural Sciences, SE-901 83 Umeå, Sweden
Corresponding author: José Alfredo Zambrano ([email protected])
Perennial plants have to adapt their growth and development to the changing
seasons. In order to survive winter, they need to anticipate and prepare for potentially
harsh conditions. Decreasing photoperiod is a good indicator for the onset of autumn
and is perceived by the CONSTANS/FLOWERING LOCUS T (CO/FT) signaling
module. This module is genetically well conserved between Arabidopsis and our
model tree species hybrid aspen (Populus tremula x tremuloides), but serves
different functions. In Arabidopsis, it controls flowering and its mode of action is fairly
well understood. In addition to photoperiod, FT is an integrator for other both internal
and environmental signals. SHORT VEGETATIVE PHASE (SVP) is a negative
regulator of flowering and suppresses FT transcription in cold temperatures. In aspen
trees, the CO/FT module is promoting vegetative growth during spring and summer.
Whether SVP has a role in the regulation of annual growth was so far unknown.
The aim of our work was to investigate the role of SVP in photoperiod signaling in
hybrid aspen trees. We used SVP RNAi and overexpressing lines to study its effect
on short day induced growth cessation. We show that SVP function is conserved and
is repressing FT transcription in short days. ChIP analyses show that SVP can
directly bind to the FT promoter and we are currently investigating whether this ability
is controlled by ambient temperature. Besides FT, gibberellins are an important
growth factor. Their biosynthesis needs to be reduced in order to stop growth in
autumn. Preliminary data shows that SVP might be involved in their regulation by
repressing GA20oxidase expression.
We conclude that SVP function is conserved between Arabidopsis and aspen trees,
even though the physiological responses it is involved in are different.
84
S-IV. Póster 18
ARABIDOPSIS SWC4 BINDS DNA AND RECRUITS THE SWR1
COMPLEX TO MODULATE HISTONE H2A.Z DEPOSITION AT
KEY REGULATORY GENES
Ángeles Gómez-Zambrano1,6, Pedro Crevillén1,6, José M. Franco-Zorrilla2, Juan
A. López3, Jordi Moreno-Romero4, Pawel Roszak4, Juan Santos-González4,
Silvia Jurado1, Jesús Vázquez5, Claudia Köhler4, Roberto Solano2, Manuel
Piñeiro1 and José A. Jarillo1
1Centro de Biotecnología y Genómica de Plantas (UPM-INIA),Campus Montegancedo
UPM, Pozuelo de Alarcón (Madrid), 28223 Spain
2Centro Nacional de Biotecnología, CSIC, Madrid, 28049,Spain.
3Proteomics Unit. CNIC, 28029, Madrid, Spain.
4Swedish University of Agricultural Sciences, Uppsala, 75652 Sweden.
5Laboratory of Cardiovascular Proteomics, CNIC, , Madrid, 28029,Spain.
6 Co-first author
Corresponding author: José A. Jarillo, [email protected]
Deposition of the H2A.Z histone variant by the SWR1 complex (SWR1-C) in
regulatory regions of specific loci modulates transcription. Characterization of
Arabidopsis thaliana mutations in homologs of the yeast SWR1-C has revealed
a role for H2A.Z exchange in a variety of developmental processes;
nevertheless, the exact composition of the plant SWR1-C and how it is recruited
to target genes remains to be established. In this work, we show that SWC4,
the Arabidopsis homolog of the yeast SANT domain protein Swc4/Eaf2, is a
DNA-binding protein that interacts with SWR1-C subunits. We demonstrate that
the swc4-1 knock-out mutant is embryo lethal, while SWC4 RNAi knockdown
lines displayed pleiotropic phenotypic alterations in vegetative and reproductive
traits, including acceleration of flowering time, indicating that SWC4 controls
post-embryonic processes. Transcriptomic analyses and genome-wide profiling
of H2A.Z indicate that SWC4 represses transcription of a number of genes
including the floral integrator FT and key transcription factors mainly by
modulating H2A.Z deposition. Interestingly, SWC4 silencing does not affect
H2A.Z deposition in the FLC locus nor expression of this gene, a master
regulator of flowering previously shown to be controlled by SWR1-C.
Importantly, we find that SWC4 is a DNA-binding protein recognizing specific
AT-rich DNA elements in chromatin regions of target genes and that SWC4
silencing impairs SWR1-C binding at FT. Collectively, our data suggest that
SWC4 regulates plant growth and development through aiding SWR1-C
recruitment and modulating H2A.Z deposition.
85
S-IV. Póster 19
GENETIC DISSECTION OF ADVENTITIOUS ROOT FORMATION
IN TOMATO (S. lycopersicum cv. Micro-Tom)
Aurora Alaguero-Cordovilla1, Sergio Ibáñez1, Ana Belén Sánchez-García1,
Paula Jadczak1, Francisco Javier Gran1, Alfonso Azorín1, Antonio Cano2,
Christophe Rothan3, Manuel Acosta2, José Manuel Pérez-Pérez1
1Instituto de Bioingeniería, Universidad Miguel Hernández, Elche, Spain,
2Departamento de Fisiología Vegetal, Universidad de Murcia, Spain, 3Dümmen
Orange, Murcia, Spain, 3Unité Mixte de Recherche 1332 Biologie du Fruit et
Pathologie, Institut national de la Recherche Agronomique (INRA), Villenave d’Ornon
Cedex, France.
Corresponding author: José Manuel Pérez-Pérez ([email protected])
Tomato is an attractive model to study the genetic basis of adventitious organ
formation. Adventitious roots (ARs) are formed from non-root tissues in
response to some abiotic stresses or after wounding (Bellini et al., 2014). We
investigated the temporal course of gene expression and of auxin and cytokinin
accumulation along the apical-basal axis of hypocotyl explants during
adventitious rooting. Gene functions related to kinase activity, cell wall
remodelling or transcription factor activity were associated to distinct
regeneration events. We have initiated both forward and reverse genetic
approaches to identify the molecular hubs required for de novo organ formation
in tomato. On the one hand, the role of selected transcription factors of the
AINTEGUMENTA-LIKE family during AR formation has been confirmed by
transient inactivation. On the other hand, we are screening a large EMS mutant
collection (Garcia et al., 2016) in search of novel gene functions related to de
novo root organogenesis. We have identified 26 monogenic mutants with
defects in AR formation that will go through a novel mapping-by-sequencing
approach to identify their casual mutations.
Bellini, C., et al. (2014). Annu Rev Plant Biol 65: 639-666
Garcia, V., et al. (2016). Nature Protocols 12: 2401-2418
Work in the laboratory of J.M.P.-P. (AGL2012-33610 and BIO2015-64255-R) is funded by the
Ministry of Economy and Competitiveness and by FEDER funds of the EC - "A way to build
Europe“.
86
S-IV. Póster 20
EPIGENETIC REGULATION BY HISTONE DEMETHYLASES IN
Brassica rapa
Laura Poza-Viejo1, Iván del Olmo1, Paxti San Martín-Uriz2, Jenifer Pozas1, Laura Castro-Labrador2, David Lara-Astiaso2 and Pedro Crevillén1
1Centro de Biotecnología y Genómica de Plantas (CBGP), Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria
(INIA), Pozuelo de Alarcón (Madrid), Spain 2Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra, Pamplona,
Spain Corresponding author: Pedro Crevillén ([email protected])
The study of the epigenome and its relation with the underlying genome sequence has become a central question in Biology nowadays. Nevertheless, epigenome studies in plant crops and its comparison with model plant systems are scarce. Polycomb protein complexes repress gene expression catalyzing the trimethylation of histone H3 lysine 27 (H3K27me3). This conserved epigenetic mark is involved in the regulation of plant gametophyte development and embryo-seedling transition or flowering induction. The methylation of H3K27 by Polycomb complexes is counteracted by the demethylase activity of specific Jumonji domain proteins. In Arabidopsis thaliana, there are two main H3K27 demethylases: EARLY FLOWERING 6 (ELF6) and RELATIVE OF ELF 6 (REF6). We are studying the role of these epigenetic factors and the significance of H3K27 methylation in the oilseed Brassica rapa R-o-18 variety. We will present data regarding the characterization of Bra.elf6 and Bra.ref6 mutant lines. We found that Bra.ELF6 and Bra.REF6 regulate the expression of key floral regulators and contribute to the the regulation of flowering time in B. rapa. We will also show novel transcriptomic and H3K27me3 epigenomic B. rapa datasets. Our data suggest that H3K27 methylation regulate a wide range of developmental responses in B. rapa. Similarities with current knowledge derived from Arabidopsis epigenomic research will be discussed. Our results improve the knowledge about the epigenetic mechanisms regulating key developmental traits in plants, and will help us to increase crop yield in Brassica crops.
Acknowledgements & Funding This work was supported by grants BIO2015-68031-R and RYC-
2013-14689 to PC, and BES-2016-078939 fellowship to LP from the Spanish Ministerio de
Economia y Competitividad (MINECO/FEDER, EU). The CBGP is a Severo Ochoa Center of
Excellence (SEV-2016-0672).
87
S-IV. Póster 21 A ROLE FOR HISTONE READERS IN THE REGULATION OF DIFFERENT
STAGES OF REPRODUCTIVE DEVELOPMENT IN ARABIDOPSIS
Dorota Komar, José A. Jarillo, Manuel Piñeiro
Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) -
Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus
Montegancedo UPM, 28223 Pozuelo de Alarcón (Madrid), Spain
Corresponding author: Manuel Piñeiro ([email protected])
Plant reproductive development is tightly controlled in response to environmental and
endogenous cues to ensure optimal production of fruits and seeds. The Arabidopsis
chromatin protein EARLY BOLTING IN SHORT DAYS (EBS) is involved in the regulation
of flowering time, and functions to directly repress the expression of the florigen gene FT.
The EBS protein contains a PLANT HOMEODOMAIN (PHD) motif that specifically
recognizes di- and trimethylated lysine 4 in histone H3 (H3K4me2/3). Furthermore, EBS
interacts physically with histone deacetylases (HDAC), and loss of function mutations in
EBS result in increased levels of histone H3 acetylation in the genomic region of FT,
suggesting that this chromatin factor is required to maintain a transcriptionally inactive
conformation in the genomic region of the florigen to prevent a premature initiation of
flowering (López-González et al., 2014).
Our recent results demonstrate that the EBS gene plays additional roles in the regulation
of later stages of reproductive development in Arabidopsis. Processes such as
inflorescence growth and flower development are conspicuously altered in the ebs mutant
grown under non-inductive short day conditions. Severe developmental defects such as
reduced apical dominance, phyllotaxy alterations, and abnormalities in floral meristem
identity, as well as problems in floral development (homeotic alterations of floral organs
and floral reversion events) are frequently observed in these mutants. Our transcriptomic
analyses have identified a number of floral identity and inflorescence architecture genes
that are differentially expressed in the ebs mutant and that could mediate the
developmental anomalies present in this mutant plants. Findings that can contribute to
reveal the function of EBS activity in the control of different aspects of reproductive
development will be discussed.
López-González, L., Mouriz, A., Narro-Diego, L., Bustos, R., Martínez-Zapater, J.M., Jarillo, J.A. and
Piñeiro, M. (2014) Chromatin-dependent repression of the Arabidopsis floral integrator genes involves plant
specific PHD-containing proteins. Plant Cell 26: 3922-3938.
Funding by BIO2013-43098-R and BIO2016-77559-R (MINECO/FEDER, EU) and EpiTRAITS (EU MSCA
316965) is acknowledged.
88
S-IV. Póster 22
DEVELOPMENT OF IMAGE ANALYSIS TOOLS TO STUDY PLANT
DEVELOPMENT
Marie-Louise Körner1, María-Victoria Díaz-Galián1, Pedro J. Navarro2, Fernándo Pérez-
Sanz1, Julia Weiss1, Marcos Egea-Cortines1
1 Instituto de Biotecnología Vegetal, Edificio I+D+I, Universidad Politécnica de Cartagena Campus
Muralla del Mar 30202, Cartagena, (Murcia), 2DSIE, Universidad Politécnica de Cartagena, Plaza
del Hospital s/n, Campus Muralla del Mar 30202, Cartagena (Murcia)
Corresponding author: Marcos Egea-Cortines [email protected]
One challenge of Developmental biology to identify the connection between genotype and
phenotype is the acquisition of data throughout developmental processes, shifting
analytics from end-point pictures to kynetics. We are interested in understanding the
timing and control of late flower development and maturation. We have developed artificial
vision systems to study growth and development in a variety of plants (Navarro et al 2012,
2016). While image acquisition is relatively straightforward, image analysis is the true
bottleneck to obtain quantitative data (Perez-Sanz et al 2017). We have started to analyze
four Arabidopsis ecotypes, Col-0 (Columbia), Ler (Landsberg erecta), Ws-2
(Wassilewskija) and En-2 (Enkheim) in order to establish a standard protocol to analyze
growth and flower development. Data on the specific requirements to perform image
acquisition, image preprocessing, segmentation and features extraction will be presented
on a set of images. Our intention is to create a set of ground-truth images of Arabidopsis
and other plants such as Antirrhinum, Petunia and strawberry for the scientific community.
References
Navarro et al (2012) Sensors. 12:15356
Navarro et al (2016) Sensors 16:641
Perez-Sanz et al (2017) Gigascience. 6:1-18
Acknowledgements & Funding.
This work was funded by the Fundación Séneca 19398/PI/14, BFU 2013-45148-R, BFU 2017 88300-C2-1-R
and BFU2017 88300-C2-2-R. Thanks to Dr. Maria Rosa Ponce Molet for Arabidopsis seed
89
S-IV. Póster 23
TERMINAL FLOWER 1 FUNCTIONS AS A TRANSCRIPTIONAL CO- REPRESSOR IN THE Arabidopsis thaliana SHOOT APICAL
MERISTEM
Marina Silvestre1*, Daniela Goretti2*, Silvio Collani2, Tobias Langenecker3, Carla Méndez1, Markus Schmid2, Francisco Madueño1, ,
1 Instituto de Biología Molecular y Celular de Plantas, CSIC-UPV, Valencia, Spain.
2 Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, Sweden.
3 Department of Molecular Biology, Max Planck Institute for Developmental
Biology,Tübingen, Germany.
Corresponding author: Marina Silvestre ([email protected]) TERMINAL FLOWER 1 (TFL1) is a key regulator of plant architecture in Arabidopsis thaliana that represses flowering and controls the identity of the shoot apical meristem (SAM). Thus, tfl1 mutant plants flower early and their inflorescence shoots are converted into terminal or axillary flowers. Conversely, overexpression of TFL1 causes an extreme delay in flowering. These phenotypes are opposite to those caused by the loss-of-function and overexpression of its homologous gene FLOWERING LOCUS T (FT), which encodes a florigen.
TFL1 is expressed in the inflorescence SAM keeping it in an undifferentiated state by repressing the expression of floral identity genes such as LEAFY and APETALA1. Though TFL1 regulates gene expression, TFL1 is a phosphatydilethanolamine- binding protein, not a transcription factor. As it has been proposed for the FT protein, it has been suggested that TFL1 regulates gene expression by acting as a transcriptional co-factor with the bZIP transcription factor FD. However, this hypothesis has not been clearly proven yet.
To obtain additional support for this hypothesis, and to answer the question of what are the TFL1 target genes, we have used a gTFL1:GFP line to identify loci bound by TFL1 by ChIP-seq, and a TFL1:GR line to identify genes regulated by TFL1 upon induction of TFL1 translocation to the cell nucleus.
Our results demonstrate that the Arabidopsis thaliana TFL1 protein does function as a transcriptional co-factor and points to several signalling pathways could be mediating the activity of TFL1.
The Laboratory of F.M. was funded by the Spanish Ministerio de Economía y Competitividad and FEDER (BFU2012-38929 and BIO2015-64307-R) and the Generalitat Valenciana (ACOMP2012-099).
90
S-IV. Póster 24 ANALYSIS OF ENDONGENOUS HORMONAL DYNAMICS DURING
ADVENTITIOUS CAULOGENESIS IN Pinus pinea l.
Natalia Bueno1, José Manuel Álvarez1, Candela Cuesta1, Isabel Feito2, Ana
Rodríguez1, Ricardo Ordás1
1Departamento Biología de Organismos y Sistemas, Universidad de Oviedo, Spain, 2Servicio
Regional de Investigación y Desarrollo Agroalimentario de Asturias (SERIDA), Spain.
Corresponding author: Ricardo Ordás ([email protected])
Pinus pinea L. (stone pine) is a coniferous species native from the Mediterranean
region that has been economically exploited for its edible seeds (pine nuts). The
establishment of high-yield P. pinea plantations requires the development of effective
procedures to select and propagate superior genotypes. To this purpose, an in vitro
micropropagation system via organogenesis, which consists in the formation of
adventitious buds in pine cotyledons cultured in the presence of the cytokinin
benzyladenine (BA), has been developed (Cuesta et al., 2009). Apart from its use in
breeding programs, this technique has been proposed as a model for the study of the
physiological and molecular basis of caulogenesis in conifers, as it is very repetitive
and cotyledons are competent to form buds per se, responding in a very
synchronous fashion to the induction.
In order to elucidate the involvement of plant growth regulators (PGRs) in the
caulogenic process, the endogenous hormonal profiles of BA-treated cotyledons and
cotyledons cultured in absence of BA (control cotyledons), which do not form
adventitious buds, were analysed by UHPLC-MS/MS and immunohistochemistry.
Expression dynamics of genes putatively involved in shoot meristem formation and
maintenance, such as members of WOX and KNOX gene families, were also studied
in both types of material with such different organogenic capacity in order to achieve
a global understanding of the process.
Hormonal analysis showed a BA peak at 12 hours in BA-treated cotyledons
coinciding with the high BA absorption rate observed during the first hours of culture.
The rise of indoleacetic acid levels observed in BA-treated cotyledons might indicate
the important role of this auxin together with BA in initiation and proliferation of
promeristemoids. On the other side, other cytokinins (free base forms and ribosides),
PGRs related to stress tolerance (abscisic acid, jasmonic acid, and salicylic acid), the
brassinosteroid castasterone, and the gibberellin A4 showed similar dynamics in both
types of material. Interestingly, some class I KNOX and WOX genes were
overexpressed in BA-treated cotyledons during shoot induction and determination,
reinforcing the role of these genes in shoot meristem establishment in conifers.
Cuesta et al. (2009). Journal of Plant Physiology, 166: 1162-1171
Project funding by “Plan Nacional I+D+I, INIA” (RTA2013-00048-C03-02). Predoctoral grant from
“Plan de Ciencia Tecnología e Innovación del Principado de Asturias” (BP10-098) to Natalia Bueno.
91
S-IV. Póster 25 THE SINGLE FLOWER GENE CONTROLS THE ACTIVITY OF THE
SECONDARY INFLORESCENCE MERISTEMS IN CHICKPEA
Ana Berbel1, Cristina Caballo2, R Ortega3, M José Domenech1, Juan Gil4, Josefa
Rubio2, Teresa Millán4, Francisco Madueño1
1Instituto de Biología Molecular y Celular de Plantas, CSIC-UPV, Valencia, Spain, 2Área de Genómica y Biotecnología, IFAPA, Córdoba, Spain, 3School of Biological Sciences,
University of Tasmania, Hobart, Australia, 4Dept. Genética, Universidad de Córdoba, Campus Rabanales , Córdoba, Spain
Corresponding author: Francisco Madueño (madueñ[email protected])
The basic type of inflorescence in legumes is the compound inflorescence, where flowers appear in lateral secondary inflorescences (I2). The number of flowers per secondary inflorescence, which depends on the activity of the I2 meristem, is characteristic of each legume species and variety. For example, the I2s of Lotus japonicus and Medicago truncatula produce 1-2 flowers, while Medicago sativa (alfalfa) I2s produce 8-12 flowers. Very little is known about the genes that control the activity of the legume I2 meristems.
Cicer arietinum (Chickpea) I2s normally produce 1 flower but mutants with more than 1 flower per I2 have been reported. Recessive mutations in the SINGLE FLOWER (SFL) locus, sfld and sflt, result in plants whose I2s produce 2 or 3 flowers.
The sfld (double-pod) mutation was mapped to a region of 92.6 Kb with 7 annotated genes. In the sfld mutant two of these genes are deleted and the other one contains a partial deletion. One of the deleted genes, CaRAX2, codes for a MYB-type transcription factor whose homologues in Arabidopsis (RAX2) and tomato (BLIND) control the activity of shoot axillary meristems, pointing CaRAX2 as a good candidate for the SFL gene. Accordingly, the CaRAX2 gene shows specific expression in the meristems of the chickpea inflorescence.
As, unfortunately, we have not got access to the sflt mutant allele, we are following several strategies to assess whether CaRAX2 is the SFL gene. Thus, from chickpea germplasm collections, we have identified three cultivars with a double-pod phenotype that do not contain the deletion present in the sfld mutant. The genetic analysis performed so far suggests that the double-pod mutations in these cultivars are allelic to sfld. We are now studying the expression of CaRAX2 in these mutants and sequencing their CaRAX2 genomic region. On the other hand, we are analysing lines from the related legumes L. japonicus and M. truncatula with insertions in the genes homologous to CaRAX2.
We hope that the identification and characterization of the SFL gene will allow us to better understand the genetic control of the development of the compound inflorescence of legumes.
The laboratory of F.M. was funded by the Spanish Ministerio de Economía y Competitividad and FEDER (BFU2012-38929 and BIO2015-64307-R) and the Generalitat Valenciana (ACOMP2012-099) and the work at the IFAPA and University of Córdoba was funded by the INIA project contract RTA2013-00025, co-financed by the European Union through the ERDF 2014-2020. Caballo C. acknowledges her Ph.D. fellowship INIA-CCAA.
92
S-IV. Póster 26
THE ARABIDOPSIS CRD3 GENE ENCODES A CHLOROPLAST
RIBOSOMAL PROTEIN INVOLVED IN EMBRYO AND VEGETATIVE
DEVELOPMENT
Pedro Robles, Laura García-Abad, Alba Valdivieso-Martínez, Gabriel Sánchez-
Martínez, Eva Núñez-Delegido, y Víctor Quesada
Instituto de Bioingeniería, Universidad Miguel Hernández, Elche, Spain
Corresponding author: Pedro Robles ([email protected])
The insertional recessive crd3 (chloroplast ribosome defective3) Arabidopsis mutant
was isolated in a reverse genetic screen to identify functions related to the genetic
information flux in the chloroplasts. Phenotypically, crd3 plants are smaller compared
with Col-0 and show a general paleness of the green tissues. Besides, crd3
vegetative leaves are pointed with toothed margins, and histological analyses show
that they are thicker than the wild-type ones and that their chloroplasts are abnormal.
Occasionally, tracheary elements of the cotyledons are disconnected giving rise to an
open venation pattern.
The CRD3 gene encodes a chloroplast ribosomal protein of the large subunit
of the chlororribosome, which was previously described as an EMB gene essential
for Arabidopsis embryo development. However, no viable alleles were so far
described. Our results reveal new functions for CRD3 besides the embryonic ones.
We have performed an allelism test between crd3 and the heterozygote emb/CRD3
and confirmed that both mutations are allelic. The compound heterozygotes
crd3/emb are viable but display a phenotype much stronger than the crd3/crd3
plants, according to the different levels of expression of the CRD3 gene in both
mutant genetic backgrounds. The vegetative leaves of crd3/emb show large air
spaces in the mesophyll and their chloroplasts are smaller and much more
disorganized than the crd3/crd3 ones. The venation pattern deviations observed in
crd3/crd3 cotyledons are also enhanced in the crd3/emb background. Those
developmental defects prompted us to study the auxin response in the crd3 mutant
using the DR5::GUS reporter construct. Our results suggest that auxin homeostasis
is altered in the crd3 mutant, since the GUS staining pattern in roots and leaves is
fainter in the mutant than in the control plants.
As a part of the molecular characterization of crd3, we have performed an
RNAseq analysis and found significant changes in the expression of 586 genes, of
which 474 are nuclear, 84 chloroplast and 28 mitochondrial. The results of the gene
ontology (GO) analysis of the differentially expressed genes between Col-0 and crd3
will be shown in the meeting.
93
S-IV. Póster 27
BIOACTIVE MOLECULES IN THE CONTROL OF FLOWERING TIME
Jesús Praena, Francisco Madueño and Reyes Benlloch
Instituto de Biología Molecular y Celular de Plantas, Ingeniero Fausto Elio s/n, 46022,
Valencia, Spain.
Corresponding author: Reyes Benlloch ([email protected])
Flowering time is one of the most relevant traits influencing crop productivity and yield. The identification of natural or synthetic compounds bioactive in the control of floral induction is of great interest. On one hand, identified compounds could be the bases for biotechnology applications to fine-tune flowering time in crops, adapting growth and reproduction to the most favourable environmental conditions. On the other hand, the characterization of the mechanism of action for each molecule could provide us with novel information on the regulation of flowering time.
In order to identify bioactive compounds in the control of flowering time we have undertaken two approaches. First, we have carried out a chemical genomics screening to identify synthetic small molecules with potential to control the expression of the florigen, FLOWERING LOCUS T (FT), in Arabidopsis. To visualize the effect of each molecule, we have used transgenic plants expressing the β- glucuronidase gene (GUS) under the control of the FT promoter region. The FT promoter integrates endogenous and environmental signals that determine the most suitable time to flower. Positive hits from the primary screening are currently being confirmed by secondary screenings in order to choose the most promising molecules. The effect of these molecules in flowering time in Arabidopsis and other species such as alfalfa or tomato will be evaluated.
Our second approach is oriented towards the characterization of endogenous molecules that would contribute to the control of flowering time. We have undertaken the characterization of metabolic profiles of leaf and shoot apical meristem tissues during floral transition. In order to do so, we have generated a line that expresses CONSTANS (CO) fused to the GR domain under the control of its own promoter, which provides a system to finely control the timing of the floral transition. By comparing induced versus non-induced plants, we will unveil metabolic changes that encompass the floral induction process and that could have a regulatory function.
In summary, we are analysing floral induction using two new approaches, chemical biology and metabolic profiling, that will provide us with candidate synthetic and endogenous molecules with potential to control flowering time in crops.
Acknowledgements & Funding. We aknowledge Dr. F. Turck for kindly providing pFT::GUS lines. The
work is supported by the Spanish Ministry of Science (BIO2015-73491-JIN to RB).
94
S-IV. Póster 28
A MOLECULAR MECHANISM REGULATING ENDOSPERM CELL EXPANSION DURING SEED GERMINATION.
Rocío Sánchez-Montesino1, Laura Bouza-Morcillo1, Melania Ghita2, Salva Duran- Nebreda3, Petra Stamm3, Luis Gómez1, Michael Holdsworth2, George Bassel3 and
Luis Oñate-Sánchez1
1Centro de Biotecnología y Genómica de Plantas, (Universidad Politécnica de Madrid – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria), Campus de
Montegancedo, Pozuelo de Alarcón, 28223, Madrid, Spain. 2Division of Plant and Crop Sciences, School of Biosciences, University of Nottingham,
Loughborough, UK. 3School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK
Corresponding author: Luis Oñate-Sánchez ([email protected])
Seed germination is an excellent system to study growth regulatory mechanisms since the developmental transition from seed to seedling is driven exclusively by cell shape change in the absence of cell division (Rombolá-Caldentey et al., 2014; Sliwinska et al., 2009). It is well known that seed germination requires endosperm weakening and embryo elongation growth, which are associated with gibberellins (GAs) and the coordinated expression of cell-wall remodelling enzymes (CWREs; Holdsworth et al., 2008). Here, we describe a GA dependent mechanism promoting endosperm cell growth during germination, which coincides with radicle growth, and demonstrate that endosperm not only facilitates seed germination but is also essential to ensure radicle integrity. By phylogenomic analyses of gene promoters (Castrillo et al., 2011), we have identified and characterized upstream regulators of an endosperm CWRE marker gene (ECMG) that integrate GA-signalling in the seed endosperm and are transcriptional activators of ECMG expression and other CWRE genes putatively involved in endosperm cell growth. Their regulatory roles only overlap partially, since single loss of function (KOs) mutants present a decrease in CWREs expression, reduced endosperm cell expansion and delayed testa rupture. Currently, we are studying to what extent this mechanism has been recruited by other developmental stages and/or plant species to control growth, as well as studying the integration of environmental signals into this regulation.
References 1. Castrillo et al. (2011) PLoS One, 6: e21524 2. Holdsworth et al. (2008) New Phytol 179, 33-54 3. Rombolá-Caldentey et al. (2014) Plant Cell, 26: 2905-2919. 4. Sliwinska et al. (2009) Experimental Botany, 60: 3587-3594
Acknowledgements & Funding This work was supported with grants to L.O.-S by the Spanish Ministry of Economy and Competitiveness (BIO2013-46076-R and BIO2016-77840-R). G.W.B. and P.S. were supported by BBSRC grant BB/J017604/1, G.W.B. by BBSRC Grants BB/L010232/1 and BB/N009754/1, and Leverhulme Trust Grant RPG-2016-049 together with S.D.N. Thanks to Anne Medhurst for making the prom:ECMG:GUS construct and the BBSRC grant BB/F006934/1 in M.H.´s lab.
95
S-IV. Póster 29
AN UPDATE ON THE ROLE OF DELAY OF GERMINATION 1 (DOG1) IN PLANTS
Néstor Carrillo-Barral1, Ángel Matilla2, María del Carmen Rodríguez-Gacio2
1Deparment of Biology, Plant Physiology Area, Coruña University, La Coruña (Spain); 2Department of Functional Biology, Plant Physiology Area, Santiago de Compostela
University, Santiago de Compostela (Spain) Corresponding author: María del Carmen Rodríguez Gacio ([email protected])
The seed germination is a crucial and strongly regulated step in the life-cycle of higher plants. Seeds act as environmental sensors integrating information like ambient temperature, humidity and light (Graeber et al., 2014). The seed ability to postpone germination (dormancy) adapting to the optimal spatiotemporal conditions is decisive for the plant survival and reproductive success (Cyrek et al., 2016). This review summarizes the current knowledge in the last years on the controlling role of DELAY OF GERMINATION 1 (DOG1) in plants. DOG1 was first identified as a quantitative trait locus (QTL) for seed dormancy in Arabidopsis thaliana, and subsequently characterized by studying the variation of dormancy between the Arabidopsis Cape Verde Island (Cvi) and Landsberg erecta (Ler-0) accessions that have high and low dormancy, respectively (Alonso-Blanco et al., 2003). DOG1 is highly conserved in both dicot and monocot, and some cereal DOG1-like genes have been described to encode proteins that are functionally orthologous to DOG1 (Ashikawa et al., 2014). Moreover, several studies have demonstrated that the role of DOG1 is conserved throughout the plant kingdom. DOG1 has been initially shown to be involved in several processes, being seed dormancy the most important; but its molecular function(s) remains unclear (Huo et al., 2016). However, many different mechanisms have been proposed to explain how DOG1 influences germination, including alteration of microRNA pathways, inhibition of auxins and GA-related genes or involvement in ABA signaling pathways (Huo et al., 2016; Bai et al., 2018). Nee et al. (2017) described that DOG1 can interact with several proteins of the PP2C family, being this fact important for the maintenance of seed dormancy. One of these PP2C interacting proteins is AHG3, which is involved in the signaling pathway of ABA.
References Alonso-Blanco et al. (2003) Genetics 164: 711–29; Ashikawa et al. (2014) Transgenic Res. 23:621–29; Bai et al. (2018) New Phytol. 217: 1077-1085; Cyrek et al. (2016) Plant Physiol. 170: 947-55; Footit et al. (2015) Plant J. 81: 413-25; Graeber et al. (2014) PNAS (USA) 111: 3571–3580; Huo et al. (2016) PNAS (USA) 113: 2199–E2206; Nee et al. (2017) Nat. Commun. doi: 10.1038/s41467-017-00113-6.
96
S-IV. Póster 30 THE CHARACTERIZATION OF TWO BASIC/HELIX-LOOP-HELIX
TRANSCRIPTION FACTORS REVEALS GENES INVOLVED IN THE
RIPENING REGULATION IN STRAWBERRY FRUIT RECEPTACLE.
Francisco Javier Molina-Hidalgo, Félix Juan Martínez-Rivas, Enriqueta Moyano, Antonio Rodríguez-Franco, José Luis Caballero, Juan Muñoz-Blanco, Rosario
Blanco-Portales. Department of Biochemistry and Molecular Biology, University of Cordoba, Córdoba, Spain.
Corresponding author: Juan Muñoz-Blanco ([email protected])
The basic helix-loop-helix (bHLH) transcription factor family is the second largest transcription factor (TF) family in plant. It is characterized by the conserved bHLH domain, that plays a central regulatory role in many biological processes. In Fragaria × ananassa have been identified putatively 94 bHLH TFs. Only two have been previously characterized. FaSPT, which is related with the fruit size and shape (Tisza et al., 2010); and FabHLH3, related with the biosynthesis of proanthocyanidin (Schaart et al., 2013). Previously, we have identified a group of TFs ripening-related (Medina-Puche et al., 2016). Among them, we identified two bHLHs, FabHLH75 and FabHLH88. Both TFs are induced during the ripen stages of the fruit receptacle, and almost specific of this fleshy tissue. The amino acid sequences show that, whilst FabHLH75 contain basic domain which binds to the promoter sequences, FabHLH88 is an atypical bHLH, with no DNA-binding ability. To understand the role played by these two TFs, we performed the silencing by RNAi of the gene expression by transiently agroinfiltration in strawberry fruit receptacles. Later, transcriptomic analysis was carried out using a custom-made oligo microarray platform (60-mer length; FraGenomics 35K). To elucidate which process could be regulated by these TFs, GO Term Enrichment were performed using the Plant Transcription Factor Database (planttfdb.cbi.pku.edu.cn/). The biological process affected by the downregulation of the FabHLH88 showed that, those genes that appear induced in RNAi::FabHLH88 fruits are involved in photosynthesis process and response to light. On other hand, the genes that are downregulated in RNAi::FabHLH88 fruits are related with morphogenesis and organ development. With these results, we could suppose that FabHLH88 could be a photosynthetic repressor and an activator of genes involved in fruit development and ripening. GO Term Enrichment of the biological process affected by the downregulation of the FabHLH75 showed that the genes that are downregulated in RNAi::FabHLH75 fruits are related with cell wall organization, metabolism and polysaccharide metabolic process. Moreover, the genes that are upregulated in RNAi::FabHLH75 fruits are mainly included in pathways involving fatty acids. Also, FabHLH75 could regulate some genes related with response to either biotic and abiotic stimulus.
• Medina-Puche L, Blanco-Portales R, Molina-Hidalgo FJ, Cumplido-Laso G, García-Caparrós N, Moyano-Cañete E, Caballero-Repullo JL, Muñoz-Blanco J, Rodríguez-Franco A. 2016. Extensive transcriptomic studies on the roles played by abscisic acid and auxins in the development and ripening of strawberry fruits. Funct Integr Genomics. 16:671-692. • Schaart JG, Dubos C, Romero De La Fuente I, van Houwelingen AM, de Vos RC, Jonker HH, Xu W, Routaboul JM, Lepiniec L, Bovy AG. 2013. Identification and characterization of MYB-bHLH- WD40 regulatory complexes controlling proanthocyanidin biosynthesis in strawberry (Fragaria × ananassa) fruits. New Phytol. 197:454-67. •Tisza V, Kovács L, Balogh A, Heszky L, Kiss E. 2010. Characterization of FaSPT, a SPATULA gene encoding a bHLH transcriptional factor from the non-climacteric strawberry fruit. Plant Physiol Biochem. 48:822-6.
This work was supported by AGL2014-55784-C2 from the Spanish Ministerio de Ciencia e Innovación. RBP was supported by “Ramón y Cajal” programme from the Spanish Ministerio de Ciencia e Innovación.
97
S-IV. Póster 31
NOVEL REGULATORS OF DE NOVO ROOT ORGANOGENESIS IN
Arabidopsis thaliana Sergio Ibáñez1, María Salud Justamante1, Helena Ruiz-Cano1, María Ángeles
Fernández-López1,2, José Luis Micol1, José Manuel Pérez-Pérez1
1Instituto de Bioingeniería, Universidad Miguel Hernández, Elche, Spain, 2Instituto de
Biología Molecular y Celular de Plantas, UPV–CSIC, Valencia, Spain.
Corresponding author: José Manuel Pérez-Pérez ([email protected])
Lateral roots (LRs) and adventitious roots (ARs) are formed de novo during post-
embryonic development, in processes that are highly dependent on environmental
inputs (Bellini et al., 2014). Wound-induced de novo root organogenesis is a suitable
model to study regeneration in plants, as it includes local hormone signalling, cell
proliferation, founder cell specification and patterning (Bustillo-Avendaño et al.,
2018). We searched for genes involved in de novo root formation by means of two
complementary approaches. First, we screened 139 T-DNA homozygous lines from
the PhenoLeaf collection (Wilson-Sánchez et al., 2014) and 23 of them (16.5%)
showed altered de novo root organogenesis. Gene products related to vesicle
trafficking and ABA signalling, among others, were confirmed to modulate AR
formation. We performed a genome-wide association study on a collection of 120
natural accessions to identify additional regulators of de novo root organogenesis.
We found statistically significant associations between several LR traits after root tip
excision and non-synonymous SNPs in the coding region of eight genes in
chromosomes 1, 3, 4 and 5. Interestingly, we found some overlap in the
developmental pathways identified by the different approaches, indicating that
wound-induced LRs and ARs might share molecular regulators.
Bellini, C., et al. (2014). Annu Rev Plant Biol 65: 639-666 Bustillo-
Avendaño, E., et al. (2018). Plant Physiol 176: 1709-1727
Wilson-Sánchez, D., et al. (2014). Plant J 75: 878-891
Work in the laboratory of J.M.P-P is funded by the Ministry of Science and Innovation (AGL2012-
33610 and BIO2015-64255-R).
98
S-IV. Póster 32
PAN INTERACTORS BOP1 AND BOP2 ARE REGULATED BY NITRIC
OXIDE IN PLANT DEVELOPMENT
Jimena Solana, María Guadalupe Fernández-Espinosa, Oscar Lorenzo
Department of Botany and Plant Physiology, Spanish-Portuguese Agricultural Research
Institute (CIALE), School of Biology, University of Salamanca, 37185 Salamanca, Spain.
Corresponding author: O Lorenzo ([email protected]) Nitric oxide (NO) is a gaseous radical with signalling functions in a variety of aspects
of plant development, among other processes1, 2. The most largely studied mode of
NO sensing is the post-translational modification of target proteins. Particularly S-
nitrosylation of key cysteine residues functions as a molecular switch suitable for
signal transduction similar to phosphorylation3. Our previous research found a
relevant role of NO during early development of primary roots in Arabidopsis through
the regulation of stem cell decisions in the meristem2.
The TGA family transcription factor PERIANTHIA (PAN), initially characterized as a
flowering regulator in Arabidopsis, has been found to play a key role upstream of
multiple signalling pathways in the root meristem stem cell niche, primarily in the
maintenance of quiescent centre (QC) function4. BLADE-ON-PETIOLE1 and 2
(BOP1/2) physically interact with PAN5, 6 and have an established function in
regulating aerial part development7. BOP1/2 are paralogs of NONEXPRESSOR OF
PATHOGENESIS-RELATED GENES1 (NPR1), a protein subjected to regulation by
specific S-nitrosylation at Cys1568. Here, we investigated whether BOP1/2 have a
function in plant development through interaction with PAN in the stem cell niche,
and their possible S-nitrosylation by NO. We first studied the plant phenotypes of
loss- and gain-of-function BOP and PAN lines in the presence and absence of NO.
Secondly, S-nitrosylation of BOP1 and BOP2 was evidenced in vitro through the
biotin switch method, and target Cys residues were predicted in silico. Insights into
the interaction of PAN – BOP1/2 regulated by NO will be presented.
References 1 Fernández-Marcos, et al (2011) PNAS, 108: 18506. 2 Sanz, et al. (2014) Plant physiology, 166: 1972. 3Astier, et al. (2017). Journal of Experimental Botany. 4de Balaguer, et al. (2017). PNAS, 114(36), E7632–E7640. 5Hepworth, et al. (2005). THE PLANT CELL, 17(5), 1434–1448. 6Xu, et al. (2010). The Plant Journal: For Cell and Molecular Biology, 63(6), 974–989. 7Khan, et al. (2014). Plant Science, 215–216, 157–171. 8Tada, et al. (2008). Science, 321(5891), 952–956.
Acknowledgements: ERC.KBBE.2012.1.1-01 (EcoSeed-311840), MINECO (BIO2017-85758-R),
CONSOLIDER (CSD2007-00057), Junta de Castilla y León (SA093U16). M.G. F-E is supported by a
JCyL and Fondo Social Europeo grant.
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S-IV. Póster 33
REDOX REGULATION OF PEP ACTIVITY DURING SEEDLING ESTABLISHMENT IN Arabidopsis thaliana
Tamara Hernández-Verdeja1, Manuel Guinea Diaz1,2, Dmitry Kremnev1, Tim
Crawford1, Carole Dubreuil1 & Åsa Strand1
1Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, Umeå,
Sweden, 2Present address: Molecular Plant Biology, University of Turku, Turku, Finland.
Corresponding author: Åsa Strand ([email protected])
Light provides plants with information to modulate developmental processes such as chloroplast development. Initiation of chloroplast development in the light, and the establishment of photosynthesis are dependent on both nuclear-encoded and plastid- encoded components, and thus requires a tight communication between the two compartments. The plastid-encoded genes of higher plants are transcribed by at least two types of RNA polymerases: the nuclear-encoded plastid RNA polymerase (NEP), and the plastid-encoded RNA polymerase (PEP). Chloroplast development is associated with a shift in the usage of the primary RNA polymerase from NEP to PEP. The mechanism(s) behind this switch is unknown. Activation of the PEP is tightly controlled and involves a network of phosphorylation and, as yet unidentified, thiol- mediated events. PLASTID REDOX INSENSITIVE2 (PRIN2) was identified as a chloroplast component involved in redox-mediated retrograde signalling, and is essential for full expression of genes assigned to be PEP-dependent (Kindgren et al, 2012). Here, we demonstrate a direct interaction between PRIN2 and a component of the PEP complex, Thioredoxin z. PRIN2 can form dimers that can be reduced into the monomeric form by thioredoxins through reduction of a disulfide bond. Our results complementing prin2-2 with mutated PRIN2 variants indicate that a reduced monomeric form of the PRIN2 protein is the active form required for light-activated PEP-dependent transcription in the plastids, and that expression of photosynthesis- associated nuclear genes is linked to the activity of PEP. In response to the establishment of electron transport activity during seedling establishment and possibly during leaf development, a thioredoxin-like protein generates a switch from dimer to monomeric form of PRIN2. Full activation of the PEP complex and plastid photosynthetic gene expression is achieved and as a consequence also full expression of the nuclear encoded photosynthesis genes. Thus, regulation of PRIN2 is the thiol-mediated mechanism required for full PEP activity, with PRIN2 monomerization via reduction by TRXs providing a mechanistic link between photosynthetic electron transport and activation of photosynthetic gene expression (Guinea Diaz et al, 2018).
Guinea Diaz, M. et al (2018) Nat Comm. 9: 50
Kindgren, P. et al. (2012) Plant J. 70: 279–291
This work was supported by grants from the Swedish research foundation, VR (Å.S.).
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S-IV. Póster 34
FUNCTIONAL ANALYSIS OF THE mTERF5 AND mTERF9 GENES OF
Arabidopsis thaliana
Víctor Quesada, Eva Núñez-Delegido, Almudena Ferrández-Ayela and Pedro Robles Instituto de Bioingeniería, Universidad Miguel Hernández de Elche, Elche, Spain
Corresponding author: Víctor Quesada ([email protected]) Unlike animals, very little is known about the functions of the mitochondrial transcription termination factors (mTERFs) in plants. To shed light into the roles of plant mTERFs we previously characterized the Arabidopsis mda1 and mterf9 mutants defective in the mTERF5 and mTERF9 chloroplast-targeted proteins, respectively (Robles et al., 2012; 2015). To advance in the functional analysis of the mTERF5 and mTERF9 genes, we have obtained overexpression constructs in which their coding sequences were put under the control of the constitutive 35S promoter and complemented the mda1-1 and mterf9 mutant phenotypes. We have also obtained mTERF5 and mTERF9-overexpressing (OE) transgenic lines in a Col-0 background and found that mTERF5 or mTERF9 overexpression does not substantially modify Arabidopsis morphology. Nevertheless, mTERF9 constitutive expression significantly reduces plant growth. We investigated the salt and abscisic acid (ABA) hormone sensitivity of the mTERF5 or mTERF9 OE lines since the mda1 and mterf9 mutants are more tolerant than Col-0 to salt stress and ABA. Our results reveal that mTERF9 overexpression significantly enhances Arabidopsis salt and ABA sensitivity.
The rRNA abundance is a proxy for the levels of the 50S and 30S plastid ribosomal subunits. Hence, we quantified the levels of the different plastid rRNA species in the mda1-1 and mterf9 mutants and the mTERF5 and mTERF9 OE lines. A differential accumulation of plastid rRNAs was found in mda1-1 and mterf9 compared with Col-0, in line with a defect in plastid ribosomal stability and/or assembly in these mutants.
A role for plant mTERFs in regulation of nuclear gene expression through plastid retrograde signalling has been proposed. We investigated by qRT-PCR whether the expression of the photosynthesis gene LHCB1, a usual marker for retrograde signalling, was affected in the mda1-1 and mterf9 mutants compared with Col-0 after treatments with inhibitors of chloroplast translation [norflurazon (NF)] or carotenoid biosynthesis [lincomycin (LIN)]. The lower inhibition of LIN or NF on LHCB1 expression in mterf9 suggests that mTERF9 might function in modulating retrograde signalling. Consistent with this, mterf9, but not mda1-1, synergistically interacts with gun1-1, a loss-of-function mutation of the GUN1 protein, a central integrator of plastid retrograde signals.
Robles P, Micol JL, Quesada V. (2012). Arabidopsis MDA1, a nuclear-encoded protein, functions in chloroplast development and abiotic stress responses. PLoS One 7(8):e42924. Robles P, Micol JL, Quesada V. (2015). Mutations in the plant-conserved MTERF9 alter chloroplast gene expression, development and tolerance to abiotic stress in Arabidopsis thaliana. Physiol. Plant. 154: 297-313.
101
S-IV. Póster 35 PcG MARKS MEDIATE NUCLEOSOME STABILIZATION AT PROMOTER REGIONS TO MAINTAIN CHROMATIN IN AN
INACCESSIBLE STATE
Wiam Merini1, Francisco J Romero-Campero1 and Myriam Calonje1
1Institute of Plant Biochemistry and Photosynthesis (IBVF-CSIC-University of Sevilla), Seville, Spain.
Corresponding authors: Francisco J Romero ([email protected]), Myriam Calonje
Nucleosome occupancy and positioning and the organization of nucleosomes into higher order chromatin structures can help to either selectively expose or hide functionally important DNA sequences required for the binding of transcription factors (TFs) and the access of the transcriptional machinery to DNA (Jiang and Pugh 2009). The formation and maintenance of accessible or inaccessible chromatin states rely on mechanisms that mediate eviction, repositioning or de/stabilization of nucleosomes, which are often related to post-translational modification of histones associated with gene regulatory elements. The evolutionary conserved Polycomb group (PcG) proteins play important roles in maintaining the repression of the genes that are not required in a specific cell fate in plants (Merini & Calonje, 2015). These proteins form multiprotein complexes with different histone modifying activities, including PcG repressive complex 2 (PRC2), which possesses histone 3 lysine 27 (H3K27) trimethyltransferase activity, and PRC1, which has histone 2A E3 ubiquitin ligase activity towards lysine 121 in Arabidopsis (Zhou, Campero et al., 2017) as well as non-enzymatic functions critical for chromatin compaction (Merini & Calonje, 2015). There is a general idea that PcG complexes create inaccessible chromatin. In vitro experiments have shown that PcG protein complexes can compact chromatin, but this activity seems to be independent of the histone modifying activities (Francis et al., 2004). Also, it has been proposed that PcG marks may interfere with transcription elongation (Zhou et al., 2008); however, the exact mechanism by which the incorporation of these histone modifications led to an inaccessible chromatin state is not clear. To address this question, we investigated the relationship between PcG marks and nucleosome occupancy in a genome-wide scale and the impact that the loss of PcG marks has on nucleosome occupancy and positioning. Our data unveil a crucial role of PcG marking in stabilizing nucleosome positioning at specific regions of the promoter of the target genes.
References:
Francis NJ, Kingston RE, Woodcock CL. Science. 2004; 306:1574–7.
Jiang C, Pugh BF. Nat. Rev. Genet. 2009;10:161–172
Merini W, Calonje M. Plant J. 2015;83:110–20.
Zhou Y., Romero-Campero F. J., Gómez-Zambrano Á., Turck F., Calonje M. (2017ª). Genome Biol. 18 69. 10.1186/s13059-017-1197-z
Zhou, W., Zhu, P., Wang, J., Pascual, G., Ohgi, K.A., Lozach, J., Glass, C.K. and Rosenfeld, M.G. Mol Cell. 2008 Jan 18;29(1):69-80.
Acknowledgements & Funding: This work is supported by BIO2016-76457-PGrant from the Spanish Ministry of Economy and Competitiveness (MINECO).
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S-IV. Póster 36
PROGRESSIVE PECTIN DE-ESTERIFICATION AND AGP INCREASE UNDERLIE CELL WALL REMODELING DURING SOMATIC
EMBRYOGENESIS IN Quercus suber
Yolanda Pérez-Pérez1, Elena Carneros1, Ivett Bárány1, M.T. Solís1,2, Beatriz Pintos2, Aránzazu Gómez-Garay2, María C. Risueño1, Pilar S. Testillano1
1Pollen Biotechnology of Crop Plants group, Biological Research Center, CIB-CSIC, Madrid, Spain. 2Dept. Plant Biology I, Plant Physiology, UCM, Madrid, Spain.
Corresponding author: Pilar S. Testillano ([email protected])
Changes in cell wall mechanics controlled by the methylesterification/de-esterification status of pectins, mediated by pectin methyl esterases (PME) and pectin methyl esterase inhibitors (PMEI) underlie organogenesis initiation and embryogenesis progression. Arabinogalactan proteins (AGPs) are present in cell walls, plasma membranes and extracellular secretions, playing a key role in several plant developmental processes. Somatic embryogenesis (SE) is a feasible system for in vitro regeneration, with many biotechnological applications in woody species, but the regulating mechanisms of the process are largely unknown.
In this study, we have investigated changes in pectin esterification and AGPs during SE in Quercus suber. Expression analysis of PMEI and AGP genes, immuno-dot-blot assays, immunofluorescence and confocal analysis were performed at specific developmental stages of SE, by using monoclonal antibodies to AGPs, high- and low-methylesterified pectins (LM6, LM2, LM19, LM20, JIM7, JIM5). Pharmacological treatments with PME and AGP inhibitors were also performed.
Results allowed the identification of the distribution patterns of AGPs and pectin esterification/de-esterification during SE progression, as well as their correlation with the expression patterns of QsPMEI, QsPMEI2, QsAGP and QsAGP-Lys-rich genes. At early SE stages, cells showed high levels of esterified pectins and AGPs. At advanced SE stages, AGP expression increased and differentiating cells exhibited walls rich in de-esterified pectins. Inhibition of PME activity and AGP blocking impaired somatic embryo development. The findings indicate a role for PME and AGPs in the cell wall remodelling associated with SE in cork oak.
References: Solís MT, Berenguer E, Risueño MC, Testillano PS [2016]. BnPME is progressively induced after microspore reprogramming to embryogenesis, correlating with pectin de-esterification and cell differentiation in Brassica napus. BMC Plant Biology 16, 176.
Funding: Supported by projects (AGL2014-52028-R, AGL2017-82447-R) funded by MINECO and ERDF/ FEDER.
103
SESIÓN V
REGULACIÓN AMBIENTAL
DEL DESARROLLO
MODERADORES:
Miguel A. Blázquez (IBMCP,
CSIC-UPV, Valencia)
Myriam Calonje (IBVF-CSIC,
Universidad de Sevilla)
104
S-V. Ponencia 1
PRC1.MEDIATED H2A.Z MONOUBIQUITINATION IS CRUCIAL
TO REGULATE DEVELOPMENTAL AND ENVIRONMENTAL
RESPONSES IN ARABIDOPSIS
Ángeles Gómez-Zambrano1, Wiam Merini1, Myriam Calonje1
1Institute of Plant Biochemistry and Photosynthesis (IBVF-CSIC-University of Sevilla), Seville, Spain
Myriam Calonje: [email protected]
The primary protein components of chromatin are the histones, which are assembled along with DNA into larger complexes known as nucleosomes. The incorporation of posttranscriptional histone modifications and histone variants can alter nucleosome structure, stability, dynamics, and ultimately DNA accessibility (Talbert and Henikoff, 2010), leading to changes in gene expression (Weber and Henikoff, 2014). Over the last years, the replication- independent replacement of nucleosomal H2A with H2A.Z variant has gained special interest due to its essential role in regulating environmental responses (Talbert and Henikoff, 2014), which is particularly important in plants since, as sessile organisms, they must rapidly respond to environmental changes in order to survive.
H2A.Z in Arabidopsis frequently co-localizes with transcription start sites but it is also found further within gene bodies (Coleman-Derr and Zilberman, 2012). The incorporation of this variant seems to have either a promoting or repressive effect on transcription depending on its location within the gene. Although the location of H2A.Z-containing nucleosomes may indeed differentially impact gene expression, the incorporation of posttranscriptional modifications to H2A.Z could also be critical to define these opposite roles. However, thus far the occurrence of H2A.Z modifications has not been investigated in plants; therefore, the role of H2A.Z in regulating gene expression remains a long- standing puzzle. Our results show that the Polycomb Repressive Complex 1 (PRC1) components AtBMI1 and AtRING1 (Merini et al., 2017; Zhou, Campero- Romero et al., 2017) are required for H2A.Z monoubiquitination and that the incorporation of this modification is crucial for the repression of a subset of genes involved in the response to endogenous and environmental signals. Our findings represent a step forward towards understanding the complex mechanism of action of this histone variant.
References:
- Coleman-Derr, D and Zilberman, D. (2012) PLoS Genet. 8,e1002988. DOI:10.1371/journal.pgen.1002988.
- Merini W, Romero-Campero FJ, Gómez-Zambrano A, Zhou Y, Turck F, Calonje M. (2017) Plant Physiol. 173(1):627-641. DOI: 10.1104/pp.16.01259.
- Talbert PB, Henikoff S (2010). Nat Rev Mol Cell Biol; 11: 264–275. DOI: 10.1016/B978-0-12- 380866-0.60005-8.
- Weber CM, Henikoff S. (2014). Genes Dev. 28(7):672-82. DOI: 10.1101/gad.238873.114. - Zhou Y, Romero-Campero FJ, Gómez-Zambrano A, Turck F, Calonje M. (2017). Genome
Biol. 18: 69.DOI: 10.1186/s13059-017-1197-z.
Acknowledgements & Funding: This work is supported by FP7-PEOPLE-2012 Marie Curie CIG Grant ID 333748 and BIO2013- 44078-P Grant from the Spanish Ministry of Economy and Competitiveness (MINECO).
105
S-V. Ponencia 2
THE COP1/SPA COMPLEX RELAYS LIGHT AND TEMPERATURE
INFORMATION ON DELLA PROTEINS IN ARABIDOPSIS
Eugenio G. Minguet1, Martina Legris2, Noel Blanco-Touriñán1, Manuel Pacín3, Elisa
Iniesto4, Antonella Locascio1, Martin Černý5, Břetislav Brzobohatý5, Henning
Frerigmann6, Vicente Rubio4, Miguel A. Blázquez1, Jorge J. Casal2,3, David Alabadí1.
1IBMCP, CSIC-UPV, Valencia, Spain. 2Fundación Instituto Leloir, Buenos Aires, Argentina.
3IFEVA, UBA-COCINET, Buenos Aires, Argentina. 4CNB, CSIC, Madrid, Spain. 5Mendel
University, Brno, Czech Republic. 6Max Planck Institute for Plant Breeding Research,
Cologne, Germany (Arial, 11 pt, italics, centered)
Corresponding author: David Alabadí ([email protected])
The correct integration of environmental information into growth and developmental
outputs is an extraordinary ability of plants that allows them to adapt and survive.
DELLA proteins are transcriptional regulators that restrain growth and adjust
development in response to environmental signals. Two characteristics of DELLAs
sustain this role: (i) they are degraded in response to the hormone gibberellin (GA),
whose metabolism is highly responsive to the environment; and (ii) they interact with
multitude of transcription factors.
Our results show that DELLAs are also regulated by COP1/SPA, a complex that
destabilizes negative regulators of growth, owing to the E3 ubiquitin ligase activity of
COP1, and that is inactivated by light and low/moderate temperature. COP1
ubiquitinates the DELLA protein GAI in vitro. Remarkably, SPA1 facilitates the
recruitment of DELLAs GAI and RGA to nuclear bodies and their interaction with
COP1. We provide evidences that the destabilization of DELLAs by COP1/SPA is
relevant to respond to light and temperature. The steady disappearance of RGA from
hypocotyls of wild type seedlings exposed to shade or warm temperature is delayed
in cop1. In agreement, GA-treatment or genetic inactivation of GAI and RGA restore
the ability of cop1 to respond to darkness, shade and temperature. We propose that
the destabilization of DELLAs by the COP1/SPA complex defines a novel entry point
to relay information about light and temperature into the growth transcriptional
network.
106
S-V. Comunicación 1 DET1 COMPLEXES CONTROL PHOTOMORPHOGENESIS
BY ACTING AT THE INTERFACE BETWEEN LIGHT SIGNALING AND
EPIGENOME DYNAMICS
Amr Nassrallah1, Sandra Fonseca1, Martin Rougée2, Clara Bourbousse2, Stéphanie Drevensek2, Elisa Iniesto1, Ouardia Ait-Mohammed2, Anne-Flore Deton-Cabanillas2,
Gerald Zabulon2, Ikhlak Ahmed2, David Stroebel2, Vanessa Masson3, Bérangère Lombard3, Dominique Eeckhout4, Damarys Loew3, Auguste Genovesio2, Cécile
Breyton5, Geert de Jaeger4, Chris Bowler2, Fredy Barneche2, Vicente Rubio1
1Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain, 2Institut de biologie de l’Ecole normale
supérieure (IBENS), Paris, France, 3 Laboratoire de Spectrométrie de Masse Protéomique, Institut Curie, Paris, France, 4Department of Plant Systems Biology. VIB-Ghent University, Ghent, Belgium,
5Institut de Biologie Structurale (IBS), Grenoble, France.
Corresponding author: Vicente Rubio ([email protected]), Chris Bowler ([email protected]), Fredy Barneche ([email protected])
Photomorphogenesis is a developmental switch that initiates upon light perception and involves a huge transcriptional reprograming. It requires a fine control of the activity of specific transcription activators and the modification of chromatin marks that facilitates Polimerase II progression, as well as intense protein turnover via the ubiquitin-proteasome system. DE-ETIOLATED1 (DET1) is an atypical DDB1- CULLIN4 Associated Factor (DCAF) that, together with DDB1, COP10 and DDA1, constitutes a substrate adaptor module of CUL4 E3 ubiquitin ligases. These complexes are well conserved among species, controlling the stability of cell proliferation factors in animals or circadian and photomorphogenesis regulators in plants. However, the exact mechanism by which DET1 affects the stability of these regulators and controls transcription has remained largely unknown. Previous work showed that DET1 displays high affinity for histone 2B but the molecular and physiological consequences of that binding remained elusive. We found that det1-1 mutant displays defects in H2B monoubiquitination (H2Bub), and concomitant dramatic alterations in gene expression. The DET1-associated protein DDA1, directly interacts with SGF11, a member of an Arabidopsis H2Bub deubiquitination module (DUBm). Besides SGF11, this atypical DUBm comprises UBP22 and ENY2 proteins, the predicted Arabidopsis orthologues of yeast Sgf11, Ubp8 and Sus1, respectively, and appears to act independently from SAGA in the absence of a plant Sgf73-like subunit. By controlling the light-dependent degradation of a H2Bub deubiquitination module, DET1 controls H2Bub homeostasis and transcriptional output. By introgressing sgf11 and ubp22 in the det1-1 background, we could ameliorate det1-1 defects in H2Bub mark, the number of genes altered, and photomorphogenic phenotypes. Our findings support a model in which H2B ubiquitination and deubiquitination dynamics impact photomorphogenesis by regulating transcription trough progression of Polymerase II. We are now characterizing the role of known light signalling components in the modulation of H2Bub homeostasis. Collectively, our studies unveil a signalling pathway employing evolutionary
conserved protein complexes that controls global H2Bub levels, potentially fine-
tuning genic transcriptional capacity or "regulatability" during developmental
responses to external cue.
107
S-V. Comunicación 2 A NIGHT HOUR COUNTER MECHANISM UNDERLIES POPLAR
SEASONAL GROWTH
Mariano Perales1, Jose Manuel Ramos-Sánchez1, Paolo M. Triozzi1, Daniel Alique1, Philip Wigge 2, Isabel Allona1,3.
1Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM,
28223 Pozuelo de Alarcón, Madrid, Spain. 2Sainsbury Laboratory, University of Cambridge,
Cambridge CB2 1LR, UK. 3Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid
(UPM), 28040 Madrid, Spain.
Corresponding author: [email protected]; [email protected] Poplar shoot apical growth is extremely sensitive to photoperiod, occurring continuously under long day conditions. When photoperiod falls below a critical day length, internode elongation and organogenesis cease, a phenomenon called growth cessation. The regulation of shoot apical growth involves a photoperiodic time measurement (PTM) mechanism, monitoring day and/or night duration. This mechanism increases plant fitness by tracking time of year and delivering the instructive information to trigger seasonal development. The module formed by CONSTANS (CO) and FLOWERING LOCUS T (FT2) was shown central into this PTM mechanism (Böhlenius et al., 2006). However, poplars overexpressing CO were not sufficient to promote SAM growth under short day (SD), indicating that a SD- repressive pathway should overcome CO activation (Hsu et al., 2012). Moreover, poplars showing reduced levels of LATE ELONGATED HYPOCOTYL (LHY2) presented delayed growth cessation under SD conditions, although its mode of action remains to be clarified (Ibañez et al., 2010).
Here we show, that poplar LHY2 transmit a repressive signal to FT2, counting night length hours. Night break experiments using red light illumination promoted poplar growth, under inductive growth cessation SD conditions, repressing LHY2 and activating FT2. Conditional activation of LHY2 coupled to RNAseq experiment, identified an APETALA 2/ETHYLENE RESPONSE FACTOR that could mediate in FT2 repression under SDs. A model including LHY2 repressive signaling into poplar PTM mechanism is proposed.
References
Böhlenius, H., Huang, T., Charbonnel-Campaa, L., Brunner, A. M., Jansson, S., Strauss, S. H., &
Nilsson, O. (2006). CO/FT regulatory module controls timing of flowering and seasonal growth
cessation in trees. Science (New York, N.Y.), 312(5776), 1040–1043.
Hsu, C. Y., Adams, J. P., No, K., Liang, H., Meilan, R., Pechanova, O., … Yuceer, C. (2012).
Overexpression of Constans Homologs CO1 and CO2 Fails to Alter Normal Reproductive Onset and
Fall Bud Set in Woody Perennial Poplar. PLoS ONE, 7(9).
Ibáñez, C., Kozarewa, I., Johansson, M., Ogren, E., Rohde, A., & Eriksson, M. E. (2010). Circadian
clock components regulate entry and affect exit of seasonal dormancy as well as winter hardiness in
Populus trees. Plant Physiology, 153(4), 1823–1833.
Acknowledgements & Funding: This study was supported by Grants AGL2011-22625, AGL2014-
53352-R, PCIG13-GA-2013-631630 awarded to I.A. and M.P.; M.P. was supported by the Ramón y
Cajal programme of MINECO (RYC-2012-10194). JM.R.-S. was funded by a FPU12/01648 fellowship
and P.M.T. Ph.D. programme of CEI campus of the Universidad Politécnica de Madrid (L1UF00-47-
JX9FYF).
108
S-V. Comunicación 3
‘BLINDED BY THE LIGHT’ - RETROGRADE SIGNALS SUPPRESS
PHOTOMORPHOGENESIS VIA GLK1 DURING HIGH LIGHT
Charlotte Gommers1, Clara Bourbousse2, Alba Ayats1, Fredy Barneche2
& Elena Monte1
1. Plant Development and Signal Transduction Program, Center for Research in Agricultural
Genomics (CRAG), Barcelona, Spain; 2. Plant and Diatom Genomics Laboratory, Institut de
Biologie de l’ENS (IBENS), CNRS UMR 8197, Paris, France
Corresponding author: Elena Monte ([email protected])
Plants obtain and process information about their light environment to optimally use
the available light for growth. Young seedlings in the dark undergo skotomorphogenic
development to facilitate emergence from the soil, after which they undergo
photomorphogenesis and develop green tissues to live as photoautotrophs. Once
exposed to the sun, high light levels damage chloroplasts, which trigger a retrograde
signal (RS) to the nucleus and suppress photomorphogenesis. These comparable
phenotypes in darkness and high light are partly regulated via suppression of
GOLDEN2-LIKE1 (GLK1). In darkness, this suppression is directly regulated by a
group of bHLH transcription factors; the PHYTOCHROME INTERACTING FACTORS
(PIFs), which bind to GLK1 promotor. During high light stress, when PIFs are
degraded by active phytochrome photoreceptors, the chloroplast-localized protein
GUN1 induces the RS which leads to GLK1 suppression. Our research focusses on
the regulation and biological function of GLK1 transcriptional suppression in these
seemingly opposite extreme light environments. We especially investigate the role of
histone tail de-acetylation, mediated by HDAC proteins, and several transcription
factors known to be involved in photomorphogenesis. This study converges plant
eco-photobiology, RS and epigenetic methods to study plant development in sub-
optimal light environments.
This project is supported by the Spanish Ministerio de Economía y Competitividad (FJCI-2016-30876 to C.G. and BIO2015-68460-P to E.M.) and the Generalitat de Catalunya (2014-SGR-1406 to E.M.). We acknowledge financial support by the CERCA Programme/Generalitat de Catalunya and from Ministerio de Economía y Competitividad through the Severo Ochoa Programme for Centers of
Excellence in R&D 2016-2019 (SEV-2015-0533).
109
S-V. Póster 1
GENOME-WIDE ASSOCIATION STUDIES OF ROOT ELONGATION IN ARABIDOPSIS NATURAL ACCESSIONS TREATED WITH THE ACTIN
DISRUPTION COMPOUND LATRUNCULIN B
Ana Paez-Garcia1, Yun Kang2, Fuqi Liao3, J. Alan Sparks1, Elison B. Blancaflor1
1Research Department-Plant Cellular Biology Laboratory, Noble Research Institute, Ardmore,
United States; 2Research Department-Functional Genomics Laboratory, Noble Research
Institute, Ardmore, United States; 3Computing Department, Noble Research Institute, Ardmore, United States
Corresponding author: Elison B. Blancaflor ([email protected])
The actin cytoskeleton plays an important role in plant growth and development. The
distribution and dynamics of the actin filament network determine multiple cellular
processes such as cell division, cell expansion, and vesicle trafficking. When plants
respond to adverse stresses, the cytoskeleton often reorients itself with the help of
cytoskeleton-associated proteins and by interacting with other molecules such as
membrane phospholipids. Cytoskeletal dynamics allows plants to modify their growth,
and improve their survival in response to abiotic stresses including drought, salinity, and
high and low temperatures.
Because the actin cytoskeleton plays a significant role in plant development and
response to the environment, we hypothesized that genetic variants in actin regulatory
components likely exist in natural accessions. To test this hypothesis, we evaluated the
sensitivity of 205 Arabidopsis thaliana natural accessions to the actin cytoskeleton-
disrupting drug, LatrunculinB (LatB). LatB binds actin monomers and prevents actin
filaments from polymerizing. When Arabidopsis seedlings are grown on LatB, primary
root elongation is inhibited. Using root elongation as a biological readout, we performed a
Genome-Wide Association Study (GWAS) on the Arabidopsis natural population growing
in media plates supplemented with or without LatB. The objective of this analysis was to
discover genetic variants associated with particular traits specified by the actin
cytoskeleton. The characterization of Arabidopsis natural accessions growing in
presence of LatB showed large variability on root length. GWAS identified significant
association between LatB-related root length reduction and a group of single nucleotide
polymorphisms (SNPs) on chromosome 5. A detailed study of these SNPs among the
Arabidopsis accessions panel will provide information on how natural selection has
preferred one allelic option to the other in specific environmental conditions. Future
validation of potential causative genes will provide insights about new players in actin
cytoskeleton function and in the role of actin in plant response to abiotic stresses.
This work was supported by Noble Research Institute, LLC.
110
S-V. Póster 2
IDENTIFICATION AND CHARACTERIZATION OF THE BCL2-
ASSOCIATED ATHANOGENE (BAG) 4 PROTEIN AS A REGULATOR
OF THE KAT1 POTASSIUM CHANNEL
Antonella Locascio, Mª Carmen Marqués, Guillermo García-Martínez, Sara Aljama
Claudia Bou, José Antonio Fernández*, José Miguel Mulet, Lynne Yenush
Department of Abiotic stress, IBMCP-Universidad Politecnica de Valencia, Valencia, Spain
Universidad de Málaga, Spain
Corresponding author: Lynne Yenush ([email protected])
Ion homeostasis is a dynamic process and a fundamental requirement for all
organisms. All living organisms have developed efficient systems to acquire and
store these elements and robust mechanisms to maintain homeostatic
concentrations to avoid toxicity and to respond to environmental changes. Potassium
is a key monovalent cation necessary for multiple aspects of cell growth and survival,
for example compensation of negative charges of macromolecules, maintenance of
electroneutrality, cell turgor and volume, enzyme activity, protein synthesis, and
maintenance of proper membrane potential and intracellular pH.
In plants, apart from the basic, general physiological functions listed above for
potassium at the cellular level, this cation also plays a key role at the whole plant
level, as it is involved in important processes such as stomatal aperture that controls
transpiration water loss and plant desiccation. Inward rectifying channels (Kin) are
responsible for potassium influx into guard cells and play a key role in stomatal
opening. KAT1, and its close homologue KAT2, are the main inward rectifying
channels expressed in guard cells. In a Split-Ubiquitin protein-protein interaction
screen searching for KAT1 potassium channel interactors. One of these candidates,
BAG4, is characterized in detail in the present study. Several biochemical and
genetic approaches have been taken to confirm this interaction and its effect on
KAT1 activity in a heterologous complementation assay and in plants. The
identification of physiologically relevant regulators of K+channels will aid in the
design of approaches that may impact both drought tolerance and pathogen
susceptibility, since these pores are responsible for CO2uptake, transpiration water
loss and are the point of entry for certain pathogens
Acknowledgements & Funding: Proyectos EXCELENCIA y Proyectos RETOS Dirección General de Investigación Científica y Técnica Subdirección General de Proyectos de Investigación, BIO2016- 77776-P, BIO2016-81957-REDT/AEI.
111
S-V. Póster 3 GENOME-WIDE SIGNATURES OF FLOWERING ADAPTATION
TO CLIMATE TEMPERATURE
Belén Méndez-Vigo1, Noelia Arteaga1, Mercedes Ramiro1, Arnald Marcer2,
F. Xavier Picó3, Carlos Alonso-Blanco1
1Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas
(CSIC), Madrid, Spain. 2CREAF, UAB, Cerdanyola del Vallès, Spain. 3Departamento de
Ecología Integrativa, Estación Biológica de Doñana (EBD-CSIC), Sevilla.
Corresponding author: Carlos Alonso-Blanco ([email protected])
Current global change is triggering the interest for understanding the genetic and
molecular mechanisms of plant adaptation to climate. In particular, genetic
modifications of the major transitions in the life cycle of annual plants, such as
flowering time or seed dormancy, are a common strategy found in nature for escape
from unfavourable climate temperature (Vidigal et al., 2016; Marcer et al., 2018).
In this study we aimed to determine the genomic bases underlying flowering
time adaptation to this climatic factor. To this end, we have carried out phenotypic
and environmental genome-wide analyses at a regional scale using a collection of
174 highly diverse A. thaliana accessions from the Iberian Peninsula. Correlations
between flowering time and temperature across this region show that the relationship
between flowering and temperature depends on the precise temperature range.
Environmental genome-wide associations (EGWA) show an overall genome
adaptation to temperature. In addition, we have obtained a catalogue of candidate
genes underlying flowering time variation by phenotypic genome-wide association
(PGWA) analyses. Finally, we have compared EGWA and PGWA genomic regions to
identify candidates for adaptation to climate temperature by altered flowering time.
Thus we have detected several known genes, such as TWIN SISTER of FT (TSF) or
FRIGIDA LIKE 1 (FRL1), as well as new ones like ESM1 or VDAC5. Therefore, this
regional collection provides a very suitable resource to address the spatial complexity
of climate adaptation in annual plants (Tabas-Madrid et al., 2018).
Marcer A. et al (2018) Plant Biology 20, 148-156.
Tabas-Madrid, et al. (2018) Plant, Cell & Environment. doi: 10.1111/pce.13189.
The 1001 Genomes Consortium (2016) Cell 166, 481-491.
Vidigal D.S., et al. (2016) Plant Cell and Environment 39, 1737-1748. This project has been funded by grant BIO2016-75754-P (AEI/FEDER, UE).
112
S-V. Póster 4
BARRIERS FORMED IN DIFFERENT WAYS – THE EXODERMIS AND
ENDODERMIS IN TOMATO
Concepcion1,2 Manzano1,2, Kaisa2 Kajala2, Robertas3 Ursache3, Carlos1 del Pozo1,
Niko3 Geldner3, Siobhan M.Brady2.
1INIA, Madrid, Spain 2 Department of Plant Biology and Genome Center, University of
California, Davis, USA
3 University of Lausanne, Switzerland
Corresponding author: Concepcion & Manzano ([email protected])
The exodermis cell type is present within most angiosperms but is understudied due
to its absence in Arabidopsis thaliana. Exodermis cells are able to form a barrier
which is thought to be analogous to the Casparian strip in the root endodermis. We
describe the morphology and the timing of the formation of this barrier in the
exodermis relative to the endodermis, the ability of the exodermis to act as a
functional barrier, and the composition of this barrier. Cell type-specific molecular
profiling and gene reporters are used to assess the degree to which the endodermis
development and Casparian strip regulatory module has been co-opted for
acquisition of exodermis identity.
Funding: Marie Sklodowska Curie Individual Fellowship
113
S-V. Póster 5
EFFECT OF SILENCING PHZTL ON GROWTH AND SCENT
EMISSION IN PETUNIA
Marta Isabel Terry1, Fernando Pérez-Sanz1, María Victoria Díaz-Galián1, Victoria Ruiz-Hernández3, Pedro J. Navarro2, Marcos Egea-Cortines1, Julia Weiss1
1Genetics, ETSIA, Instituto de Biotecnología Vegetal, UPCT, Cartagena, Spain, 2Tecnologías de la Información y las Comunicaciones, ETSIT, UPCT, Cartagena, Spain
Corresponding author: Marcos Egea-Cortines ([email protected])
The circadian clock is an adaptation that allows organisms to anticipate periodic changes. In plants, this biological clock is comprised by a set of genes which form feedback interlocked loops. Zeitlupe (ZTL) codifies an F-box protein that interacts with other two clock components, TIMING OF CAB EXPRESSION 1 (TOC1) and PSEUDO-RESPONSE REGULATOR 5 (PRR5), playing a role in TOC1 and PRR5 degradation (Más et al., 2003; Kiba et al., 2007); additionally ZTL has a PAS domain, present in blue-light receptor molecules (Somers et al., 2000). In Arabidopsis, ztl mutants displayed a longer period, for example, flowering was delayed (Somers et al., 2004). Moreover, the role of circadian clock genes in several processes such as organ development, movement or scent emission is still poorly known (Fenske et al, 20015; Yon et al., 2016). In the present work, we silenced the expression of ZTL by RNAi in Petunia (PhZTL) and we analysed growth and scent emission. Growth was significantly shorter or slower depending on the organ analysed. Most volatile organic compounds showed a rhythmic emission. Time of maximum emission was delayed or advanced in RNAi plants depending on the volatile. The relative contribution of every compound to scent varied between non-transgenic and transgenic Petunia flowers resulting in quantitative changes in scent blends at different times of the day. These results suggest that PhZTL plays a role, directly or indirectly, in biological rhythms in plants, affecting flowering time, stem and flower growth and scent emission.
Fenske, M.P., Hazelton, K.D.H., Hempton, A.K., Shim, J.S., Yamamoto, B.M., Riffell, J.A., & Imaizumi, T. (2015). Circadian clock gene LATE ELONGATED HYPOCOTYL directly regulates the timing of floral scent emission in Petunia. Proceedings of the National Academy of Sciences, 112(31), 9775- 9780. Kiba, T., Henriques, R., Sakakibara, H., & Chua, N.H. (2007). Targeted degradation of PSEUDO- RESPONSE REGULATOR5 by an SCFZTL complex regulates clock function and photomorphogenesis in Arabidopsis thaliana. The Plant Cell, 19(8), 2516-2530. Más, P., Kim, W.Y., Somers, D.E., & Kay, S.A. (2003). Targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis thaliana. Nature, 426(6966), 567. Somers, D.E., Kim, W.Y., & Geng, R. (2004). The F-box protein ZEITLUPE confers dosage-dependent control on the circadian clock, photomorphogenesis, and flowering time. The Plant Cell, 16(3), 769- 782. Somers, D.E., Schultz, T.F., Milnamow, M., & Kay, S.A. (2000). ZEITLUPE encodes a novel clock- associated PAS protein from Arabidopsis. Cell, 101(3), 319-329.
Yon, F., Joo, Y., Cortés Llorca, L., Rothe, E., Baldwin, I.T., & Kim, S.G. (2016). Silencing Nicotiana attenuata LHY and ZTL alters circadian rhythms in flowers. New Phytologist, 209(3), 1058-1066.
Acknowledgements & Funding This work was funded by the Fundación Séneca 19398/PI/14, BFU 2013-45148-R, BFU 2017 88300- C2-1-R and BFU2017 88300-C2-2-R
114
S-V. Póster 6
DEPENDENCE OF PLANT MOVEMENT ON LIGHT SIGNALLING
María-Victoria Díaz-Galián1, Pedro J. Navarro2, Fernándo Pérez-Sanz1, Julia Weiss1, Marcos Egea-Cortines1
1 Instituto de Biotecnología Vegetal, Edificio I+D+I, Universidad Politécnica de Cartagena Campus Muralla del Mar 30202, Cartagena, (Murcia), 2DSIE, Universidad Politécnica de Cartagena, Plaza del Hospital s/n, Campus Muralla del Mar 30202, Cartagena (Murcia)
Corresponding author: Marcos Egea-Cortines [email protected]
Plant organ movement is considered a surrogate trait linked to circadian regulation. Indeed, leaf movement has been used to identify natural variation in circadian clock genes (Müller et al 2016). However, control of organ movement maybe a combination of light inputs and/or circadian clock driving. Plant organ movement makes the study of organ growth specially challenging due to changes in organ positioning. We analysed organ movement and growth in the trailing species Antirrhinum linkianum versus the erect lab line A. majus 165E (Ruiz-Hernández et al 2017). We found that both species display similar growth rates at neutral photoperiods of 12:12 DL. Movement speed and circadian amplitude of A.linkianum were 2.33 and 1.75 fold higher than in A.majus. They showed similar speeds of movement during the day but during the night A.linkianum increased movement while A.majus decreased. Maximal speed of movement for A.linkianum was close to 2 cm/h while A.majus was below 1 cm/h. We used Petunia x hybrida to assess leaf movement and found that lower leaves show a stronger movement than higher, with peak speed values up to 2.5 cm/h average values of 1-1.5 cm/h. We analysed the effect of light signalling and found that leaves display a high angle related to horizontal during the night and a lower angle during the day. Putting the plants under free running conditions showed that leaves remain in the “day angle” under LL conditions and in the “night angle” during the DD conditions. Recovery of leaf angle back to normal cycling took a single day for DD conditions but took 2 days for LL conditions. Our results show that leaf and stem movement displays a clear diel pattern that is species specific. In the case of Petunia, movement is completely dependent on light signalling. As seen in tomato, wild species (A.linkianum) show highly differing organ movement as compared to laboratory lines (A.majus 165E). The use of organ movement to screen for circadian related changes maybe performed under day:night schemes but not under free running conditions of light or dark.
References Müller et al (2016) Nature Genetics. 48: 89-93 Ruiz-Hernández et al (2017) Frontiers in Plant Sciences. 8: 27-40
Acknowledgements & Funding
This work was funded by the Fundación Séneca 19398/PI/14, BFU 2013-45148-R, BFU 2017 88300- C2-1-R and BFU2017 88300-C2-2-R
115
S-V. Póster 7 A ROLE FOR YUCCA9 IN PLANT DEFENSE
Marta-Marina Pérez Alonso1, Beatriz Sánchez-Parra1, Víctor Carrasco Loba1, M. Estrella Santamaria1, Ronald Pierik2, Julia Kehr3, Francisca Blanco-Herrera4, Isabel Diaz1, Stephan
Pollmann1,*
1 Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus de Montegancedo UPM, Pozuelo de Alarcón
(Madrid), Spain, 2 Plant Ecophysiology, Institute of Environmental Biology, Utrecht University, Utrecht, The Netherlands, 3 Molekulare Pflanzengenetik, Universität Hamburg, Biozentrum Klein Flottbek, Hamburg, Germany,
4 Centro de Biotecnología Vegetal, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile * Corresponding author: Stephan Pollmann, e-mail: [email protected]
Indole-3-acetic acid (IAA), the main naturally occurring auxin in plants, is an essential plant hormone that has been associated with the regulation of many aspects of plant development and growth (Hentrich et al., 2013a; Woodward & Bartel, 2005). Nonetheless, insight into the role of auxin in plant stress responses is just emerging. Recently, Hentrich et al. (2013a) demonstrated that exogenous application of methyl jasmonate (MeJA) or endogenous production of jasmonic acid (JA), a hormone with a prevalent role in plant defense to necrotrophic pathogens and insect herbivores (Studham & MacIntosh, 2012), is sufficient to trigger IAA production through the up-regulation of YUCCA9; a flavin- like monooxygenase gene whose product is involved in the rate-limiting step of the IPyA pathway of auxin biosynthesis (Mashiguchi et al., 2011; Stepanova et al., 2011; Won et al., 2011; Zhao 2010). Using whole-genome transcriptomic analysis of wild-type Col-0 and 35S::YUCCA9 (YUC9ox) Arabidopsis transgenic lines, which exhibit increased IAA levels (Hentrich et al., 2013b), we revealed a group of XYLOGLUCAN ENDOTRANS-GLYCOSYLASE/HYDROLASE (XTH) cell wall-related genes that were up-regulated in the YUC9ox mutant. In addition, our histochemical analyses and quantitative cell wall assays performed in mature stems showed that, in comparison to Col-0, the overexpression of YUC9 leads to stimulated xylem and interfascicular fibers differentiation, as well as increased lignification, which is enriched in H-lignin deposition. These alterations are likely to be triggered by elevated ethylene (ET) levels, as we demonstrated that ET production is significantly increased in the YUC9ox line. On the other hand, the analysis of differential gene expression levels in YUC9ox provided first evidence for the existence of an IAA-dependent negative feedback loop that controls chitin-triggered cellular defenses, and an implication of YUC9 in water deficit responses. Our experiments exposing YUC9 mutants to the two-spotted spider mite Tetranychus urticae and to drought conditions revealed that the overexpression of the YUC9 gene rendered the mutant plants more resistant toward T.urticae and more tolerant to drought. Taken together, the presented findings suggest that the induction of YUC9 gene expression plays a role in the regulation of the complex response to herbivore predators and possibly to water deprivation. References - Hentrich M, Böttcher C, Düchting P, Cheng Y, Zhao Y, Berkowitz O, Masle J, Medina J, Pollmann S. 2013a
The jasmonic acid signaling pathway is linked to auxin homeostasis through the modulation of YUCCA8 and YUCCA9 gene expression. Plant Journal, 74, 626-637. doi: 10.1111/tpj.12152
- Hentrich M, Sánchez-Parra B, Pérez Alonso MM, Carrasco Loba V, Carrillo L, Vicente-Carbajosa J, Medina J, Pollmann S. 2013b. YUCCA8 and YUCCA9 overexpression reveals a link between auxin signaling and lignification through the induction of ethylene biosynthesis. Plant Signaling & Behavior, 8, e26363. doi:10.4161/psb.26363
- Mashiguchi K, Tanaka K, Sakai T, Sugawara S, Kawaide H, Natsume M, Kasahara H. 2011. The main auxin biosynthesis pathway in Arabidopsis. Proceedings of the National Academy of Sciences, 108, 18512–18517. doi:10.1073/pnas.1108434108
- Stepanova AN, Yun J, Robles LM, Novak O, He W, GUO H, Ljung K, Alonso JM. 2011. The Arabidopsis YUCCA1 Flavin Monooxygenase Functions in the Indole-3- Pyruvic Acid Branch of Auxin Biosynthesis. The Plant Cell, 23(11), 3961–3973. doi: 10.1105/tpc.111.088047
- Studham ME & MacIntosh GC. 2012. Phytohormone signaling pathway analysis method for comparing hormone responses in plant-pest interactions. BMC Research Notes, 5, 392. doi:10.1186/1756-0500-5-392
- Won C, Shen X, Mashiguchi K, Zheng Z, Dai X, Cheng Y, Zhao Y. 2011. Conversion of tryptophan to indole-3- acetic acid by TRYPTOPHAN AMINOTRANSFERASES OF ARABIDOPSIS and YUCCAs in Arabidopsis. Proceedings of the National Academy of Sciences, 108(45), 18518–18523. doi:10.1073/pnas.1108436108
- Woodward AW & Bartel B. 2005. Auxin: Regulation, action, and interaction. Annals of Botany, 95, 707–735. doi:10.1093/aob/mci083
- Zhao Y. 2010. Auxin biosynthesis and ist role in plant development. Annual Reviews of Plant Biology, 2(61), 49–64. doi:10.1146/annurev-arplant-042809-112308.
ACKNOWLEDGMENTS This work was supported by MINECO grant BFU2014-55575-R and Marie-Curie grant FP7-PEOPLE-CIG-2011- 303744 to SP. We thank Yunde Zhao for providing the yuc9ko, mutant.
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S-V. Póster 8
USING NATURAL VARIATION TO IDENTIFY EPIGENETICALLY REGULATED GENES INVOLVED IN ROOT DEVELOPMENT
Luis Valledor1, Lara Garcia1, Iris Martínez1, Sara Martín1, María Jesús Cañal1 Mónica
Meijón1
1Plant Physiology, Faculty of Biology, Department of Organisms and Systems Biology,
University of Oviedo, Asturias, Spain
Corresponding author: Mónica Meijón ([email protected])
The root of Arabidopsis thaliana can be used as a model system to study epigenetic
regulation in plants and adaptive processes to changing environmental conditions. In
recent years, genome wide association studies (GWAS) have been positioning in
plant species research as a simple and inexpensive tool allowing to associate
specific genes with certain processes (Meijón et al., 2014), opening the possibility of
studying the epigenetic regulation of root development using quantitative genetics. In
this context, a novel approach combining the use of epigenetic drugs and GWAS was
employed to unmasked novel genes involved in root development. Sixteen root
development traits related to root growth and gravitropism were quantified for 5 days
using a semi-automatic phenotyping platform in 160 different natural accessions,
accounting the worldwide distribution of Arabidopsis considering clinal and soil
conditions. Different epigenetic drugs targeting DNA methylation and Histone
acetylation were employed to unmask new genes implied in root development.
GWAS analysis in these conditions revealed, notably polymorphisms related to CG
islands and presence/lack of deletions of transcription factor binding sequences in the
promoter zone or even in the coding region involved in natural variation observed
both in root growth and gravitropism traits across the accessions. Some of the most
promising candidates identified through these analysis, were: ROOT HAIR SPECIFIC
(RSH), SUN-DOMAIN PROTEIN (SUN), MYB FAMILY TRANSCRIPTION FACTOR
and SHORT ROOT IN SALT MEDIUM (RSA). These candidates are currently being
validated as master regulators of root development by classic and novel approaches,
as well as deepening in the study of their epigenetic regulation not only from a
mechanistic perspective, but also considering the link between epigenetics and
environment, and how epigenetics is related to ecological adaptive processes
(Kawakatsu et al., 2016).
References
Kawakatsu et al., (2016). Cell, 166: 492-505
Meijón et al., (2014). Nature Genetics, 46(1): 77-81
Acknowledgements & Funding
This work was supported by Ramón y Cajal program from Spanish Ministry of Economy and
Competitiveness (RYC-2014-14981) and University of Oviedo (PAPI-17-PEMERG-1).
117
S-V. Póster 9
DELLA PROTEINS CONTROL GENE EXPRESSION BY
INTERACTING WITH HISTONE MONOUBIQUITINATION1 IN
ARABIDOPSIS
Noel Blanco-Touriñán1, David Esteve-Bruna1, Clara Bourbousse2, Miguel de Lucas3,
Fredy Barneche2, Miguel Á. Blázquez1, David Alabadí1.
1 Instituto de Biología Molecular y Celular de Plantas (IBMCP), CSIC-UPV, Valencia, Spain, 2 Institut de Biologie d´Ecole Normale Supérieure (IBENS), CNRS, INSERM, PSL Université
Paris, Paris, France, 3Department of Biosciences, Durham University, Durham, UK.
Corresponding author: David Alabadí ([email protected])
DELLA proteins are transcriptional regulators that in higher plants act as negative
elements in the gibberellin (GA) signalling pathway. The current model for DELLA
action is that they regulate the activity of many transcription factors, either inhibiting
their activity by sequestration away from the chromatin, or by acting as co-regulators
in the context of the target promoters. Interestingly, the identification of an atypical
interactor, the prefoldin complex, suggested that DELLAs might regulate gene
expression through other mechanisms as well (Locascio et al, 2013), since prefoldins
show extensive interactions with proteins involved in various stages of gene
expression in other model organisms.
One of these interactors is the yeast Bre1 that monoubiquitinates H2B (H2Bub), a
critical mark for transcription elongation (Wood et al, 2003). Here, we show that
HISTONE MONOUBIQUITINATION1 (HUB1), a functional homolog of the Bre1,
interacts with DELLAs and prefoldins in Arabidopsis. The similar phenotypes of
dellaKO, pfd and hub1 mutants, for instance in response to stress, suggested to us
that DELLA and prefoldin might promote the activity of HUB1. To test this idea, we
have first compared the transcriptional profiles of seedlings deficient in prefoldin,
HUB1 and DELLA activities. We have found many genes that are similarly
misregulated in all genotypes, suggesting that these proteins might participate in the
same mechanism to regulate these genes. Gene ontology analysis indicated
enrichment in categories related to the response to stimuli, including abiotic and
biotic stresses, which agrees with the decreased salt tolerance manifested by these
mutants. To further test our hypothesis, we are currently studying the contribution of
DELLA and prefoldin to the genome-wide distribution of H2Bub. Our results so far
suggest that DELLA proteins regulate different steps of gene expression.
References: Locascio et al (2013). Curr Biol 23(9):804-9; Wood et al (2003). Mol Cell 11(1):267-74.
Acknowledgements & Funding (Arial, 10 pt, justified): NB-T is supported by a fellowship from MINECO
[BES-2014-068868]. This work was funded by grants BIO2013-43184-P and BIO2016-79133-P from
the Spanish Ministry of Economy and Innovation.
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S-V. Póster 10
INCREASED AMBIENT TEMPERATURE REGULATES FLOWERING
TIME THROUGH CHROMATIN-MEDIATED REGULATION OF
Brassica rapa FT
Iván del Olmo, Laura Poza, Jenifer Pozas, Manuel Piñeiro, José A. Jarillo and Pedro
Crevillén
Centro de Biotecnología y Genómica de Plantas (CBGP), Universidad Politécnica de Madrid
(UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA),
Pozuelo de Alarcón (Madrid), Spain
Corresponding author: Pedro Crevillén ([email protected])
Plants are sessile organisms that synchronize their development with a changing environment to maximize reproductive success. The transition from vegetative to reproductive development is crucial for the proper formation of fruits and seeds. Flowering time is a key agronomic trait since it is essential for crop yield and quality. A complex regulatory network regulates this developmental process and converges in a small number of floral integrator genes. Among them FLOWERING LOCUS T (FT) has a particular relevance because it is part of the so called ‘florigen’ mobile signal. Despite all our knowledge in Arabidopsis, studies about the regulatory pathways of flowering time in Brassica crops are scarce. Brassica rapa is a diploid species that comprises different morphotypes that are cultivated as vegetables, condiments, fodder and oilseeds due to a long history of breeding and selection. To better understand flowering time regulation in B. rapa, we are characterizing this developmental transition in the oilseed B. rapa R-o-18 variety. We found that B. rapa R-o-18 FT expression pattern is consistent with its role as floral promoter and that Br.ft.a-1 loss-of-function mutant plants display extremely late flowering phenotype. These data support that B. rapa FT function is crucial to regulate flowering time in this crop. In addition, we explored the response to high ambient temperature in B. rapa. Contrary to what has been reported in Arabidopsis thaliana, high ambient temperature delays flowering in B. rapa R-o-18, and this late flowering response is associated with reduced FT expression. We will present data regarding the chromatin conformation of B. rapa FT in response to high ambient temperature in relation to results obtained with Arabidopsis laboratory accessions. These results support that Br. rapa FT is a major target of the thermosensory pathway and that different Brassica species use conserved chromatin regulatory mechanisms but in various ways to modulate flowering time. Our results will help to breed new Brassica varieties with different flowering time requirements to cope with global warming.
Acknowledgements & Funding: This work was supported by grants BIO2015-68031-R and RYC-2013- 14689 to PC, and grants BIO2016-77559-R and ERA46-SYBRACLIM to M.P and J.A.J, and BES- 2016-078939 fellowship to LP from the Spanish Ministerio de Economia y Competitividad (MINECO/FEDER, EU). The CBGP is a Severo Ochoa Center of Excellence (SEV-2016-0672).
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S-V. Póster 11
ELUCIDATING PIF7 MOLECULAR MECHANISMS IN THE RESPONSE
OF SEEDLINGS TO PLANT SHADE
Pedro Pastor-Andreu 1, Jordi Moreno-Romero1, Jaime F Martínez-García 1,2
1Center for Research in Agricultural Genomics (CRAG), CSIC- IRTA-UAB-UB, Campus UAB, Bellaterra, Barcelona, Spain, 2Institució Catalana de Recerca i Estudis Avançats (ICREA),
Barcelona, Spain Corresponding author: Pedro Pastor-Andreu ([email protected])
One of the factors that strongly influences plant development is light availability, which can be limited when growing in high vegetation density. Arabidopsis thaliana is a shade avoider species that when facing vegetation proximity initiates a set of responses collectively known as the shade avoidance syndrome (SAS). These responses include the induction of hypocotyls elongation, apical dominance and flowering, and a decrease of leaf expansion and seed yield (Roig-Villanova & Martinez-Garcia, 2016). Plants are able to distinguish between unshaded and shaded conditions through detecting the Red (R) to Far-Red light (FR) ratio (R:FR). When plants grow in low vegetation density (unshaded), the sunlight has a high R:FR; but when growing surrounded by vegetation (shaded), the R:FR ratio decreases.
Because phytochomes exist in two photoconvertible forms, they are in charge of detecting the changes in the R:FR ratio, and then vegetation proximity and shade. Under low R:FR ratio (shaded conditions) the photoequilibrium is displaced towards the inactive form and SAS is induced. This reduces the interaction of phytochromes with various PHYTOCHROME INTERACTING FACTORs (PIFs) (Lorrain et al., 2008), which alters PIF DNA-binding activity and results in rapid changes in the expression of PHYTOCHROME RAPIDLY REGULATED (PAR) genes, postulated to be instrumental in implementing the SAS responses (Leivar et al., 2012). PIFs promote the shade-triggered hypocotyl elongation. From the different PIFs, PIF7 has a dominant role in this process [e.g., pif7 mutant hypocotyls are almost unresponsive to simulated shade, the laboratory conditions that mimic plant proximity, i.e., white light (W) enriched with FR, (W+FR)]. PIF7 contains 2 characteristics domains: a conserved active phytochrome B binding (APB) motif, required for binding to the active form of phytochrome B, and a basic-helix-loop-helix domain that provides DNA-binding activity as dimers (Leivar & Quail, 2011). The aim of this work is to elucidate the molecular action mechanism of PIF7 by transforming A. thaliana pif7-1 plants with wild-type and mutant forms (with altered molecular functions) of PIF7. Our latest results will be presented.
Leivar, Tepperman, Cohn, Monte, Al-Sady, Erickson & Quail PH (2012) Plant Cell 24, 1398–1419 Leivar & Quail (2011) Trends Plant Sci 16, 19–28. Lorrain, Allen, Duek, Whitelam & Fankhauser (2008) Plant J 53, 312-323. Roig-Villanova & Martínez-García (2016) Front Plant Sci 7: 236.
Our research is supported by grants from MINECO-FEDER (BIO2014-59895-P; BIO2015-72093-EXP; BIO2017-85316-R) and AGAUR (2017-SGR1211 and Xarba).
120
121
SESIÓN VI
SEÑALIZACIÓN
MODERADORES:
Andrea Chini (CNB-CSIC,
Madrid)
Salomé Prat (CNB-CSIC,
Madrid)
122
S-VI. Ponencia 1
WHEN CHEMISTRY MEETS BIOLOGY: IDENTIFICATION OF A
NOVEL OPR3-INDEPENDENT PATHWAY FOR JA
BIOSYNTHESIS.
Chini A1, Monte I1, Zamarreño AM2, Hamberg M3, Lassueur S4, Reymond P4, Weiss S5,
Stintzi A5, Schaller A5, García-Mina JM2, Solano R1.
1 Department of Plant Molecular Genetics, National Centre for Biotechnology, Consejo Superior
de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.
2 Environmental Biology Department, University of Navarra, Navarre, Spain.
3 Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics,
Karolinska Institutet, Stockholm, Sweden.
4 Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland.
5 Institute of Plant Physiology and Biotechnology, University of Hohenheim, Stuttgart, Germany.
6 Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Halle, Germany.
Biosynthesis of the phytohormone jasmonoyl-isoleucine (JA-Ile) requires reduction of
the JA precursor 12-oxo-phytodienoic acid (OPDA) by OPDA reductase 3 (OPR3).
Previous analyses of the opr3-1 Arabidopsis mutant suggested an OPDA signaling role
independent of JA-Ile and its receptor COI1; however, this hypothesis has been
challenged because opr3-1 is a conditional allele not completely impaired in JA-Ile
biosynthesis. To clarify the role of OPR3 and OPDA in JA-independent defenses, we
isolated and characterized a loss-of-function opr3-3 allele. Strikingly, opr3-3 plants
remained resistant to necrotrophic pathogens and insect feeding, and activated COI1-
dependent JA-mediated gene expression. Analysis of OPDA derivatives identified 4,5-
didehydro-JA in wounded wild-type and opr3-3 plants. In addition, OPR2 was found to
reduce 4,5-didehydro-JA to JA, explaining the accumulation of JA-Ile and activation of
JA-Ile-responses in opr3-3 mutants. Our results demonstrate that in the absence of
OPR3, OPDA enters the β-oxidation pathway to produce 4,5-ddh-JA as a direct
precursor of JA and JA-Ile, thus identifying an OPR3-independent pathway for JA
biosynthesis.
Work in RS’s lab was funded by the Spanish Ministry for Science and Innovation grant
BIO2016-77216-R (AEI/FEDER, EU)
123
S-VI. Ponencia 2 PIF4-INDUCED BR-SYNTHESIS IS CRITICAL TO DIURNAL AND
THERMOMORPHOGENIC GROWTH
Cristina Martínez1, Ana Espinosa-Ruíz1, Miguel de Lucas1,a, Stella Bernardo-García1, José M. Franco-Zorrilla1 and Salomé Prat1
1 Department of Plant Molecular Genetics, Centro Nacional de Biotecnología-CSIC, Darwin
3, 28049 Madrid, SPAIN, aCurrent address: Department of Biosciences, Durham University, Stockton Road, DH1 3LE Durham, UK
Corresponding author: Salome Prat ([email protected])
Arabidopsis PIF4 and BES1/BZR1 transcription factors antagonize light signaling by facilitating co-activated expression of a large number of cell-wall loosening and auxin-related genes. While PIF4 directly activates these targets, BES1 and BZR1 activity switch from a repressive to an activator function, depending on interaction with TOPLESS (Oh et al., 2014; Espinosa-Ruiz, et al., 2017), and other families of regulators, like BIM1 or PIFs (Yin et al., 2005; Oh et al., 2012). However, combinatorial complexity of this regulation and its role in diurnal control of plant growth and BR levels is little understood. Here, by using protein array hybridization, we show that BES1, PIF4, and the BES1-PIF4 complex recognize different DNA elements, thus revealing a distinctive cis-regulatory code beneath BES1 repressive (BRRE- and G-box) and PIF4 co-activation (PBE-box) function. BES1 homodimers bind to conserved BRRE- and G-box elements in the BR-biosynthetic promoters and inhibit their expression during the day, while elevated PIF4 competes for BES1 homodimer formation, which results in de-repressed expression of these genes at dawn. High ambient temperatures were shown to stabilize PIF4, leading to enhanced auxin biosynthesis and response gene activation (Koini et al., 2009; Stavang et al., 2009; Franklin et al., 2011). We demonstrate that in addition to auxin, PIF4 plays a central role in the control of BR synthesis, induction of BR levels being essential to thermomorphogenic hypocotyl elongation.
References Espinosa-Ruiz A, Martinez C, de Lucas M, Fabregas N, Bosch N, Cano-Delgado AI, Prat S (2017) TOPLESS mediates brassinosteroid control of shoot boundaries and root meristem development in Arabidopsis thaliana. Development 144: 1619-1628 Franklin KA, Lee SH, Patel D, Kumar SV, Spartz AK, Gu C, Ye S, Yu P, Breen G, Cohen JD, Wigge PA, Gray WM (2011) Phytochrome-interacting factor 4 (PIF4) regulates auxin biosynthesis at high temperature. Proc Natl Acad Sci U S A 108: 20231-20235 Koini MA, Alvey L, Allen T, Tilley CA, Harberd NP, Whitelam GC, Franklin KA (2009) High temperature-mediated adaptations in plant architecture require the bHLH transcription factor PIF4. Curr Biol 19: 408-413 Oh E, Zhu JY, Wang ZY (2012) Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nature Cell Biology 14: 802-809 Oh E, Zhu JY, Ryu H, Hwang I, Wang ZY (2014) TOPLESS mediates brassinosteroid-induced transcriptional repression through interaction with BZR1. Nature communications 5: 4140 Stavang JA, Gallego-Bartolome J, Gomez MD, Yoshida S, Asami T, Olsen JE, Garcia-Martinez JL, Alabadi D, Blazquez MA (2009) Hormonal regulation of temperature-induced growth in Arabidopsis. Plant J 60: 589-601
Yin Y, Vafeados D, Tao Y, Yoshida S, Asami T, Chory J (2005) A new class of transcription factors mediates brassinosteroid-regulated gene expression in Arabidopsis. Cell 120: 249-259
Acknowledgements & Funding
This work was supported by grant BIO2014- 60064-R from the Spanish Ministerio de Economía y
Competitividad.
124
S-VI. Comunicación 1
NITRIC OXIDE REGULATES MAINTENANCE OF THE ROOT APICAL
MERISTEM BY CONFINING WOX5 EXPRESSION TO THE
QUIESCENT CENTER
Tamara Lechón1, Noel Blanco-Touriñán2, Virginia Palomares1, Miguel A. Blázquez2,
Walter Dewitte3, Ivett Bárány4, Miguel A. Moreno-Risueño5, Mari C. Risueño4, Pilar S.
Testillano4, Óscar Lorenzo1, Luis Sanz1. 1Spanish-Portuguese Agricultural Research Institute (CIALE), University of Salamanca (USAL), Salamanca,
Spain, 2Institute for Plant Molecular and Cell Biology (IBMCP), Polytechnic University of Valencia (UPV),
Valencia, Spain, 3School of Biosciences-James Murray Lab, Cardiff University, Cardiff, United Kingdom, 4Centre
for Biological Research (CIB), Spanish National Research Council (CSIC), Madrid, Spain, 5Centre for Plant
Biotechnology and Genomics (CBGP), Polytechnic University of Madrid (UPM), Madrid, Spain.
Corresponding authors: Oscar Lorenzo ([email protected]), Luis Sanz ([email protected])
Development of the Arabidopsis root is a dynamic process that involves the
integration of different signals to regulate complex gene networks. Our recent
research has uncovered a role for nitric oxide (NO) on primary root growth
(Fernández-Marcos et al., 2011). NO accumulates in cortex/endodermis stem cells
and their immediate progeny, generating endodermal and cortical tissues, suggesting
that NO could play an important role in regulating stem cell decisions, as has been
reported in animals (Sanz et al., 2014).
PROHIBITIN3 (PHB3), a protein with wide implications in plant development, has
been reported to have a role in NO homeostasis during oxidative stress (Wang et al.,
2010) and is highly expressed in the root apical meristem (Van Aken et al., 2007;
Kong et al., 2018). We have observed that loss-of-function mutant atphb3 presents
defects in root development, decreased meristematic cell production and loss of root
cell identity, including ectopic expression of WUSCHEL-related homeobox5 (WOX5),
an inhibitor of cell differentiation and master regulator of stem cell activity, which
ultimately results in the concomitant displacement of the meristem. We demonstrate
that the activity of PHB3 is necessary for maintaining WOX5 expression confined to
the quiescent center of the root apical meristem during post-embryonic development
through a NO-dependent mechanism.
Fernández-Marcos et al. (2011) Proc Natl Acad Sci USA, 108, 18506-11.
Kong et al. (2018) Cell Rep, 22(5), 1350-63.
Sanz et al. (2014) Plant Phys, 166(4), 1972-84.
Van Aken et al. (2007) Plant J, 52(5): 850-64.
Wang et al. (2010) Plant Cell, 22(1): 249-59.
This work is financed by grants: ERC.KBBE.2012.1.1-01 (EcoSeed-311840). MINECO: (BIO2017-
85758-R), CONSOLIDER (CSD2007-00057). Junta de Castilla y León (SA093U16). Fundación
Solórzano (FS/12-2017). TL is supported by a fellowship from MEC (FPU13/05569).
125
S-VI. Comunicación 2 ORIGIN OF THE GIBBERELLIN-DEPENDENT TRANSCRIPTIONAL
REGULATION BY MOLECULAR EXPLOITATION OF A
TRANSACTIVATION MOTIF IN DELLA PROTEINS
Jorge Hernández-García1, Asier Briones-Moreno1, Renaud Dumas2, Miguel A
Blázquez1
1 Instituto de Biología Molecular y Celular de Plantas (IBMCP, CSIC-UPV), Valencia, Spain,
2CNRS, LPCV / IRTSV, Grenoble, France.
Corresponding author: Miguel A Blázquez ([email protected])
DELLA proteins are land-plant specific transcriptional regulators known to interact
through their C-terminal GRAS domain with over 150 transcription factors in
Arabidopsis thaliana. Besides, DELLAs from vascular plants can interact through the
N-terminal domain with the gibberellin receptor encoded by GID1, through which
gibberellins promote DELLA degradation. However, this regulation is absent in non-
vascular land plants, which lack active gibberellins or a proper GID1 receptor, but the
features needed for DELLA interaction with the receptor were already present in the
ancestor of all land plants. Current knowledge indicates that DELLAs are important
pieces of the signalling machinery of vascular plants, especially angiosperms, but
nothing is known about DELLA function during early land plant evolution or if they
exist at all in charophytan algae. We have now elucidated the evolutionary origin of
DELLA proteins, showing that algal GRAS proteins are monophyletic and evolved
independently to those of land plants, which explains why there are no DELLAs
outside land plants. DELLA genes have been maintained throughout land plant
evolution with only two major duplication events kept among plants. Furthermore, we
propose that the DELLA N-terminal motifs needed for GID1 interaction are highly
conserved in non-vascular land plants for their function in transcriptional co-
activation, and this allowed subsequent co-option and molecular exploitation for the
interaction with the GID1 receptor when vascular plants developed gibberellin
synthesis and the corresponding perception module.
Work in the laboratory was funded by grants BFU2016-80621-P of the Spanish Ministry of Economy,
Industry and Competitiveness, and H2020-MSCA-RISE-2014-644435 of the European Union. JH-G
and AB-M hold Fellowships of the Spanish Ministry of Education, Culture and Sport FPU15/01756 and
FPU14/01941, respectively.
126
S-VI. Comunicación 3
IS S-CYANYLATION A NOVEL PROTEIN POST-TRANSLATIONAL
MODIFICATION?
Irene García, Lucía Arenas-Alfonseca, Inmaculada Moreno, Cecilia Gotor and Luis
C. Romero.
Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-Universidad de Sevilla, Sevilla, Spain
Corresponding author: Irene García ([email protected]) Beyond its recognized toxic capacity, cyanide has a regulatory function in several
biological processes in the different organisms producing it. In Arabidopsis thaliana,
cyanide modulates germination, the morphogenesis of the root hair and the immune
response, suggesting that cyanide should act as a signal molecule in these biological
processes (Siegien and Bogatek, 2006; García et al., 2010, 2013; Arenas-Alfonseca
et al., 2018). However, the mechanisms underlying it are unknown. Cyanide shares
some characteristics with other signaling molecules of the gasotransmitter type as
nitric oxide or hydrogen sulfide, such as its low molecular weight and its toxicity at
moderate concentrations. The action mechanisms of these gasotransmitters include
the post-translational modification of cysteine (Cys) (Aroca et al., 2015, 2017;
Yamasaki et al., 2016). We have deepened into the function of cyanide and the
enzyme ß-cyanoalanine synthase, which regulates its accumulation or detoxification,
in the physiology of the cell through a proteomic approach. In addition, we have
analyzed the molecular mechanism by which the cyanide molecule can act as a
signaling molecule and considered the reaction of cyanide ions with disulfide bridges
within polypeptides originating S-cyanylated Cys. By using two different
physicochemical approaches, the 2-imino-thiazolidine method and direct untargeted
analysis of proteins by LC-MS/MS, we have identified that this modification occurs
under physiological growth condition in leaf and root tissues. Protein activity
measurements have been conducted in order to determine the biochemical effect of
this novel protein modification.
References: Arenas-Alfonseca et al. (2018) Plant Cell Physiol. in press; Aroca et al (2015) Plant
Physiol. 168:334-42; Aroca et al. (2017) J Exp Bot 68:4915-4927; García et al. (2010) Plant Cell
22:3268-3279; García et al. (2013) Plant Physiol 162:2015-27; Siegien and Bogatek (2006) Acta
Physiologiae Plantarum 28:483-497; Yamasaki et al. (2016) Methods Mol Biol 1424,1-14.
Acknowledgements & Funding: We acknowledge the European Regional Development Fund,
Ministerio de Economía y Competitividad and the Agencia Estatal de Investigación (grants BIO2013-
44648-P and BIO2016-76633-P) for funding.
127
S-VI. Póster 1
DISSECTING THE FUNCTION OF GENES ENCODING YTH
PROTEINS IN Arabidopsis thaliana
Ainara Pérez-Morón, Eva Rodríguez-Alcocer, Natalia Gómez-Peral, Erundina Ruiz,
Carlos Hernández-Cortés, Sara Jover-Gil and Héctor Candela.
Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche,
Spain
Corresponding author: Héctor Candela ([email protected])
The most prevalent internal post-transcriptional modification in eukaryotic
messenger RNAs is N6-methyladenosine (m6A). This modification functions in a wide
range of different biological processes. Recent studies have shown that m6A reader
proteins contain an RNA-binding domain, the YTH domain, that physically interacts
with m6A residues in the mRNA. The genome of Arabidopsis thaliana has previously
been reported to contain as many as thirteen RNA binding proteins containing a YTH
domain, but their functions remain mostly unknown. In line with the research interests
of our group, we have undertaken a systematic approach to investigate the specific
functions performed by individual members of this small family of RNA-binding
proteins. We are characterizing transgenic lines carrying T-DNA insertions residing
within or near the coding regions of these genes. Our preliminary results show that
most of the examined lines lack a mutant phenotype, suggesting extensive functional
redundancy among the members of the family. To overcome this problem, we have
initiated the isolation of double and triple mutants involving loss-of-function alleles of
the phylogenetically closest genes. In addition to this, we have prepared Gateway-
compatible constructs to investigate the consequences of overexpressing the YTH
genes of Arabidopsis thaliana and their normal expression patterns. Furthermore, we
have also performed several yeast two-hybrid screens using YTH proteins as baits.
128
S-VI. Póster 2
UNCOVERING THE ROLE OF PIFS IN THE REGULATION OF ABA-
MEDIATED RESPONSES
Arnau Rovira1, Gianfranco Diretto2, Pablo Leivar3, Elena Monte1*
1Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra, Catalonia,
Spain
2Italian National Agency for New Technologies, Energy and Sustainable Development
(ENEA), Roma, Italy
3Bioengineering Department, IQS School of Engineering, Barcelona, Catalonia, Spain
Corresponding author: Elena Monte ([email protected])
Phytochrome-interacting factors (PIFs) are members of the Arabidopsis thaliana bHLH family
of transcriptional regulators that interact specifically with the active Pfr conformer of
phytochrome (phy) photoreceptors. PIFs induce photomorphogenic development and are
involved in regulation of different developmental processes under diurnal conditions in short-
day (SD). We know that members of the PIF quarte (PIFq; PIF1, PIF3, PIF4, and PIF5)
collectively contribute to the induction of growth in Arabidopsis seedlings under SD,
specifically promoting elongation at dawn. In our group, we recently published a genomic
analysis defining SD-regulated genes at dawn followed by identification of those SD-
regulated genes whose expression depended on the presence of PIFq. Interestingly,
promoter analysis showed that most PIF/SD-repressed genes were indirectly regulated by
the PIFs and might be more enriched in ABA-regulated genes. Recently, significant progress
has been made in defining the PIF-regulated transcriptional network, showing their
interaction with a number of hormone-related genes. However, the interaction of PIF-
repressed genes and ABA is still unclear. We will present recent progress aimed to unravel
the relevance of this interaction.
This project is supported by the Spanish Ministerio de Economía y Competitividad (BIO2015-68460-P to E.M.) and the Generalitat de Catalunya (2014-SGR-1406 to E.M.). We acknowledge financial support by the CERCA Programme/Generalitat de Catalunya and from Ministerio de Economía y Competitividad through the Severo Ochoa Programme for Centers of Excellence in R&D 2016-2019
(SEV-2015-0533).
129
S-VI. Póster 3
PREFOLDIN MODULATES CHROMATIN REMODELLING THROUGH
THE INTERACTION WITH THE SWR1 COMPLEX IN ARABIDOPSIS
Cristina Marí-Carmona1, David Esteve-Bruna1, Noel Blanco-Touriñán1,
David Alabadí1, Miguel Á. Blázquez1
1 Instituto de Biología Molecular y Celular de Plantas (IBMCP), CSIC-UPV, Valencia, Spain,
Corresponding authors: Miguel A. Blázquez ([email protected])
David Alabadí ([email protected])
PREFOLDIN (PFD) is an evolutionarily conserved heterohexameric chaperonin that
delivers unfolded tubulin to the main cytosolic chaperone CCT. The observation that
PFD can accumulate in the nucleus in a DELLA-dependent manner (Locascio et al.,
2013) sparked the possibility that PFD might have additional nuclear functions. In
fact, several nuclear interactors of the six PFD subunits were components of the
SWR1 complex that exchanges H2A with the histone variant H2A.Z, thereby
regulating gene expression (March-Díaz and Reyes, 2009).
We have now experimentally confirmed in planta that at least PFD6 interacts
with ARP6SWR1, and comparative transcriptomic analysis indicates a strong positive
correlation between PFD and SWR1 activity on the regulation of gene expression,
with 20% of the genes misregulated in pfd6, also differentially expressed in the
swc6SWR1 and arp6 SWR1 mutants. These defects also correlate with a genome-wide
alteration of H2A.Z deposition in the pfd6 mutant. Moreover, all pfd mutants tested
displayed early flowering even at lower temperatures, as described for loss of SWR1
function, and this effect is associated with changes in FLC expression and in H2A.Z
distribution at the FLC locus. We propose that PFD is a positive regulatory
component of the SWR1 complex.
Locascio, A., et al. (2013) Curr Biol 23: 804-809. March-Diaz, R. and J. C. Reyes (2009) Mol Plant 2: 565-577.
Acknowledgements: CM-C and NB-T are supported by fellowships from the Universidad Politécnica
de Valencia and from MINECO [BES-2014-068868], respectively. This work was funded by grant
BIO2013-43184-P of the Spanish Ministry of Economy and Innovation.
130
S-VI. Póster 4
STUDIES OF AtDWARF14, THE STRIGOLACTONE RECEPTOR
Elena Sánchez1, Aitor Muñoz1 and Pilar Cubas1
1Plant Molecular Genetics Department, Centro Nacional de Biotecnología-Consejo Superior
de Investigaciones Científicas, Madrid, Spain
Corresponding author: Elena Sánchez ([email protected])
Strigolactones (SL) are recently discovered phytohormones involved in shoot
branching inhibition. Its perception and signalling is mediated by DWARF14 (D14),
which acts as the SL receptor, the F-Box protein MORE AXILLARY GROWTH2
(MAX2) and the repressors of axillary bud dormancy SUPRESSOR OF MORE
AXILLARY GROWTH (SMXL) (Nakamura et al., 2013; Yao et al., 2016; Waters et al.,
2012; Liang et al., 2016; Wang et al., 2015; Soundappan et al., 2015). After SL
perception, the AtD14-SCFMAX2 complex recognises SMXL6,7,8 and targets them for
proteasomal degradation, which activate SL responses.
In our lab, we identified a D14 degradation mechanism in the presence of SL
(Chevalier et al., 2014). D14 degradation is slower than that of the SMXL, needs
MAX2 and it´s mediated by the proteasome. This negative feedback loop limits SL
perception, and may be mediated by the same SCFMAX2 involved in SL signalling.
We are currently studying in more detail DWARF14 degradation mechanism. Does
D14 degradation require interaction with the SMXL-SCFMAX2 complex? Does it
require SL binding and hydrolysis? And finally, what is the biological significance of
D14 degradation? To answer these question we´re using a collection of AtD14
mutant proteins that may be impaired in the SL binding, interaction with the repressor
proteins or D14 ubiquitination. Moreover, we have generated 35S:AtD14:GFP and
UBp:AtD14:LUC Arabidopsis transgenic lines and we are using them to carry out in
vivo studies of D14 degradation dynamic.
References:
· Chevalier, F., Nieminen, K., Sánchez-Ferrero, J., et al. (2014). Plant Cell 26: 1134–50.
. Liang, Y., Ward, S., Li, P., et al. (2016). Plant Cell 28: 1581–601.
· Nakamura, H., Xue, Y.-L., Miyakawa, T., et al. (2013). Nat. Commun. 4: 2613.
· Soundappan, I., Bennett, T., Morffy, N., et al. (2015).. Plant Cell 27: 3143–59.
· Wang, L., Wang, B., Jiang, L., et al. (2015). Plant Cell 27: 3128–42.
· Waters, M.T., Nelson, D.C., Scaffidi, A., et al. (2012).
. Yao, R., Ming, Z., Yan, L., et al. (2016). Nature 536: 469–473.
131
S-VI. Póster 5
STUDYING THE FUNCTION OF GENES INVOLVED IN ADENOSINE
METHYLATION IN THE ARABIDOPSIS TRANSCRIPTOME
Eva Rodríguez-Alcocer, Natalia Gómez-Peral, Erundina Ruiz, Ainara Pérez-Morón,
Carlos Hernández-Cortés, Eduardo Burillo, Alejandro Peñín, Sara Jover-Gil and
Héctor Candela.
Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche,
Spain
Corresponding author: Héctor Candela ([email protected])
The reversible methylation of adenosine residues is the most abundant
internal modification in the messenger RNA of eukaryotes. Recent work has shown
that N6-methyladenosine (m6A) plays important roles in the regulation of the
transcription, translation and stability of cellular transcripts. To further our
understanding of this post-transcriptional modification, we are systematically
characterizing the functions of genes that encode m6A writers (subunits of the
methyltransferase complex), erasers (enzymes with putative m6A demethylase
activity) and readers (proteins containing a YTH RNA-binding domain). Using the
combinatorial power of the Gateway cloning technology, we have generated a large
collection of constructs that should help us to address questions about the
consequences of experimentally increasing or reducing the levels of m6A in the
Arabidopsis transcriptome. To investigate the function of these genes, we are
studying single, double and higher-order mutants that carry loss-of-function alleles,
as well as transgenic plants that carry constructs aimed at overexpressing their
coding sequences. We are also systematically performing yeast two-hybrid screens
using eraser and reader proteins as baits, which have yielded some promising
protein-protein interactions. We are establishing an in vivo RNA tagging protocol that
we plan to apply to the high-throughput identification of mRNA molecules targeted by
proteins from the three functional categories. Our long-term objective is to make a
significant contribution to this new field by identifying the protein-protein and RNA-
protein interactions that shape the m6A epitranscriptome in Arabidopsis thaliana.
This work has been funded by MINECO and European Regional Development Fund (“Una manera de
hacer Europa”; BFU2012-31719) and Generalitat Valenciana (ACOMP/2015/042) grants to H.C. The
contract of E.R.-A. was supported by a MINECO grant (PEJ-2014-A-21398).
132
S-VI. Póster 6
PERIANTHIA (PAN) IS A MASTER REGULATOR BY NITRIC
OXIDE OF ROOT STEM CELL NICHE
María Guadalupe Fernández-Espinosaa, María Angels de Luis Balaguerb, Adam
P. Fisherb, Rosangela Sozzanib and Oscar Lorenzoa.
aDepartment of Botany and Plant Physiology, Spanish-Portuguese Agricultural
Research Institute (CIALE), School of Biology, University of Salamanca, 37185
Salamanca, Spain. bPlant and Microbial Biology Department, North Carolina State
University, Raleigh, NC 27695.
The stem cell niche (SCN) has critical roles in apical meristems and specially
for the proper plant root system development. Unraveling genetic
regulatory networks of SCN specific cells is a crucial goal to understand the root
organ growth. In our previous work, different stem cell populations have been
transcriptionally profiled to develop a gene regulatory network (GRN) inference
algorithm (GENIST), which combines spatial and temporal transcriptomic
datasets, to identify relevant stem cell-related transcription factors (TFs) and
infer interactions between them1. In this study, PERIANTHIA (PAN), a member
of the TGA group of bZIP (basic region/leucine zipper) TFs has been identified
as an important molecular regulator of stem cell maintenance and quiescent
center function1.
Our previous research highlighted an important role of NO in the regulation of
root stem cell decisions2, 3. Here, we show the involvement of PAN in root
development and stem cell maintenance and their link with NO. To investigate
the mechanism by which NO modulates these processes, we first evaluated the
phenotypes of loss- and gain-of-function PAN lines in the presence and
absence of NO. Secondly, in silico analysis to predict S-nitrosylation sites and in
vitro analysis of specific Cys residues after GSNO treatment highlighted PAN is
S-nitrosylated. Finally, we carried on studying in vivo protein modifications and
possible PAN-regulated targets.
References 1 de Luis Balaguer MA, et al (2017) PNAS, 114: E7632. 2 Fernández-Marcos, et al (2011) PNAS, 108: 18506. 3 Sanz, et al. (2014) Plant Physiology, 166: 1972.
Acknowledgements
ERC.KBBE.2012.1.1-01 (EcoSeed-311840), MINECO (BIO2014-57107-R), CONSOLIDER
(CSD2007-00057), Junta de Castilla y León (SA093U16). M.G. F-E is supported by a JCyL and
Fondo Social Europeo grant.
133
S-VI. Póster 7 EMBRYO FATTY ACID STORAGE REGULATION BY bZIP
REVERSIBLE S-NITROSYLATION
Inmaculada Sánchez Vicente1, Pablo Albertos2, Isabel Mateos1, Carlos Sanz3, Brecht Wybouw4, 5, Bert De Rybel4, 5, Juan C. Begara-Morales6, Mounira Chaki6,
Juan B. Barroso6 and Oscar Lorenzo1
1Dept. of Botany and Plant Physiology, Instituto Hispano-Luso de Investigaciones Agrarias (CIALE), Universidad de Salamanca. Salamanca, Spain
2Biotechnology of Horticultural Crops, TU München, Freising, Germany 3Department Biochemistry and Molecular Biology of Plant Products, Instituto de la Grasa-CSIC,
Campus Universidad Pablo de Olavide, Sevilla, Spain 4Dept. of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052
Ghent, Belgium 5VIB Center for Plant Systems Biology, Technologiepark 927, 9052 Ghent, Belgium
6Dept. of Experimental Biology, Facultad de Ciencias Experimentales, Campus Universitario “Las Lagunillas” s/n, Universidad de Jaén, E-23071 Jaén, Spain
Corresponding author: Oscar Lorenzo ([email protected]) Nitric oxide (NO) is a key gasotransmitter involved in a plethora of physiological events along the whole plant life, controlling not only growth and developmental processes (Albertos et al., 2016, Sanz et al., 2015), but also biotic and abiotic stresses. We analyze the NO signalling mechanisms involved in the regulation of early plant development, including seed maturation, dormancy and germination where some members of the basic region/leucine zipper motif (bZIP) transcription factor family constitute important players. NO exerts its function mainly through S-nitrosylation, defined as the specific posttranslational modification by which NO is attached covalently to a cysteine residue, modifying the protein properties. Recently, S- nitrosylation has been deciphered as a reversible modification by an enzymatic process called denitrosylation (Kneeshaw et al., 2014).Our results show NO functions during embryo fatty acid storage regulation through the specific and reversible S-nitrosylation of bZIP transcription factor bZIP67, taking part in seed fatty acid accumulation and promotion of germination. bZIP67 is the closest homolog of ABI5, previously characterized as a key seed germination repressor, also modified by S-nitrosylation (Albertos et al., 2015). Likewise, this posttranslational modification can also be reversed by the trans-denitrosylation activity of redoxins, suggesting a novel mechanism of bZIP regulation. In addition, bZIP67 can be modified by nitro-fatty acids, recently discovered as potential signalling molecules (Mata-Pérez et al., 2016). These results led us to understand a molecular framework for NO control of seed fatty acid
profile during maturation and germination.
Albertos, P., Romero-Puertas, M. C., Tatematsu, K., Mateos, I., Sanchez-Vicente, I., Nambara, E., and Lorenzo O. (2015). S-nitrosylation triggers ABI5 degradation to promote seed germination and seedling growth. Nat. Commun. 6, 8669.
Albertos, P., Sanz, L., Mateos, I., Sánchez-Vicente, I., Lechón, T., Fernández-Espinosa, G., Rodríguez, D., and Lorenzo O. (2016). Gasotransmission of nitric oxide (NO) at early plant developmental
stages. Ed. Lamattina, L. and C. García-Mata. Springer. 978-3-319-40713-5. Kneeshaw, S., Gelineau, S., Tada, Y., Loake, G. J., and Spoel, S. H. (2014). Selective protein
denitrosylation activity of thioredoxin-h5 modulates plant immunity. Mol. Cell 56, 153-162. Mata-Perez, C., Sanchez-Calvo, B., Padilla, M. N., Begara-Morales, J. C., Luque, F., Melguizo, M.,
Jimenez-Ruiz J., Fierro-Risco, J., Peñas-Sanjuán, A., Valderrama, R. et al. (2016b). Nitro-fatty acids in plant signaling: nitro-linolenic acid induces the molecular chaperone network in Arabidopsis. Plant Physiol. 170, 686-701.
Sanz, L., Albertos, P., Mateos, I., Sanchez-Vicente, I., Lechón, T., Fernández-Marcos, M., and Lorenzo, O.
(2015). Nitric oxide (NO) and phytohormones crosstalk during early plant development. J. Exp. Bot. 66, 2857-2868.
This work is financed by grants: ERC.KBBE.2012.1.1-01 (EcoSeed-311840). MINECO: (BIO2017-85758- R), CONSOLIDER (CSD2007-00057). Junta de Castilla y León (SA093U16). Fundación Solórzano (FS/26-2017).
134
S-VI. Póster 8
ANAC089 IS A NITRIC OXIDE MASTER REGULATOR DURING
SEED GERMINATION AND STRESSES IN ARABIDOPSIS
Isabel Mateos1, Pablo Albertos1, Kiyoshi Tatematsu2, Alejandro Fernández-
Arbaizar1, Kazumi Nakabayashi3, Eiji Nambara4, Marta Godoy5, José M.
Franco5, Roberto Solano5, Carlos Perea6, Julio Salinas6, Thomas Roach7,
Erwann Arc7, Ilse Kranner7 and Oscar Lorenzo1
1Dpto. de Botánica y Fisiología Vegetal, CIALE, Universidad de Salamanca,
Salamanca, Spain, 2Laboratory of Plant Organ Development, National Institute for
Basic Biology, Okazaki, Japan, 3School of Biological Sciences, University of London,
Egham, United Kingdom, 4Department of Cell & Systems Biology, University of
Toronto, Toronto, Canada, 5Dpto. de Genética Molecular de Plantas. CNB-CSIC,
Madrid, Spain, 6Dpto. de Biología Medioambiental. CIB-CSIC, Madrid. Spain.7 Institut
für Botanik, Innsbruck, Austria)
Corresponding author: Oscar Lorenzo ([email protected])
Seed dormancy and germination are complex traits regulated by the interaction
of different signalling molecules such as abscisic acid (ABA) and nitric oxide
(NO). To elucidate this process, a genetic screening in presence of (+)-S-ABA
coupled to NO scavenger (cPTIO) was performed. In this screening we
identified gap1 mutants (gap1-1 and gap1-2) that show ABA and cPTIO-
insensitive phenotypes in the transition from dormancy to germination. These
mutants lack the transmembrane domain of ANAC089 protein and this delection
confers the protein constitutive nuclear localization. The activity of ANAC089 is
regulated by mono-ubiquitination and this modification probably could regulate
its traffic to the nucleus. Furthermore, mutants exhibited higher endogenous NO
levels avoiding the effect of NO-depletion during seed germination. In addition,
whole-genome transcriptional profiling indicates that different groups of ABA-
and redox-related genes are differentially regulated by ANAC089. According to
this idea, mutants present GSH/GSSG levels altered. This transcription factor
can specifically bind to the core cis-regulatory element GCGTCAGC harbour in
the promoters of ANAC089 regulated genes. Consistently, translocation of
ANAC089 protein to the nucleus was directed by changes in cell redox status
after NO- and redox-related compound treatments. Collectively, our results
indicate that ANAC089 transcription factor integrates ABA signalling with NO
levels to modulate redox homeostasis as a novel master regulator during seed
germination and stresses in Arabidopsis.
Acknowledgment: ERC.KBBE.2012.1.1-01 (EcoSeed-311840), Fundación memoria de Don
Manuel Solorzano Barruso (FS/12-2017), Junta de Castilla y León (SA093U16), MINECO
(BIO2017-85758-R).
135
S-VI. Póster 9
EXOGENOUS STRIGOLACTONE APPLICATION AFFECTS
TRANSCRIPTIONAL AND METABOLIC LEVELS IN TOMATO
Jordi Gamir1, Rocio Torres-Vera1, Estefanía Berrio1, María J. Pozo1, Juan A.
López-Ráez1.
1Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del
Zaidín (EEZ-CSIC), Granada, Spain
Corresponding author: Juan A. López-Ráez ([email protected])
Strigolactones (SLs) are the latest plant hormones described that act as modulators of plant responses under nutrient deficient conditions, mainly phosphorous (P) and nitrogen, and other abiotic stresses such as drought and salinity (Albabili and Bouwmeester 2015; Andreo-Jiménez et al., 2015). In addition to their role as phytohormones, they are important ex-planta signalling molecules in the rhizosphere, promoting the association with beneficial microorganisms, but also with harmful parasitic plants (López-Raez et al., 2017). In the present study, we explore the plant response to exogenous SL application under both normal and limiting P conditions. For that, a pulse (10 nM for 1 h) of the synthetic SL analogue GR24 was applied to tomato plants grown hydroponically, and its effects evaluated locally on roots and systemically on shoots. Strikingly, GR24 application triggered a huge transcriptional and metabolic reprograming in plants grown under normal P conditions, leading to profiles similar to those in plants grown under P- limitation. Therefore, the pulse of GR24 mimicked, to some extent, the effect of P starvation in plants grown under normal P conditions. Moreover it enhanced the response in P-starved plants, suggesting that SLs might act as early signals upon sensing the limitation of this nutrient in the soil. Their interaction with other phytohormones as jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA) was also assessed. The results will be discussed and future prospects proposed to get further insights on the role of SLs as modulators of plant responses to P deprivation.
References
- Al-Babili, S. and Bouwmeester, H.J. Strigolactones, a novel carotenoid-derived plant hormone. Ann Rev Plant Biol, 2015, 161-186.
- Andreo-Jiménez, B. et al. Ecological relevance of strigolactones in nutrient uptake and other abiotic stresses, and in plant-microbe interactions below-ground. Plant Soil 2015, 394: 1-19.
- López-Ráez, J.A. et al. Strigolactones in plant interactions with beneficial and detrimental organisms: the yin and yang (2017). Trends Plant Sci, 2017, 22: 527-537.
Acknowledgements & Funding
Research carried out by the authors is supported by the grants AGL2015-64990-C2-1-R from the National R&D Plan of the Ministry of Economy and Competitiveness (MINECO).
136
S-VI. Póster 10 MODULATION OF S-NITROSOTHIOL LEVELS
BY NITRO-FATTY ACIDS IN ARABIDOPSIS PLANTS
Mounira Chaki, Juan Carlos Begara, Raquel Valderrama, Capilla Mata-Pérez,
Beatriz Sánchez-Calvo, María N. Padilla, Lorena Aranda-Caño, Juan B. Barroso
Group of Biochemistry and Cell Signalling in Nitric Oxide, Department of Experimental Biology, Center for Advanced Studies in Olive Grove and Olive Oils, Faculty of Experimental Sciences, University of Jaén, Jaén, Spain.
Corresponding author: Juan Bautista Barroso Albarracín ([email protected])
S-nitrosothiols (SNOs) result from the reaction of nitric oxide (NO) with thiol groups present in the cysteine residues of proteins. SNOs can act as intracellular NO reservoir and vehicle. These molecules are modulated in response to the intracellular redox status, conferring them the capacity to act as molecular cues involved in signalling processes (Begara-Morales et al., 2018). On the other hand, the reaction between NO and unsaturated fatty acids generates nitro-fatty acids (NO2-FAs), which are more stable and have a longer half-life than other NO-derived molecules. Recently, it was reported the endogenous occurrence of nitro-linolenic acid (NO2-Ln), a type of nitrated fatty acid in Arabidopsis plants and it was demonstrated its signalling role in plant defence against diverse abiotic stress conditions (Mata-Pérez et al., 2016a, 2016b, 2017, 2018). Furthermore, it was reported its ability to modulate the generation of NO both in vitro and in vivo (Mata-Pérez et al., 2016b; Padilla et al., 2017). However, the mechanisms by which this NO2-FA is able to induce these responses remain poorly understood in plant systems. To assess the capacity of NO2-Ln to generate SNOs, wild type Arabidopsis plants were treated with 100 µM NO2-Ln and an increase of SNOs by ozone chemiluminiscence was detected. To get more information about this mechanism, overexpressing- GSNOR Arabidopsis mutant that exhibits low levels of SNOs and high GSNOR activity were used. Interestingly, the treatment of this mutant with NO2-Ln increased SNO levels and decreased GSNOR activity. Additionally, the Arabidopsis recombinant GSNOR activity was reduced after the incubation with NO2-Ln and restored by a NO scavenger (cPTIO). Moreover, the recombinant GSNOR was S-nitrosylated by this nitro fatty acid. Therefore, these findings suggest the capacity of NO2-Ln to modulate the SNO levels by inhibiting GSNOR activity.
References
Begara-Morales et al., 2018. J Exp Bot doi: 10.1093/jxb/ery072. Mata-Pérez et al., 2016a. Plant
Physiology, 170: 686 –701. Mata-Pérez et al., 2016b. Nitric Oxide. 57:57-63.
Mata-Pérez et al., 2017. Redox Biol. 11:554-561.
Mata-Pérez et al., 2018. Nitric Oxide. doi: 10.1016/j.niox.2018.03.011. Padilla et al., 2017. Nitric
Oxide. 68: 14 –22.
Acknowledgements & Funding
This work was supported by an ERDF grant co-financed by the Ministry of Economy and
Competitiveness (project BIO2015-66390-P) and the Junta de Andalucía (group BIO286) in
Spain.
137
S-VI. Póster 11
INSIGHTS INTO LIGHT AND ABA-GA RESPONSES DURING
SEED GERMINATION.
Gerardo Carrera-Castaño1, Rocío Sánchez-Montesino1, Christoph Weiste2,
Wolfgang Dröge-Laser2, Luis Gómez1, Luis Oñate-Sánchez1
1Centro de Biotecnología y Genómica de Plantas, (Universidad Politécnica de Madrid – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria), Campus de
Montegancedo, Pozuelo de Alarcón, 28223, Madrid, Spain. 2Julius-von-Sachs-Institut, Pharmazeutische Biologie, Julius-Maximilians-Universität
Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany. Corresponding author: Luis Oñate-Sánchez ([email protected])
Seed germination is a crucial process in spermatophytes with an impact on crop establishment and yield. This process depends on hormonal regulation, being abscisic acid (ABA) and gibberellins (GAs) key regulators playing antagonistic roles. Several environmental factors may compromise seed germination and plant growth, mainly through the regulation of ABA-GA metabolism and signalling pathways (Colebrook et al., 2014). By screening a seed library (Weiste et al., 2007) we have identified Arabidopsis lines constitutively overexpressing the single transcription factor LID1 (light-in-darkness1) with improved germination under conditions known to decrease GA biosynthesis (paclobutrazol; PAC). Whereas this phenotype is compatible with enhanced GA levels/signalling or reduced ABA sensitivity, the overexpression lines (LID1oex) show reduced germination in the presence of ABA and drought-like conditions (high mannitol levels) known to increase ABA levels. Loss-of-function lid1 mutants show opposite phenotypes, with reduced germination in the presence of PAC and enhanced germination in ABA and mannitol, thus pointing to a possible role of LID1 in GA-ABA crosstalk. Light is known to influence ABA-GA balance and growth (de Lucas et al., 2008; Feng et al., 2008; Oh et al., 2009), and lid1 mutants germinate in the dark more rapidly than WT seeds. Under light conditions, they also germinate and develop into photomorphogenic seedlings more quickly than WT plants. Molecular studies indicate that LID1 is expressed specifically in the seed, is a transcriptional repressor and interacts with DELLA proteins. Our molecular studies indicate that LID1 is a novel regulator integrating light and growth responses in seeds with a promising biotechnological potential.
References 1. Colebrook et al (2014). J Exp Biol 217: 67-75. 2. Weiste et al (2007) Plant J 52:382-390. 3. De Lucas et al (2008) Nature 480-484 4. Feng et al (2008) Nature 451: 475-479 5. Oh et al (2009) Plant Cell 21: 403-419
Acknowledgements & Funding This work was supported with grants to L.O.-S by the Spanish Ministry of Economy and Competitiveness (BIO2013-46076-R and BIO2016-77840-R). We thank Dr. José Jarillo for advice with experiments involving light conditions and Dr. Ángela Contreras and Natalia Pozuelo for their help with plasmid constructions and generation of transgenic plants.
138
S-VI. Póster 12
IDENTIFYING KEY GENES DEPENDENT ON PEROXISOMAL
ROS
Laura C. Terrón-Camero1, Coral Del Val2, Luisa M. Sandalio1, María C.
Romero-Puertas1.
1Department of Biochemistry, Cell and Molecular Biology of Plants, Estación
Experimental del Zaidín, CSIC, C/Profesor Albareda 1, E-18008 Granada, Spain; 2Department of Artificial Intelligence, University of Granada, E-18071 Granada, Spain.
Corresponding author: María C. Romero-Puertas ([email protected])
Enhanced reactive oxygen species (ROS) production in plant cells is a common
physiological event that takes place in response to biotic and abiotic
perturbations. Disruption of ROS cellular homeostasis is caused by the
excessive reduction of activities in different organelles, and future research
needs to clarify the structure of gene networks and to identify downstream
responses induced by H2O2 originating from specific organelles (Suzuki et al.,
2012). Peroxisomes are highly dynamic, metabolically active organelles which
used to be regarded as a hydrogen peroxide sink generated in different
organelles. However, these organelles, which are now considered to be more
complex, contain different metabolic pathways and are an important source of
reactive oxygen species (ROS), nitric oxide (NO) and reactive nitrogen species
(RNS). Thus, peroxisomes have been shown to play a key role in many aspects
of plant development and acclimation to stress conditions. These organelles
can sense ROS/redox changes in the cell and therefore trigger rapid and
specific responses to environmental cues involving changes in peroxisomal
dynamics as well as ROS and NO-dependent signalling networks, although the
mechanisms involved have not yet been established (Sandalio and Romero-
Puertas, 2015; Rodríguez-Serrano et al., 2016). In this study, we have
examined both in-house and public data sets derived from the profiling of
Arabidopsis gene expression in mutants and treatments with peroxisomal-
dependent ROS levels altered in order to identify a data set of genes regulated
by peroxisomal ROS which will enable us to gain a deeper understanding of the
role played by peroxisomes as stress sensors and regulators of cellular
responses to adverse conditions resulting in plant acclimation and resistance.
Rodríguez-Serrano et al (2016) Plant Physiol., 171: 1665-1674
Sandalio and Romero-Puertas (2015) Annals of Botany, 116: 475-85
Suzuki, N., et al (2012).Plant Cell Environ. 35, 259–270.
This study was funded by an ERDF grant co-financed with the Spanish Ministry of Economy,
Industry and Competitiveness (BIO2015-67657-P). L.C. T-C was supported by an University
Staff Training Program (FPU) fellowship from the Spanish Ministry of Education, Culture and
Sports
139
S-VI. Póster 13 POST-TRANSLATIONAL MODIFICATIONS OF PROTEINS BY
NITRATED FATTY ACIDS: NITROALKYLATION OF
ANTIOXIDANT SYSTEMS IN PLANTS
Raquel Valderrama, Beatriz Sánchez-Calvo, Juan Carlos Begara-Morales, Capilla Mata-Pérez, María N. Padilla, Lorena Aranda-Caño, Juan Bautista
Barroso and Mounira Chaki
Group of Biochemistry and Cell Signalling in Nitric Oxide, Department of Experimental Biology, Center for Advanced Studies in Olive Grove and Olive Oils, Faculty of Experimental Sciences, University of Jaén, Jaén, Spain.
Corresponding author: Juan Bautista Barroso ([email protected])
Nitro-fatty acids (NO2-FAs) result from the interaction of non-saturated fatty acids and nitric oxide (NO) or NO-derived species. Knowledge concerning NO2- FAs has significantly increased within a few years ago and the beneficial actions of these species uncovered in animal systems have led to consider them as molecules with therapeutic potential (Kansanen et al., 2017; Trostchanski y Rubbo, 2017). Recently, the endogenous occurrence of nitrated- derivatives of linolenic acid (NO2-Ln) was detected in plant species (Sánchez- Calvo et al., 2013; Mata-Pérez et al., 2016a; 2017; 2018). Based on their nature and structure, NO2-FAs have the ability to release NO in aqueous environments (Mata-Pérez et al., 2016b, c) and the capacity to mediate post-translational modifications (PTMs) by nitroalkylation. In this work, the modulating capacity of nitrolinolenic fatty acid (Ln-NO2) on the activity of different antioxidant systems, including the Ascorbate-Glutathione cycle, in the plant species Arabidopsis thaliana and Pea (Pisum sativum L.) was analyzed. At the same time, the nitroalkylated residues responsible for the detected modifications in the enzymatic systems were identified by liquid chromatography techniques coupled to mass spectrometry (LC-MS/MS). In this sense, in vitro treatment with Ln-NO2 compromised the functioning of the main antioxidant systems studied (catalase, ascorbate-glutathione cycle enzymes), NADP-dependent dehydrogenases, as well as the NO metabolism by inhibition of GSNO reductase. The analysis of these results suggests that the functional modulation of the main antioxidant defense systems by NO2-FAs-related nitroalkylation can constitute a key control mechanism of these systems in situations of cell damage.
References
Kansanen E. et al. (2017) Mol. Pharmacol. 117.109751. Mata-Pérez, C. et al. (2016a) Plant Physiol. 170(2):686-701. Mata-Pérez, C. et al. (2016b) Plant Signal Behav. 11(3):e1154255. Mata-Pérez, C. et al. (2016c) Nitric Oxide 57:57-63. Mata-Pérez, C. et al. (2017) Redox Biol. 11:554-561. Mata-Pérez, C. et al. (2018) Nitric Oxide. doi: 10.1016/j.niox.2018.03.011. Sánchez-Calvo, B. et al. (2013) Plant Science 199-200:1-6. Trostchansky A. y Rubbo H. (2017) Arch. Biochem. Biophys. 617:155-161.
Acknowledgements & Funding We would like to thank the Technical Services Department of the University of Jaén for the LC- MS/MS analyses. LA-C thanks the University of Jaén for funding the Ph.D. fellowship. This work was supported by an ERDF grant co-financed by the Ministry of Economy and Competitiveness (Project BIO2015-66390-P) and the Junta de Andalucía (Group BIO286) in Spain.
140
S-VI. Póster 14
DE NOVO ASSEMBLY OF THE MITOCHONDRIAL GENOME OF
Allium Sativum
Ricardo Parreño-Montoro1, Eva Rodríguez-Alcocer1, Álvaro Ferriz1, Felipe
Gómez del Castillo2, Purificación Castillo Martínez2, and Héctor Candela1
1 Instituto de Bioingeniería, Universidad Miguel Hernández, 03202, Elche, Alicante.
2 Coopaman, S.C.L., c/ General Borrero, s/n., 16660, Las Pedroñeras, Cuenca.
Corresponding author: Héctor Candela ([email protected])
The genus Allium comprises several species of cultivated plants, such as
garlic (Allium sativum) and onion (Allium cepa), which are highly appreciated for
the commercial value of their bulbs. Overcoming the infertility of garlic cultivars
is the most important challenge in garlic cultivation and would be a solution to
problems such as the loss of genetic variation, the accumulation of pathogens
in propagules or the difficulties to develop new cultivars, all of them occurring
directly or indirectly as a consequence of the vegetative mode of propagation. In
order to generate resources for developing garlic as a modern crop, we have
carried out the sequencing, de novo assembly and annotation of the complete
mitochondrial genome of Allium sativum. Our efforts to annotate the garlic
mitochondrial genome have allowed us to identify more genes than in the not-
so-closely related mitochondrial genome of onion. In agreement with current
models of mitochondrial genome structure in plants, our results demonstrate
that the garlic mitochondrial genome consists of numerous subgenomic
molecules that undergo a complex recombination pattern at specific sequences.
The recombination sites can be effectively detected as shared paths in the
assembly graph. We have experimentally validated these paths by means of
PCR amplification and Sanger sequencing. Our findings suggest that
recombination actively and specifically occurs at short repeated sequences,
which shape a dynamic genomic landscape where up to four distinct
recombination products co-exist at each repeat. Our in-depth analysis of the
structural variation of the garlic mitochondrial genome has been performed with
unusual definition and sets the basis for further experiments on the dynamics of
mitochondrial genome dynamics in other plants.
The authors wish to thank Coopaman, S.C.L. for funding this project.
141
S-VI. Póster 15 EMS MUTAGENESIS AND SCREENING FOR GENES INVOLVED
IN THE DEGRADATION OF D14 AND SMXL7, KEY
COMPONENTS OF THE STRIGOLACTONE PATHWAY
Rodrigo García-del Campo, Elena Sánchez Martín-Fontecha, Aitor Muñoz, Pilar
Cubas
1Plant Molecular Genetics Department, National Center of Biotechnology-CSIC,
Campus of Madrid Autonoma University; Madrid, Spain,
Corresponding author: Rodrigo García, [email protected]
Strigolactones (SL) are phytohormones that control, among others processes,
lateral shoot growth. Knowledge about the synthesis, transport, perception and
signalling of SL may have important implications for agriculture, and it is also
essential to understand how plants control their architecture (Waters et al.
2017).
Recent studies showed that SL signalling involves the F-box protein MAX2 (a
key component of an SCF E3-ubiquitin ligase complex), the α/β hydrolase
DWARF14 (D14) the SL receptor, and SMXL6, 7, and 8 repressors of the
pathway. The max2 or d14 mutants have a bushy phenotype and smxl6, 7, 8
mutants suppress these phenotypes reverting plants to a wild-type phenotype.
SL cause MAX2-dependent proteasome-dependent degradation of SMXLs.
Remarkably they also cause MAX2-dependent proteasome-dependent D14
degradation.
In this study, we have mutagenized Ub:D14:LUC and Ub:SMXL7:LUC
transgenic seeds with ethyl methanesulfonate (EMS), a mutagen that induces
random point mutations, and we have generated M2 plants. In our mutant
screening we will look for seedlings that display abnormal D14:LUC or
SMXL7:LUC degradation patterns respectively. Mutant genes in those
individuals are expected to be involved directly or indirectly in the strigolactone
signalling pathway.
Chevalier F., Nieminen K., Sánchez-Ferrero J.C., Rodríguez M.L., Chagoyen M., Hardtke C.S.,
Cubas P. 2014. Strigolactone promotes degradation of DWARF14, an α/β hydrolase essential
for strigolactone signaling in Arabidopsis. Plant Cell. 2014 Mar;26(3):1134-50.
Waters M. T., Gutjahr C., Bennett T. and Nelson D.C.. 2017. Strigolactone Signaling and
Evolution. Annual Review of Plant Biology Vol. 68:291-322.
142
143
SESIÓN VII
ESTRÉS ABIÓTICO
MODERADORES:
Rafael Catalá (CIB-CSIC,
Madrid)
Juan Carlos Del Pozo
(CBGP, UPM-INIA, Madrid)
144
S-VII. Ponencia 1
NEW INSIGHTS ON THE DETERMINATION OF SPLICEOSOME
SPECIFICITY
Rafael Catalá1, Raúl Huertas1,2, José M Jiménez-Gómez3, Mª Mar
Castellanos1,4, Pedro Crevillén4, Manuel Piñeiro4, José Jarillo4 and Julio
Salinas1
1Departamento de Biotecnología Microbiana y de Plantas, Centro de Investigaciones
Biológicas, Madrid, Spain, 2 Noble Research Institute, Admore, Oklahoma, USA 3
Institut Jean-Pierre Bourgin, Versailles, France. 4Centro de Biotecnología y Genómica
de Plantas, Universidad Politécnica de Madrid (UPM), Pozuelo de Alarcón, Spain.
Corresponding author: Rafael Catalá, [email protected]
For many years, it has been assumed that the molecular mechanisms governing plant response to abiotic stress were mainly controlled at the transcriptional level. However, recent studies have revealed that posttranscriptional regulatory mechanisms, such as pre-mRNA splicing, also play a pivotal role. In eukaryotes, pre-mRNA splicing is governed by the activity of a multimegadalton complex known as the spliceosome. The core of the spliceosome is composed by five small nuclear ribonucleoproteins (snRNPs), where the Sm ring and the related Sm-like (LSM) 2-8 complex constitute their protein moieties. We have previously demonstrated that the Arabidopsis LSM2- 8 complex dynamically determines spliceosome specificity. Whether the Sm ring has a similar function remains unknown. To answer this question, we have characterized SmE1, an Arabidopsis gene encoding a protein with high identity to the SmE subunit of eukaryotic Sm ring. We determined that SmE1 is actively expressed under control conditions and specifically up-regulated by low temperature. The characterization of two null mutant alleles revealed that SmE1 positively controls snRNA accumulation, demonstrating that, as predicted, it is a functional component of the Arabidopsis Sm ring. Furthermore, our results evidenced that this protein imposes the appropriate pre-mRNA splicing pattern to regulate plant growth and development, and to modulate plant response to low temperature. These data, together with our previous results on the LSM2-8 complex, constitute a change of paradigm in the regulation of eukaryotic spliceosome activity: the protein moieties of the snRNPs are no longer important exclusively because of their structural function, but they also play a crucial role as determinants of the spliceosome specificity.
145
S-VII. Ponencia 2
ROOT RESPONSES TO PHOSPHATE STARVATION: NOVEL
FUNCTIONS OF cisZeatin AND BiAux
Angela Saez1,2, Juan C. del Pozo 2, Javier Silva-Navas2, Sara Navarro-Neila2, Jose M. Garcia-Mina3, Angel M. Angel M. Zamarreño3, Roberto Baigorri1, Pilar Hoyos4,
Maria J. Hernaiz4
1CIPAV, Timac Agro Int-Roullier Group, Orcoyen, Spain; 2Centro de Biotecnología y Genómica de Plantas (CBGP). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid,
Spain. 3Environmental Biology Department, University of Navarra, Navarra, Spain. 4Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad Complutense de
Madrid, Spain
Corresponding author: Angela Saez: [email protected]
Root system development and responses to environmental changes are crucial for whole plant adaptation. Deficiency of phosphate (Pi), an essential nutrient, restricts plant growth and production. Pi starvation triggers adaptive mechanisms that range from improvements of Pi-uptake, changes in metabolism, differential usage of Pi- compounds, differential gene expression and morphological changes in root architecture. Root are underground organs that grow in darkness. However, root responses and molecular analyses during Pi starvation have been mainly analyzed in light-grown roots. Here, we show that root illumination significantly modified the Pi- starvation response at the molecular, physiological and morphological level. Analyzing dark-grown roots, we identified many transcripts that were not previously described as Pi-starvation response (PSR) genes. Pharmacological treatments show that cytokinin regulates Pi-accumulation in roots in detriment of shoot. Moreover, a quantitative hormonal analysis show that Pi-starved plants tend to accumulate higher levels of cis-Zeatin (cZ) than trans-Zeatin (tZ). Morphological and transcriptomic analyses demonstrated that cZ induces the typical cytokinin response but maintaining higher rates of cell division and root growth as well as increasing root hair length than tZ in low-Pi mediums. Thus, we postulate cZ as Pi-starvation response hormone that stimulates root growth and root hairs elongation as well as P- organic mobilization to accumulate free Pi in roots during Pi starvation. This higher phosphate content favors root growth to increase absorbing surface and improve Pi uptake.
We also have carried out a root metabolomic analysis in Pi starved plants, identifying a metabolite (BiAux) that significantly increased its levels. We were able to synthesize it in vitro. Application of BiAux to plants significantly increases the number of lateral roots and total root system in Arabidopsis and tomato plants. Molecular analyses show that BiAux partially regulates auxin signaling by controlling the degradation of Aux/IAA repressors. Currently, in order to understand the role of BiAux we are carrying out transcripotmic analyses. We found that BiAux induces genes involved in electron transport and energy as well as PSR genes. In fact, application of BiAux to Pi starved plant enhances the phenotypic PSR. Taken together, BiAux might be a second messenger of Pi starvation signaling response.
This work has been funded by grants BIO2014- 52091-R (JCP) and PTQ- 15-07915 (AS) by the
MINECO.
146
S-VII. Comunicación 1 BPM3 AND BPM5 SUBUNITS OF Cullin3-RING E3 UBIQUITIN
LIGASES TARGET CLADE A PP2CS FOR DEGRADATION
Jose Julián1, Alberto Coego1 Jorge Lozano-Juste1, Borja Belda-Palazón1, Qian Wu2, Xu Zhang2, Esther Lechner3, Pascal Genschik3, Pedro L. Rodríguez1.
1Instituto de Biología Molecular y Celular de Plantas, CSIC-UPV, Valencia, Spain, 2The State Key Laboratory of Protein and Plant Gene Research, Peking University, Beijing, China,
3Institut de Biologie Moléculaire des Plantes, CNRS, Strasbourg, France. Corresponding author: Pedro L. Rodríguez ([email protected])
Plants are sessile organisms, so they need to adapt to environmental changes. Climate change will increase drought periods, which will bring plants beyond their capacity of adaptation, decreasing crop yield (Cassia et al., 2018). The plant hormone abscisic acid (ABA) regulates many key processes in plants, including abiotic stress responses (Cutler et al., 2010; Finkelstein, 2013). Recently, studies that address the turnover of core ABA signalling components have opened new avenues of research in ABA signalling (Bueso et al., 2014; Irigoyen et al., 2014; Kong et al., 2015; Belda-Palazón et al., 2016; Wu et al., 2016; Yu et al., 2016); however, a comprehensive understanding of the mechanisms that regulate receptor and clade A protein phosphatases type-2C (PP2Cs) protein levels is still lacking. These mechanisms can contribute to the modulation of ABA signalling in different tissues and developmental stages as well as in response to environmental cues. Recently, we identified RGLG1 and RGLG5 E3 ligases as regulators of the turnover of PP2CA (Wu et al., 2016). Additionally, during the course of proteomic studies aimed to identify components that mediate the turnover of clade A PP2Cs, we identified BTB/POZ AND MATH DOMAIN proteins (BPMs), substrate adaptors of the multisubunit cullin3 (CUL3)-RING based E3 ligases (CRL3s), as PP2CA-interacting proteins. BPMs belong to a six-member BPM1-6 protein family in Arabidopsis. The BTB domain binds CUL3, a highly conserved CULLIN family member that is present in CRL3s. The MATH domain serves as the substrate receptor
of the CRL3MATH subfamily of CRLs. Indeed, BPM3 and BPM5 interact in the nucleus with PP2CA as well as with ABI1, ABI2 and HAB1. BPM3 and BMP5 gain-of-function promotes PP2C degradation in vivo, leads to enhanced sensitivity to ABA and enhances drought tolerance. Conversely, reducing BPM function leads to higher accumulation of PP2Cs, which impairs stomatal closure, enhances water transpiration and leads to insensitivity to ABA in germination, root growth and gene expression assays. Therefore, degradation of clade A PP2Cs through different E3 ligases is a complementary mechanism to PYR/PYL/RCAR mediated inhibition of PP2C activity to relieve repression of ABA signalling.
· Belda-Palazon, B., Rodriguez, L., Fernandez, M. A., Castillo, M.-C., Anderson, E. M., Gao, C., and Rodriguez,
P. L. (2016). FYVE1/FREE1 Interacts with the PYL4 ABA Receptor and Mediates Its Delivery to the Vacuolar Degradation Pathway. The Plant Cell, 28(9), 2291–2311. · Bueso, E., Rodriguez, L., Lorenzo-Orts, L., Gonzalez-Guzman, M., Sayas, E., Muñoz-Bertomeu, J., Ibañez, C., Serrano, R., Rodriguez, P.L. (2014). The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signalling. Plant Jounal, 80, 1057–1071.
· Cassia, R., Nocioni, M., Correa-Aragunde, N., and Lamattina, L. (2018). Climate Change and the Impact of Greenhouse Gasses: CO2 and NO, Friends and Foes of Plant Oxidative Stress. Frontiers in Plant Science, 9, 273. · Kong, L., Cheng, J., Zhu, Y., Ding, Y., Meng, J., Chen, Z., and Gong, Z. (2015). Degradation of the ABA co- receptor ABI1 by PUB12/13 U-box E3 ligases. Nature Communications, 6, 8630. · Wu, Q., Zhang, X., Peirats-Llobet, M., Belda-Palazon, B., Wang, X., Cui, S., Rodriguez, P.L., and An, C. (2016). Ubiquitin Ligases RGLG1 and RGLG5 Regulate Abscisic Acid Signaling by Controlling the Turnover of Phosphatase PP2CA. The Plant Cell, 28(9), 2178–2196. · Yu, F., Lou, L., Tian, M., Li, Q., Ding, Y., Cao, X., Wu, Y., Belda-Palazon, B., Rodriguez, P.L., Yang, S., and Xie,
Q. (2016) ESCRT-I Component VPS23A Affects ABA Signaling by Recognizing ABA Receptors for Endosomal Degradation. Molecular Plant, 9(12), 1570 – 1582.
This work was funded by grants BIO2014-52537-R and BIO2017-82503-R to P.L.R., JJ was funded by BIO2014- 52537-R from Ministerio de Economía, Industria y Competitividad (MINECO).
147
S-VII. Comunicación 2 ARABIDOPSIS SYNAPTOTAGMINS 1 AND 3 ARE INVOLVED IN
LIPID HOMEOSTASIS AT ER-PM CONTACT SITES UNDER COLD
STRESS.
Noemí Ruiz-López1, Jessica Pérez-Sancho1, Arnaldo Schapire1, Vítor Amorim-Silva1,
Alicia Esteban1, Araceli Castillo2, Abel Rosado3, Sonia Osorio1, Steffen Vanneste4,
Lothar Willmitzer5, Carlos Perea6, Julio Salinas6, Miguel Angel Botella1
1Dpto. Biología Molecular y Bioquímica y 2Dpto. Genética, UMA-IHSM(CSIC), Málaga, Spain;
3Dpt of Botany, Univ of British Columbia, Vancouver, Canada; 4Dpt of Plant Systems Biology,
VIB-Ghent University, Ghent, Belgium; 5Max Planck Institute of Molecular Plant Physiology,
Potsdam-Golm, Germany; 6Dpto Biología Medioambiental, CIB-CSIC, Madrid, Spain.
Corresponding author: Noemí Ruiz-López ([email protected])
As sessile organisms, plants must cope with abiotic stress such as soil salinity,
drought, and extreme temperatures. This stress signal can activate a phospholipase
C (PLC), which hydrolyses PIP2 to generate IP3 and diacylglycerol (DAG). ER-PM
contact sites are conserved structures defined as regions of the endoplasmic
reticulum (ER) that tightly associate with the plasma membrane (PM). Our recent
data suggest that the constitutively expressed Arabidopsis Synaptotagmin 1 (SYT1)
and the cold-induced homolog SYT3 are proteins located in these ER-PM contact
sites that are essential for freezing tolerance. Additionally, like mammalian Extended
Synaptotagmins, membrane tethering is mediated by C2-domains which interact with
acidic phospholipids (enhanced by Ca2+). Our experiments of depletion of PM PI(4)P
triggers loss of SYT1 and SYT3 at ER-PM CS. Moreover, our analysis in SYT1 and
SYT3 proteins predicted a SMP domain like the recently crystalized E-SYT2 which
exhibits a hydrophobic groove capable of harbouring phospholipids, suggesting that
SYT1 and SYT3 mediate lipid exchange between the ER and the PM. This idea is
supported by the over-accumulation of saturated DAG found in SYT1 after a high-
resolution lipidome analysis. Additionally, we have identified DGK2 (diacylglycerol
kinase 2) as an interactor of SYT1. In summary, our recent studies suggest that
SYT1 and SYT3 are ER-PM tether components responsible for the elimination of
excess DAG from the PM after its acute generation by PLC in cold conditions.
The authors acknowledge the support by the Plan Propio from University of Malaga, Campus de Excelencia Internacional de Andalucía and by the Redes of Excelencia (BIO2014-56153-REDT) and BIO2017-82609-R & BIO2014-55380-R of the Ministerio de Economía, Industria y Competitividad.
148
S-VII. Comunicación 3
UNCOUPLING GROWTH REPRESSIVE AND ABIOTIC-STRESS
TOLERANCE EFFECTS of DELLAs
Alejandro Navarro-Galiano, Pilar Lasierra and Salomé Prat1.
Department of Plant Molecular Genetics, Spanish National Centre for Biotechnology (CNB-
CSIC), Madrid, Spain
Corresponding author: Salomé Prat ([email protected])
Environmental stresses affect agronomic production worldwide. Understanding
the mechanisms that determine the balance between plant growth and their ability to
adapt to environmental stresses is essential for the selection of more productive
varieties and sustainable crop production. One of the main key regulators of this
trade-off is the DELLA group of transcriptional repressors. DELLAs were shown to
interact with phytochrome interacting transcription factors (PIFs) and sequester these
regulators to arrest growth. However, these repressors have also been described to
enhance plant survival under salt or drought stress.
In this work we aimed to identify DELLA mutations that impaired interaction with
the growth-related factors and did not affect their binding to other stress-related
regulators. To do so, we carried out a directed mutagenesis of the LHRI motif, which
mediates PIFs interaction, and confirmed by yeast two-hybrid (Y2H), BiFC and co-
immunoprecipitation assays that this mutation disrupts PIF3 and PIF4 interaction, but
does not affect binding to several stress-related factors. Transgenic Arabidopsis rga-
24 gai-t6 mutants expressing a stable form of this DELLA variant showed enhanced
survival to salt stress, but did not display the dwarf phenotype of pGAI::ΔGAI plants.
While gene expression studies are required to define which pathways are affected
by this mutation, our findings underscore that genome editing of DELLAs can be an
excellent approach to improve stress tolerance of agricultural relevant crops.
This research was supported by the Spanish Ministry of Economy, Industry and Competitiveness (FPI
grant BES-2015-073518)
149
S-VII. Comunicación 4 EPIGENETIC REGULATION OF HEAT STRESS RESPONSE IS
REVEALED THROUGH SUBCELLULAR PROTEOMICS ANALYSIS IN
Pinus radiata
Laura Lamelas1, Luis Valledor1, Sara Guerrero1, Víctor Roces1, Mónica Meijón1,
María Jesús Cañal1
1Plant Physiology, Faculty of Biology, Department of Organisms and Systems Biology,
University of Oviedo, Asturias, Spain
Corresponding author: Mónica Meijón ([email protected])
Forest species are negatively affected by rising temperatures. In this context of
climate change, increasing our knowledge of heat-stress tolerance and adaption
mechanisms is particularly interesting. Although responses to heat stress have been
characterized at system and cellular levels in several forest species, the dynamics of
nuclear proteome triggered in this situation remains unknown, despite its key role in
heat-stress response modulation. To this end, Pinus radiata seedlings were
subjected to 45 ºC in a 10 days course experiment and a recovery step of 30 days
under control conditions to elucidate the possible acquired memory after heat
exposure.
Nuclear proteins were isolated and analyzed by nLC-MS / MS (Orbitrap Fusion)
which allowed the identification of 862 nuclear protein species. After multivariate and
integrative statistical analyses, a set of 309 specific nuclear heat response proteins
were identified and evaluated from the functional biology approach, revealing in
addition to HSPs (HEAT SHOCK PROTEINS) and HSE (HEAT SHOCK ELEMENTS)
various clusters involved in different regulation steps, as epigenomic-drived gene
regulation, several transcription factor families and a variety of RNA-associated
functions (spliceosome, proteasome and mRNA surveillance). The result of all these
changes was detectable in the overaccumulated traslasome machinery (ribosomal
proteins and eukaryotic translation initiation factors), needed to carry out the required
cell reorganization. It is also described differential tendency in short, mid and long
term heat exposure, as well as, stable H2A HISTONE and methyl cycle enzymes as.
S-ADENOSYL-L-METHIONINE SYNTHASE and ADENOSYLHOMOCYSTEINASE
heat-induced lost and its following accumulation after the recovery step. These
results suggest a relevant role of epigenetic mechanisms in Pinus radiata related to
adaptation process and a possible thermopriming efect. Chloroplast proteome is also
being analyzed to identify new signaling communication channels between these two
organelles and the nuclear-plastid heat crosstalk response, and thereby allowing to
reach a complete view of thermopriming adquision in conifers.
Acknowledgements & Funding
This work is an output of the AGL2016-77633-P project financed by the Spanish Ministry of Economy
and Competitiveness.
150
S-VII. Comunicación 5 ESSENTIAL REGIONS INVOLVED IN FUNCTION AND REGULATION
OF THE ARABIDOPSIS HIGH-AFFINITY K+ TRANSPORTER ATHAK5.
Reyes Ródenas, Jesús Amo, Rosa M. Rivero, Vicente Martínez, Manuel Nieves-
Cordones, Francisco Rubio
1Departamento de Nutrición Vegetal, CEBAS-CSIC, 30100 Murcia, Spain
Corresponding author: Reyes Ródenas ([email protected] )
Potassium (K+) is an important macronutrient for plants present in the soil solution
at a wide range of concentrations. It has been shown in Arabidopsis (Arabidopsis
thaliana) that when the external K+ concentration is very low (10 µM), K+ nutrition
depends exclusively on the high-affinity K+ transporter 5 (HAK5). Low K+ induced
transcriptional activation of the gene encoding HAK5 has been previously reported 1. - -2
Deficiency of other nutrients such as nitrate (NO3 ) or phosphate (PO4 ) also induced
the gene, although no AtHAK5-medianted K+ uptake was observed. This suggested
a posttranslational activation of the transporter that takes place exclusively upon K+
starvation. In fact, Ragel et al (2015) 2 reported that AtHAK5 was activated by
phosphorylation at its N-terminal upon K+ starvation by the protein kinase CIPK23.
To gain insights into the AtHAK5 functional domains involved in K+ transport and its
activation, a mutagenesis approach was undertaken. AtHAK5 mutants were
generated and expressed in yeast to determining their functional complementation
capabilities. Two main regions of the protein were studied: (i) The putative pore-
forming region GVVYGD and the long C-terminus. The results obtained showed that
the G, Y, G and D residues of the putative pore-forming region are individually
essential for AtHAK5 functionality. A serial deletion study of the C-teminus showed
that this domain is essential for functionality and that an auto-inhibitory domain is
probably contained in this region.
References
1 Nieves-Cordones, M., Frontiers in Plant Science 7, doi:10.3389/fpls.2016.00992 (2016). 2 Ragel, P. et al.. Plant Physiol 169, 2863-2873, doi:10.1104/pp.15.01401 (2015).
Acknowledgements & Funding
This work was supported by grant numbers AGL2015-66434-R (to F. R.) from Ministerio de Economía
y Competitividad, Spain. A. L.-H. and R. R. are recipients of a F. P. U. Fellowship from Ministerio de
Educación, Cultura y Deporte, Spain. This work was also supported by the Red de Excelencia
BIO2014-56153-REDT.
151
S-VII. Póster 1
A ROLE FOR ATHAK5 AND AKT1 IN K+ TRANSLOCATION TO THE SHOOT IN ARABIDOPSIS
Alberto Lara, M. Ángeles Botella, Vicente Martínez, Francisco Rubio, Manuel Nieves- Cordones.
1Departamento de Nutrición Vegetal, CEBAS-CSIC, 30100 Murcia, Spain
Corresponding author: ([email protected]) Potassium is an essential major inorganic constituent of plant cells acquired from the
soil solution by the root HAK5 and AKT1 transport 1. In different experiments carried
out by 2 and 3 in rice it was confirmed that OsHAK1 and OsHAK5 were expressed in
the stele of the root. Besides, Lagarde, et al., (1995) 4 observed GUS staining of
pAKT1-GUS transgenic lines inside the root central cylinder. Thus, the hypothesis
that both HAK and AKT1 transport systems might be involved in K+ translocation to
the shoot was put forward 2 3. In our work, by using pHAK5::GUS transgenic lines,
expression of AtHAK5 was deduced to take place in the vascular cells, although this
expression pattern depended on the nutritional state of the plant. Thus, under
potassium starvation, GUS staining was observed in the cortex and the epidermis,
while in potassium-replete plants, GUS staining appeared in root vascular bundles.
Rubidium absorption experiments made with different line mutants of Arabidopsis,
hak5, akt1, hak5akt1 and wild type, showed that the translocation of rubidium from
the root to shoots was greater in the hak5akt1 line than in the rest of the lines, and
that the difference between hak5akt1 and WT was of a higher magnitude in K+-
replete plants. This information suggests that HAK5 and AKT1 may be involved in the
reabsorption of potassium from vascular tissues.
References
1 Nieves-Cordones, et al. Frontiers in Plant Science 7, doi:10.3389/fpls.2016.00992 (2016).
2 Chen, G. et al. Plant, cell & environment 38, 2747-2765, doi:10.1111/pce.12585 (2015).
3 Yang, T. et al.. Plant physiology 166, 945-959 (2014).
4 Lagarde, D. et al.. The Plant journal : for cell and molecular biology 9, 195-203 (1996). Acknowledgements & Funding This work was supported by grant numbers AGL2015-66434-R (to F. R.) and AGL2015-74011-JIN (to M. N.-C.) from Ministerio de Economía y Competitividad, Spain. A. L.-H. and R. R. are recipients of a F. P. U. Fellowship from Ministerio de Educación, Cultura y Deporte, Spain.
152
S-VII. Póster 2
ANTIOXIDANT ENZYMATIC RESPONSES OF H. annuus AND S.
lycopersicum vs D. viscosa AGAINST ANTIMONY TOXICITY
Alfonso Ortega1, Inmaculada Garrido2, Francisco Espinosa2
1Department Plant Physiology, UCLM, Av. Carlos III s/n, 45071, Spain
2Research Group FBCMP, UEx, Campus Av. Elvas s/n, 06011 Badajoz, Spain
Heavy metals are mainly derived from geochemical materials and they may act
as pollutants by increasing their presence in the biosphere in a natural way such as
surface mineralization, volcanic degassing, combustion or forest fires, among other
causes, or as a consequence of human activities (industrialization, mining and
urbanization) constituting a potential threat to human health and ecosystems. By
these reasons, it is necessary to find mechanisms to reduce its presence in the
environment.
Our aim in this work is to study in plants the changes caused by Antimony
(Sb), a metalloid present in mining areas with a geochemical behavior and
characteristics similar to arsenic. For this reason, we have performed a comparative
study between Dittrichia viscosa, which is able to colonize highly degraded soils with
high amounts of Sb, another plant from the same family (Asteraceae), Helianthus
annuus and Solanum lycopersicum. In both cases, the seedlings were cultivated in a
hydroponic medium with 0.0, 0.5 and 1.0 mM Sb with the aim of determining, after 17
days of treatment, the expression of genes that encode defense enzymes and their
antioxidant activities in leaves and roots. In this way, each plant response to stress
could be assessed by comparing the possible differences in their defence systems
and its relationship with their ability to tolerate soils with high levels of Sb, and their
possible use in phytoremediation. In preliminary studies, we have seen that on the
one hand, SOD, APX and DHAR activities showed alterations due to treatment with .-
Sb, which were attenuated in Dittrichia and, on the other hand, the O2 generation
was similar between Dittrichia and their control and other control plants, which has
not occurred for sunflower and tomato with Sb treatment. In both cases, the
compounds that constitute the AsA/GSH cycle were altered.
Acknowledgments: This study was made possible thanks to the Junta de Extremadura/FEDER for
the Research Project IB16078 and the support given to the Research Group FBCMP.
153
,
S-VII. Póster 3
ttl MUTANTS ARE IMPAIRED IN CELLULOSE BIOSYNTHESIS
UNDER OSMOTIC STRESS
Álvaro García-Moreno1, Vitor Amorim-Silva1, Araceli Castillo2 Victoriano Valpuesta1,
Alberto Macho3, Yvon Jaillais4, Miguel A. Botella1
1IHSM-UMA-CSIC, Departamento de Biología Molecular y Bioquímica, Universidad de
Málaga, Málaga, Spain.
2IHSM-UMA-CSIC, Departamento de Genética, Universidad de Málaga, Málaga, Spain.
3Shangai Center for Plant Stress Biology (PSC), SIBS CAS, Shangai, China.
4Laboratoire Reproduction et Développement des Plantes (SICE), ENS Lyon, Lyon, France.
Corresponding author: Miguel Ángel Botella ([email protected]) As sessile organisms, plants require mechanisms to sense and respond to the
challenging environment, that encompass both biotic and abiotic factors that results
in differential development. In these conditions is essential to balance growth and
stress responses. As cell walls shape plant growth, this differential growth response
cause alterations to the plant cell wall and cellulose is a major component. Therefore,
understanding the mechanisms that regulate cellulose biosynthesis is essential to
develop strategies to improve plant production. Previous studies have shown that the
GSK3 kinase BIN2 modulate cellulose biosynthesis through phosphorylating
cellulose synthases and that the expression of cellulose synthases are regulated by
Brassinosteroids. Our previous work reveals that the tetratricopeptide-repeat
thioreoxin-like (TTL) TTL1, TTL3, and TTL4 genes, in addition to their reported role in
abiotic stress tolerance, are positive regulators of BR signaling. We observe
association of TTL3 with most core components in traducing BR signalling, such as
LRR-RLK BRI1, BIN2 and the transcription factor BES1 that positively regulate
cellulose biosynthesis. We show that ttl mutants are affected in cellulose
biosynthesis, particularly in osmotic stress conditions. Furthermore, TTL3 associates
with LRR-RLKs that have been shown to be important for cellulose biosynthesis such
as FEI1 in the FEI1/FEI2/SOS5 pathway. We aim to investigate the mechanisms by
which TTL proteins regulate cellulose biosynthesis using a combination of genetics,
biochemical, and molecular and cell biology approaches.
This work was supported by grants from: (1) Ministerio de Ciencia e Innovación BIO2014-55380-R,
BIO2014-56153-REDT; (2) Ministerio de Economía, Industria y Competitividad (BES-2015-071256);
(3) Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.
154
S-VII. Póster 4 STRUCTURE-BASED APPROACH TO ENHANCE PLANT
RESISTANCE UNDER DROUGHT STRESS
Lourdes Infantes1, Juan Luis Benavente1, Jorge Lozano-Juste2, Pedro L. Rodriguez2
and Armando Albert1
1Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas,
ES-28006 Madrid, Spain. 2Instituto de Biología Molecular y Celular de Plantas, Consejo
Superior de Investigaciones Científicas-Universidad Politécnica de Valencia, ES-46022
Valencia, Spain.
Corresponding author: Armando Albert ([email protected])
Abscisic acid (ABA) is the main phytohormone involved in the adaptive crop
responses to drought. ABA signaling relies on the family of pyrabactin resistance
1/PYR1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) receptors,
which upon ABA binding form high-affinity ternary complexes with clade A protein
phosphatases type 2C (PP2Cs) and inhibit them. Our current understanding on the
structural mechanism of ABA signaling relies exclusively on crystallographic analyses
performed with Arabidopsis thaliana ABA receptors. Although mechanistic insights
obtained in Arabidopsis are usually translated to other plant species, studies in crops
might overturn or update the dogma established in Arabidopsis. As a result from
structural studies performed with crop ABA receptors, we have identified ABA-bound
intermediates that provide novel mechanistic insight on ABA signaling. This highlights
the role of the PP2C as necessary ABA co-receptor and provides the basis for
structure-based genetic and chemical approaches for the improvement of plant
resistance under drought stress.
The structure of ligand-bound intermediates of crop ABA receptors highlights the role of the PP2C as
necessary ABA co-receptor. Moreno-Alvero M, Yunta C, Gonzalez-Guzman, Lozano-Juste J,
Benavente JL, Arbona V, Menéndez M, Martinez-Ripoll M, Infantes L, Gomez-Cadenas A, Rodriguez
PL, Albert A. Molecular Plant (2017) 10, 1250–1253
Acknowledgements & Funding. . This work was funded by grants from MINECO (BFU2014-59796-R,
BIO2017-89523-R and BIO2014-56153-REDT to A.A.; BIO2014-52537-R and BIO2017-82503-R to
P.L.R. and EU grant H2020-MSCA-707477 to J.L.J
155
S-VII. Póster 5
ARSENIC, THE PHANTOM MENACE
Cristian Mateo-Elizalde, Micaela Navarro, Cristina Navarro, José Pruneda-Paz,
Yogev Burko, José Manuel Franco, Yolanda Leo-del Puerto, Javier Paz-Ares,
Joanne Chory and Antonio Leyva
Genética Molecular de Plantas, CNB-CSIC, Cantoblanco (Spain)
Corresponding author: Antonio Leyva ([email protected])
Arsenic is a metalloid classified as one of the main carcinogenic compounds
on Earth. It is widely distributed in nature, compromising the health of more than 200
million people due to its presence in water and crops. As sessile organisms, plants
have developed efficient strategies to survive in the presence of arsenic, mainly, the
accumulation into the vacuole or its extrusion. Indeed, some plants have an
extraordinary ability to extract and accumulate high amounts of arsenic.
In order to clean up arsenic from the environment, we aim to characterize the
signal transduction pathway involved in arsenic perception and accumulation in
Arabidopsis thaliana. For that purpose, we have developed two main approaches:
First, we mutagenized a transgenic line carrying the arsenate reductase promoter
fused to the GUS reporter gene with the objective to identify mutants altered in the
arsenic response. Secondly, we have performed yeast one hybrid screening to
identify transcription factors that bind to two arsenic inducible promoters and have
conducted an in-silico characterisation of enrichment of transcription factor binding
sites in clusters of genes differentially expressed under arsenic stress. Our studies
have allowed us to identify three mutants that have altered the arsenic response
using the first approach, and three additional mutants using the in-silico assays.
In this communication we will present our advances towards the identification
of master regulator/s of the arsenic signalling cascade.
Work funded by the Spanish Minister BIO2014-55741-R and the Howard Hughes Medical Institute
156
S-VII. Póster 6 PREFOLDIN IS INVOLVED IN ALTERNATIVE SPLICING IN
ARABIDIOPSIS
David Esteve-Bruna1, Cristian Carrasco-López2, Noel Blanco-Touriñán1, Carlos
Perea-Resa2, Javier Iserte3, Claudio Novella-Rausell1, Julián Calleja-Cabrera1,
Marcelo J Yanovsky3, Miguel A Blázquez1, Julio Salinas2, David Alabadí1
1Instituto de Biología Molecular y Celular de Plantas, CSIC-UPV, Valencia, Spain,
2Centro de Investigaciones Biológicas, CSIC, Madrid, Spain,
3Fundación Instituto Leloir, CONICET, Buenos Aires, Argentina.
Corresponding author: David Alabadí ([email protected])
PREFOLDIN (PFD) is an evolutionarily conserved heterohexameric co-chaperon that
presents unfolded tubulin to the main cytosolic chaperone CCT. This function takes
place in the cytosol, but PFD can also accumulate in the nucleus of Arabidopsis cells
through the interaction with the DELLA transcriptional regulators. This interaction, in
addition to impair PFD function in the cytosol, allows it to also having a role in the
nucleus.
The yeast, fly and human interactomes revealed extensive interaction between all six
PFD subunits and nuclear proteins, including members of the LSM2-8 complex
involved in mRNA splicing. Interestingly, several Arabidopsis PFD genes are
extraordinarily coexpressed with genes coding for LSM proteins, suggesting
functional relationship. The LSM2-8 complex binds and stabilizes the U6 snRNA
forming one of the five small nuclear ribonucleoproteins of the spliceosome.
Here we show that the interaction between PFDs and LSM8 is conserved in
Arabidopsis. Moreover, loss-of-function alleles of PFDs and LSM8 interact
genetically. Importantly, loss of PFD alters the levels of both LSM8 and U6 snRNA,
the effect being greater in high order pfd mutants, which suggests functional
redundancy between PFDs. Remarkably, a high-coverage RNA-seq analysis
revealed alternative splicing (AS) alterations in lsm8 and pfd2 pfd4 pfd6 triple
mutants, but not in the single pfd4 mutant. Since pfd4 and lsm8 plants shows an
anomalous response to low temperature, we performed the same RNA-seq analysis
in these mutants exposed to cold and found hundreds of AS events altered in both
mutants. These results suggest that (i) the regulation of LSM8 levels by PFD is
important for the proper control of AS, and (ii) PFD4 performs a limiting role in the
control of AS under cold stress.
157
S-VII. Póster 7
INVOLVEMENT OF TWO TRANSCRIPTION FACTORS IN ZUCCHINI
FRUIT RESPONSE TO COLD STRESS
Raquel Jiménez-Muñoz1, Fátima Carvajal1,2, Raquel Rosales1, Francisco Palma1,
Amada Pulido1, Manuel Jamilena3, Dolores Garrido1
1Department of Plant Physiology, Facultad de Ciencias, University of Granada, Granada,
Spain 2 Plant Research International, 6700 AA Wageningen, The Netherlands 3Department
of Biology and Geology, Agrifood, (ceiA3), University of Almería, Spain,
Corresponding author: Dolores Garrido ([email protected])
Zucchini (Cucurbita pepo L.) is a very important greenhouse crop in South-eastern
Spain. The fruits are harvested and consumed at an immature stage, and almost the
entire production is exported to other European countries, which entails a long period
of storage at low temperature until the fruit reaches the market. The subtropical origin
of this fruit makes zucchini very susceptible to develop chilling injury, a physiological
and metabolic disorder that contributes to the loss of quality and therefore results in
important economic losses. After a transcriptomic analysis comparing fruit of two
cultivars of zucchini showing contrasting resistant to chilling injury (Carvajal et al,
2011, 2018), a set of candidate genes related to chilling tolerance have been
selected for analysis. In this work, two candidate genes encoding two transcription
factors: a MYB106-like and ZAT10-like factors, are described during fruit postharvest
in the two varieties Natura (chilling resistant) and Sinatra (chilling sensitive). Their
differential expression by qRT-PCR shows that these genes undergo changes in their
expression during cold storage that are different in the cold resistant fruit of Natura
and in the sensitive Sinatra. Future experiments involving these genes and their
implication in cold resistance will be discussed in this study.
Carvajal, F., Martinez, C., Jamilena, M., & Garrido, D. (2011). Differential response of zucchini
varieties to low storage temperature. Scientia Horticulturae, 130(1), 90-96.
Carvajal, F., Rosales, R., Palma, F., Manzano, S., Cañizares, J., Jamilena, M., & Garrido, D. (2018).
Transcriptomic changes in Cucurbita pepo fruit after cold storage: differential response between two
cultivars contrasting in chilling sensitivity. BMC genomics, 19(1), 125.
Acknowledgements & Funding
This research has been funded by FPI Grant (MEC) in the Project AGL2014-54598-C2-R.
158
S-VII. Póster 8
METACASPASES AND AUTOPHAGY IN STRESS-INDUCED
MICROSPORE EMBRYOGENESIS OF Brassica napus
Eduardo Berenguer1, María-Teresa Solís1, 2, Ivett Bárány1, Yolanda Pérez-Pérez1, Elena Minina3, María C. Risueño1, Peter Bozhkov3, Pilar S. Testillano1
1Pollen Biotechnology of Crop Plants, Biological Research Center, CIB-CSIC, Madrid, Spain,
2Dep. Plant Biology I, Plant Physiology, UCM, Madrid, Spain, 3Dep. Molecular Sciences, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant
Biology, Uppsala, Sweden
Corresponding author: Pilar S. Testillano ([email protected])
Microspore embryogenesis is a fascinating system of cell reprogramming, totipotency acquisition and embryogenesis initiation, induced by in vitro stress treatment and very useful for rapid production of doubled-haploid plants in crop breeding programs. Increasing evidence has revealed that the efficiency of the system is limited by the occurrence of cell death triggered by the stress treatment (Bárány et al., 2018) but their underlying mechanisms remain unknown.
Autophagy plays crucial roles in response to various types of stress and in plant cell death. Metacaspases are major plant proteases involved in programmed cell death, which also regulate autophagy in some systems (Minina et al., 2013). We have studied the activation of autophagy and metacaspases in cell death occurring during stress-induced microspore embryogenesis of Brassica napus. After cell isolation and stress treatment autophagy flux was activated, BnATG5 was upregulated and autophagic structures increased in cytoplasm. Cell-death proteases with specific activity of caspase 3-like and metacaspase were induced, and type I and II metacaspase genes were upregulated. Treatments with autophagy, caspase-3 and metacaspase specific inhibitors reduced cell death levels and increased embryogenesis induction efficiency. Our findings indicate the involvement of metacaspases and autophagy in the initiation and/or execution of cell death during the induction of microspore embryogenesis in B. napus, giving new insights into the stress-induced cell death pathways in plants.
References: Bárány et al., 2018. J Exp Bot 69 (6): 1387-1402 Minina et al., 2013. J Cell Biol 203 (6): 917-927
Funding: Supported by project grants AGL2014-52028-R and AGL2017-82447-R of the Spanish MINECO, the European ERDF/FEDER and STSM grant of Transautophagy COST action CA15138.
159
S-VII. Póster 9
TRANSCRIPTOME ANALYSES DISCOVER CANDIDATE DROUGHT
TOLERANCE-ASSOCIATED GENES IN COMMON VETCH (Vicia
sativa L.)
Elena Ramírez-Parra 1, Encarnación Zambrana2, Lucía de la Rosa2
1 Centro Biotecnología y Genómica de Plantas, CBGP, INIA-UPM, Pozuelo de Alarcón,
Madrid; 2 Centro de Recursos Fitogenéticos, CRF, INIA, Alcalá de Henares, Madrid
Elena Ramírez-Parra ([email protected])
The losses in crop yield due to climate change and associated drought is one of the
main problems faced by current crops, including the legume common vetch (Vicia
sativa L.). Vetch is a forage grain legume of high protein content, which its high
nitrogen fixation ability makes its cultivation suitable in sustainable agriculture
systems.
To better understand the vetch drought tolerance mechanism in Vicia sativa, we used
high-throughput de novo RNA sequencing to assess the global changes in the
transcriptomes of plants of drought-susceptible and drought-tolerant vetch
accessions previously screened from a preliminary core collection of 50 vetch
accessions.
A total of 2367 significantly differentially expressed unigenes (DEGs) were detected,
including 1786 up-regulated unigenes and 418 down-regulated unigenes. Our
transcriptomic analyses revealed important differences in the expression of genes
involved in redox homeostasis, cell wall modifications and stress-response between
the tolerant and susceptible accessions. The presence of polymorphic variants in
these DEGs has also been analyzed. This information will be essential for the design
of molecular markers potentially associated with drought.
In addition, we broaden our transcriptomic studies comparing DEGs in control and
drought conditions. These analyses provided valuable information for identifying and
characterizing drought response networks and their underlying molecular
mechanisms in the common vetch.
These transcriptomic analyses were completed with different physiological studies
(osmolytes levels, stomatal opening, epicuticular waxes…). Taken together, our data
gives first insights into the molecular basis associated with drought tolerance in
common vetch, and provides new genetic resources for adaptation research in this
species.
Acknowledgements & Funding
This research was supported by grants from the Instituto Nacional de Investigación y Tecnología
Agraria y Alimentaria; INIA –(AT2016-009).
160
S-VII. Póster 10 DECIPHERING MOLECULAR COMPOSITION OF mRNP
COMPLEXES IN Arabidopsis thaliana THROUGH ISOLATION OF
TSN-INTERACTING PROTEINS
Emilio Gutierrez-Beltran1,*, Pernilla H. Elander2, Kerstin Dalman2, Panagiotis N. Moschou3 and Peter V. Bozhkov2
1Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla / Consejo Superior de
Investigaciones Científicas, 41092 Seville, Spain 2Department of Molecular Sciences, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, PO Box 7015, SE-75007 Uppsala, Sweden
3Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, PO Box 7080, SE-75007 Uppsala, Sweden
*Corresponding author: Emilio Gutierrez-Beltran ([email protected])
In natural conditions, all organisms are exposed to a variety of environmental stresses, adaptation to which depends on the modulation of gene expression. Basic cellular processes such as regulation of mRNA stability, degradation and translation taking place in cytoplasmic messenger ribonucleoprotein complexes (mRNP) known as stress granules (SGs) and processing bodies (PBs) play an important role in fine tuning gene expression (Chantarachot and Bailey-Serres, 2018). Molecular composition, structure, and function of SGs and PBs in plants are largely unknown. Recently, we have established Tudor Staphylococcal Nuclease (TSN or Tudor-SN; also known as SND1) proteins TSN1 and TSN2 as integral functional components of SGs and PBs in Arabidopsis thaliana (Gutierrez-Beltran et al., 2015, Gutierrez- Beltran et al., 2016). Since TSN proteins in Arabidopsis are stably associated with the mRNP complexes and may in this way play scaffolding role to recruit other proteins, we used TSN1 and TSN2 as baits in alternative tandem affinity purification (TAPa) of plant mRNP complex-associated proteins. We found that interactomes of both TSN1 and TSN2 are enriched with proteins featuring low-complexity regions (LCRs) and possessing intrinsically disordered domains (IDRs) typically found in proteins associated with SGs. Localization of identified proteins to stress-induced cytoplasmic foci in vivo has been further verified using a combination of biochemical and live imaging techniques. As a result, we have produced a list of TSN-interacting proteins sharing punctate cytoplasmic localization under stress conditions. Some of these proteins have previously been found in animal and/or yeast stress-induced mRNP complexes, while others appear to be novel or plant-specific mRNP components.
Chantarachot, T. and Bailey-Serres, J. (2018) Polysomes, Stress Granules, and Processing Bodies:
A Dynamic Triumvirate Controlling Cytoplasmic mRNA Fate and Function. Plant physiology, 176, 254-269.
Gutierrez-Beltran, E., Denisenko, T.V., Zhivotovsky, B. and Bozhkov, P.V. (2016) Tudor staphylococcal nuclease: biochemistry and functions. Cell death and differentiation, 23, 1739- 1748.
Gutierrez-Beltran, E., Moschou, P.N., Smertenko, A.P. and Bozhkov, P.V. (2015) Tudor Staphylococcal Nuclease Links Formation of Stress Granules and Processing Bodies with mRNA Catabolism in Arabidopsis. The Plant cell.
Acknowledgements & Funding This work was supported by grants from Knut and Alice Wallenberg Foundation, the Swedish Research Council, the Swedish Foundation for Strategic Research, Olle Engkvist Foundation, and Trees and Crops for the Future Research Programme and from the European Commission (Marie Curie IEF-reSGulating- 702473).
161
S-VII. Póster 11
WHEAT GENETIC DIVERSITY PERFORMANCE AT THE HIGH
TEMPERATURE AND CO2 CONCENTRATION FORESEEN WITH
CLIMATE CHANGE. IDENTIFICATION OF KEY MARKERS OF
GROWTH AND CENTRAL METABOLISM
Emilio Marcos-Barbero1, Pilar Pérez1, Rafael Martínez-Carrasco1, Juan-Bautista
Arellano1, Yves Gibon2 and Rosa Morcuende1
1Abiotic Stress Department, Institute of Natural Resources and Agrobiology of Salamanca,
CSIC, Cordel de Merinas 40-52, Salamanca, 37008 Spain
2UMR 1332 Biologie du Fruit et Pathologie, INRA Bordeaux-Aquitaine-Université de
Bordeaux, F-33882 Villenave d´Ornon, France
Agricultural production is highly dependent on climate, and improved crop varieties
that can counteract the effects of climate change will be required in the future. To
date, the evaluation of the natural genetic diversity for the improvement of crop yield
in the future climatic scenario is largely unexplored. We assessed the natural
variation in tolerance to high temperature and CO2 concentration in a population of
60 wheat genotypes from the CIMMYT 8TH HTWSN collection, preselected at high
temperature, with rapid and non-destructive phenotyping techniques. Considerable
variations among genotypes in photosynthetic capacity, biomass and grain yield were
detected in response to the future climatic scenario, although no consistent
correlation was found between grain yield and photosynthetic rate per unit area when
all genotypes were compared. The main factor contributing to yield variability was the
growth lenght, the higher yielding genotypes being those with faster development,
which avoid the increase of temperatures during grain filling. We also explored the
variation in performance under elevated CO2 and high temperature across a set of
ten wheat lines with different productivity using an integrated approach, combining
the study of the relationship of physiological characteristics with transcript, metabolite
and enzyme activity profiles at critical developmental stages. At ear emergence, the
connectivity between traits showed that an increase in starch and nitrate content in
the flag leaf, together with a lower activity of enzymes involved in glycolysis,
tricarboxylic acid cycle and N assimilation could be considered as positive markers
for selection of high yielding wheat lines under climate change. Finally, the transcript
abundance of primary C and N metabolism genes will be also analysed in order to
identify which C-N metabolic traits and transcripts are co-regulated under conditions
anticipated with climate change.
Acknowledgements & Funding Grants, AGL2013-41363-R, CSI083U16, AGL2016-79589-R (ERDF).
Marcos-Barbero had a JCyL fellowship. Boyero, Verdejo, Calvo and González for technical
assistance.
162
S-VII. Póster 12
EFFECT OF ABA APPLICATION ON THE BIOSYNTHESIS OF
CUTICULAR WAXES IN POSTHARVEST OF ZUCCHINI FRUIT
Francisco Palma1, Fátima Carvajal1,2, Raquel Jiménez-Muñoz1, Amada Pulido1,
Manuel Jamilena3, Dolores Garrido1
1Department of Plant Physiology, University of Granada, Granada, Spain 2 Plant Research
International, 6700 AA Wageningen, The Netherlands 3Department of Biology y Geology,
Agrifood, (ceiA3), University of Almería, Spain,
Corresponding author: Dolores Garrido Garrido ([email protected])
The plant hormone abscisic acid (ABA) plays a crucial role in different processes of
plant development and it is a central regulator of the resistance and adaptive
response against several environmental stresses, especially those that induce
dehydration such as drought, high salinity, and low temperature. In zucchini fruit, it is
known that ABA is mediating in the induction of chilling tolerance; and that an
increase in the amount of ABA during postharvest has a significant negative
correlation with weight loss and chilling damage, and a positive correlation with
firmness (Carvajal et al, 2017). During tomato leaf development, ABA regulates the
structure of the cuticle, the amount of cutin, and the composition of cutin and waxes
(Martín et al, 2017). In fruit, the cuticle plays a central role modulating the growth and
development, being also important for the quality of the fruit during its postharvest
life. In this work, we have investigated the implication of a treatment with ABA (1
mM), that improves cold storage resistance in zucchini fruit, on the expression of
genes related with the biosynthesis of cuticular waxes. During postharvest, the fruit
treated with ABA showed a higher expression of some of the genes that form the
Fatty Acid Elongase (FAE) complex: β-ketoacyl-CoA synthase (CER6 and FDH), β-
ketoacyl-CoA reductase (KCR), β-hydroxyacyl-CoA dehydratase (PAS2), and enoyl-
CoA reductase (CER10) genes. Overall, we provide evidences supporting the
involvement of ABA in the regulation of the metabolism of cuticular waxes.
Carvajal, F., Palma, F., Jiménez-Muñoz, R., Jamilena, M., Pulido, A., & Garrido, D. (2017). Unravelling the role of abscisic acid in chilling tolerance of zucchini during postharvest cold storage. Postharvest Biology and Technology, 133, 26-35.
Martin, L. B., Romero, P., Fich, E. A., Domozych, D., & Rose, J. K. (2017). Cuticle biosynthesis is developmentally regulated by abscisic acid. Plant physiology, pp-00387.
Acknowledgements & Funding
This research has been funded by Project AGL2014-54598-C2-R.
163
S-VII. Póster 13
THE CYSTEINE PROTEASES HvPap-1 AND HvPap-19 ARE
RELEVANT IN ABIOTIC AND BIOTIC STRESS IN BARLEY
Andrea Gómez-Sánchez1, Pablo González-Melendi1,2, Mª Estrella Santamaría1,2,
Manuel Martínez1,2, Isabel Díaz1,2
1 Centro de Biotecnología y Genómica de Plantas (CBGP). Universidad Politécnica
de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y
Alimentaria (INIA). Campus Montegancedo UPM. 28223-Pozuelo de Alarcón
(Madrid), Spain.
2 Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior de
Ingeniería Agronómica, Alimentaria y de Biosistemas, UPM, 28040-Madrid, Spain.
Corresponding author: Andrea Gómez Sánchez ([email protected])
Almost four-tenths of the world's agricultural fields lie in arid or semi-arid regions.
These areas are stringy expanding due to climate change. Besides, extreme
temperatures and drought are the most harmful stresses for plant productivity. Under
these conditions, plants respond developing some molecular and physiological
mechanisms. This strategy includes, among other important events, the dismantling
of the leaf cell organization, the closure of stomata, changes in cell cuticles,
alterations in the photosynthetic efficiency and modifications in proteolytic processes
like macromolecule degradation and mobilization of degraded compounds.
Cysteine Proteases (Cys-Prot) C1A of the papain family are the most abundant
enzymes involved in several physiological processes related to stresses. They are
induced by both biotic and abiotic stresses, and particularly by drought conditions. To
decipher the contribution of these proteases under different stresses is crucial due to
their impact on plant growth and grain yield and quality.
RT-PCR demonstrated the induction of HvPap-1 and HvPap-19 C1A proteases,
cathepsins F-like and B-like respectively, under drought conditions. In addition,
alterations in their proteolytic activities, modifications in the leaf protein content and
changes in some physiological parameters related to drought stress, highlighted their
function.
We used amiRNA knocked down lines of each cathepsin-encoding genes to unveil
their role when barley plants were subjected to abiotic (drought stress) and biotic
(mite infestation and fungal infection) stresses. To accomplish these goals, we used
a multidisciplinary approach including the measure of biochemical parameters,
molecular markers, proteomics and microscopy characterization of stress-challenged
leaves.
164
S-VII. Póster 14 DEVELOPMENT OF A HIGH THROUGHPUT BIOCHEMICAL
SCREENING TO DISCOVER NEW ABA RECEPTOR AGONISTS.
Irene García-Maquilón1, Andrea Chini2, Armando Albert3, Pedro L. Rodríguez1 and
Jorge Lozano-Juste1
1Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de
Investigaciones Científicas (CSIC), Universidad Politécnica de Valencia (UPV), Valencia,
Spain, 2Plant Molecular Genetics Department, National Centre for Biotechnology (CNB),
Consejo Superior de Investigaciones Científicas (CSIC), Campus University Autónoma,
Madrid, Spain, 3Instituto de Química Física Rocasolano, Consejo Superior de
Investigaciones Científicas (CSIC), Madrid, Spain.
Corresponding author: Jorge Lozano-Juste ([email protected])
Drought is one of the most important causes of productivity loss in crops worldwide.
Understanding the processes of adaptation and tolerance to water stress, especially
in plants with agronomic interest, it is a very important goal. The plant hormone
abscisic acid (ABA) regulates many developmental and stress responses in plants,
playing a fundamental role in the adaptation to water stress conditions. In order to
improve drought tolerance in economically important crops, we have developed a
novel chemical genetics screening aimed to identify ABA receptor agonists in the
recently established C4-crop model Setaria viridis. An in vitro biochemical assay
used to identify ABA binding to ABA receptors has been adapted to a 96-well plate
format allowing high-throughput screening of small molecules. The method has been
optimized considering the reproducibility of each test and the required amount of
protein in order to minimize variability and screening time. A library of ≈1000 small
molecules was screened along with proper controls using the S. viridis SvPYL1 ABA
receptor and the PP2C phosphatase HAB1. The screening is very reproducible
showing low variability among control data points. As a result, we have identified
IRE1, a compound able to mimic ABA both in vitro tests and in vivo experiments on
S. viridis and Arabidopsis thaliana seedlings. IRE1 binds to the S. viridis ABA
receptor, SvPYL1, inhibiting the phosphatase activity of HAB1 and other PP2Cs,
leading to inhibition of seed germination. Additionally, a second compound has been
identified, IRE2, which can act as a specific inhibitor of the phosphatase HAB1 and
enhances sensitivity to ABA in S. viridis and A. thaliana seedlings. This is the first
specific inhibitor of a PP2C phosphatase identified to date. S. viridis is a plant with
high agronomic interest since it is related to staple crops like maize and sorghum,
making these discoveries more relevant and eventually closer to be transferred to the
field. The method developed in this work, as well as the results obtained represent an
advance in the development of chemical molecules able to promote drought
tolerance.
Acknowledgements & Funding. Funding from MINECO (BIO2014-52537-R and BIO2017-82503-R) to
Pedro L. Rodríguez, EU grant H2020-MSCA-707477 to Jorge Lozano-Juste and Pedro L. Rodríguez,
and MINECO (BIO2017-89523-R) to Armando Albert is acknowledged.
165
S-VII. Póster 15
HISTONE H3K9 METHYLATION REDUCTION BY BIX-01294 PROMOTES CELL TOTIPOTENCY AND IMPROVES STRESS- INDUCED MICROSPORE EMBRYOGENESIS INITIATION IN
RAPESEED AND BARLEY
Eduardo Berenguer, Ivett Bárány, Teresa Solís, Yolanda Pérez-Pérez, María C. Risueño, Pilar S. Testillano
Pollen Biotechnology of Crop Plants, Biological Research Center, CIB-CSIC, Madrid, Spain.
Corresponding author: Pilar S. Testillano ([email protected])
After external stress signals, the microspore can reprogram to become a totipotent cell that develops into an embryo through in vitro microspore embryogenesis, a widely used biotechnological process in plant breeding for rapid production of doubled-haploids, but its regulating mechanisms are still largely unknown. Increasing evidence has revealed epigenetic reprogramming during microspore embryogenesis, through DNA methylation, but less is known about the involvement of histone modifications. In this work, we have analyzed the dynamics and possible role of histone H3K9 methylation, a major repressive modification, as well as the effects on microspore embryogenesis initiation of BIX-01294, an inhibitor of histone methylation, tested for the first time in plants, in Brassica napus and Hordeum vulgare. Results revealed that microspore reprogramming and embryogenesis initiation involved low levels of H3K9 methylation. With the progression of embryogenesis, methylation of H3K9 increased, correlating with gene expression profiles of histone methyl transferase BnHKMT SUVR4-like and histone demethylase BnLSD1-like. BIX-01294 treatments promoted cell reprogramming, totipotency and embryogenesis induction, while diminishing bulk H3K9 methylation. By contrast, long BIX-01294 treatments impaired embryogenesis progression, indicating that H3K9 methylation is required for embryo differentiation. These findings open up new possibilities to enhance microspore embryogenesis efficiency in recalcitrant species through pharmacological modulation of histone methylation by using the bioactive small molecule BIX-01294.
Reference: Berenguer E, Bárány I, Solís MT, Pérez-Pérez Y, Risueño MC,Testillano PS (2017) Inhibition of histone H3K9 methylation by BIX-01294 promotes stress-induced microspore totipotency and enhances embryogenesis initiation. Front. Plant Sci. 8, 1161.
Funding:
Supported by projects (AGL2014-52028-R, AGL2017-82447-R) funded by MINECO and ERDF/ FEDER.
166
S-VII. Póster 16
GENOME-WIDE ASSOCIATION STUDIES REVEAL NEW GENETIC
COMPONENTS INVOLVED IN SEED DETERIORATION RESISTANCE
Joan Renard Meseguer, Regina Niñoles Rodenes, Eduardo Bueso Ródenas,
Rubén Mateos Fernández, Irene Martínez Almonacid, Jose Gadea Vacas and
Ramón Serrano Salom
Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-
Consejo Superior de Investigaciones Científicas, 46022 Valencia, Spain
Joan Renard Meseguer ([email protected])
Seed life span, defined as the total time that seeds remain viable, from seed
dispersal until germination, is a crucial trait to understand the ecological mechanisms
of dispersion and also to preserve germplasm collection of endangered and
cultivated species. Seed longevity is influenced by environment but also by
endogenous seed factors, as protection structures, repair of non-functional molecules
and detoxification of toxic compounds, among others. In the last years, our laboratory
screened activation-tagging mutant collections of Arabidopsis thaliana in search of
dominant contributors to seed longevity and revealed the important role of the seed
coat in this process. We now extend the search of new actors contributing to this trait
by exploring natural variation. 280 Arabidopsis accessions have been tested for
resistance to natural and artificial seed aging treatments. Our data reveal high
variation in seed longevity among the different natural accessions. Correlations
between natural aging and the different artificial aging assays described in the
literature highlight the relevance of oxidation events in the natural seed deterioration
process. Genome-Wide Association Studies (GWAS) have been performed in order
to identify new genetic components involved in seed longevity. Our results points to a
multigenic trait with small effects. Knock-out lines of candidate genes are revealing
new genetic components involved in this important process of plant biology.
Funding still didn’t arrived.
167
S-VII. Póster 17
CHEMICAL ACTIVATION OF Setaria viridis ABA RECEPTORS
Irene Garcia-Maquilon1, Alfredo Manicardi1, Sttefany Rosario1,2, Andrea Chini3,
Armando Albert4, Pedro L. Rodriguez1, Jorge Lozano-Juste1.
1Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de
Investigaciones Científicas (CSIC), Universidad Politécnica de Valencia (UPV), Valencia,
Spain, 2Actual address: Laboratorio de Biología Molecular, Facultad de Agronomía,
Universidad Autónoma de Santo Domingo (UASD), Santo Domingo, República Dominicana, 3Plant Molecular Genetics Department, National Centre for Biotechnology (CNB), Consejo
Superior de Investigaciones Científicas (CSIC), Campus University Autónoma, Madrid,
Spain, 4Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones
Científicas (CSIC), Madrid, Spain.
Corresponding author: Jorge Lozano-Juste ([email protected])
Drought is the most important stress affecting crop yield worldwide. The plant
hormone abscisic acid (ABA) coordinates multiple physiological responses to tolerate
reduced water soil content. The PYR/PYL/RCAR family of ABA receptors are
responsible for ABA perception and activation of ABA responses and have been
described in detail in the eudicot model plant Arabidopsis thaliana. However, the
information about this protein family in crop plants remains scant. Setaria viridis
(setaria) has emerged lately as a great model system for C4-monocot crops like
maize or sorghum. Here, we describe the identification and characterization of S.
viridis ABA receptors. Additionally, we have leveraged this knowledge to design a
drug discovery approach to identify small molecules able to activate setaria ABA
receptors and stress tolerance. We have found one molecule, IRE1, able to engage
and activate SvPYL1 in vitro and in vivo. IRE1 is not related to any previously
described ABA receptor agonist and binds to SvPYL1 with an IC50 in the micromolar
range, being able to activate the ABA response in planta i.e. inhibition of seed
germination. Additionally, we also report on the identification of a second small
molecule, IRE2, able to trigger ABA responses by inhibiting HAB1, a key negative
regulator of ABA signalling. Finally, we have also developed a genome-editing
pipeline to generate mutants that will help us to understand ABA signalling and the
effect of ABA agonists in Setaria viridis. Future work is proposed for the improvement
of IRE1 and IRE2 in order to use them as agrochemicals to control stress tolerance
in the field.
Acknowledgements & Funding.
Funding from MINECO (BIO2014-52537-R and BIO2017-82503-R) to Pedro L. Rodriguez, EU grant
H2020-MSCA-707477 to Jorge Lozano-Juste and Pedro L. Rodriguez and MINECO (BIO2017-89523-
R) to Armando Albert is acknowledged.
168
S-VII. Póster 18
NEW ROLES OF ZF1-3 TRANSCRIPTION FACTORS IN THE
CONTROL OF NITROGEN ASSIMILATION AND EXPORT DURING
VEGETATIVE GROWTH
José Domínguez-Figueroa1, Begoña Renau-Morata2, Laura Carrillo1, Sharareh
Ghasemi1, Alba-R Corrales1, Mar González1, Stephan Pollmann1, Rosa-V Molina2,
Daniel Marino3, Javier Canales4, Martin Weih5, Jesús Vicente-Carbajosa1, Sergio G.
Nebauer2, Joaquín Medina1
1Centro de Biotecnología y Genómica de Plantas (UPM-INIA). Madrid, Spain, 2Universitat Politècnica de València. Valencia, 3Universdad del Pais Vasco (UPV/EHU) Bilbao, Spain, 4Universidad Austral de
Chile, Valdivia, Chile, 5Department of Crop Production Ecology, Swedish University of Agricultural
Sciences, Uppsala, Sweden. Corresponding author: Joaquín Medina. ([email protected])
Nitrogen is an essential macronutrient whose availability in soil is crucial for plant
growth and crop productivity. Nitrate is the main source of inorganic nitrogen for land
plants. However, it is usually limiting in the majority of the ecosystems, due to a
variety of biotic and abiotic factors like microbial consumption, water leaching and
soil erosion. Besides a macronutrient, nitrate is also a signal molecule that regulates
the expression of a variety of genes that modulate many aspects of metabolism,
growth and development. Here we identify the roles of ZF1 and ZF3, Arabidopsis
DOF transcription factors in nitrate responses. Loss-of-function null mutant zf3-1 and
zf1R exhibit nitrate-dependent root phenotypes, whereas ZF1 and ZF3
overexpression lines showed increased biomass and altered root architecture.
Transcriptomic analyses of overexpressor lines indicated that both factors regulate
the expression an important set of carbon and nitrogen regulated genes.
Consistently, metabolite profiling disclosed that the total amount of key nitrogen
metabolites including glutamate and glutamine were higher in ZF1/3-overexpressing
plants in N-sufficient and limiting conditions. By contrast, zf3-1 and zf1R mutants
exhibited lower amounts of those metabolites. In tomato, the overexpression of ZF1
and ZF3 genes modified amino acid and sugar content, improving biomass and yield.
These results identify ZF1 and ZF3 as important regulatory factors of the nitrate
response in Arabidopsis, suggesting that they are potential targets for improving
nutrient use efficiency in crops.
Funding: RTA2015-00014-c02-01 (INIA); REDI170024 (CONICYT).
169
S-VII. Póster 19
ROLE OF FATTY ACID DESATURASES AND UNSATURATED
FATTY ACIDS IN THE RESPONSE TO ABIOTIC STRESSES IN OLIVE
(Olea europaea) MESOCARP
M. Luisa Hernández, M. Dolores Sicardo, Patricia M. Arjona, José M. Martínez-Rivas
Department of Biochemistry and Molecular Biology of Plant Products, Instituto de la Grasa
(IG-CSIC), Sevilla, Spain.
Corresponding author: José M. Martínez-Rivas ([email protected])
Plants modify the unsaturated fatty acid content of their membranes and storage
lipids in order to adapt to environmental changes. This response is largely controlled
by the activity of fatty acid desaturases. In higher plants, the first desaturation step is
catalysed by the stearoyl-acyl carrier protein desaturase (SAD), leading to oleic acid,
which can be further desaturated to linoleic and α-linolenic acids by membrane-
bound fatty desaturases, that differ in their cellular localization, lipid substrates, and
electron donor system. The microsomal oleate (FAD2) and linoleate (FAD3)
desaturases are located in the endoplasmic reticulum, whereas the plastidial oleate
(FAD6) and linoleate (FAD7/8) desaturases are located in the plastid. The aim of the
present study was to investigate the regulation of olive fatty acid desaturases by
different environmental factors. To this end, their gene expression levels and the
unsaturated fatty acid composition in different lipid classes were determined in the
mesocarp tissue of olive fruit from Picual and Arbequina cultivars subjected to
different abiotic stresses. The results showed that SAD, FAD2, FAD6 and FAD7
genes are transcriptionally regulated by low and high temperatures, and light. In
addition, SAD, FAD2 and FAD7 are involved in the wounding response of olive
mesocarp. Finally, water deficit also regulates the expression levels of FAD2 and
FAD7 genes. The physiological roles of fatty acid desaturases and unsaturated fatty
acids in the olive mesocarp stress response will be discussed.
170
S-VII. Póster 20
SINGLET OXYGEN-MEDIATED CELL DEATH IN THE ZEAXANTHIN
EPOXIDASE DEFECTIVE MUTANT aba1 OF Arabidopsis thaliana
UNDER HIGH LIGHT
Álvaro Sánchez-Corrionero1,2,3, Inmaculada Sánchez-Vicente2, Sergio González1,
Ascensión Corrales1,2, Anja Krieger-Liszkay4, Óscar Lorenzo2 and Juan B. Arellano1
1Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Salamanca,
Spain, 2Instituto Hispano-Luso de Investigaciones Agrarias (CIALE), Universidad de
Salamanca, Salamanca, Spain, 3Center for Plant Genomics and Biotechnology, Universidad
Politécnica de Madrid, Pozuelo de Alarcón, Spain. 4Institute for Integrative Biology of the
Cell, Université Paris-Saclay, Gif-sur-Yvette cedex, France.
Corresponding author: Juan B. Arellano ([email protected])
The two Arabidopsis thaliana mutants, aba1 and max4, were previously identified as
sharing a number of co-regulated genes with both the flu mutant and Arabidopsis cell
suspension cultures exposed to high light (HL). On this basis, we investigated
whether aba1 and max4 were generating high amounts of singlet oxygen (1O2) and
activating 1O2-mediated cell death. Thylakoids of aba1 produced twice as much 1O2
as thylakoids of max4 and wild type (WT) plants when illuminated with strong red
light. Up-regulation of the 1O2 responsive gene AAA-ATPase was only observed with
statistical significant in aba1 under HL. Two early jasmonate (JA)-responsive genes,
JAZ1 and JAZ5, encoding for two repressor proteins involved in the negative
feedback regulation of JA signalling, were not up-regulated to the WT plant levels.
Chloroplast rupture and eventual cell death was observed in aba1 by confocal
imaging. In conclusion, aba1 may serve as an alternative model to other 1O2-
overproducing mutants of Arabidopsis for investigating 1O2-mediated cell death.
EcoSeed-311840, AGL2013-41363-R, BIO2017-85758-R, CSI083U16, SA239U13 and SA093U16.
171
3
3
S-VII. Póster 21
NRT2.5 A PUTATIVE SODIUM DEPENDENT HIGH AFFINITY
NITRATE TRASNPORTER OF Zostera marina L.
Lourdes Rubio1, Jordi Díaz-García1, Miguel A. Botella2, José A. Fernández1. 1Department of Biología Vegetal, University of Málaga, Málaga, Spain, 2Deparment of
Bioquímica y Biología Molecular, University of Málaga, Málaga, Spain. Corresponding author: Lourdes Rubio ([email protected])
Seagrasses are the only group of vascular plants that recolonized the marine environment, possibly the most severe habitat shift ever accomplished by flowering plants. These plants have regained functions enabling them to thrive in liquid medium with an extremely high salinity (0.5 M NaCl), high alkaline conditions (pH 8.2) and
- very low concentration of essential nutrients as NO3 or Pi. Despite this, seagrasses form one of the highest productive and widespread ecosystems of the planet (Larkum et al., 2006). Zostera marina (L.) was the first seagrass fully sequenced and its genome reveals important insights about this secondary adaption (Olsen el al., 2016). Comparison with land plants indicates that less than 20 % of the genes families are specific in the genome of seagrasses (Olsen et al., 2016). Thus, adaptation to marine environment seems to be due to molecular changes of the same family genes rather that the speciation of pre-existing genes. This appears to be the case of the high affinity nitrate transporter belonging to the NRT family. In contrast to terrestrial vascular plants, where NRT2 encode high affinity NO -
transporters that operate as H+ symporters, our electrophysiological analysis indicate that in Z. marina high affinity NO -
uptake is mediated by a Na+-dependent mechanism (Rubio et al., 2005). A detailed analysis of the Z. marina genome indicates the presence of only one gene encoding for this type of transporter: Zosma70g00300.1. Phylogenetic analysis shows that this high affinity nitrate transporter is more related to NRT2.5 than to NTRT2.1, sharing a common ancestor with both, monocot and dicot plants. We have cloned Zosma70g00300.1 and the high-affinity nitrate transporter accessory protein NAR2 (Zosma63g00220.1) in order to characterize the specific transport mechanism mediated by these proteins in Z. marina. Thus, the putative Z. marina NRT2.5 transporter could have evolved to use
Na+ as a driving ion, which might be an essential adaptation of seagrasses to colonize the marine environment.
References. Larkum, W. D., Orth, R. J. & Duarte, C. M. 2006. Seagrasses: Biology, Ecology and Conservation Springer, Netherlands,
Olsen, J.L., Rouzé, P., Verhelst, B., Lin, Y-C., Bayer, T. et al. 2016. The genome of the seagrass Zostera marina reveals angiosperm adaptation to the sea. Nature 530, 331–335.
Rubio, L., Linares-Rueda, A., García-Sánchez, M.J. & Fernández J.A. 2005. Physiological evidence for a sodium-dependent high-affinity phosphate and nitrate transport at the plasma membrane of leaf and root cells of Zostera marina L. Journal of Experimental Botany 56, 613-622.
Acknowledgements & Funding This work is financed by the Spanish MICINN (BFU2017-85117-R and BIO2016-81957-REDT).
172
S-VII. Póster 22 ROLE OF SLSKOR IN SHOOT K+ TRANSLOCATION AND
ADAPTATION TO SALT STRESS IN TOMATO PLANTS
Manuel Nieves-Cordones1, Jesus Amo1, Alberto Lara1, Reyes Ródenas1, Francisco
Rubio1.
1Departamento de Nutrición Vegetal, CEBAS-CSIC, 30100 Murcia, Spain
Corresponding author: Manuel Nieves-Cordones ([email protected] )
Among the essential elements for plants, potassium (K+) plays a crucial role in crop
growth and yield and its correct supply and distribution in plants allow them to be
more tolerant to different abiotic and biotic stresses. K+ is taken up by roots and then
loaded into the xylem (together with other nutrients and water) to be distributed in
aerial parts (Nieves-Cordones, et al., 2016). In Arabidopsis it was shown that the K+
channel SKOR plays an important role in xylem K+ loading (Gaymard, et al., 1998). In
this work we have gained insights into the function of SlSKOR (tomato homolog of
SKOR) in tomato plants following two approaches: (i) functional characterization of
SlSKOR in Xenopus oocytes with the two-electrode voltage clamp technique (TEVC)
(Miller and Zhou, 2000) and (ii) the study of their contribution to xylem K+ load by
generating loss-of-function (slskor plants) mutants with the CRISPR-Cas technique
(Scheben, et al., 2017). Analyses of oocytes injected with SlSKOR cRNA revealed
that it encodes a voltage-dependent K+-selective channel with strong outward-
rectification. In parallel, the SlSKOR locus was efficiently edited with the CRISPR-
Cas technology which led to bi-allelic frame-shift mutations in the coding sequence
that produced KO mutants. Phenotyping of slskor plants revealed a significant
reduction in the stem K+ concentration in comparison to WT plants when grown
under salt stress. This information will allow the design of strategies aiming at
improving K+ circulation and allocation in tomato plants based on the role of SlSKOR.
References
Gaymard, F. et al. Cell 94, 647-655, (1998). Miller, A. J. et al. Biochim Biophys Acta 1465, 343-358, (2000). Nieves-Cordones, M. et al. in Metal ions in life sciences Vol. 16 291-324 (2016). Scheben, A. et al. New Phytol 216, 682-698, (2017).
Acknowledgements & Funding
This work was supported by grant numbers AGL2015-66434-R (to F. R.) and AGL2015-74011-JIN (to
M. N.-C.) from Ministerio de Economía y Competitividad, Spain. A. L.-H. and R. R. are recipients of a
F. P. U. Fellowship from Ministerio de Educación, Cultura y Deporte, Spain. This work was also
supported by the Red de Excelencia BIO2014-56153-REDT.
173
S-VII. Póster 23
HSF4 PARTICIPATES IN THERMOINHIBITION OF SEED
GERMINATION IN ARABIDOPSIS
Isabel Mateos1, Luis Quintero1, María Dolores Rodríguez1 y Oscar Lorenzo 1
1Dpto. de Botánica y Fisiología Vegetal, CIALE, Universidad de Salamanca, Salamanca,
Spain
Corresponding author: Oscar Lorenzo ([email protected])
Nitric oxide (NO) is a reactive nitrogen molecule that participates in a plethora of
physiological processes during whole plant life. In our lab, we searched out a role of
NO on germination (1) and primary root growth in Arabidopsis (2). However, despite
its relevance as a plant growth and stress regulator, the NO signalling pathway is still
unknown. Therefore, our main objective in this work is the identification and
characterization of transcription factors (TFs) involved in the NO signal transduction
pathway. To achieve this goal, we used a collection of Arabidopsis transgenic plants
conditionally expressing TFs under a β-estradiol inducible promoter (3), and we
studied their germination and root phenotype in the presence and absence of the NO
donor SNAP. In this screening a total of 1261 transgenic lines corresponding to 516
TFs were analyzed. Some of these lines displayed differential root phenotypes in the
presence of NO at early plant development as compared to the wild type. One of the
NO-insensitive lines overexpresses HSF4 gene. This TF has been described as a
repressor of the expression of heat inducible genes and a regulator of acquired
thermotolerance (4), a molecular switch for plant growth to defense transition (5) and
as a component of the unfolded protein response (6). In our lab, we are especially
interested in the response to heat during germination. During this stress an increase
in NO production occurs. In fact, a deficient-NO mutant is able to germinate faster
than wild type after heat stress. After HSF4 overexpression seeds can germinate
faster than the wild type. This result provides us a new function of this gene during
germination at high temperature.
(1) Albertos et al., (2015) Nat. Comm. 6; (2) Fernández-Marcos et al. (2011) PNAS 108, 18506-18511; (3). Coego et al. (2014) Plant J 77, 944-53; (4) Ikeda el al., (2011) Plant Phys. 157, 1243-1254; (5) Pajeroska-Mukhtar et al., (2012) Current Biol. 22, 103:102; (6) Hossain et al., (2016) Front. Plant Science 7, 650.
Acknowledgment: TRANSPLANTA CONSOLIDER (CSD2007-00057), Fundación memoria de Don
Manuel Solorzano Barruso (FS/12-2017), Junta de Castilla y León (SA093U16) and MINECO
(BIO2017-85758-R).
174
S-VII. Póster 24 THE UB-CONJUGATING ENZYME UBC26 AND THE E3 UBIQUITIN
LIGASE RFA4 TARGET ABA RECEPTORS IN NUCLEUS
María A. Fernández1, Borja Belda-Palazon1, Jose Julián1, Alberto Coego1, Jorge Lozano- Juste1, Sabrina Iñigo2, Alain Goossens2 and Pedro L. Rodríguez1
1Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones
Científicas-Universidad Politécnica de Valencia, Valencia, Spain 2Department of Plant Systems
Biology, VIB, Ghent University, Ghent, Belgium
We have identified a 10-member family of single-subunit E3 ubiquitin ligases, named RFA for RING finger ABA-related, acting as E3 ligases of the PYR/PYL/RCAR ABA receptors and therefore controlling their ubiquitination and half-life. The RFA family is different from the CULLIN4-RING E3 ubiquitin ligase (CRL4) complex that interacts with ABA receptors through the substrate adapter DDB1-ASSOCIATED1 (Irigoyen et al., 2014). RFAs are characterized by the presence of three putative RING domains in tandem (Bueso et al., 2014) and according to this structure they belong to RBR (RING between RING fingers) E3 ligases (Marin et al., 2010). Very little is known regarding the biological function RBR-type E3 Ub ligases in plants. In contrast, the human RBR Parkin has been extensively studied because of its association with Parkinson’s disease. Here we demonstrated that ABA receptors are targeted by RFA1 and RFA4 RBR-type E3 ligases, which are members of the RSL1/RFA family not associated to plasma membrane as RSL1. RFA4 shows specific nuclear localization whereas RFA1 is localized both in nucleus and cytosol, where they interact with ABA receptors. Therefore, different members of the RSL1/RFA family interact with ABA receptors in different subcellular locations, targeting them for degradation via the endosome/vacuolar or 26S proteasome degradation pathways. RFA1 and RFA4 promote degradation of ABA receptors in vivo, and for instance, we demonstrated that endogenous levels of PYR1 and PYL4 ABA receptors are increased in the rfa1 rfa4 double mutant compared to wt plants. We also identified UBC26 as the cognate nuclear E2 enzyme that interacts with the RFA4 E3 ligase and accordingly, we observed UBC26-RFA4-ABA Receptor complexes in nuclear speckles. Loss-of-function ubc26 alleles and rfa1 rfa4 double mutant showed enhanced sensitivity to ABA and accumulation of ABA receptors compared to wt. Altogether our results reveal a sophisticated Ub targeting of ABA receptors at different subcellular locations, which involves different members of the RBR- type RSL1/RFA family and other previously described E3 ligases.
References: Bueso E, Rodriguez L, Lorenzo-Orts L, Gonzalez-Guzman M, Sayas E, Muñoz-Bertomeu J, Ibañez C, Serrano R, Rodriguez PL.(2014). The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling. Plant J. Dec;80(6):1057-71.
Irigoyen M.L., Iniesto E., Rodriguez L., Puga M.I., Yanagawa Y., Pick E., Strickland E. et al (2014) Targeted degradation of abscisic acid receptors is mediated by the ubiquitin ligase substrate adaptor DDA1 in Arabidopsis. Plant Cell, 26, 712–728.
Marín I. (2010) Diversification and Specialization of Plant RBR Ubiquitin Ligases. PLoS One. 2010 Jul 14;5(7): e11579.
Acknowledgements: This work was supported by Ministerio de Ciencia e Innovacion, Fondo Europeo de Desarrollo Regional and Consejo Superior de Investigaciones Cientificas (gants BIO 2014-52537 to P.L.R., BIO 2017-82503 to P.L.R) and Ministerio de Educacion, Cultura y Deporte fellowships FPU15/05677 to M.A.F.L
175
S-VII. Póster 25 EXPLORING THE USE OF RECOMBINANT IMBRED LINES TO
IMPROVE NITROGEN USE EFFICIENCY UNDER ABIOTIC STRESS
COMBINATION IN TOMATO PLANTS
Maria Lopez de la Calle1, Vicente Martínez1, Rosa M Rivero1*. 1Department of Plant Nutrition, CEBAS-CSIC, Campus Universitario Espinardo, Ed 25,
Espinardo, Murcia, SPAIN *Corresponding author: Rosa M Rivero ([email protected])
The development of varieties with a better nitrogen use efficiency (NUE) is a means for modern agriculture to decrease environmental pollution by nitrates and to maintain a sufficient net income. On the other side, environmental conditions for agriculture tends to be more adverse in the coming years, with increases of temperatures, water scarcity and salinity being at the top of the productivity constrains for plants. NUE is inherently a complex trait, as each step-including N uptake, translocation, assimilation, and remobilization-is governed by multiple interacting genetic and environmental factors. In this study, 3 recombinant lines (RL) from a cross between S. lycopersicum and S. pimpinellifoilum with different degree of tolerance to the combination of salinity and heat were subjected to an ionomic, metabolomic and transcriptomic study to better understand how nitrogen metabolism is affected in tolerant plants as compared to
sensitive ones. Ionomics showed a different profile among the 3 RL, being K+ and Mg2+
significantly lower in RL18 (low tolerant) as compared to RL66 (medium tolerant) and RL76 (high tolerant) under salinity and heat combination. No differences were shown
-
among the 3 RL in N total content; however, N-NO3 was significantly higher in RL18 whereas N-Norg was lower as compared to the other genotypes. RL76 showed a significant increase in N-Norg which could be correlated with its tolerance to the combination of salinity and heat. Total proteins and total amino acids concentration were significantly higher in RL76 as compared to the others RL under these conditions. Glutamate, but more importantly glutamine, was also highly synthesized and accumulated in RL76 under the combination of salinity and heat, which was in concordance with the total amino acids and proteins found in this line, since glutamine is the precursor in their synthesis, this was also in agreement with the upregulation found for the nitrogen-related transcripts studied (SlNR, SlNiR, SlGDH, SlGS) in this RL as
compared to the other two lines. This study empathized the importance of studying abiotic stress in combination and how recombinant material with different degrees of tolerance can be highly important for the improvement of NUE in horticultural plants.
References
1. Anwar, F., M. H. Siddiqui, S. A. Salem, M. H. Al-Whaibi, and C. Abhishek. 2011. Nitrogen Use- Efficiency and Crop Production - A Mini Review. Environment We International Journal Science Technology 6: 167–74
2. Dechorgnat, J., C. T. Nguyen, P. Armengaud, M. Jossier, E. Diatloff, S. Filleur, and F. Daniel-Vedele.
2011. From the soil to the seeds: the long journey of nitrate in plants. Journal of Experimental Botany 62: 1349–59.
3. Masclaux-Daubresse, C., F. Daniel-Vedel, J. Dechorgnat, F. Chardon, and L. Gaufichon. 2010.
Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture. Annals of Botany 105: 1141– 57.
Acknowledgements & Funding. We thank the Metabolomics Core for the quantification of amino acids and other compounds, and the Ionomics Core of CEBAS-CSIC for the quantification of cations, ions and N forms. This work was supported by the Ministry of Economy and Competitiveness from Spain (GrantNo.AGL2015-66033-R).
176
S-VII. Póster 26
GENETIC DETERMINANTS OF VITAMIN C CONTENT IN HIGHER
PLANTS.
Mario1 Fenech-Torres1, Vitor1 Amorim-Silva1, Nicholas2 Smirnoff2, Victoriano1
Valpuesta1, Miguel Ángel1 Botella1
1Laboratorio de Bioquímica y Biotecnología Vegetal, Instituto de Hortofruticultura Subtropical
y Mediterránea (IHSM), Universidad de Málaga-Consejo Superior de Investigaciones
Científicas. Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, UMA,
Málaga, Spain, 2Biosciences, College of Life and Environmental Sciences, University of
Exeter, United Kingdom.
Corresponding author: Miguel Ángel Botella ([email protected])
Ascorbic acid (AsA, VitC) is the most abundant water-soluble antioxidant in plants
and it plays a plethora of biological roles including resistance to abiotic stress.
Hence, including VitC as a trait to improve in breeding programs is not only a way to
enhance food quality but also to increase resistance to expected environmental
alterations due to global change like drought, salinity or heat. Although all
components of the Smirnoff-Wheeler pathway of AsA in plants are known, little
information is available about how their regulation at the biochemical and cellular
levels is. We have generated a number of molecular tools such as tagged constructs,
stable transgenics and mutant lines with the aim of getting detailed information about
how this pathway operate in plants. We will present data on protein localization,
interaction among different components and their role in affecting VitC levels using a
heterologous system such as Nicotiana bethamiana.
This research was supported by a grant from the Spanish Ministerio de Educación, Cultura y Deporte
para la formación del Profesorado Universitario (FPU014/01974), as well as by a project funded by the
Spanish Ministerio de Ciencia e Innovación (BIO2014-55380R; BIO2014-56153-REDT). We also
acknowledge the support by the Plan Propio from University of Malaga, Campus de Excelencia
Internacional de Andalucía.
177
3
3
S-VII. Póster 27 CHARACTERIZATION OF AtSLAH4, A ROOT ANION CHANNEL
INVOLVED IN NET CHLORIDE UPTAKE
Paloma Cubero-Font1, Pablo Díaz-Rueda1, Miguel A. Rosales1, Tobias Maierhofer2,
Julian Lehmann2, Juan D. Franco-Navarro1, Carlos Rivero1, Dietmar Geiger2, José
M. Colmenero-Flores1
1Plant Biotechnology Department, Instituto de Recursos Naturales y Agrobiología (IRNAS-
CSIC), Sevilla, España, 2Molecular Plant Physiology and Biophysics Department, Julius-von-
Sachs Institute, University of Würzburg, Würzburg, Germany.
Corresponding author: José M. Colmenero-Flores ([email protected])
Although generally considered a toxic anion, we have shown that besides of an
essential micronutrient, chloride (Cl−) is a beneficial macronutrient (Franco-Navarro
et al., 2016). Optimal growth of plants requires the synchronic supply of both Cl− and − −
nitrate (NO3 ) molecules, but salt stress causes excessive accumulation of Cl in
leaves that can produce ionic toxicity in Cl−-sensitive crops such as citrus and
grapevine.
Apart from the control of stomatal closure, members of the SLAC/SLAH family of
slow-type anion channels are involved in the regulation of NO −
− and Cl−
− homeostasis
in the root, including root-to-shoot transport and NO3 versus Cl discrimination
according to environmental cues (Cubero-Font et al., 2016). AtSLAH3 mediates the
loading of anions into the xylem vessels, with a high discrimination of NO − over Cl−.
Under optimal growing conditions, high expression of AtSLAH1 and SLAH1/SLAH3
heteromerization increase by seven times the Cl− conductance of SLAH3. In contrast,
salinity and water deficit abolish AtSLAH1 expression in an ABA-dependent manner,
strongly reducing root-to-shoot translocation of Cl− under abiotic stress (Cubero-Font
et al., 2016).
AtSLAH4, a highly homologous family member of AtSLAH1, is expressed in
peripheral root cell layers (Cubero-Font, 2017). Root anions uptake results from the
combined activity of active (influx) and passive (efflux) plasma membrane
transporters. Therefore, SLAH4 is expected to have an important contribution to the −
regulation of net Cl− and NO3 uptake. Data concerning the functional
characterization of AtSLAH4 (cell-type expression, developmental and stress-
dependent expression as well as knock-out lines phenotypes) will be presented.
Cubero-Font (2017) PhD Thesis, Universidad de Sevilla, Seville, Spain.
Cubero-Font P, Maierhofer T, Jaslan J, Rosales MA, Espartero J, Díaz-Rueda P, Müller HM, Hürter A-
L, Al-Rasheid KAS, Marten I, Hedrich R, Colmenero-Flores JM, Geiger D (2016). Current Biology 26
(16): 2213-2220.
Franco-Navarro JD, Brumos J, Rosales MA, Cubero-Font P, Talon M, Colmenero-Flores JM (2016).
Journal of Experimental Botany 67 (3): 873-891.
This work has been supported by the MINECO-FEDER grants AGL2009-08339/AGR and AGL2015-
71386-R and by the National Network BIO2014-56153-REDT. PC-F has been supported by the CSIC
JAE-PreDoc and the German Academic Exchange Service (DAAD) fellowships.
178
S-VII. Póster 28 RAV1 DOSE IS REQUIRED FOR POPLAR ADAPTATION TO LOW
AMBIENT TEMPERATURE DURING GROWTH-DORMANCY
TRANSITION
Paolo M. Triozzi1, Daniel Conde2, Alicia Moreno-Cortés1, Miguel Angel Torres1, Pablo González-Melendi1,3, Jerald D. Noble2, Chris Dervinis2, Matias Kirst2, Isabel Allona1,3
and Mariano Perales1. 1Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto
Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM,
28223 Pozuelo de Alarcón, Madrid, Spain,2School of Forest Resources and Conservation (SFRC),
University of Florida (UF), Gainesville, Florida, USA,3Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad
Politécnica de Madrid (UPM), 28040 Madrid, Spain. Corresponding author: [email protected]; [email protected]
Fine-tuning of environmental signals maximize plant growth and guarantee their survival. To anticipate the winter, poplar uses photoperiodic information as a warning system to trigger cold hardiness and dormancy. When photoperiod fall below a critical day-length, poplar ceases growth and initiates a cascade of physiological and morphological transformation leading to dormancy. Temporal transcriptome analyses unraveled the molecular framework operating during the photoperiodic-dependent induction of growth cessation, cold acclimation and bud set (Ruttink et al., 2007; Howe et al., 2015). However, functional characterization of potential regulatory candidate genes and molecular mechanisms involved are very little investigated. Among these candidates, a related to APETALA2 and Viviparous transcription factor, RAV1, was previously characterized in our laboratory as a regulator of sylleptic branching development in poplar (Moreno-Cortes et al., 2012; Moreno-Cortes et al., 2017). Additionally, we found that RAV1 is accumulated at the RNA and protein level during growth- dormancy transition. In this work, we show that poplar response to short days and low ambient temperature depends on RAV1 doses. RAV1 overexpressing transgenic plants showed earlier bud set and higher leaf turgor pressure than wild type and RAV1 knock down plants, suggesting that RAV1 mediates adaptation to low ambient temperature. RNA-seq analyses showed that RAV1 constitutive expression induces poplar orthologs to ETHYLENE RESPONSE FACTOR TYPE VII transcription factor and plasma membrane RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN-LIKE (RBOH-like), which have been shown to create ROS-dependent tolerance to abiotic stress in Arabidopsis (Yao et al., 2017). Moreover, annual transcriptional profiling showed RAV1, ERF and RBOH transcripts are induced in the transition from summer to winter. Thus, we propose that RAV1-ERF-RBOH module contributes to seasonal plant adaptation to low ambient temperature. References Howe, G. T., Horvath, D. P., Dharmawardhana, P., Priest, H. D., Mockler, T. C., & Strauss, S. H. (2015). Extensive Transcriptome Changes During Natural Onset and Release of Vegetative Bud Dormancy in Populus. Frontiers in Plant Science, 6. Moreno-Cortés, A., Hernández-Verdeja, T., Sánchez-Jiménez, P., González-Melendi, P., Aragoncillo, C., & Allona, I. (2012). CsRAV1 induces sylleptic branching in hybrid poplar. New Phytologist, 194(1), 83–90. Moreno-Cortés, A., Ramos-Sánchez, J. M., Hernández-Verdeja, T., González-Melendi, P., Alves, A., Simões, R.,
… Allona, I. (2017). Impact of RAV1-engineering on poplar biomass production: A short-rotation coppice field trial. Biotechnology for Biofuels, 10(1). Ruttink, T., Arend, M., Morreel, K., Storme, V., Rombauts, S., Fromm, J., … Rohde, A. (2007). A molecular timetable for apical bud formation and dormancy induction in poplar. The Plant Cell, 19(8), 2370–90. Yao, Y., He, R. J., Xie, Q. L., Zhao, X. hai, Deng, X. mei, He, J. bo, … Wu, A. M. (2017). ETHYLENE RESPONSE FACTOR 74 (ERF74) plays an essential role in controlling a respiratory burst oxidase homolog D (RbohD)- dependent mechanism in response to different stresses in Arabidopsis. New Phytologist, 213(4), 1667–1681.
Acknowledgements & Funding This study was supported by Grants AGL2014-53352-R, PCIG13-GA-2013-631630 awarded to I.A. and M.P.;
M.P. was supported by the Ramón y Cajal programme of MINECO (RYC-2012-10194). P.M.T. Ph.D. programme of CEI campus of the Universidad Politécnica de Madrid (L1UF00-47-JX9FYF).
179
S-VII. Póster 29
UNCOVERING THE ROLE OF FLAVONOIDS IN SEED LONGEVITY
Regina1 Niñoles1, Raquel1 Bertí1, Thomas2 Roach2, Ilse2 Kranner2, Ramón1 Serrano1, Jose1 Gadea1
1Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Valencia, España.
2Institute of Botany, Functional Plant Biology, University of Innsbruck, Innsbruck, Austria Corresponding author: Jose Gadea ([email protected])
Seed longevity is defined as the total time span during which seeds remain viable. As seeds constitute the main system for plant propagation, increasing their longevity is a major challenge for the conservation of plant biodiversity and for crop success. Therefore, it is an important trait for ecology, agronomy and economy.
In order to optimize their life span, seeds possess complex systems of protection, detoxification and repair (Rajjou and Debeaujon, 2008). The protective mechanisms include the presence of the seed coat, which performs important functions to protect the embryo from biotic and abiotic stresses during storage, the formation of a glassy cytoplasm to reduce cellular metabolic activities and the production of antioxidants, that prevent accumulation of oxidized macromolecules during seed storage. Enzymatic antioxidants also play a role in ROS detoxification. The repair system removes damage accumulated in DNA, RNA and proteins upon seed imbibition.
Flavonoids are antioxidant secondary metabolites that accumulate in most plant seeds (testa and embryo) and are involved in many physiological functions, including seed longevity (Debeaujon et al. 2000). The protective role of these secondary metabolites, such as vitamin E or flavonoids, during oxidative stress is well documented. However, it is very likely that flavonoid protective role at the cell level is not due exclusively to their antioxidant properties as there is increasing evidence that flavonoids may also have signaling functions (Stevenson, R.D. Hurst, 2008) . On the other hand, testa flavonoids such as proanthocyanidins also increase impermeability to tetrazolium salts (Debeaujon et al., 2000).
In this work we study the role of flavonoids in seed longevity using flavonoid biosynthetic mutants and analysing their performance after accelerated ageing treatments, their behaviour during oxidative stress and their seed permeability.
References 1- I. Debeaujon K.M., Léon-Kloosterziel, M., Koornneef, Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis, Plant Physiol. 122 (2000) 403–413
2- Rajjou L, Debeaujon I. Seed longevity: survival and maintenance of high germination ability of dry seeds.C R Biol. (2008) Oct;331(10):796-805. doi: 10.1016/j.crvi.2008.07.021.
3- Stevenson D.E., Hurst R.D., Polyphenolic phytochemicals – just antioxidants or much more? Cel. Mol. Life Sci. 64 (2007) 2900–2916.
Funding: Ministerio de Economía, Industria y Competitividad. Ref:BIO2014-52621-R
180
S-VII. Póster 30
IDENTIFICATION OF NITRATE-DEPENDENT Na+ UPTAKE IN
ARABIDOPSIS ROOTS THROUGH TRANSCRIPTOMIC PROFILING
Rocío Álvarez-Aragón, Lorena Blázquez and Begoña Benito
Centro de Biotecnología y Genómica de Plantas (CBGP), Universidad Politécnica de Madrid
(UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA).
Campus Montegancedo UPM. 28223-Pozuelo de Alarcón (Madrid), Spain.
Corresponding author: Begoña Benito ([email protected])
Despite intensive research for more than 50 years, how Na+ enters the plant is still unknown. Previously we found out that under saline conditions Na+ entrance in Arabidopsis takes place largely in a nitrate-dependent manner (Álvarez-Aragón and Rodriguez-Navarro, 2017), suggesting that Na+ may enter the root through a nitrate transporter. A large family of at least 53 genes in Arabidopsis encodes nitrate transporters. Therefore, the identification of which transporters are involved in Na+
uptake may be a hard task. In this study we performed a transcriptomic analysis to identify transporters differentially expressed during Na+ root entry. Using Illumina RNA-Seq transcriptome profiling, we compared the expression of genes encoding putative transporters or channels in hydroponic-grown plants in control conditions (MES without Na+) or in two different saline conditions, nitrate independent Na-MES (MES with 20 mM Na+) or nitrate dependent NaNO3 (20 mM NO3 and 20 mM Na+). A total of 53 and 59 differentially expressed genes (DEGs) were identified under Na-MES and NaNO3
treatments compared to control with less than 20% overlap between both gene sets. Indeed, 42 DEGs encoding transporters were identified in a comparison between NaNO3 and Na-MES conditions. We hypothesized that the transporters responsible for nitrate-dependent Na+ entrance will be among this latter group and therefore we have selected some genes for further study. In addition, we also identified candidates for nitrate-independent Na+ uptake among DEGs from Na-MES vs control comparison. We are carrying out a phenotypic study of Na+ accumulation of the respective Arabidopsis defective mutants and a functional characterization in Na+
defective yeast mutants that should allow us to identify the transporters involved in the Na+ root entrance by plant roots.
References
Álvarez-Aragón and Rodríguez-Navarro, 2017. The Plant Journal, 91: 208-219
Funded by grants AGL2012-36174, AGL2016-80593-R and BIO2014-56153-REDT from MINECO.
181
S-VII. Póster 31
FIP1-MEDIATED ALTERNATIVE POLYADENYLATION MEDIATE ROOT DEVELOPMENT AND SALT RESPONSES
Sara Navarro-Neila1, Barbara Telléz-Robledo1, Concepcion Manzano1, Angela Saez1, 3, Javier Silva-Navas1, Laura de Lorenzo2, Arthur G. Hunt2, Roberto Baigorri3, Carlos
del Pozo1
1 Centro de Biotecnología y Genómica de Plantas (CBGP). Instituto Nacional de
Investigación y Tecnología Agraria y Alimentaria. Madrid, Spain.2 Department of Plant and
Soil Sciences, University of Kentucky, Lexington, KY, 40546-0312, USA.3 CIPAV, Timac Agro Int-Roullier Group, Orcoyen, Spain.
Corresponding author: Carlos del Pozo: [email protected]
Root system development and responses to environmental changes, including
excess of salt in the environment is crucial for whole plant adaptation. Lateral roots
are postembryonic organs that develop from the main root with a well-defined
genetic-temporal program. The formation of lateral roots imply specific changes in
gene transcript, accompanying with alternative splicing, as well as posttranslational
modification of proteins. Here we report an additional level of regulation, the
alternative polyadenylation (APA) of mRNA. We isolated a viable mutant that affects
FIP1 activity, a component of the polyadenylation machinery. The fip1-2 mutant has
fewer emerged lateral roots and shows a lateral root primordia arrest in early stages.
In addition, this mutant shows defects in cell division and elongation in the root apical
meristem and in leaves. Mutation in FIP1 also affects ion accumulation and
potentiates root growth in medium without phosphate. Genomic studies of poly(A)
site usage show that mutation in FIP1 increases the amount of mRNA with
preferential polyadenylation signal in the coding region, introns and 5´-UTR. Gene
ontology analyses of root deregulated transcripts in fip1-2 mutant and genes with
differential APA shows an enrichment in genes involved in ABA and salt responses,
indicating a role of APA in these responses. In fact, fip1-2 has an altered response to
ABA and is more tolerant to salt than control plants. Our data indicate that FIP1-
mediated alternative polyadenylation is important for plant development as well as for
stress responses.
This work has been funded by grants from the Spanish Government BIO2011-28184-C02-01, CSD2007-00057 and BIO2014-52091-R to Carlos del Pozo.
182
S-VII. Póster 32
UNRAVELING THE ROLE OF NITRIC OXIDE IN SEEDLING GROWTH UNDER CADMIUM STRESS
Laura C. Terrón-Camero1, Coral Del Val2, Luisa M. Sandalio1, María C. Romero-
Puertas1.
1Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental
del Zaidín, CSIC, C/Profesor Albareda 1, E-18008 Granada, Spain; 2Department of Artificial
Intelligence, University of Granada, E-18071 Granada, Spain.
Corresponding author: María C. Romero-Puertas ([email protected])
The heavy metal Cadmium (Cd) enters the environment mainly via phosphate
fertilizers as well as industrial and mining activity. The principal problem with Cd
accumulation is that the metal is taken up by plants through the roots and is rapidly
transferred to the food chain since it can be loaded into the xylem and then
transported to the leaves and fruits. Nitric oxide (NO), a hydrophobic gaseous
molecule, is a diffusible free radical which acts as a biological mediator in key
physiological processes such as plant adaptive responses to both biotic and abiotic
stresses (Delledonne 2005; Romero-Puertas et al., 2013). Although NO has
recently been reported to be involved in Cd responses in plants, the molecular
mechanism(s) underpinning its function and sources remain(s) unclear (Besson-
Bard et al. 2009). In this study, we have cross-examined both public and in-house
data sets derived from the profiling of Arabidopsis gene expression in response to
Cd and NO to determine the main functional categories in which the free radical
may be involved. We also investigated the functions of different NO-related
pathways in Arabidopsis by studying the response to Cd stress of mutants with
altered NO levels. Thus, the effects of different NO levels and the involvement of
various NO sources in seedling growth in Cd will be discussed with the aid of a
bioinformatic analysis of our findings.
Besson-Bard, A., et al. (2009) Plant physiology 149, 1302-1315
Delledonne (2005) Current opinion in plant biology, 8:390-6
Romero-Puertas, et al. (2013) Frontiers in Plant Science, 373.
This study was funded by an ERDF grant co-financed by the Ministry of Economy, Industry and
Competitiveness (BIO2015-67657-P). L.C. T-C was supported by a University Staff Training
Program (FPU) fellowship from the Spanish Ministry of Education, Culture and Sports.
183
S-VII. Póster 33
NATURAL VARIATION OF TRANSCRIPTOMIC AND PHYSIOLOGICAL RESPONSES TO PHOSPHATE STARVATION:
DIFFERENT DISTRIBUTION OF CIS AND TRANS EQTLS OVER THE ARABIDOPSIS GENOME AND IDENTIFICATION OF A MAJOR QTL
CONTROLLING ROOTSOOT GROWTH RATIO
Sergio Diaz*, Miguel Miñambres*, Erica Gil Velasco, David San León, Antonio
Leyva, Carlos Alonso, Javier Paz-Ares.Centro Nacional de Biotecnología-CSIC, Campus
Univ Autónoma, 28049Madrid (* co-first authors). Corresponding author: Javier Paz-Ares
Phosphorus (P) is one of the most important nutrients for all living organisms.
Although it is abundant in ecosystems, plants suffer from P limitation in many soils
because phosphate (Pi), the form in which plants take P, is quite insoluble. To cope
with growth under low Pi conditions, plants have evolved different adaptative
responses. These are known as Pi starvation responses (PSR) and consist of
physiological and morphological adaptations, which are associated with changes in
around twenty percent of the transcriptome. Several Pi starvation signaling
components have been identified, including key transcription factors; components of
Pi sensing; Pi starvation induced microRNAs; noncoding RNAs regulating
microRNAs; components of the ubiquitination system; and a endoplasmic reticulum
(ER) exit cofactor and a protein kinase that positively and negatively control the exit
from the ER of Pi transporters, respectively. Natural variation can be exploited to
analyze traits that display quantitative variation. In this context, we performed an
expression quantitative trait locus (eQTL) analysis of the Pi starvation transcriptome
in Arabidopsis thaliana using the recombinant inbred line (RIL) population Ct-1 x Col-
0. We identified more than 10000 eQTLs (8800 cis and 3300 trans eQTLs). Trans
eQTLs are more highly enriched in Pi starvation responsive genes compared to cis
eQTLs (2x vs 1.2x, respectively), which may suggest that the effect of trans eQTLs
displays higher specificity than that of cis eQTLs. cis-eQTLs are distributed uniformly
over the entire genome whereas this is not the case for major trans eQTLs. In fact,
we found seven major hot spot trans eQTLs that account for 44, 107, 42, 66, 1766,
204 and 125 of the trans eQTLs, respectively. Parallel physiological analysis revealed
that the major trans eQTL –named VULCANO- coassociates with a major QTL that
affects root-shoot growth ratio.
184
185
SESIÓN VIII
METABOLISMO
MODERADORES:
Manuel Rodríguez
Concepción (CRAG, CSIC-
IRTA-UAB-UB, Barcelona)
Francisco J. Cejudo (IBVF-
CSIC, Universidad de Sevilla)
186
S-VIII. Ponencia 1
THE ROLE OF 2-CYS PEROXIREDOXINS IN THE OXIDATION OF
CHLOROPLAST REDOX-REGULATED ENZYMES IN THE DARK
Juan Manuel Pérez Ruiz, Valle Ojeda and Francisco Javier Cejudo
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla and CSIC
Chloroplasts harbor a complex redox network formed by two systems: a large
set of Thioredoxins (Trxs) and NTRC, an NADPH-dependent Trx reductase.
While Trxs rely on photosynthetically produced reducing equivalents, NTRC
allows the use of NADPH for redox modulation of several Trx-regulated
enzymes including thiol-dependent peroxidases such as 2-Cys peroxiredoxins
(2-Cys Prxs) (Perez-Ruiz et al., 2006). Interestingly, Arabidopsis mutants that
combine the deficiencies of NTRC and Trxs have a very severe phenotype
(Ojeda et al., 2017), suggesting that both redox systems act concertedly to
regulate chloroplast metabolism. We have recently proposed that the action of
NTRC and Trxs is integrated via the hydrogen peroxide scavenging activity of 2-
Cys Prxs (Perez-Ruiz et al., 2017). Our model establishes that NTRC controls
the redox balance of 2-Cys Prxs, maintaining the reducing capacity of Trxs and,
consequently, the proper regulation of chloroplast processes such as the light-
activation of photosynthetic carbon assimilation enzymes. It is well-known that
upon darkness these enzymes become rapidly deactivated due to the oxidation
of thiol groups, but the mechanism that allows this deactivation remains
unknown. Because chloroplast redox regulation and hydrogen peroxide
reduction are linked by the action of NTRC and 2-Cys Prxs (Perez-Ruiz et al.,
2017), in this work we have explored the role of 2-Cys Prxs in the deactivation
of chloroplast metabolism during the night. Overall, our results indicate that 2-
Cys Prxs enable oxidation of photosynthetic carbon assimilation enzymes
during light-to-dark transitions.
Ojeda, V., Perez-Ruiz, J.M., Gonzalez, M., Najera, V.A., Sahrawy, M., Serrato, A.J.,
Geigenberger, P., and Cejudo, F.J. (2017). NADPH Thioredoxin Reductase C and Thioredoxins
Act Concertedly in Seedling Development. Plant physiology 174, 1436-1448.
Perez-Ruiz, J.M., Spinola, M.C., Kirchsteiger, K., Moreno, J., Sahrawy, M., and Cejudo, F.J.
(2006). Rice NTRC is a high-efficiency redox system for chloroplast protection against oxidative
damage. Plant Cell 18, 2356-2368.
Pérez-Ruiz, J.M., Naranjo, B., Ojeda, V., Guinea, M., and Cejudo, F.J. (2017). NTRC-
dependent redox balance of 2-Cys peroxiredoxins is needed for optimal function of the
photosynthetic apparatus. Proceedings of the National Academy of Sciences 114, 12069-
12074.
187
S-VIII. Ponencia 2 FINDING THE ENZYMES THAT FEED THE CAROTENOID
PATHWAY IN DIFFERENT PLANT TISSUES
M. Victoria Barja1, M. Águila Ruiz-Sola2, Rumyana Karlova3, Jules Beekwilder3
and Manuel Rodríguez-Concepción1. 1Centre for Research in Agrigenomics (CRAG) CSIC-IRTA-UAB- UB, Campus UAB
Bellaterra, Barcelona, Spain, 2Institut de Biosciences et Biotechnologies de Grenoble (BIG), Grenoble, France, 3Plant Research International, Wageningen, The Netherlands.
Corresponding author: Manuel Rodríguez-Concepción (manuel.rodrí[email protected])
Isoprenoids represent a hugely diverse class of biological molecules, among which carotenoids are of special industrial interest since they are a source of natural pigments and beneficial nutrients for human health (e.g. beta-carotene as pro- vitamin A). Carotenoids are synthesized in plastids and play fundamental roles in plants such as photoprotection (in leaves), signalling (e.g. as precursors of abscisic acid and strigolactone synthesis in roots), and pigmentation (in flowers and fruits). They derive from geranylgeranyl diphosphate (GGPP) that is synthesized by a family of GGPP synthase (GGPPS) enzymes in plants (Ruiz-Sola and Rodríguez- Concepción, 2012). Arabidopsis thaliana has 5 GGPPS isoforms distributed in different cellular compartments (Beck et al., 2013). From these, 2 isoforms are located in plastids, but only one (GGPPS11) is essential for carotenoid production (Ruiz-Sola et al., 2016a, 2016b). There is a gap of knowledge about GGPPS families in other plants, including those with interest as a source of dietary carotenoids. Tomato (Solanum lycopersicum) fruits accumulate high amounts of carotenoids that provide orange and red colors to ripe tomatoes. We speculate that carotenoid production in different tomato tissues most likely involves the activity of specific GGPPS paralogs, depending on the carotenoid function (photoprotection, signalling, or pigmentation). We found 5 putative GGPPS isoforms in the tomato genome, 3 of which are localized in plastids. All 3 plastidial isoforms are expressed in leaves but only one is primarily expressed in roots and the other 2 are up- regulated during fruit ripening. Work is in progress to identify which of the plastidial GGPPS paralogs is responsible for carotenoid biosynthesis in particular organs. This information will be very useful for future metabolic engineering approaches, as it will allow us to specifically improve the nutritional quality of fruits without interfering with photoprotection, mycorrhization, or tolerance to stress.
References Beck G, Coman D, Herren E, Ruiz-Sola MA, Rodríguez-Concepción M, Gruissem W and Vranová E (2013). Characterization of the GGPP synthase gene family in Arabidopsis thaliana. Plant Mol. Biol. 82: 393–416. Ruiz-Sola MA, Coman D, Beck G, Barja MV, Colinas M, Graf A, Welsch R, Rütimann P, Bühlmann P, Bigler L, Gruissem W, Rodríguez-Concepción M and Vranová E (2016a). Arabidopsis GERANYLGERANYL DIPHOSPHATE SYNTHASE 11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids. New Phytol. 209(1):252-264. Ruiz-Sola MÁ, Barja MV, Manzano D, Llorente B, Schipper B, Beekwilder J and Rodriguez- Concepcion M (2016b). A single arabidopsis gene encodes two differentially targeted geranylgeranyl diphosphate synthase isoforms. Plant Physiol. 172(3):1393-402. Ruiz-Sola MÁ and Rodríguez-Concepción M (2012). Carotenoid Biosynthesis in Arabidopsis: A Colorful Pathway. Arab. B. 10: e0158.
Acknowledgements & Funding. We are very grateful to Francel Verstappen and Bert Schipper (Wageningen University&Research, The Netherlands) for their support on the GGPP analysis. We also thank Jordi Perez-Gil for his assistance with the GGPPS biochemical assays and M. Rosa Rodríguez-Goberna and members of the CRAG Services for their technical support. This work was supported by MECD FPU fellowship and by grants from MINECO (BIO2014-59092-P and BIO2015- 71703-REDT).
188
S-VIII. Comunicación 1 ESTERYL ESTER BIOSYNTHESIS IN TOMATO
Alma Burciaga-Monge1 Juan Alejandro Lara1, Angel Chávez1, Monserrat Arró1,2, Albert Boronat1,3, Teresa Altabella1,4, Albert Ferrer1,2
1Plant Metabolism and Metabolic Engineering Program, Centre for Research in
Agricultural Genomics (CRAG) (CSIC-IRTA-UAB-UB), Bellaterra, Spain, 2Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Sciences, 3Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, 4Department of Biology, Healthcare and the Environment, Faculty of Pharmacy and Food Sciences, University
of Barcelona, Barcelona, Spain. Corresponding author: Albert Ferrer ([email protected])
Esteryl esters (SE) are a storage pool of sterols that serves to maintain free sterol (FS) homeostasis in cell membranes throughout plant growth and development. SE are also involved in the recycling of FS and fatty acids released from disorganized cell membranes in senescing tissues (Bouvier-Navé et al., 2010), and a drastic increase of SE content has been associated to tomato fruit ripening (Whitaker, 1988). Sterol acyltransferases, the enzymes that transfer long-chain fatty acil groups to the free hydroxyl at C-3 position of the sterol backbone, are categorized into acyl-CoA:sterol acyltransferases (ASAT) and phospholipid:sterol acyltransferases (PSAT) depending on whether the fatty acyl donor is an acyl-CoA or a phospolipid. We have identified, cloned and functionally characterized tomato (Solanum lycopersicum cv, Micro-Tom) SlPSAT1 and SlASAT1 enzymes, which restored SE to wild type levels in the Arabidopsis psat1-2 and asat1-1 knock-out mutants (Bouvier-Navé et al., 2010), respectively. Expression of SlPSAT1 also reversed the toxicity caused by an external supply of mevalonate and the early senescence phenotype in detached leaves of the psat1-2 mutant. Expression of SlASAT1 in the asat1-1 mutant revealed a clear substrate preference of the tomato enzyme for the sterol precursors cycloartenol and 24-methylene cycloartanol. Subcellular localization studies showed that SlPSAT1 localize in cytoplasmic lipid droplets while SlASAT1 localize in the plasma membrane (PM), which suggests that PM-localized SlASAT1 may act catalytically in trans on their sterol substrates, which are presumably embedded in the ER membrane. SlPSAT1 and SlASAT1 genes are widely expressed in different tomato organs and display a moderate transcriptional response to several stresses, suggesting a dual role of these enzymes in tomato plant and fruit development, and the adaptive responses to stress. SlPSAT1 and SlASAT1 gene expression is detected in all organs analyzed, including fruits at different developmental and ripening stages. Their overlapping but largely complementary expression patterns throughout fruit development suggests a concerted transcriptional regulation of these genes in opposite direction during fruit development and ripening. Work is currently in progress using the available Arabidopsis knock-out mutants and tomato CRISPR-Cas9 mutants generated in our laboratory to establish the role of SE in these biological processes.
References
Bouvier-Navé, P., Berna, A., Noiriel, A., Compagnon, V., Carlsson, A. S., Banas, A., Stymne, S., and Schaller, H. (2010). Involvement of the phospholipid sterol acyltransferase1 in plant sterol homeostasis and leaf senescence. Plant Physiol. 152, 107–119. doi: 10.1104/pp.109.145672
Whitaker, B. D. (1988). Changes in the steryl lipid content and composition of tomato fruit during ripening. Phytochemistry, 27, 3411–3416. doi: 10.1016/0031-9422(88)80740-4
Acknowledgements & Funding
AB-M, JAL and AC received Ph.D. fellowships from the CONACYT (México). This work was funded by grants AGL2013-43522-R and AGL2017-88842-R from the Spanish Government, 2014SGR-1434 from the Generalitat de Catalunya, and by the CERCA Programme/ Generalitat de Catalunya. We also acknowledge financial support from the Spanish Ministry of Economy and Competitiveness through the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016-2019 (SEV-2015- 0533).
189
4
4
S-VIII. Comunicación 2 MYB28 AND MYB29 GENES PARTICIPATE IN THE
REGULATION OF ARABIDOPSIS RESPONSE TO AMMONIUM
STRESS
Inmaculada Coleto1, Iraide Bejarano1, Meike Burow2, Daniel Marino1,3
1Department of Plant Biology and Ecology, University of the Basque Country
(UPV/EHU), 48940-Leioa, Spain. 2Department of Plant and Environmental Sciences,
Faculty of Science, DynaMo Center, University of Copenhagen, Frederiksberg,
Denmark. 3Ikerbasque, Basque Foundation for Science, E-48011 Bilbao, Spain
Corresponding author: Daniel Marino ([email protected])
Ammonium (NH +) is a major nitrogen (N) source taken up by plants. However,
+ when NH4 is present as the sole N source or at high concentrations in the soil it
becomes toxic for most plant species (Britto and Kronzucker, 2002). Molecular +
mechanisms determining plant tolerance/sensitivity to NH4 are still not
completely clear. For instance, no transcription factor has been identified in
Arabidopsis in relation with ammonium stress (Liu and von Wirén, 2017). In
previous studies, we observed that glucosinolate (GLS) biosynthesis increased +
in Arabidopsis, broccoli and oilseed rape when exposed to NH4 (Marino et al.,
2016; Coleto et al., 2017). To further explore the link between GLS metabolism
and ammonium nutrition, in this study we analyzed how different Arabidopsis
knockout mutants related to GLS metabolic pathway responded to NH +.
Among them, the double mutant of the R2R3-Myb transcription factors Myb28
and Myb29 (myb2829), which is deficient in aliphatic GLS biosynthesis (Hirai et
al., 2007) was hypersensitive to ammonium nutrition. Individual myb28 and
myb29 mutants showed a similar phenotype to wild type plants, suggesting that
Myb28 and Myb29 genes played a redundant role in the control of ammonium
response. To understand the function of Myb28 and Myb29 genes in
ammonium stress we have carried out different metabolic, biochemical and
molecular approaches that will be discussed.
Coleto, I., de la Peña, M., Rodríguez-Escalante, J., Bejarano, I., Glauser, G., Aparicio-Tejo,
P.M., González-Moro, M.B. and Marino, D. (2017) Leaves play a central role in the adaptation
of nitrogen and sulfur metabolism to ammonium nutrition in oilseed rape (Brassica napus). BMC
Plant Biol 17. 157.
Hirai, M.Y., Sugiyama K., Sawada Y., Tohge T., Obayashi, T., Suzuki, A., Araki, R., Sakurai, N.,
et al., (2007) Omics-based identification of Arabidopsis Myb transcription factors regulating
aliphatic glucosinolate biosynthesis. Proc Natl Acad Sci USA 104: 6478-6483.
Liu, Y and von Wirén, N. (2017) Ammonium as a signal for physiological and morphological
responses in plants. J Exp Bot 68: 2581-2592.
Marino, D., Ariz, I., Lasa, B., Santamaría, E., Fernández-Irigoyen, J., González-Murua, C. and
Aparicio-Tejo, PM. (2016) Quantitative proteomics reveals the importance of nitrogen source to
control glucosinolate metabolism in Arabidopsis thaliana and Brassica oleracea. J Exp Bot 11:
3313-3323.
This work was supported by the Basque Government [IT932-16] and the Spanish Ministry of
Economy and Competitiveness [BIO2014-56271-R] co-funded by FEDER
190
S-VIII. Comunicación 3
INSIGHTS INTO THE MEMBERS OF THE MICROSOMAL
OLEATE DESATURASE (FAD2) GENE FAMILY INVOLVED IN
LINOLEIC ACID SYNTHESIS IN OLIVE (Olea europaea)
M. Luisa Hernández, M. Dolores Sicardo, Patricia M. Arjona, José M. Martínez-
Rivas
Department of Biochemistry and Molecular Biology of Plant Products, Instituto de la
Grasa (IG-CSIC), Sevilla, Spain.
Corresponding author: M. Luisa Hernández ([email protected])
Plant lipids contain polyunsaturated fatty acids, mainly linoleic and α-linolenic
acids, which play crucial roles in plant metabolism as storage compounds
mainly in the form of triacylglycerols, as structural components of membrane
lipids, and as precursors of signalling molecules involved in plant development
and stress response. Linoleic acid, together with oleic acid, is a major fatty acid
in vegetables oils and its content greatly affects technological properties such
as their oxidative stability, and nutritional characteristics. In higher plants, the
microsomal oleate desaturase (FAD2) is the main responsible for the
conversion oleic acid to linoleic acid. Multiple members of the FAD2 gene family
are known to occur in several oilseed species. However, unlike oilseed, little
information is available on the regulation of linoleic acid synthesis in oil fruits. In
olive, two genes encoding FAD2 have been previously reported, and FAD2-2
was described as the main gene involved in the linoleic acid accumulation in
olive mesocarp. In this study, three novel full length cDNA sequences have
been isolated and characterized in olive. Sequence analysis indicated that they
code for microsomal oleate desaturases and were named as FAD2-3, FAD2-4
and FAD2-5. The identity of the novel FAD2 genes was confirmed by functional
expression in yeast, with the three proteins showing a temperature-dependent
conversion of oleic to linoleic acid. Genomic structure showed an intron in the
5’-UTR of four of them, which is characteristic of many plant FAD2 genes. We
have also determined the expression levels of olive FAD2 genes and the lipids
linoleic acid content in different olive tissues. The physiological role of each
microsomal oleate desaturase in olive, including the specific contribution of
each gene to the linoleic acid content in the olive oil will be discussed.
189
S-VIII. Póster 1
199
S-VIII. Póster 2 THE COMBINED ACTION OF PLASTIDIAL PHOSPHOGLUCOSE
ISOMERASE AND GLUCOSE-6-P/PHOSPHATE TRANSLOCATOR2 IS AN IMPORTANT DETERMINANT OF THE
RESPONSE OF PLANTS TO MICROBIAL VOLATILES
Adriana Ricarte-Bermejo1, Ángela María Sánchez-López1, Marouane Baslam1,2, Edurne Baroja-Fernández1, Francisco José Muñoz1, Abdellatif Bahaji1, Lydia Ugena3, Kinia Ameztoy1, Goizeder Almagro1, Nuria De Diego3, Lukás Spíchal3, Karel Dolezal3
and Javier Pozueta-Romero1
1Instituto de Agrobiotecnología (CSIC/UPNA/Gobierno de Navarra). Mutilva, Navarra, Spain, 2Graduate School of Science and Technology and Department of Applied Biological Chemistry, Niigata University,
Japan. 3Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Olomouc, CZ-78371, Czech Republic.
Corresponding author: Javier Pozueta-Romero; E-mail: [email protected]
Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of fructose-6- phosphate and glucose-6-P (G6P). It is widely accepted that the plastidic PGI isoform (PGI1) plays an important role in connecting the Calvin-Benson cycle with the starch biosynthetic pathway in mesophyll cells of leaves. Recent studies have provided evidence that PGI1 is an important determinant of photosynthesis, growth and starch production, likely as a consequence of its involvement in the synthesis of metabolic intermediates required for the synthesis of plastidial isoprenoid compounds such as hormones and photosynthetic pigments (Bahaji et al. 2015). We have recently shown that volatile compounds (VCs) emitted by microbial phytopathogens promote growth (Sánchez-López et al. 2016b). When Arabidopsis plants were exposed to airborne signals released by the fungal phytopathogen Alternaria alternata, growth promotion was accompanied by enhanced levels of plastidial cytokinins (CKs), augmented photosynthesis, and accumulation of exceptionally high levels of starch in leaves (Sánchez-López et al. 2016b). This response was PGI1-independent, as fungal VCs exposed PGI1 null pgi1-2 plants grew faster and accumulated exceedingly higher levels of starch and CKs than plants not exposed to VCs. The most up-regulated gene in leaves of fungal VCs exposed plants was At1g61800, encoding the GPT2 glucose-6- phosphate (G6P)/phosphate translocator that is necessary for dynamic photosynthetic acclimation to increased irradiance. This indicated that the response of pgi1-2 to fungal VCs could be due to the GPT2-mediated transport of cytosolic G6P into the chloroplast. To test this hypothesis we produced and characterized pgi1-2/gpt2-2 PGI1- and GPT2-null double mutants cultured in the presence or absence of VCs emitted by A. alternata. We found that photosynthesis, active CK content, growth and leaf starch content in pgi1-2/gpt2-2 plants exposed to fungal VCs were lower than in VCs exposed WT, gpt2-2 and pgi1-2 plants. Isobaric labeling based differential proteomic analyses revealed that fungal VCs strongly up-regulate the expression of proteins involved in photosynthesis in WT, gpt2-2 and pgi1-2 plants, but in much lower extent in pgi1- 2/gpt2-2 plants. The overall data show that the combined action of PGI1 and GTP2 is an important determinant of the plant´s response to microbial VCs. The possible involvement of these functions in the production of growth- and foliar metabolism- regulating isoprenoid hormones in heterotrophic organs is discussed.
Bahaji A, et al (2015b) Plastidic phosphoglucose isomerase is an important determinant of starch accumulation in mesophyll cells, growth, photosynthetic capacity, and biosynthesis of plastidic cytokinins in Arabidopsis. PLoS One Doi:10.1371/journal.pone.0119641
Sánchez-López, Á.M. et al. (2016a) Arabidopsis responds to Alternaria alternata volatiles by triggering plastid phosphoglucose isomerase-independent mechanisms. Plant Physiol. 172: 1989-2001.
Sánchez-López A.M., et al. (2016b) Volatile compounds emitted by diverse phytopathogenic microorganisms promote plant growth and flowering through cytokinin action. Plant Cell Environ. 39: 2592–260
191
S-VIII. Póster 3
NON-TARGETED METABOLITE PROFILE ANALYSIS OF Quercus ilex ACORNS THROUGH A LC-MS/MS APPROACH
Cristina López-Hidalgo1, Macedonia Trigueros1, Macarena Menéndez-García2,
Luis Valledor3, Jesús V. Jorrín-Novo1
1Agroforestry and Plant Biochemistry, and Systems Biology; Department of
Biochemistry and Molecular Biology. University of Córdoba; UCO-CeiA3, Córdoba,
Spain.2Unidad de Metabolómica; SCAI-UCO; Córdoba, Spain. 3Department of
Organisms and Systems Biology. University of Oviedo, Oviedo, Spain.
Corresponding author: Cristina López-Hidalgo ([email protected])
The metabolite profiles of acorns in Holm oak (Quercus ilex L. subsp. ballota
[Desf.] Samp.) have been analyzed from different trees by a non-targeted LC-
MS approach. This work has a double objective, first to characterize population
variability and diversity through the chemical compositions of acorns, and
second to search for bioactive compounds. Fruits showing different
morphometric characteristics (size, weight, and color) were collected from trees
located at the “Aldea de Cuenca” (Fuente Obejuna, Córdoba, South-Western
Spain). The metabolites were extracted using aqueous and organic solvents
from acorn flour, to cover a wide range of compounds (Valledor et al., 2014).
Then, final extracts were subjected to UHPLC-ESI-qTOFa LC-MS analysis. A
total of 4022 molecular masses (2611 positive mode and 1411 negative mode)
were reliably quantified, out of which 35% of metabolite features were putatively
identified using the mass spectra through a search in several plant databases.
After a PCA statistical analysis, qualitative and quantitative differences were
observed among acorn types. Relative quantitation of 20 metabolites with
higher loadings over the first two PCA components, explaining most of the
experimental variability, included alkaloids (N-Methylmescaline), terpenoids
(Rehmaionoside A), phytohormones (Cis-Zeatin-9-glucoside) and
carbohydrates, revealing clear variations between acorn types. This work is in
progress trying to further our knowledge of the acorn metabolome in parallel to
the bioactivity characterization of extracts.
Valledor, L., Escandón, M., Meijón, M., Nukarinen, E., Cañal, M. J., and Weckwerth, W. (2014).
A universal protocol for the combined isolation of metabolites, DNA, long RNAs, small RNAs,
and proteins from plants and microorganisms. Plant J. 79, 173–180. doi:10.1111/tpj.12546.
The research work is part of the Doctoral Thesis of Cristina López Hidalgo and is integrated into
the funded research project STUDIES OF POPULATION VARIABILITY AND RESPONSE TO
STRESSES IN HOLM OAK THROUGH A MULTI-OMIC APPROACH (TRANSCRIPTOMICS,
PROTEOMICS AND METABOLOMICS). ENCINOMICA BIO2015-64737-R. Programa Estatal
de I+D+i Orientada a los Retos de la Sociedad-Ministerio de Economía y Competitividad.
Convocatoria 2015.
a Ultrahigh-performance liquid chromatography, electrospray ionization, quadrupole time-of-
flight
192
S-VIII. Póster 4
193
S-VIII. Póster 5 THE RESPONSE OF TOMATO LEAF TRANSCRIPTOME TO AMMONIUM
NUTRITION IS ENHANCED BY ELEVATED CO2
Izargi Vega-Más1, Teresa Fuertes-Mendizábal1, Carmen Pérez-Delgado2,
Rodrigo Sagaceta1, Daniel Marino1,3, Carmen González-Murua1, Jose Mª
Estavillo1,Mª Begoña González-Moro1
1Departamento de Biología Vegetal y Ecología, Universidad del País Vasco UPV/EHU,
Leioa, Spain 2Departamento de Bioquímica Vegetal y Biología Molecular, Universidad
de Sevilla, Sevilla, Spain, 3Ikerbasque, Basque Foundation for Science, Bilbao, Spain.
Corresponding author: Mª Begoña González-Moro ([email protected]).
Crop plants are normally adapted to nitrate nutrition, whose fertilization
generates many environmental pollution problems in soils, in fresh waters and
in the atmosphere. As alternative, the use of ammonium-based fertilizers is
known to mitigate these negative effects. Since ammonium-fed plants have a
huge carbon demand to sustain nitrogen (N) assimilation, for this study, we
hypothesized that the increment of atmospheric CO2 in the context of climate
change may be of prominent relevance to improve the productivity of
ammonium-fed plants. In this regard, to identify genes that may be important for
plant response to climate change in relation with the N source, we analysed the
leaf transcriptome of tomato plants grown under nitrate or ammonium nutrition,
at ambient (400 ppm) or elevated (800 ppm) atmospheric CO2 conditions. At
ambient CO2, 133 genes were differentially expressed in leaves of ammonium
vs. nitrate grown plants. Interestingly, N source effect was clearly exacerbated
at elevated CO2, where 305 genes were differentially expressed. Out of them,
56 genes were commonly regulated at both CO2 conditions by the N source.
Among the genes commonly down-regulated by ammonium nutrition we found
10 genes encoding monothiol glutaredoxins-S1 and 4 genes related to
gluconeogenesis/glyoxylate cycle. Regarding the commonly up-regulated genes
by ammonium nutrition we found one gene encoding for carbonic anhydrase
and one for zinc induced facilitator like 1-protein. Among the specific genes
down-regulated by ammonium and exclusively at elevated CO2, we found a
number of genes involved in the metabolism of pyruvate and glycolate
(photorespiratory pathway). To go in-depth into the regulation of C metabolizing
pathways in tomato leaves, we also measured the activity of carbonic
anhydrase, enzymes of the tricarboxylic acid cycle, photorespiration and
glyoxylate cycle, together with the main metabolites linked to these pathways.
The overall results reveal that fine regulation of these routes of carbon
metabolism could be essential in order to optimize N use efficiency in a context
of increasing environmental CO2 conditions.
Acknowledgements & Funding: Consolidated Group IT-932-16 funded by Basque Government.
Project MINECO AGL2015-64582-C3-2-R
194
4
S-VIII. Póster 6 TRANSCRIPTOMIC ANALYSIS TO DECIPHER THE ROLE OF
AtMYB28 AND AtMYB29 GENES IN AMMONIUM STRESS
RESPONSE
Iraide Bejarano1, Inmaculada Coleto1, Daniel Marino1,2
1Department of Plant Biology and Ecology, University of the Basque Country
(UPV/EHU), 48940-Leioa, Spain.2Ikerbasque, Basque Foundation for Science, E-
48011 Bilbao, Spain
Corresponding author: Daniel Marino ([email protected])
Most plant species develop stress symptoms when ammonium (NH +) is the
sole nitrogen source or when it is present at high concentrations in the soil.
Understanding the molecular mechanisms involved in the response of plants to +
NH4 is crucial to reduce its negative impact and until now, only a few genes
participating in the response of plants to ammonium stress have been identified
(Liu and von Wirén, 2017). Arabidopsis Myb28 and Myb29 genes encode
R2R3-Myb transcription factors, which are widely known as master regulators of
aliphatic glucosinolate biosynthesis (Hirai et al., 2007). We observed that
Arabidopsis double knocked-out mutant myb2829 showed a more sensitive
phenotype to ammonium than the wild type plants Col-0. In this study, as a
means of identify the function of Myb28 and Myb29 genes in plant response to
ammonium, we analyze the differential gene expression in Col-0 and myb2829
plants under ammonium nutrition using a microarray analysis. Microarray data
revealed a differential expression of about 2500 genes between myb2829 and
wild type plants. Gene enrichment analysis of down and up-expressed genes
highlighted 39 metabolic processes differentially regulated in myb2829 which
were mainly related with defense response, sulfur metabolism, mineral nutrition
and hormonal response.
Hirai, M.Y., Sugiyama K., Sawada Y., Tohge T., Obayashi, T., Suzuki, A., Araki, R., Sakurai, N.,
et al., (2007) Omics-based identification of Arabidopsis Myb transcription factors regulating
aliphatic glucosinolate biosynthesis. Proc Natl Acad Sci USA 104: 6478-6483.
Liu, Y and von Wirén, N. (2017) Ammonium as a signal for physiological and morphological
responses in plants. J Exp Bot 68: 2581-2592.
This work was supported by the Basque Government [IT932-16] and the Spanish Ministry of
Economy and Competitiveness [BIO2014-56271-R] co-funded by FEDER
195
S-VIII. Póster 7 β-GLUCOSIDASE GENE FAMILY FROM OLIVE FRUIT
INVOLVED IN THE PHENOLIC PROFILE OF VIRGIN OLIVE OIL
Lourdes García-Vico1, Rosario Sánchez1, Ana María Herruzo1, Carlos Sanz1
and Ana Gracia Pérez1
1Department of Biochemistry and Molecular Biology of Plant Products, Instituto de la Grasa (CSIC), Seville-Spain
Corresponding author: Lourdes García-Vico ([email protected])
Phenolic compounds present in Virgin Olive Oil (VOO) are involved in its nutritional and organoleptic properties (Konstantinidou et al., 2010; Mateos et al., 2004). Phenolic composition of VOO is determined by the enzymatic reactions that take place during olive fruit processing. On these enzymes, β- glucosidase (EC 3.2.1.21) activity plays a main role in the transformation of the phenolic glycosides present in the olive fruit (Romero-Segura et al., 2012). In a previous work, the molecular and biochemical characterization of one gene coding for a β-glucosidase (β-GLUC1A) from olive (Olea europaea L.) Picual cultivar has been described (Velázquez-Palmero et al., 2017). In this work, we have performed a transcriptomic analysis in seven olive cultivars and two developmental stages of the olive fruit. This study has revealed the presence of seven β-glucosidase genes which can be classified into three groups, stated β- GLUC1 (1A; 1B), β-GLUC2 (2A; 2B) and β-GLUC3 (3A; 3B; 3C). Gene expression analysis by real time PCR in this harvesting season confirmed previous season´s transcriptomic data showing differential expression of these genes along cultivars and fruit maturation stages. β-GLUC1A gene showed the higher level of expression in all samples and all β-GLUC genes displayed a similar expression pattern resulting the main messenger detection in mid fruit development stages. Olive cultivar analysis revealed those cultivars whose gene expression is markedly higher for some members of the β-GLUC family, as for β-GLUC1B gene. We have also measured the main phenolic compounds present in fruits (oleuropein, demethyloleuropein and ligstroside) and oil samples (aldehydic/dialdehydic forms of oleuropein/demethyloleuropein and ligstroside aglycones) by High-Performance-Liquid-Chromatography (HPLC) and analyzed the total β-glucosidase activity in fruits by a spectrophotometric method. Global data analyses have been carried out to evaluate the correlation of cultivar and fruit development stage with their oil phenolic compound profile, the total β-glucosidase activity and the gene expression data of each β- glucosidase family member. The information obtained in this study could help in the design of molecular markers for the marker-assisted selection of novel olive cultivars with improved phenolic content in their oils.
-Mateos, R., Cert, A., Pérez-Camino, M.C., and García, J.M. (2004). Evaluation of virgin olive oil bitterness by quantification of secoiridoid derivatives. J. Am. Oil Chem. Soc. 81, 71-75. Doi:10.1007/s11746-004- 0859-x. -Kontantinidou, V., Covas, M-I., Muñoz-Aguayo, D., Khymenents, O., de la Torre, R., Saez, G., et al. (2010). In vivo nutrigenomic effects of virgin olive oil polyphenols within the frame of the Mediterranean diet: a randomized controlled trial. FASEB J. 24, 2546-2557. doi:10.1096/fj.09-148452.
-Romero-segura C., García-Rodríguez R., Sánchez-Ortíz, A., Sanz, C., and Pérez, A.G. (2012). The role of olive ß-glucosidase in shaping the phenolic profile of virgin olive oil. Food Res. Int. 45, 191-196. doi:10.1016/j.foodchem.2014.11.033. -Velázquez-Palmero D., Romero-Segura, C., García-Rodríguez, R., Hernández, M.L., Vaistij, F.E., Graham, I.A., Pérez, A.G., and Martínez-Rivas, J.M. An oleuropein ß-glucosidase from olive fruits is involved in determining the phenolic composition of virgin olive oil.
196
S-VIII. Póster 8
THIOREDOXIN-LINKED REDOX REGULATION IN OXYGENIC
PHOTOSYNTHETIC ORGANISMS WITHOUT
FERREDOXIN:THIOREDOXIN REDUCTASE (FTR)
Mónica Balsera1, Rubén M. Buey2, Peter Schürmann3, Bob B. Buchanan4
1Instituto de Recursos Naturales y Agrobiología (IRNASA-CSIC), Salamanca, Spain., 2
Dpto. Microbiología y Genética. Universidad de Salamanca, Salamanca, Spain,
3Laboratoire de Biologie Moléculaire et Cellulaire, Université de Neuchâtel, Neuchâtel,
Switzerland, 4Department of Plant & Microbial Biology, University of California,
Berkeley, USA
Corresponding author: Mónica Balsera ([email protected])
Thioredoxins (Trxs) are small oxidoreductases that regulate the redox state of
target proteins through dithiol-disulfide exchange reactions. Trx-linked redox
regulation is known to play a central role in a spectrum of processes in living
cells. In oxygenic photosynthetic organisms, a key enzyme
ferredoxin:thioredoxin reductase (FTR) links light (via photoreduced ferredoxin
or Fdx) to the regulation of target enzymes (via Trx), such as enzymes of the
Calvin-Benson (CB) cycle or the oxidative pentose phosphate pathway (OPPP)
(Balsera M et al, 2014). We have found that FTR is not universally present in
oxygenic photosynthetic organisms (Balsera M et al, 2013). This observation
raises the question of how these organisms link the CB cycle and related
metabolic processes to light and other changing environmental conditions. We
have identified a protein that possibly functions in this connection. The enzyme,
provisionally named DTR for deeply rooted bacterial thioredoxin reductase,
shows high similarity to NADP-dependent thioredoxin reductases (NTR) but
exhibits unique functional and structural features (Buey RM et al, 2017a). In this
meeting, we will present our latest findings on the structural and functional
diversity of the thioredoxin system in oxygenic photosynthetic organisms
(Balsera M et al 2013; Buey RM et al, 2017a; Buey RM et al, 2017b).
Balsera M et al (2014), ARS, 21:1327-1355
Balsera M et al (2013), Planta, 237:619-635
Buey RM et al (2017a), Mol Plant, 10:212-215
Buey RM et al (2017b), PNAS, 2017b, 114:12725-12730
197
S-VIII. Póster 9
FUNCTIONALITY OF MONOCOTS TRANSIT PEPTIDES IN
DICOTS
M. Teresa Ruiz*, Tomás Albi*, Federico Valverde, José Mª Romero
Plant Development Unit, Plant Biochemistry and Photosynthesis Institute (Uni. Sevilla-
CSIC), Sevilla, Spain
Corresponding author: Teresa Ruiz ([email protected]) *Equal authorship
Starch is the main carbon reserve in plants. Transitory starch is synthesised in
photosynthetically active tissues while storage starch is accumulated in sink
tissues as seeds, roots, etc. Appropriate levels of both kinds of starch are
crucial for plant fitness and reproductive success (Ortiz-Marchena et al., 2014).
In green tissues, ADPGase, the first enzyme of starch synthesis, is chloroplastic
and regulated by the levels of 3PGA (activator) and PPi (inhibitor) as sensors of
photosynthetic activity, while the isoforms present in storage tissues such as
cereal endosperm are mostly insensitive to these effectors (Lee et al., 2007).
Arabidopsis thaliana aps1 mutant lacks of AGPase activity, and thus starch
levels are very low (Ventriglia et al., 2008). We expressed the ADPGase small
subunit isoforms from rice (amyloplastidic (OsAPS1), chloroplastidic (OsAPS2a)
and cytoplasmic (OsAPS2b)) in the Arabidopsis aps1 mutant. All isoforms, both
in their native form or in constructs where the rice TP (Transit Peptide) has
been replaced with that from Arabidopsis (a TP from either rice or Arabidopsis
was fused to the cytoplasmic OsAPS2b) were able to recover starch synthesis
in aps1 and therefore complement the phenotypic characteristics related to lack
of starch. These results indicate that isoforms from storage tissues could be
expressed in green tissues to increase transitory starch synthesis and as a
consequence to improve plant fitness. On the other hand, the results suggest
that TPs from monocots are functional in dicots. To determine the motifs that
might be involved in TP functionality, an in silico analysis is being carried out to
determine putative motives responsible for the functionality of a monocot TP in
a dicot.
References
- S-K. Lee et al.., Plant Mol. Biol. 65(4):531-46, 2007. - M.I. Ortiz-Marchena et al., Plant Cell, 26: 565-584,2014. - T. Ventriglia et al., Plant Physiol. 148(1): 65-76, 2008.
Acknowledgements & Funding.
The authors would like to thank funding from projects BIO2014-52425-P and BIO2017-83629-R
(Spanish Ministry of Economy and Competitiveness, MINECO) partially supported by FEDER
funding.
198
S-VIII. Póster 10
NITRIC OXIDE (NO) INVOLVEMENT IN THE REGULATION OF
THE BIFUNCTIONAL ENO2/MBP1 LOCUS
Pablo Albertos1, Svenja Bomers1 and Brigitte Poppenberger1
1Biotechnology of Horticultural Crops, Center for Life and Food Sciences
Weihenstephan, Technische Universität München, D-85354 Freising, Germany.
Corresponding author: Brigitte Poppenberger ([email protected])
Glycolytic enzymes are highly conserved in prokaryotic and eukaryotic
organisms and enolase (ENO2) is among the most abundant cytosolic proteins.
ENO2 is essential for the growth and development of plants (Eremina et al.,
2015). Defects in ENO2 function yield plants with severe developmental defects
and constitutively reduced growth (Eremina et al., 2015). ENO2 can be
regulated by different post-translational modifications (PTMs) (Pearlman et al.,
2011). In particular ENO2 S-nitrosylation seems to play a major role in the
activity of this enzyme (Zhang et al., 2017). However how this PTM, which is
directed by nitric oxide (NO), affects ENO2 activity and possibly also redox
stability in plants is still to be deciphered. Using different genetic and
pharmaceutical approaches, we show that clear changes in the enzymatic
activity of ENO2 are correlated with alterations in NO levels in the plant. In
addition to an enolase the ENO2 locus also encodes the transcription factor
AtMBP-1, which feedback regulates ENO2 abundance. Studies of how NO
regulates AtMBP1 to maintain ENO2 homeostasis are on their way to a better
understanding of this crucial enzyme in plants.
Eremina et al., 2015. The Plant Journal, 81, 895–906.
Pearlman et al., 2011. Cell, 147 (4), 934-946.
Zhang et al., 2017. Plant, Cell and Environment, 40, 1834–1848.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB924 TP-A12 to B.P.)
and the
Alexander von Humboldt Foundation (Alexander von Humboldt fellowship to P.A.).
199
S-VIII. Póster 11
PLASTIDIAL LIPOYLTRANSFERASES FROM Helianthus annuus
Raquel Martins-Noguerol, Rafael Garcés, J.J. Salas and Enrique Martínez-Force
Instituto de la Grasa CSIC. Sevilla, Spain
Corresponding author: Raquel Martins-Noguerol ([email protected])
In most prokaryotic and eukaryotic organisms and some archaea, prosthetic lipoyl
groups are essential for the activity of several multienzymatic complexes in central
metabolism. In plants very little is known about lipoic acid biosynthesis but
lipoyltransferase (LIP2) and lipoyl synthase (LIP1) enzymes have been proposed to
be involved in de novo-synthesis and attachment of this cofactor to their specific
targets. These target enzymes use to be located only in mitochondria but in plants
PDH complex also occurs in plastids, where catalyzes first step in fatty acids
biosynthesis. Since no transport of lipoic acid has been demonstrated into plastids,
these organelles needs specific plastidial LIP1 and LIP2 activities. An essential LIP1
and two redundant LIP2 enzymes have been elucidated in chloroplasts from
Arabidopsis (Ewald et al., 2014). Here, we report two LIP2 isoforms in plastids from
Helianthus annuus. Both enzymes were identified (HaLIP2p1 and HaLIP2p2),
sequenced and cloned in a heterologous system (Escherichia coli). Phylogenetic
studies were performed and the expression of these enzymes in different sunflower
tissues was analyzed. Tertiary structure for both enzymes was modelled. The
contribution of this cofactor to sunflower plant development and oil synthesis is
discussed in order to provide substantial knowledge for further biotechnological
approaches in this important oil crop.
References:
Ewald R, Hoffmann C, Neuhaus E, Bauwe H (2014). Two redundant octanoyltransferases and one
obligatory lipoyl synthase provide proteinlipoylation autonomy to plastids of Arabidopsis. Plant Biology,
16: 35-42.
200
S-VIII. Póster 12
DIVERSIFYING BIOSYNTHESIS AND STORAGE CAPACITY OF
TERPENOIDS IN PLANTS
Trine Bundgaard Andersen, Briardo Llorente & Manuel Rodriguez-Concepcion
Centre for research in agricultural genomics (CRAG), CSIC-UAB-IRTA, Campus UAB
Bellaterra, Cerdanyola del Vallès, Barcelona, Spain
Corresponding author: Trine Andersen ([email protected])
Many specialized plant metabolites, including terpenoids, are valued for their function as nutrients, pharmaceuticals and fragrances. Specialized terpenoids are often biosynthesized by the plants in very small amounts, in specific tissues and/or only under certain (a)biotic stress conditions. To reach the full potential of these metabolites it is essential to develop methods to boost not only the biosynthesis but also the capacity of the plants to store these valuable metabolites. However, modifying a plant to produce any metabolite in large amounts can have toxic effects on the plant. This is especially true for metabolites whose production or/and accumulation can interfere with the metabolic homeostasis of the plant. The focus of the research is on terpenoid metabolites derived from C20 geranylgeranyl diphosphate (GGPP), i.e. C20 diterpenes (such as the anticancer drug taxol or the sweetener steviol) and C40 tetraterpenes (such as the health- promoting carotenoid pigments). The biosynthesis of these terpenoids includes the production of GGPP in the plastids followed by downstream steps in the plastid (carotenoids) or the ER/cytosol (diterpenes). Previous results have shown that carotenoids can be produced in the cytosol of plant cells engineered with bacterial genes. However, high carotenoid levels caused necrosis in the leaves, most likely because in the absence of proper storage structures (e.g. vesicles or lipid bodies) they are inserted into the cell membranes and compromise their normal function. In this project, the supply of GGPP in the cytosol and the abundance of ER-derived endomembrane structures will be increased by expressing a bacterial gene encoding GGPP synthase fused to an ER-anchoring domain demonstrated to stimulate ER membrane proliferation. In these plants, ER-derived vesicles are expected to contain the engineered enzymes but also endogenous enzymes catalyzing downstream steps of diterpene biosynthesis. The vesicles should provide a proper storage compartment for diterpenes but also for extraplastidial carotenoids. As a proof of concept, results from transient expression experiments and stable transformation of the model plant Nicotiana tabacum (tobacco) will be shown.
201
SESIÓN IX
INTERACCIÓN PLANTA –
OTROS ORGANISMOS
MODERADORAS:
Isabel Díaz (CBGP, UPM-INIA
Madrid)
Ainhoa Martínez-Medina
(IDIv HALLE-JENA-LEIPZIG,
Alemania)
202
S-IX. Ponencia 1 ROOT MUTUALISTIC SYMBIONTS: PLANT ALLIES IN A
MULTITROPHIC CONTEXT
Ainhoa Martinez-Medina1, Javier Rivero Bravo2, Alexander Weinhold1, Maria J.
Pozo2 and Nicole M. van Dam1.
1Molecular Interaction Ecology, German Centre for Integrative Biodiversity Research
(iDiv), Institute of Biodiversity, Friedrich Schiller University; Deutscher Pl. 5E, 04103
Leipzig, Germany. 2Department of Soil Microbiology and Symbiotic Systems, Estación Experimental del
Zaidín, CSIC, Profesor Albareda 1, 18008 Granada, Spain.
Corresponding author: Ainhoa Martinez Medina ([email protected])
Plants nurture a vast community of mutualistic microbes. In analogy to the
microbial communities in the human gut, they provide their host with essential
functions related to nutrient acquisition and protection against infections.
Arbuscular mycorrhizal (AM) and Trichoderma fungi are widespread soil
inhabitants that establish symbiosis with the vast majority of terrestrial plants,
conferring positive effects on growth and fitness. Despite the clear potential of
root mutualistic microbes for sustainable pest management strategies, the
mechanisms underlying their impact on plant-insect interactions are still unclear.
Besides improving plant growth and inducing defenses against herbivorous
insects, root mutualistic microbes may also affect plant-insect performance via
interactions at higher trophic levels. However, the effects of root mutualists on
the recruitment of natural enemies of herbivores (indirect defenses) are largely
unknown. In our study, we take an ‘integrative multiomics approach’, combining
untargeted transcriptomics and metabolomics with performance and behavioral
studies to uncover key traits driving the impact of AM and Trichoderma fungi on
the interaction of tomato plants with specialist and generalist herbivores. We
found that root interactions with both fungal symbionts, influences the shoot
metabolome and transcriptome, and primes the plant for an enhanced induction
of different defense-related compounds. In consequence, root inoculation with
the microbial symbionts altered the dynamics of the plant-herbivore interaction
by negatively affecting the growth and survival rates, pupation success and sex
ratio of a lepidopteran herbivore; which eventually translated into improved plant
performance. Moreover, the microbial symbionts strongly affected host
preference by the herbivore’s natural enemies, via changes in volatiles released
by the plants after herbivory, indicating that the effect of root symbionts on
herbivores can scale up to further trophic levels.
Acknowledgements: Program to support junior researchers to obtain third-party funding from
Friedrich-Schiller-Universität Jena (DRM/2015-02); German Centre for Integrative Biodiversity
Research (iDiv) Halle-Jena-Leipzig funded by the German Research Foundation (FZT 118); and
Grant AGL2015-64990-C2-1-R from the Spanish MINECO.
203
S-IX. Ponencia 2 ROS HOMEOSTASIS ESSENTIAL IN PLANT DEFENCE
RESPONSES TO PESTS
Ana Arnaiz1,2, Mª Estrella Santamaria1,2, Maria C. Romero-Puertas3, Pablo Gonzalez-Melendi1,2, Vojislava Grbic4, Manuel Martinez1,2, Luisa M. Sandalio3,
Isabel Diaz1,2
Ana Arnaiz: [email protected]
1Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM, 28223-Pozuelo de Alarcón (Madrid), España. 2Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, UPM, 28040-Madrid, España. 3Departamento de Bioquímica, Biología Molecular y Celular de Plantas. Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas. C/Profesor Albareda 1, 18008 Granada, España. 4Department of Biology, University of Western Ontario, 1151 Richmond St, London, ON N6A 5B7, Canada.
The two-spotted spider mite Tetranychus urticae is an important agricultural pest worldwide. It is a polyphagous acari that has a wide host range, feeding on more than 1,100 plants including agricultural crops. Spider mite is considered a model specie because of its rapid life cycle, the genome availability and because of they can be easily rear in the laboratory. Arabidopsis-T. urticae interaction concerns a complex network of molecules and signals for overwhelming the defences developed by each other. Early events in plant–herbivore interactions start with membrane potential depolarization, change in ion fluxes followed by alterations in the intracellular Ca2+ and production of reactive oxygen and/or nitrogen species (ROS/RNS). ROS and RNS are molecules that play a significant role in plant-herbivore interactions. These reactive molecules are related to cell death but also they have an important role in transmitting information to allow appropriate cellular responses. For this reason, a dual role has been associated to these molecules as direct defenses and as signaling molecules. Despite its importance, no information has been reported on the relationships between ROS/RNS and plant defence responses to mites. The aim of our research is deciphering the role of ROS homeostasis in plant defence to pests. Particularly, we have focused our attention on one oxidoreductase, the thioredoxin h-5, in the plant-spider mite interphase. Thioredoxins are important enzymes that reduce oxidized cysteine residues and cleavage disulfide bonds of proteins. TRX h-5 reduces the disulphide bonds of non-expressor of pathogenesis-related (NPR1) oligomer realising NPR1 monomers that are able to enter in the nucleus and activate the expression of pathogenesis-related gene (PR1), which is important for defense against biotrophic pathogens. Recently, this thioredoxin has emerged as important player to elicit defence response against necrotrophic pathogens. We found that the overexpression of TRX h-5 confers more plant resistance to T. urticae and reduce their fecundity compared to wild type plants and TRX h-5 silenced plants. These results will allow us to go into detail about the molecular signaling of TRX h-5 in defence against T. urticae and to unveil the physiological pathways involved in.
204
S-IX. Comunicación 1
PARTIAL RESISTANCE IN WHEAT IS TRIGGERED UPON
RECOGNITION OF AN AVIRULENCE GENE
Lukas Meile, Clémence Plissonneau, Fanny Hartmann, Patrick Brunner,
Marcello Zala, Parvathy Krishnan, Daniel Croll, Bruce McDonald and Andrea
Sánchez-Vallet
1Plant Pathology Group, USYS, ETH, Zurich, Switzerland
Corresponding author: Andrea Sánchez-Vallet ([email protected])
Adaptation of fungal pathogens to colonize a plant often involves escape from
host surveillance. This can be mediated by sequence polymorphism of
avirulence genes that evolve to prevent recognition by host resistance genes.
Despite the ubiquity and importance of avirulence genes for the infection
outcome, the mechanisms behind their evolution remain unknown. The causal
agent of Septoria Tritici Blotch on wheat, Zymoseptoria tritici, is a necrotrophic
pathogen that is globally distributed. Resistance is mediated by 21 mapped
major resistance genes, of which many lead to partial resistance. It still remains
unknown what components are recognized by these particular resistance
genes. In order to elucidate the genetic basis of quantitative virulence, a genetic
mapping approach was undertaken. Differences between two isolates were
mapped to a transposable element-rich and highly dynamic genomic region that
included a cluster of four genes encoding putative effectors. We confirmed that
one of the genes, Avr3D1, encodes an avirulence protein that is specifically
recognized by some wheat cultivars. Disruption of Avr3D1 in the avirulent
isolate led to an increase in virulence on the resistant hosts. Complementation
experiments demonstrated that polymorphism in the coding sequence is
responsible for the differences in virulence between the two isolates. Population
genetics analyses showed that Avr3D1 is present in all of 132 investigated
isolates from around the world and that it has evolved under diversifying
selection. In contrast, the transposable elements and the putative effector
genes surrounding Avr3D1 are under presence/absence polymorphism. Genes
in the cluster were shown to be silenced in vitro but highly up-regulated during
infection. Thus, we identified a novel avirulence gene whose recognition leads
to partial resistance. Its high controlled gene expression regulation, its clear
signs of diversifying selection and its localization in a highly dynamic genomic
environment provide us with evidences of the evolution of this avirulence gene
to escape recognition.
Meile, L. , Croll, D. , Brunner, P. C., Plissonneau, C. , Hartmann, F. E., McDonald, B. A. and
Sánchez-Vallet, A. (2018), A fungal avirulence factor encoded in a highly plastic genomic region triggers partial resistance to septoria tritici blotch. New Phytologist, doi:10.1111/nph.15180
205
S-IX. Comunicación 2
KEY GENES FOR ROOT DEVELOPMENT ARE ALSO RELEVANT FOR GALL FORMATION BY ROOT- KNOT
NEMATODES
Javier Cabrera1, Rocío Olmo1, Alejandra García1, Miguel Ángel Moreno- Risueño2, Maria Fe Andrés3, Carmen Fenoll1 and Carolina Escobar1
1Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La
Mancha, Toledo, Spain, 2Departmento de Biotecnología, Centro de Biotecnología y
Genómica de Plantas, Universidad Politécnica de Madrid INIA, Madrid, Spain 3Departamento Protección Vegetal, Instituto Ciencias Agrarias, CSIC, Madrid, Spain.
Corresponding author: Carolina Escobar ([email protected])
Root-knot nematodes (RKNs; Meloidogyne spp.) are a group of plant endoparasitic nematodes that cause a negative impact on plant productivity. The search for control methods based on biotechnological alternatives is pertinent because of the gradual banishment of effective but contaminant chemical nematicides. RKNs form feeding cells, giant cells (GCs), induced from vascular cells by nematode effector molecules. GCs increase notably their volume and suffer other changes, e.g., a modified cell cycle with repeated mitosis and partial cytokinesis. Additionally, a knot or gall is formed around the GCs by the divisions and hypertrophy of several tissues within the root. The transcriptomes of early-developing Arabidopsis GCs/galls in Arabidopsis (Barcala et al., 2010) were compared to those characteristics of different root cell types (Brady et al., 2007). Galls/GCs transcriptomes showed a clear enrichment in genes characteristics of undifferentiated root cell types, as those from the quiescent centre (QC), protoxylem and the lateral root initial cells (Cabrera et al., 2014). Therefore, we studied the expression and function of key genes for the development of these cell types. Genes with essential roles stem cell maintenance and root QC establishment like SCHIZORIZA (SCZ), SCARECROW (SCR), SHORT ROOT (SHR) and 5 (WUSCHEL-RELATED HOMEBOX WOX5) were induced in Arabidopsis galls. Loss of function lines of these genes showed a decrease in the number of infections and in some cases in the reproduction parameters as compared to the wild type lines. Furthermore, HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), a protoxylem marker in the root apical meristem, was also induced in galls and the mutant line ahp6 presented a significant reduction in the number of galls formed by RKNs. Our main conclusion is that genes characteristics of root developmental processes are also relevant during gall development.
Barcala et al., 2010. Plant J 61: 698; Brady et al., 2007. Science 318: 801; Cabrera et al.,
2014. New Phytol. 203: 632
206
S-IX. Comunicación 3
UNEXPECTED NEGATIVE EFFECT OF Watermelon mosaic virus
(WMV) P1 GENE PRODUCT ON THE SILENCING SUPPRESSOR
ACTIVITY OF Cucurbit yellow stunting disorder virus (CYSDV) P25
Maria Luisa Domingo-Calap1, Ana Beatriz Moreno1, Mariona Estapé1, Juan José
López-Moya1
1Centre for Reserach in Agricultural Genomics, CRAG, CSIC-IRTA-UAB-UB, Campus UAB
Bellaterra, Cerdanyola del Valles, Barcelona, Spain
Corresponding author: Juan José López-Moya ([email protected])
While numerous viral diseases are known to be caused by a single virus, field surveys and
metagenomics studies are revealing the frequent occurrence of mixed viral infections in
plants. In most cases when a potyvirid is involucrated in mixed infections, the load of the
unrelated virus partners showed significant increases, resulting in synergism. This effect was
suggested to be related with the highly efficient activity of the RNA silencing suppresors
(RSS) of the potyvirus, the HCPro gene product. Our current project is studying mixed
infection caused by the potyvirus Watermelon mosaic virus (WMV) and the crinivirus
Cucurbit yellow stunting disorder virus (CYSDV), both being relevant pathogens affecting
cucurbit crops in the Mediterranean bassin area. In this pathosystem, we saw indeed an
increase in the viral load of CYSDV in melon during mixed infections with WMV (Domingo-
Calap, 2018).
In an attempt to characterise at a molecular level this synergism, we performed transient co-
expression assays of viral proteins with RSS activity (P25 of CYSDV, and HCPro of WMV),
together with proteins no exhibiting this activity (such as P22 of CYSDV, and P1 of WMV).
The purpose was to evaluate possible interferences, and the influence of different
heterologous gene products that could co-exist in mixed infections. Our results showed that
the presence of the P1 protein of WMV had an unexpected negative effect on the RNA
silencing suppression activity of the P25 protein of CYSDV. This effect had not been
previously described in other combinations of criniviruses and potyviruses, and we also
confirmed that the interference was dose-dependant. Regarding the subcellular localization
of each of the proteins involved, we found that both the proteins P1 of WMV and P25 of
CYSDV were detectable in both the nucleus and the cytoplasm. Microscopy observations
also confirmed that both proteins can interact in vivo. The implications of our findings to the
specific responses during mixed viral infections will be discussed.
Domingo-Calap, M.L. (2018) Efectos biológicos en infecciones mixtas de crinivirus y virus de la familia
Potyviridae. Tesis Doctoral, UAB.
This work was funded by grant AGL2016-75529-R (Mineco, Spain).
207
S-IX. Comunicación 4
NAHG INCREASES TRANSIENT TRANSFORMATION OF
ARABIDOPSIS LEAVES BY Agrobacterium
Pepe Cana-Quijada1, Tábata Rosas-Díaz2, Vitor Amorim-Silva1, Miguel A Botella1, Rosa Lozano-Durán2, Eduardo R Bejarano1
1Instituto de Hortofruticultura Subtropical y Mediterránea ‘‘La Mayora’’, (IHSM-UMA-CSIC),
Universidad de Málaga, Málaga, Spain, 2Shanghai Center for Plant Stress Biology/CAS Center for Excellence in Molecular Plant Science, Chinese Academy of Sciences, Shanghai,
China Corresponding author: Eduardo R Bejarano ([email protected])
Agrobacterium tumefaciens-mediated transient transformation has demonstrated to be an
invaluable tool in plant molecular biology studies. However, low efficiency and inconsistency
of this method in Arabidopsis has forced the implementation of Nicotiana benthamiana as a
surrogate system, limiting applicability. One of the main reasons to underlie the recalcitrance
of Arabidopsis to Agrobacterium-mediated transformation is the activation of plant immune
responses upon perception of the bacteria. Perception of bacterial pathogen-associated
molecular patterns (PAMPs), including EF-Tu, leads to activation of PAMP-triggered
immunity (PTI). Activation of PTI ultimately induces salicylic acid (SA) accumulation, which in
turn shuts down expression of the vir genes, potentially interfering with the transfer of the T-
DNA, and therefore T-DNA gene expression (Yuan et al., 2007; Anand et al., 2008).
However, previous results suggested that besides depletion of SA other hormone-mediated
defence responses, including jasmonic acid (JA), might be responsible for the low efficiency
of transient transformation in Arabidopsis (Tsuda et al., 2012).
In this work, we evaluate the efficiency of Agrobacterium-mediated transient transformation
in Arabidopsis genotypes affected in JA perception or signalling (coi1, jin1), or with low SA or
JA content (sid2, NahG, aos). The results show that impairment of JA signalling reduces or
does not affect transient expression in mature leaves, but expression of the NahG transgene
dramatically improves this process. We demonstrate that Arabidopsis NahG plants can be
efficiently used for transient expression-based functional assays routinely performed in N.
benthamiana, such as determination of subcellular localization of GFP-fused proteins or
analysis of protein-protein interactions by Bimolecular Fluorescent Complementation.
Considering the wide-spread use of Agrobacterium-mediated transient transformation, this
system can enormously facilitate research in this model plant species, allowing for an
efficient use of the full potential of the numerous tools and resources currently available to
the community.
Anand, A et al. (2008). Plant Physiol. 146:703–715.
Tsuda, K et al. (2012). Plant J. 69:713–719.
Yuan, Z.C. et al. (2007). Proc. Natl. Acad. Sci. USA 104:11790–11795.
This work was supported by the Spanish Ministerio de Ciencia y Tecnología (AGL2016-75819-C2 and
BIO2014-55380R)
208
S-IX. Póster 1
GEMINIVIRUS REPLICATION PROTEIN IMPAIRS SUMO
CONJUGATION OF PCNA AT TWO ACCEPTOR SITES Blanca Sabarit1, Manuel Arroyo-Mateos1,2, Francesca Maio2, Miguel Sánchez- Durán1, Tábata Rosas-Díaz1,4, Alejandro F. Uceta-Gamaza1, Marcel Prins3, Javier Ruiz -Albert1, Ana P. Luna1, Harrold A. van den Burg2 and Eduardo R. Bejarano1.
1Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, (IHSM-UMA-CSIC),
Málaga, Spain. 2Molecular Plant Pathology, Swammerdam Institute for Life Sciences, Faculty
of Science University of Amsterdam, Amsterdam, The Netherlands. 3Keygene NV,
Wageningen, The Netherlands. 4 Shanghai Center for Plant Stress Biology/CAS Center for
Excellence in Molecular Plant Science, Chinese Academy of Sciences, Shanghai, China.
Corresponding author: Eduardo R. Bejarano ([email protected])
Geminiviruses are plant viruses with circular, single-stranded DNA (ssDNA) genomes
that infect a broad range of plants causing substantial crop diseases worldwide. They
replicate in nuclei of infected cells by using host DNA replication machinery and an
essential protein encoded in their genome designated Rep (replication-associated
protein). This multifunctional protein induces the accumulation of the host factors
involved in replication and it is capable of interacting with a lot of plant proteins
including PCNA (Proliferating Cell Nuclear Antigen), a processivity factor that
coordinates a wide range of processes involved in maintenance, duplication and
transmission of the genome, and the sumoylation enzyme that conjugates SUMO to
target proteins (SUMO-conjugating enzyme- SCE). PCNA modification by SUMO,
and also ubiquitin, has long been known to be of key importance for determining how
DNA damage is processed by the replisome and for maintenance of overall genome
integrity. In yeast, PCNA sumoylation has been associated to DNA repair involving
homologous recombination (HR). Previously, we reported that Rep ectopic
expression does not result in broad changes in the sumoylation pattern of plant cells,
but it modifies the sumoylation state of selected host proteins. In this work, we show,
using a reconstituted sumoylation system in Escherichia coli, that tomato PCNA is
sumoylated at two residues, K254 and K164, and that co-expression of the Rep
protein suppresses PCNA sumoylation at these lysines. Finally, we confirm that
PCNA is sumoylated and that Rep also interferes with PCNA sumoylation in planta.
This work was supported by AGL2016-75819-C2 and BES-2014-069064
209
S-IX. Póster 2
GENOME-WIDE ASSOCIATION STUDY UNRAVEL CYTOKININS AS
A MAJOR COMPONENT IN THE ROOT DEFENSE RESPONSES
AGAINST BACTERIAL WILT CAUSED BY Ralstonia solanacearum
Alejandro Alonso-Díaz1, Santosh B Satbhai3, Christian Göschl3, Wolfgang Busch3,
Marc Valls1,2 & Núria S. Coll1
1 Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra, Spain
2 Genetics Department, University of Barcelona, Barcelona, Spain 3 Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna,
Austria
Corresponding author: Núria Sánchez Coll ([email protected])
The causing agent of bacterial wilt, Ralstonia solanacearum, is a soilborne pathogen
that invades plants through their roots, traversing many tissue layers until it reaches
the xylem, where it multiplies and causes plant collapse. The effects of R.
solanacearum infection are devastating and no effective approach to fight the
disease is so far available. Leaf wilt has been typically used to score R.
solanacearum infection, with few studies analyzing the very early steps of infection,
essential for colonization. We have developed an in vitro system that allows studying
the interaction of R. solanacearum with plants precisely focusing on distinctive root
phenotypes associated to the infection1.
Here, we have used genome-wide association study (GWAS) to identify genomic
regions associated with one of the root phenotypes caused by R. solanacearum
infection: root growth inhibition. Using a panel of 480 Arabidopsis thaliana
accessions, we have identified allelic variation at several loci related to cytokinin
metabolism (synthesis and degradation). Cytokinins have been previously shown to
play a fundamental role in plant defense, although its mechanisms details are far
from being elucidated. Our data show that i) R. solanacearum infection induces
cytokinin signaling in the root and ii) cytokinin deficiency results in enhanced
susceptibility to R. solanacearum infection. R. solanacearum also has the ability to
synthesize cytokinin. Interestingly, the cytokinin-deficient R. solanacearum strain is
more virulent than the wild-type, indicating that R. solanacearum-derived cytokinin
could also be perceived by the plant as a cue to activate immunity against the
pathogenic bacteria. Taken together, these preliminary data underscores the
importance of cytokinin in defense against the vascular pathogen R. solanacearum.
Our goal is to better understand the mode of action of this hormone in root defense
and its interplay with other defense hormones. In addition, this work pinpoints the
value of natural variation studies to identify genetic determinants of complex traits. 1. Lu, H. et al. Type III Secretion–Dependent and –Independent Phenotypes Caused by Ralstonia
solanacearum in Arabidopsis Roots. Mol. Plant-Microbe Interact. 31, 175–184 (2018).
210
S-IX. Póster 3 UNRAVELLING THE EPIGENETIC BASIS OF PLANT IMMUNITY
Ana López Sánchez1,2, Joost Stassen2, Ritushree Jain3,2, Leonardo Furci2, Carmen
Castresana1 and Jurriaan Ton2.
1Dept. of Plant Molecular Genetics- C. Castresana´s Lab, Spanish National Center for
Biotechnology (CNB-CSIC), Madrid, Spain, 2Animal and Plant Sciences Dept- P3 Institute,
The University of Sheffield, Sheffield, UK, 3AgriBio, La Tobe University, Bundoora, Australia.
Corresponding author: Ana López Sánchez ([email protected])
In response to a pathogen attack, plants induce a variety of different defence
mechanisms. In addition, after an infection, plants respond more effectively against a
second pathogen encounter. This is known as acquired immunity or priming. Over
recent years, evidence has accumulated that this priming of defence can be
transmitted to the next generation of plants, resulting in transgenerational acquired
resistance (TAR). As epigenetics refers to the study of inheritable changes that
modify the phenotype of the organisms in the absence of changes in the DNA
sequence, this discovery prompted further research to resolve the role of epigenetic
mechanisms in plant immunity. Here, we present our latest results and future plans to
decipher the epigenetic basis of plant immunity and priming.
Methylation at the level of the DNA is one of the most relevant and stable epigenetic
mechanisms. Analysis of Arabidopsis mutants has shown that TAR is regulated by
RNA-directed DNA methylation (RdDM) and ROS1-dependent DNA (de)methylation.
This work also revealed that changes in DNA methylation affect nearly half of the
pathogenesis-related transcriptome, illustrating the importance of DNA methylation in
plant immunity. In addition, by analysing the DNA methylome of progenies from
disease-exposed Arabidopsis plants, we found that the methylome responds
dynamically to disease encountered in previous generations. This supports the notion
that DNA methylation provides transgenerational phenotypic plasticity to biotic stress.
Finally, we have studied the parental contributions and specificity of TAR in response
to different (a)biotic stresses. Amongst others, this research has revealed that the
durability of TAR depends on the intensity of stress exposure in the previous
generations. Lastly, we will introduce some current work focused in analysing the
interaction of specific defence pathways (oxylipins mediated pathways) and the
epigenetic machinery, in order to position them in the plant canonical defence
scenario at a molecular level
211
S-IX. Póster 4 MicroRNA-INDUCED GENE REPRESSION ON PLANT-ROOT-KNOT NEMATODE
INTERACTION
Ángela Martínez-Gómez*1, Ana Cláudia Silva*1; Virginia Ruiz-Ferrer1; Javier Cabrera1, Fernando E. Díaz- Manzano1, Rocío Olmo1, Marta Barcala1, Isabel Martínez2, Carmen Fenoll1 and Carolina Escobar1 1Department
of Environmental Sciences, Faculty of Environmental Sciences and Biochemistry, Universidad de Castilla-La Mancha, Avenida Carlos III, s/n, 45071, Toledo, Spain 2Department of Agroforestry Science and Technology
and Genetics, Faculty of Environmental Sciences and Biochemistry, Universidad de Castilla-La Mancha, Avenida
Carlos III, s/n, 45071, Toledo, Spain Corresponding author: [email protected] *Both authors have contributed equally to this work
Plant-parasitic nematodes represent a major threat to the agricultural production and economy worldwide. Within them, the root-knot nematodes (RKN), Meloidogyne spp., are sedentary obligate parasites that need their host plants to complete their life cycles. In the presence of their host plant, these nematodes enter the root by the elongation zone and migrate intercellularly until they reach the root tip, where they turn around and stablish in the differentiation zone within the vascular cylinder. RKNs feed from specialized feeding cells, the giant cells (GCs), that they induce by repeated mitosis without cytokinesis. The cells around the GCs start to proliferate and the cortex cells undergo hypertrophy, developing a pseudo-organ called gall. We have analysed the transcriptomes of the early-developing giant cells and galls induced by Meloidogyne javanica in Arabidopsis roots. At 3 days post infection (dpi), generalised gene repression is observed (Barcala et al., 2010). One of the mechanisms of gene repression in galls could be mediated by small RNAs (sRNAs), through RNA silencing. We have described the tight regulation and role of miR390, miR172 and miR159 during the RKN interaction with Arabidopsis (Cabrera et al., 2016; Díaz-Manzano et al., 2017; Medina et al., 2017). Furthermore, after sRNA-seq we found that a group of 24-nt- sRNAs were highly abundant in galls compared to non-infected tissue. From those 24-nt- sRNAs, most of them were rasiRNAs, described to regulate gene expression through RNA- directed DNA methylation pathways (RdDM; Cabrera et al., 2016). Therefore, we aimed then to deepen in the rasiRNA distribution along the Arabidopsis genome, as well as obtaining data on epigenetic marks (cytosine methylation) in galls and GCs through MethylC-seq and immunolocalization, respectively. For this reason, we are also developing a simultaneous method for extraction of DNA and RNA from the same biological replicate from galls and control root tissue to compare in the same sample the differential methylome and transcriptome of galls formed by Meloidogyne javanica, versus non infected roots at early infection stages.
References Barcala, M., García, A., Cabrera, J., Casson, S., Lindsey, K., Favery, B., et al. 2010. Early transcriptomic events in microdissected Arabidopsis nematode-induced giant cells. The Plant Journal, 61, 698–712.
Cabrera, J., Barcala, M., García, A., Rio-Machín, A., Medina, C. et al. 2016. Differentially expressed small RNAs in Arabidopsis galls formed by Meloidogyne javanica: a functional role for miR390 and its TAS-3 derived tasiRNAs. New Phytologist, 209: 1625–1640.
Díaz-Manzano, F.E., Cabrera, J., Ripoll, J., del Olmo, I., Andrés, M.F., Silva, A.C. et al. 2017. A role for the gene regulatory module microRNA172/TARGET OF EARLY ACTIVATION TAGGED 1/FLOWERING LOCUS T (miRNA172/TOE1/FT) in the feeding sites induced by Meloidogyne javanica in Arabidopsis thaliana. New Phytologist, 217: 813-827.
Medina, C., da Rocha, M., Magliano, M., Ratpopoulo, A., Revel, B. Marteu, N., et al. 2017. Characterization of microRNAs from Arabidopsis galls highlights a role for miR159 in the plant response to the root-knot nematode Meloidogyne incognita. New Phytologist, 216: 882-896.
Acknowledgements & Funding. The work was supported by the Spanish Government AGL2016-75287-R to CE, and PCIN-2013-053 to CF. AMG is supported by a fellowship from Fundación Tatiana Peréz de Guzmán El Bueno. ACS is supported by a fellowship from the University of Castilla-La Mancha, co-founded by the European Social Fund.
212
S-IX. Póster 5
STUDY OF Na+ AND K+ TRANSPORTERS IN PLANTS AND
ASSOCIATED MICROORGANISMS: SYMBIOSIS BETWEEN RICE
AND Piriformospora indica
Mónica Lanza, Rosario Haro, Laura Jaén and Begoña Benito
Centro de Biotecnología y Genómica de Plantas (CBGP), Universidad Politécnica de Madrid
(UPM) - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA).
Campus Montegancedo UPM. 28223-Pozuelo de Alarcón (Madrid), Spain.
Corresponding author: Begoña Benito ([email protected])
Our group is interested in the study of the role of the transporters involved in maintaining the Na+ and K+ homeostasis in fungi and plant cells. For a long time, we have used the yeast Saccharomyces cerevisiae as a model organism to clone and functionally characterize Na+ and K+ transporters expressing in different cellular compartments, initially, its own transporters and later, homologous transporters of others fungi and plants, from the primitive moss Physcomitrella patens to several monocot plants. One aspect that lacks so far is to know whether any of these Na+
and K+ transporters have any relevant role during symbiotic associations. The current goal of our project is to identify the Na+ and K+ transport systems that operate during the symbiosis between plants and microorganisms associated and to determine their contribution to the mineral nutrition and salt tolerance of plants growing in saline conditions. The model organisms used are the endophyte Piriformospora indica in interaction with rice. In a first attempt we have initiated the characterization of the ENA ATPases of the fungus. These are proteins involved in Na+ efflux from cells that have demonstrated to be very effective systems to increase salt tolerance in fungi. Two genes, PiENA1 and PiENA5 have been identified in the Piriformospora genome (https://genome.jgi.doe.gov/Pirin1/Pirin1.home.html). Their cloning and heterologous expression in yeast defective mutants in their own Na+ efflux systems indicated that PiENA1 preferentially transports K+ and PiENA5 Na+. Both genes are constitutively expressed but at very low levels in Piriformospora growing in control conditions and both are induced in saline conditions and during the fungal growth at alkaline pH. The inoculation of Arabidopsis and rice roots with the fungus induced an increase in the growth of both the roots and aerial part of the plants growing in control medium but also exposed to salinity. During infection, PiENA1 and PiENA5 are expressed. Now we want to studied a putative role of these transporters in the increased salt tolerance of the infected plants.
Funded by grant AGL2016-80593-R and BIO2014-56153-REDT from MINECO.
213
S-IX. Póster 6
HISTONE EPIGENETIC MARKS IN ARABIDOPSIS AND TOMATO GENES IN THE EARLY RESPONSE TO Botrytis cinerea
Carmen González Bosch, Óscar Crespo Salvador, Mónica Escamilla Aguilar, Jaime
López Cruz, Lorena Sánchez Giménez.
1Departamento de Bioquímica y Biología Molecular, Universitat de València; Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Valencia, Spain.
Corresponding author: Carmen González Bosch ([email protected])
Our previous studies have provided valuable data on the molecular mechanisms that
underlie basal and induced resistance against B. cinerea in Arabidopsis and tomato
plants (Aranega-Bou et al, 2014). B. cinerea is a broad-host necrotroph that requires
the involvement of many genes and pathways for full resistance. Recent studies have
linked epigenetic modifications with plant responses to biotic stresses. However,
Information about specific histone marks upon necrotrophic pathogens is scarce. Here
we analyzed the histone epigenetic marks associated with gene activation or
repression in Arabidopsis in the differentially expressed genes during the early
response of Arabidopsis plants to B. cinerea. Using an adapted chromatin
immunoprecipitation (ChIP) protocol we obtained differential epigenetic signature of
activating marks H3K4me3, H3K9ac, and the repressor one H3K27me3 on both the
promoter and the body of the highly induced PR1 in Arabidopsis plants infected with B.
cinerea at 24 and 33 h after inoculation. These epigenetic marks were also determined
in two differentially expressed genes in response to B. cinerea, as well as to oxidative
stress, given its relevance in this infection. These are both the induced CYP71A13,
which encodes a cytochrome P450 involved in camalexin synthesis, and is essential
against this necrotroph and the repressed EXL7 (Exordium-like 1). An adapted protocol
allowed determine the presence of the activating marks H3K4me3 and H3K9ac in
genes involved in the tomato basal and induced response to B. cinerea, but also to
changes in the oxidative environment (Finiti et al. 2014; Lopez-Cruz et al. 2017). These
genes are DES (divinyl ethyl synthase), LoxD (lipoxygenase D), DOX1 (α-dioxygenase
1), PR2 (pathogenesis-related protein2), WRKY53 and WRKY33. The obtained results
will allow future studies on epigenetic marks and their transgenerational inheritance in
plants infected with B. cinerea. The information provided may also contribute to find
biomarkers of the early detection of this devastating pathogen.
References Aranega-Bou P, Leyva MO, Finiti I, Garcia-Agustin P, Gonzalez-Bosch C (2014) Front Plant Sci 5:488 Finiti I, Leyva MO, Vicedo B, Gómez-Pastor R, López-Cruz J, García-Agustín P, Real MD, González-Bosch C. (2014). Mol. Plant Pathol. 15, 550–562 Lopez-Cruz J, Crespo O, Fernández E, García-Agustin P, González-Bosch C (2017) Mol Plant
Pathol 18:16–31
Acknowledgements & Funding This work has been supported by grants from the Spanish
Ministry of Science and Innovation (AGL2010-22300-C03-01 and AGL2013-49023-C03-01), co-
funded by European Regional Development Funds (ERDF), and by Generalitat Valenciana
Grupos de Excelencia PROMETEO/2012/066.
214
S-IX. Póster 7
miRNA/phasiRNA MEDIATED REGULATION OF PLANT DEFENSE
RESPONSE AGAINST P. syringae
Diego López-Márquez1, Ángel Del-Espino Pérez1, Nieves López-Pagan1, Edgar A.
Rodríguez-Negrete1, Adela Zumaquero1, Javier Ruíz-Albert1, Ignacio Rubio-
Somoza2, Eduardo R. Bejarano1 and Carmen R. Beuzon1
1Department of Cellular Biology, Genetics and Physiology. IHSM-UMA-CSIC, Málaga, Spain.
2 Molecular Reprogramming and Evolution Laboratory. Centre for Research in Agricultural
Genomics, Barcelona, Spain
Corresponding author: Diego López Márquez ([email protected]) Gene silencing is a mechanism of regulation of gene expression where the small
RNAs (sRNAs) are key components for giving specificity to the system. In plants, two
main types of noncoding small RNA molecules have been found: microRNAs
(miRNAs) and small interfering RNAs (siRNAs). DCL proteins acting on large RNA
precursors produce the mature forms of sRNAs (20-24nt) that can act as negative
regulators of gene expression. In recent years, the role of miRNAs in regulation of
gene expression in plant responses against bacterial pathogens is becoming clearer.
Comparisons carried out in our lab between expression profiles of different
Arabidopsis thaliana mutants affected in gene silencing, and plants challenged with
Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of
uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, as
differentially expressed in these conditions. Through the use of bioinformatics tools,
we found a miRNA* of 22 nt putatively responsible for down-regulating expression of
these R genes. We have validated this regulation, and have also established that the
corresponding pri-miRNA is down-regulated upon PAMPs or bacteria perception.
Using GUS reporters, we have characterized the expression pattern of both pri-
miRNA and its best target R genes. We demonstrate that plants with altered levels of
miRNA* (knockdown or overexpression lines) exhibit altered PTI-associated
phenotypes, supporting a role for this miRNA* in the defence response against this
bacterial pathogen. Finally, we identify phasiRNAs that arise from the transcript of
one of the R target genes in a miRNA*-RDR6-DCL4-dependent manner.
215
S-IX. Póster 8
MODELLING OF PLANT CELL WALL COMPOSITION LINKS
SPECIFIC WALL STRUCTURES WITH DISEASE RESISTANCE AND
FITNESS
Eva Miedes1,2, Nicolas Denance3, Tinguaro Rodríguez4, Laura Bacete1,2, Hugo Mélida1, Andrea Sánchez-Vallet1, Marie Pierre Rivieré1, Gemma López1, Philippe Ranocha3, Xavier Bartel3, Yves Marco3, Michael Hahn5, Deborah Goffner2, and
Antonio Molina1,2
1Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM)-
Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus
Montegancedo UPM, Pozuelo de Alarcón, Madrid, Spain. 2Departamento de Biotecnología- Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de
Biosistemas, Universidad Politécnica de Madrid, Madrid, Spain. 3Unité Mixte de Recherche. Centre National de la Recherche Scientifique. Univ Toulouse III. Castanet Tolosan, France.
4Departamento de Estadística e Investigación Operativa, Facultad de Mataméticas,
Universidad Complutense de Madrid, Madrid, Spain. 5Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.
Corresponding author: Eva Miedes ([email protected])
Plant cell wall is a complex structure subjected to dynamic remodeling in response to internal and external cues, and plays an essential function in plant disease resistance. We have determined the specific contribution of plant cell wall to pathogens resistance by testing the susceptibility of a diverse set of Arabidopsis thaliana cell wall mutants (cwm) to three pathogens with different colonization styles: the necrotrophic fungus Plectosphaerella cucumerina, the vascular bacterium Ralstonia solanacearum, and the biotrophyc oomycete Hyaloperonospora arabidopsidis. We found that a remarkable 60% of the cwm mutants tested showed, in comparison to wild-type plants, alterations in their disease resistance to at least one of these pathogens, illustrating the relevance of plant cell wall in pathogens colonization. Notably, the enhanced resistance of cwm plants to the necrotrophic fungus and the vascular bacterium, but not to the biotrophic oomycete, impacts negatively on their fitness traits, such as biomass and seed production, though resistance-fitness uncoupling examples were also found. De-regulation of canonical immune pathways, like those modulated by defensive phytohormones or triggered by Microbe-Associated Molecular Patterns, was not observed in the cwm plants showing enhanced resistance or susceptibility, indicating that novel, uncharacterized defensive pathways would contribute to cwm-mediated resistance. Chemically extracted wall fractions from cwm plants showed a high diversity of alterations in their composition as revealed by Fourier-transform infrared spectroscopy and biochemical analyses, and by glycome profiling using specific wall antibodies. Mathematical modeling of the glycome data from a core set of eleven cwm mutants and from wild- type plants, identified specific variations in cell wall carbohydrate moieties which explain a significant proportion of cwm disease resistance and fitness phenotypes, further supporting the relevant function of plant cell wall in regulating these processes.
216
S-IX. Póster 9
CONTRIBUTION OF THE NRAMP6 METAL ION TRANSPORTER TO
DISEASE RESISTANCE IN RICE
Ferran Sánchez-Sanuy1, Cristina Peris-Peris1, Albert Serra-Cardona2, Joaquin
Ariño2, Sonia Campo1, and Blanca San Segundo1
1 Plant responses to stress, Centre for Research in Agricultural Genomics (CRAG) CSIC-
IRTA-UAB-UB. Edifici CRAG, Campus UAB, Bellaterra (Cerdanyola del Vallés), Barcelona,
Spain; 2 Departament de Bioquímica i Biologia Molecular, Institut de Biotecnologia i
Biomedicina and, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona,
Spain
Corresponding author: Blanca San Segundo ([email protected])
Metal ions are essential elements for all living organisms. However, metals can be
toxic when present in excess. In plants, metal homeostasis is partly achieved through
the function of metal transporters, including the diverse natural resistance-associated
macrophage proteins (NRAMP). Among them, the OsNramp6 gene encodes a
previously uncharacterized member of the rice NRAMP family that undergoes
alternative splicing to produce different NRAMP6 proteins. In this work, we
determined the metal transport activity and biological role of the full-length and the
shortest NRAMP6 proteins (l-NRAMP6 and s-NRAMP6, respectively). Both l-
NRAMP6 and s-NRAMP6 are plasma membrane-localized proteins that function as
iron and manganese transporters. The expression of l-Nramp6 and s-Nramp6 is
regulated during infection with the fungal pathogen Magnaporthe oryzae, albeit with
different kinetics. Rice plants grown under high iron supply show stronger induction
of rice defense genes and enhanced resistance to M. oryzae infection. Also, loss of
function of OsNramp6 results in enhanced resistance to M. oryzae, supporting the
idea that OsNramp6 negatively regulates rice immunity. Furthermore, nramp6 plants
showed reduced biomass, pointing to a role of OsNramp6 in plant growth. A better
understanding of OsNramp6-mediated mechanisms underlying disease resistance in
rice will help in developing appropriate strategies for crop protection.
This project was funded by the MINECO (BIO2015-67212 to Blanca San Segundo and BFU2014-
54591-C2-1-P to Joaquin Ariño)
217
S-IX. Póster 10
COMPLETE GENOME SEQUENCE OF A NOVEL VIRUS OF THE
POTYVIRIDAE FAMILY THAT INFECTS PASSION FRUIT (Passiflora
edulis)
Sara Jover-Gil1, Avital Beeri2, Patricia Fresnillo2, Alon Samach2, and Héctor Candela1
1Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, Elche, Spain,
2The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Robert H.
Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem,
Rehovot, Israel.
Corresponding author: Héctor Candela ([email protected])
Passion fruit (Passiflora edulis Sims) is a crop native to Brazil and Paraguay that is
cultivated for its edible fruit, which is highly appreciated for fresh consumption and
the production of juices by industry. Researchers have defined two forms within this
species, yellow and purple, that can be easily crossed to create hybrids such as the
‘Passion Dream’ cultivar (PD). While performing a de novo assembly of the PD
transcriptome, we identified two positive-strand RNA viruses in an RNA sample
isolated from mixed vegetative and reproductive tissues of asymptomatic plants
grown in 2013 in the experimental farm of the Faculty of Agriculture in Rehovot,
Israel. The genomes of both viruses were assembled with the Oases pipeline using
paired-end 51 bp reads (Illumina), and corrected by aligning the reads with Bowtie2.
The genome sequence of the second virus (PeSV) was most similar to the sequence
of the rose yellow mosaic virus (RoYMV), a recently described member of the
Potyviridae family that is sufficiently distinct as to define a new genus on its own. Our
data indicate that PeSV defines a novel species in the same (yet unnamed) genus as
RoYMV. The genome of PeSV is 9,928 nucleotides long, excluding the poly(A) tail,
and contains a long open reading frame (ORF) encoding a 3,173 aa polyprotein
predicted to be proteolytically processed into ten mature peptides (P1, HC-pro, P3,
6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb and CP) at consensus cleavage sites similar to
those found in the polyproteins of other potyviruses.
218
S-IX. Póster 11
THREE-STEP SCREENING OF BACTERIA-ORIGINATED
COMPOUNDS FOR IDENTIFICATION OF NEW MODULATORS OF
EARLY PLANT DEVELOPMENT.
Isabel Manrique Gil1, Pilar Martínez Hidalgo2, Raúl Rivas González1,2, Óscar Lorenzo Sánchez1,3.
1Spanish-Portuguese Agricultural Research Institute (CIALE), University of Salamanca, Salamanca.
2Microbiology and Genetics Department, University of Salamanca, Salamanca 3Botany and Plant Physiology Department, University of Salamanca, Salamanca
Corresponding author: Óscar Lorenzo Sánchez ([email protected])
Germination and seedling establishment are vital processes during plant
development where hormonal signalling (i.e. abscisic acid, gibberellins) must be
properly regulated to guarantee the survival of the plant. The gasotransmitter nitric
oxide (NO) also plays an important role in these processes through its interaction
with many hormonal signalling pathways (Bethke et al, 2004).
Chemical genomics offer the possibility to identify novel organic molecules that can
modulate plant development (Robert et al, 2008). In this regard, bacteria are a useful
resource for searching new bioactive compounds.
The purpose of this work is the identification of new molecules produced by bacteria
that promote early plant development and could be used as plant growth promoter
compounds. To achieve this objective, a three-step screening strategy is performed
with a library of more than 100 bacteria crude extracts. This library includes crude
extracts from different species of Paenibacillus, Pseudomonas, Rhizobium or
Micromonospora; genera previously described as plant growth-promoting
rhizobacteria (PGPR).
First step consists on the analysis of the phenotypes observed during germination
and seedling establishment caused by these crude extracts in wild type seeds of
Arabidopsis thaliana. After this preliminary selection, we continued studying the
crude extracts that positively affected the germination and seedling development. To
assess if a specific phytohormone or NO signalling is involved in the observed
phenotype, selected crude extracts are tested with mutants impaired in hormone
perception and signalling pathways and in NO homeostasis. To further confirm the
specific hormonal pathway and if there is a perturbation in cell cycle activity, different
GUS reporter lines will be employed in the third step. Last, we will advance in the
identification of individual pure compounds with plant-growth promotion properties for
biotechnological applications. References Bethke et al. (2004) Planta 219:847–855 Robert et al. (2009). The Arabidopsis Book 7: e0109.
Acknowledgements & Funding (Arial, 10 pt, justified). This work is financed by Unidad de Excelencia del CIALE: “Producción Agrícola y Medioambiente”.
219
S-IX. Póster 12
FUNCTIONAL ANALYSIS OF NITRIC OXIDE (NO) HOMEOSTASIS
DURING THE Botrytis cinerea-HOST PLANT INTERACTION
Isabel Torres-Quezada1, Ernesto Pérez Benito2, María Teresa Escribano3 and Óscar Lorenzo1.
1Dpto. de Botánica y Fisiología Vegetal, Instituto Hispano-Luso de Investigaciones Agrarias (CIALE), Facultad de Biología, Universidad de Salamanca, C/ Río Duero 12, Salamanca
37185, Spain. 2Dpto. de Microbiología y Genética, Instituto Hispano-Luso de Investigaciones Agrarias (CIALE), Facultad de Biología, Universidad de Salamanca, C/ Río Duero 12,
Salamanca 37185, Spain. 3Dpto. de Química Analítica, Nutrición y Bromatología, Grupo de Investigación en Polifenoles, Facultad de Farmacia, Universidad de Salamanca, Campus
Miguel de Unamuno, 37007, Salamanca, Spain. Corresponding author: Óscar Lorenzo ([email protected])
Nitric oxide (NO) is a gaseous free radical involved in a great variety of physiological processes related not only to growth and development, but also to abiotic and biotic stresses (Sanz et al. 2015). It has been described that during plant–pathogen interactions, the hydrogen peroxide and the NO produced by the plant play a crucial role on the activation of plant cell death leading to the hypersensitive response (HR) (Delledonne et al., 1998). Botrytis cinerea is a necrotrophic plant pathogen with a wide range of hosts. Experimental evidence indicates that this fungus exploits the hypersensitive cell response of healthy plant tissues for a successful infection (Govrin and Levine, 2000). In addition, B. cinerea survives in this hostile environment and is certainly exposed to toxic reactive oxygen species (ROS) and to NO itself. The pathogen would be expected to benefit from mechanisms limiting damage caused by ROS, NO, and NO-derived reactive nitrogen species (RNS). It has been described that flavohemoglobins are the only NO detoxification method in B. cinerea (J.L Turrión- Gómez et al. 2010) and it has been demonstrated that B. cinerea not only can detoxify but also produce NO both during saprophytic growth and during interaction with its host (Turrión-Gómez and Benito, 2011). Despite all the above, research about the presence and function of NO in phytopathogenic fungi is scarce. This work aims to decipher the implication of NO through the colonization process of B. cinerea and the plant response to the infection through metabolic and pharmacologic approaches by using genetic tools available in Arabidopsis impaired in NO homeostasis.
References: Delledonne, M., Xia, YJ, Dixon, RA, Lamb, C (1998). Nature. 394: 585-588. Govrin, EM and Levine, A (2000) Curr. Biol. 10(13): 751-7. Sanz L, et al. (2015). J Exp Bot. 66, 2857-68. Turrión-Gómez JL, and Benito, EP (2011) Mol. Plant. Pathol. 12: 606-616. Turrión-Gómez, JL, Eslava, AP, Benito, EP (2010) Fungal Genet. Biol. 2010, 47(5): 484-96.
Acknowledgements & Funding Unidad de Excelencia “Producción agrícola y medio ambiente”, Universidad de Salamanca.
220
S-IX. Póster 13
THE BACTERIAL EFFECTOR HopZ1a ACETYLATES THE ZIP1
KINASE TO SUPPRESS ARABIDOPSIS DEFENCE RESPONSES
Javier Rueda-Blanco, Jose S. Rufián, Diego Lopez-Marquez, Carmen R. Beuzón,
Javier Ruiz-Albert
Instituto de Hortofruticultura Subtropical y Mediterránea, Universidad de Málaga-Consejo
Superior de Investigaciones Científicas (IHSM-UMA-CSIC). Dpto. Biología Celular, Genética
y Fisiología, Campus de Teatinos, Málaga, E-29071, Spain
Corresponding author: Javier Rueda Blanco ([email protected]) During the plant-pathogen interaction, disease or resistance are determined in the
plant by a series of molecular events. The plant detects Pathogen-Associated
Molecular Patterns (PAMPs), such as flagellin, triggering a defence response called
PTI (PAMP-Triggered Immunity). Bacterial pathogens can in turn suppress such
defence response through the translocation into the plant cell cytosol of virulence
proteins, called effectors, via a Type Three Secretion System (T3SS). In resistant
plants, intracellular receptors known as R proteins recognize these effectors,
triggering a second line of defence, more specific and intense, called ETI (Effector-
Triggered Immunity), which usually leads to programmed cell death known as HR
(Hypersensitive Response). Pseudomonas syringae is a phytopathogenic bacterium
whose virulence depends on a T3SS and its effector repertoire. Some strains include
HopZ1a, an unusual effector which is able to suppress in Arabidopsis both local (PTI
and ETI), and systemic (SAR, for Systemic Acquired Resistance) defences, by
means of its acetyltransferase activity. In resistant Arabidopsis plants, HopZ1a
acetylates the ZED1 pseudokinase, which is proposed to function as a decoy
mimicking HopZ1a target in the plant: ZED1 modification activates an R-protein
(ZAR1) to trigger HopZ1a-dependent ETI. None of the Arabidopsis proteins proposed
to date as HopZ1a targets is a kinase, nor fully explains the effector´s defence
suppression abilities. In this work we identify an Arabidopsis kinase that functions as
a positive regulator of PTI, ETI and SAR, which interacts with HopZ1a and is
acetylated by this effector in lysine residues essential for its kinase activity. Further,
HopZ1a can specifically suppress the defence phenotypes resulting from ZIP1
expression in Arabidopsis. We propose that ZIP1 acetylation by HopZ1a interferes
with its kinase activity, and consequently with positive defence signalling.
221
S-IX. Póster 14
UNCOVERING Arabidopsis thaliana IMMUNE RESPONSES
REGULATED BY PLANT CELL WALL
Laura Bacete1,2, Hugo Mélida1 Eva Miedes1,2, Antonio Molina1,2
1Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM) -
Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus de
Montegancedo UPM, 28223-Pozuelo de Alarcón (Madrid), Spain. 2Departamento de
Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica,
Alimentaría y de Biosistemas, UPM, 28040-Madrid, Spain.
Plant cell wall, a complex cellular structure that surrounds all plant cells, is an
important component of plant disease resistance. Besides its essential role as a
physical barrier for pathogens, plant cell wall is also a dynamic structure whose
integrity is modified during the infection process or in response to environmental
stresses. These changes in cell wall integrity cause the release of wall molecules –
known as Damage Associated Molecular Patterns (DAMPs)– which are perceived by
complex sets of plasma membrane proteins receptors, activating immune responses
conferring enhanced disease resistance. In previous studies performed in our
research group, we have demonstrated that a set of Arabidopsis thaliana mutants
with alterations in their cell wall composition showed enhanced resistance or
susceptibility to biotrophic and necrotrophic pathogens. The characterization of the
molecular bases of the differential disease resistance of these wall mutants proved
that these defensive responses are largely due to the active role of the plant cell wall
as a source of DAMPs. We found that pectin enriched wall fractions from these
mutants were more active than the corresponding fractions from wild-type plants in
modulating immunity responses, such as increase in cytoplasmic [Ca2+], the
phosphorylation of Mitogen-Activated Protein Kinases (MAPKs) cascades and the
expression of defence genes. In-depth biochemical characterization of these active
fractions revealed that they contained a significant amount of pentose
oligosaccharides. Among them, an active novel wall DAMP was found that activated
immune responses similar to those observed after treatments with the active pectin-
enriched fractions. Remarkably, we found that the responses activated by this novel
wall DAMP depend on receptors of the Lysine Motif family, such as CERK1.
Moreover, the immune responses activated by this novel wall DAMP differ from those
activated by other cell wall DAMPs, such as pectin-derived oligogalacturonides. We
will present our recent progresses in the characterization of the mechanisms
underlying the active role of Arabidopsis thaliana cell wall in the regulation of plant
immunity.
This work was supported by Spanish Ministry of Economy and Competitiveness (MINECO) grants
BIO2015-64077-R and BIO2012-32910 to AM. LB was recipient of a FPI fellow (BES-2013-065010)
from MINECO and HM was funded by an IEF grant (SignWALLINg-624721) from the European Union.
222
S-IX. Póster 15
DYNAMICS OF RNA SILENCING SUPPRESSION UPON PATHOGEN INFECTIONS
Álvaro de Andrés-Pablo, Luis Villar-Martín, Ignacio Rubio-Somoza
Molecular Reprogramming and Evolution (MoRE) Laboratory, Centre for Research in Agricultural Genomics (CRAG), Cerdanyola del Vallés, Spain.
Corresponding author: Ignacio Rubio-Somoza ([email protected]) RNA silencing is an antiviral defence mechanism in plants and animals. In order to
evade that layer of defence, viruses produce counteracting effectors, known as
silencing suppressors. It has been recently found that pathogens other than viruses,
such as bacteria and oomycetes, also produce silencing suppressors as part of their
infection strategy. Despite of that evolutionary convergent strategy, the ways those
different classes of pathogens infect and spread in the host are diverse. Those
dissimilarities might encompass a differential regulation of the production of their
specific silencing suppressors and the host cells target of their action. The
intracellular presence of silencing suppressors does not only disrupt a host´s defence
system but interferes with other endogenous processes orchestrated by this
universal gene regulatory system. Of especial interest is their impact on micro RNA-
mediated gene regulation (miRNA, a subclass of small RNAs). Plant miRNAs tend to
regulate the expression of genes with pivotal roles in plant development and stress
responses. Since the repertoire of miRNAs and their targets is cell-type specific, the
molecular events conducting to effector-mediated transcriptional reprogramming in
plant cells might differ. Establishing the dynamics of pathogen-triggered miRNA
dysfunction is a first key step to broaden our knowledge about the subsequent
molecular events led by pathogen-deployed silencing suppressors. To reveal at
which point of the infections and in which cell-types different pathogens interfere with
miRNA function, we have combined fluorescently-labeled pathogens and plants
bearing a sensor of miRNA activity. Therefore, in those specific cells in which RNA
silencing suppressors impair the normal functioning of the miRNA machinery
luminescence signal should be recovered.
Results from experiments with the single stranded RNA (ssRNA) virus Plump Pox
Virus (PPV), and the bacterium Pseudomonas syringae will be discussed.
Acknowledgements and funding: We acknowledge the generous donation of GFP labeled pathogens to Juan Antonio García (PPV) and Sheng-Yang He (Pseudomonas syringae DC3000). The work in the MoRE group is funded by Spanish Ministry of Economy and Competitiveness (I.R-S is supported by BFU-2014-58361-JIN, RYC-2015-19154; L. V-M is supported by BES-2016-076986) and through the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016-2019 (SEV-2015- 0533) and the CERCA programme from the Generalitat de Catalunya.
223
S-IX. Póster 16
PHOSPHORYLATION CONTRIBUTES TO THE COAT PROTEIN-
MEDIATED REGULATION IN PLUM POX VIRUS INFECTION
Marta Hervás, Juan Antonio García and Sandra Martínez-Turiño
Department of Plant Molecular Genetics, Centro Nacional de Biotecnología, Madrid, Spain
Corresponding autor: Sandra Martínez-Turiño ([email protected])
Plum pox virus (PPV) (genus Potyvirus) causes sharka, one of the most damaging diseases affecting Prunus production worldwide. PPV genome is a positive-sense single-stranded RNA encapsidated by a single type of capsid protein (CP) in flexuous rod particles. Potyviral CP contains a core region highly conserved and two more variable N-t and C-t exposed protrusions. Roles of potyviral CP go beyond encapsidation of viral genome and virus spread within and between plants, since also extend to genome replication and translation.
Our previous studies had shown PPV CP is modified by O-GlcNAcylations and phosphorylations in the N-proximal segment of CP, and we postulated that the joint actions of both post-translational modifications (PTMs) modulates the protein stability, providing the fair amount of CP required in each step of viral infection. A further characterization of the phosphorylation pattern of PPV virions, allowed to detect additional phospho-targets along the whole protein. At least, two of them, in the core ending (pThr254) and in the C-terminus region (pThr313) seem to be crucial for viral infection.
To enquire the putative role of phosphorylation at the C-t region of the CP, viral mutants to independently mimic phosphorylation at Thr254 and Thr313 were used. Thus, cell-to-cell movement, virion formation and negative RNA strand synthesis was analysed for both mutants. Additionally, we found that the deleterious effect concerning virion structure/stability, as consequence of emulating CP phosphorylation in the N-t part, can be partially restored through spontaneous mutations arising not only in a specific phospho-target in this region but also in the distant Thr254. This last finding, together with some competitions assays carried out with properly mutants, allowed to reveal the existence of a functional interplay between phosphorylations at N-t and C-t segments of PPV CP.
Interestingly, phosphorylations at the N-t and C-t regions of CP seem to separately work in different functions during viral cycle. However, there are also evidences showing that phosphorylations at these two regions could share certain role during infection. Finally, we postulate PPV CP behaves as a dynamic hub, able to participate in a wide range of viral steps by means of a phospho-dependent finest- tuning.
224
S-IX. Póster 17 MITOGEN-ACTIVATED PROTEIN KINASE PHOSPHATASE 1 (MKP1)
NEGATIVELY REGULATES MICROBE-ASSOCIATED MOLECULAR
PATTERN-TRIGGERED IMMUNITY IN ARABIDOPSIS
Miguel Ángel Torres1,2 , Viviana Escudero1,2, Magdalena Delgado1,2, Sara Sopeña-
Torres1,2, Sanjay Swami1,2, Jorge Morales1,2, Antonio Muñoz-Barrios1,2, Hugo Mélida1,2,
Lucía Jordá1,2 and Antonio Molina1,2
1Department-Laboratory, Institution, City, Country, 2Deparment-Laboratory, Institution, City,
Country, 3Deparment-Laboratory, Institution, City, Country. (Arial, 11 pt, italics, centered) 1Centro de Biotecnología y Genómica de Plantas (CBGP), Centro Severo Ochoa, Universidad
Politécnica de Madrid (UPM) – Instituto Nacional de Investigación y Tecnología Agraria y
Alimentaria (INIA), Campus Montegancedo, 28223-Pozuelo de Alarcón (Madrid), Spain. 2Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingeniería
Agronómica, Alimentaria y de Biosistemas, 28040-Madrid, Spain.
Corresponding author: Miguel Ángel Torres1 ([email protected])
The heterotrimeric G protein complex is an essential regulator of Arabidopsis immune
responses. Genetic ablation of G protein function, as it occurs in the agb1-2 mutant, which
lacks the β subunit protein (AGB1) of this complex, confers defective activation of
Microbe-Associated Molecular Pattern (MAMP)-triggered immunity resulting in
susceptibility to different pathogens, including the necrotrophic fungus Plectosphaerella
cucumerina BMM (PcBMM). A mutant screen for suppressors of agb1-2 susceptibility (sgb
mutants) to PcBMM identified the sgb10 mutant, which harbours a null allele (mkp1-2) of
the mitogen-activated protein kinase (MAPK) phosphatase 1 gene (MKP1). MKP1 is a
negative regulator of the activity of MAPKs during MAMP-triggering immunity and the
salicylic acid (SA) signalling pathway. We found that the SA pathway is constitutively
active in the mkp1-2 agb1-2 double mutant and that the enhanced susceptibility of agb1-2
to the bacterium Pseudomonas syringae pv. tomato DC3000 and the oomycete
Hyaloperonospora arbidopsidis is restored to wild-type levels by the mkp1-2 allele. The
production of reactive oxygen species (ROS) is enhanced upon MAMP treatment in mkp1
mutants compared to wild-type plants, indicating that MKP1 negatively balances the
production of ROS triggered by MAMPs. This MKP1 function takes place through the
transcriptional modulation of the NADPH oxidase gene RbohD, which is enhanced in
mkp1 plants upon MAMP treatment or pathogen infection. Moreover, MKP1 regulates
ROS production by modulating RBOHD activity, since mkp1 plants constitutively
overexpressing RbohD (35S::RbohD mkp1) display elevated ROS levels upon MAMP
treatment. In addition, a significant reprograming of the metabolic profile occurs in mkp1
plants, with more than 200 metabolites (including antimicrobial metabolites) showing
differential accumulation in comparison to wild-type plants. These results indicate that
MKP1 functions downstream of the heterotrimeric G protein during MAMP-triggered
immunity, directly regulating the activity of RboHD, as well as other immune responses
required for disease resistance.
This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) grant
BIO2015-64077-R.
225
S-IX. Póster 18
ANALYSIS OF THE CELLULAR LOCALIZATION OF MELON VPS41,
A PROTEIN THAT CONFERS RESISTANCE TO Cucumber mosaic
virus
Nùria Real1 and Ana Montserrat Martín Hernández1,2
1Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, C/ Vall
Moronta, Edifici CRAG, Bellaterra (Cerdanyola del Vallès), 08193 Barcelona, Spain. 2IRTA
(Institut de Recerca i Tecnologia Agroalimentàries), Barcelona, Spain.
Corresponding author: Ana Montserrat Martín Hernández ([email protected])
Recessive Resistance to CMV is usually oligogenic, governed by several
Quantitative Trait Loci (QTLs). In the Korean melon accession "Songwhan Charmi"
(SC), we have identified a gene, cmv1, which confers resistance to strains of
subgroup II, like CMV LS by preventing viral transport from the bundle sheath cells,
that surround the vein, to the phloem. However, it cannot restrict the phloem invasion
of strains of subgroup I, like CMV-FNY. This discrimination occurs depending on the
Movement Protein (MP) of the virus. cmv1 encodes a Vacuolar Protein Sorting 41
(VPS41), a protein involved in intracellular trafficking from late Golgi to the vacuole.
By comparing the VPS41 sequence from the susceptible Piel de Sapo and resistant
SC melon genotypes, we have identified the polymorphism L348R as the causal
mutation. Further analysis of VPS41 genes from 54 new exotic melon genotypes
showed that those resistant to CMV-LS did not carry the L348R polymorphism, but a
different causal mutation (G85E) that causes a strong theoretical effect on the
protein.
To analyze more in detail the difference between VPS41 from resistant and
susceptible accessions, we have transiently overexpressed them in N. benthamiana.
VPS41 from both the susceptible PS and the resistant SC genotypes are expressed
in the cytoplasm of the epithelial cells. However, there are some differences between
them, such as formation of speckles on the nuclear and plasma membranes and
"walls" through the vacuole in PS that are absent in SC. These special structures co-
localize with late endosomes. VPS41 from two exotic melon genotypes carrying the
polymorphism G85E have the same cellular localization than that of SC. Therefore,
transient expression of VPS41 from resistant melon genotypes correlates with the
lack of particular structures that are present when VPS41 from a susceptible
genotype is expressed and that might have a role in the infection by CMV.
Further experiments will address the relationship between the cellular localization of
VPS41 and the MP of the virus.
226
S-IX. Póster 19
MAJOR PHENOTYPIC TRAITS UNDER SELECTIVE PRESSURE IN
Antirrhinum
Victoria1 Ruiz-Hernández1, Fernando1 Pérez-Sanz1, Lize2 Jourbert2, Amador3
Rodríguez-Gómez3, Julia1 Weiss1, Pablo3 Bielza3, Beverley4 Glover4, Marcos1 Egea-
Cortines1
1Genética Molecular, Universidad Politécnica de Cartagena, Cartagena, Spain, 2Deparment
of Plant Sciences, University of the Free State, Bloemfontein, South Africa, 3Protección de
Cultivos, Universidad Politécnica de Cartagena, Cartagena, Spain, 4Department of Plant
Sciences, University of Cambridge, Cambridge, United Kingdom
Corresponding author: Victoria Ruiz-Hernández ([email protected])
Plant aromas are composed by independent Volatile Organic Compounds (VOCs) in
different proportions. Scent profiles are distinctive blends that can characterize
species as has been published for Antirrhinum (Weiss et al. 2016). The genetic
structure of the emission of independent VOCs in this genus appears to be
composed of single genes coding for VOC synthesis enzymes (Ruiz-Hernández et al.
2017). We developed a recombinant inbred line of A.majus x linkianum, and found
segregating lines for ocimene, methyl benzoate, methyl cinnamate and
acetophenone. We have assessed preferences of pest Frankliniella occidentalis and
pollinator Bombus terrestris for different floral scent profiles. Our results indicate that
changes in scent profiles have an important role in pest and pollinator choices,
therefore having an impact on natural and agricultural ecosystems.
Ruiz-Hernández V, Hermans B, Weiss J, Egea-Cortines M. 2017. Genetic analysis of natural variation
in Antirrhinum scent profiles identifies BENZOIC ACID CARBOXYMETHYL TRANSFERASE as
the major locus controlling methyl benzoate synthesis. Front. Plant Sci. 8:27–40.
Weiss J, Mühlemann JK, Ruiz-Hernández V, Dudareva N, Egea-Cortines M. 2016. Phenotypic space
and variation of floral scent profiles during late flower development in Antirrhinum. Front. Plant
Sci. 7:1903.
Acknowledgements & Funding: MECD (FPU13/03606) and UPCT (PMPDI) to VRH,
This work was funded by the Fundación Séneca 19398/PI/14, BFU 2013-45148-R, BFU 2017 88300-
C2-1- and BFU2017 88300-C2-2-R.
227
S-IX. Póster 20
ARABIDOPSIS GCN1 MEDIATES NON-CANONICAL TRANSLATION
REGULATION AND STRESS ADAPTATION
Yovanny Izquierdo1, Satish Kulasekaran1,2, Pablo Benito1, Bran López1, Ruth
Marcos1, Tomás Cascón1, Mats Hamberg3, Carmen Castresana1.
1Plant Molecular Genetics, Centro Nacional de Biotecnología, Madrid, Spain. 2School of Life
Sciences, University of Warwick, Coventry, UK. 3Deparment of Medical Biochemistry and
Biophysics, Karolinska Institutet, Stockholm, Sweden.
Corresponding author: Carmen Castresana ([email protected])
Stress adaptation and translational regulation was studied using noxy7 (non-
responding to oxylipins7) from a series of Arabidopsis thaliana mutants. We identified
the noxy7 mutation in At1g64790, which encodes a homolog of the yeast
translational regulator GENERAL CONTROL NON-DEREPRESSIBLE1 (GCN1).
Yeast GCN1 functions as a scaffold to bind the ABC protein GCN20 and the kinase
GCN2, which upon activation phosphorylates the α subunit of the translation initiation
factor eIF2 (P-eIF2α) to facilitate stress adaptation by inhibiting protein synthesis.
This regulatory circuit is conserved in yeast and mammals, although its role in plants
is still controversial. Our studies with Arabidopsis mutants showed that GCN1 and
GCN2 mediate P-eIF2α formation in response to different stresses that inhibit protein
synthesis but, unlike in yeast, the Arabidopsis GCN20 homolog was not required for
this process. In these responses P-eIF2α formation did not cause the observed
protein synthesis inhibition. Phenotypic analyses nonetheless showed that GCN1
and GCN2 promote adaptation to amino acid deprivation, suggesting that plant P-
eIF2α may affect translation of specific stress-responsive messengers. Additional
evidence indicated that GCN1 but not GCN2 regulates activation of plant immunity
against Pseudomonas syringae pv tomato (Pst), adaptation to mitochondrial
dysfunction and to high boron concentration. In these responses GCN1 acts with
GCN20 to regulate translation, independently of GCN2 and P-eIF2α. We thus
propose that plant GCN1 and GCN20 integrate a non-canonical pathway to control
protein synthesis in response to biotic and mitochondrial stress, and suggest the
lesser functional relevance of GCN2 and P-eIF2α in plants relative to other
eukaryotes.
Acknowledgements & Funding. YI and SK were PhD fellows of the La Caixa Foundation International
Fellowship Programme (La Obra Social La Caixa/CNB). BL is a fellow of the Severo Ochoa/CNB FPI
Programme of the Spanish Ministry of Economy and Competitiveness. This work was supported by
grants from the Spanish Ministry of Economy and Competitiveness/FEDER (BIO2015-68130-R) and
from the Community of Madrid (P2013/ABI-2734) to CC.
228
S-IX. Póster 21
POST-TRANSLATIONAL MODIFICATIONS AT THE COAT PROTEIN OF THE PLUM POX VIRUS OCCUR IN NATURAL HOST
INFECTIONS AND ARE SHARED BY UNRELATED VIRAL STRAINS
Sandra Martínez1-Turiño1, Marta1 Hervás1, Sergio2 Ciordia2, Rosana2 Navajas2 and
Juan Antonio1 García1
1Department of Plant Molecular Genetics and, 2Proteomics Unit, Centro Nacional de
Biotecnología, Madrid, Spain
Corresponding author: Sandra Martínez-Turiño ([email protected])
Phosphorylation and O-GlcNAcylation are two widespread posttranslational modifications (PTMs), often affecting same target protein. Although phosphorylation has been studied extensively in animals and plants, research on O-GlcNAcylation in plants lags far behind that in other eukaryotic organisms. Plum pox virus (PPV) (genus Potyvirus) infects a wide range of plant species and causes sharka, one of the most damaging diseases affecting stone fruit trees. PPV genome is a positive-sense single-stranded RNA encapsidated by a single type of capsid protein (CP) in flexuous rod particles.
Previously, we have reported that PPV virions (isolate Rankovic, strain D), obtained from herbaceous hosts, are extensively modified in vivo by O-GlcNAcylation in the N-terminus of CP and by phosphorylation along the whole protein. These PTMs are carried out by Secret Agent, one of the two O-linked N-acetylglucosamine transferases identified in Arabidopsis thaliana, and by still non-identified kinase(s). O-GlcNAcylation of CP has a positive role during infection, intervening in virion assembly and/or stability; while phosphorylation seems to modulate, on its own or together with O-GlcNAcylation, different CP-mediated processes during viral cycle.
In this study, a combination of proteomics approaches, MALDI-TOF and ESI MS/MS, plus protein immunodetection techniques were applied to study the phosphorylation and O-GlcNAcylation pattern of PPV virions purified from the natural host Prunus persica. Additionally, viruses belonging to strains C and Rec propagated in the experimental host Nicotiana benthamiana were examined. Confirmation that PPV virions are O-GlcNAcylated and phosphorylated in its natural host, suggests the regulation role attributed to these modifications is part of an overall strategy occurring during plant-pathogen interaction. Likewise, the fact that both PTMs are not restricted to a specific viral strain, once they have been found in isolates as diverse as those from Rec and C strains, points out contribution of these modifications is a general feature of PPV infections.
Finally, a battery of mutants and PPV-subviral constructs, impaired in several points of viral cycle, were used to study the relationship between O-GlcNAcylation and different viral functions. Preliminary results suggest that impairing replication, cell-to-cell movement or virion assembly do not prevent O-GlcNAcylation; but the abolishment of CP self-assembly does so.
Alfonso Muñoz
SIV. C2
Alfonso Ortega
SII. p1 SII. p9 SVII.p2
Alfredo Manicardi
SVII.p17
Alicia Esteban
SVII.C2
Alicia Moreno-Cortés
SVII.p28
Alisdair R. Fernie
SI. P1 SI. p3 SII. p10
Alma Burciaga-Monge
SVIII. C1
Almudena Ferrández- Ayela
SIV. p34
Almudena Molla-Morales
SII. p2
Almudena Trapero- Mozos
SIV. P1 SIV. p12
Alon Samach
SIX. p10
Álvaro de Andrés-Pablo
SIX. p15
Álvaro Ferriz
SVI. p14
Álvaro García-Moreno
SVII.p3
Álvaro Sanchez- Corrionero
SI. C1 SVII.p20
AM Zamarreño
SVI. P1 SVII.P2
Amada Pulido
SII. C2 SVII.p7 SVII.p12
Amador Rodríguez- Gómez
SIX. p19
Amanda F. Martín-Forero
SII. P9
Amelia Felipo-Benavent
SIV. p10
Amit Gal-On
SII. C2
Amparo Primo
SIV. p2
Amr Nassrallah
SV. C1
Ana Alarcia-García
SIV. p2
Ana Arnaiz
SIX. P2
Ana Beatriz Moreno
SIX. C3
Ana Belén Sánchez- García
SIV. p19
Ana Berbel
SIV. p25
Ana Casañal
SII. p3 SIV. p7
Ana Cláudia Silva
SIX. p4
Ana Conesa
SI. P1
Ana Espinosa-Ruíz
SVI. P2
Ana Gracia Pérez
SVIII. p7
Ana I. Caño-Delgado
SI. P1 SIII. C6
Ana Isabel González- Hernández
SIV. p14
Ana López Sánchez
SIX. p3
Ana M. Maldonado- Alconada
SI. C3 SI. p9
Ana María Herruzo
SVIII. p7
Ana Montserrat Martín Hernández
SIX. p18
Ana P. Luna
SIX. p1
Ana Paez-Garcia
SV. p1
Ana Rodríguez
SIV. p24
Andrea Chini
SVI. P1 SVII.p14 SVII.p17
Andrea Gómez-Sánchez
SVII.p13
Índice autores A Bovy
SII. P1
A Medina
SII. P1
A Schaller
SVI. P1
A Stintzi
SVI. P1
A Urbaneja
SII. P1
Abdellatif Bahaji
SII. P2 SVIII. p1 SVIII. p2 SVIII. p4
Abel Rosado
SVII.C2
Adam P. Fisher
SVI. p6
Adela Zumaquero
SIX. p7
Adrian A. Valli
SIII. C4
Adrián Cabezas-Fuster
SIV. p1
Adriana Ricarte-Bermejo
SII. P2 SVIII. p2
Agnieszka Masny
SII. p13
Ainara Pérez-Morón
SVI. p1 SVI. p5
Ainhoa Martinez-Medina
SIX. P1
Aitor Muñoz
SVI. p4 SVI. p15
AJ Monforte
SII. P1 SIV. p4
Alain Goossens
SVII.p24
Alba Ayats
SV. C3
Alba Rivas-Sendra
SVIII. p1
Alba Valdivieso-Martínez
SIV. p26
Alba-R Corrales
SVII.p18
Albert Bernal
SII. p7
Albert Boronat
SVIII. C1
Albert Ferrer
SVIII. C1
Albert Serra-Cardona
SIX. p9
Alberto Carbonell
SIII. C3
Alberto Coego
SVII.C1 SVII.p24
Alberto de Marcos
SII. P9 SII. p1
Alberto Lara
SVII.p1 SVII.p22
Alberto Macho
SVII.p3
Alejandra García
SIX. C2
Alejandro Alonso-Díaz
SIX. p2
Alejandro F. Uceta- Gamaza
SIX. p1
Alejandro Fernández- Arbaizar
SVI. p8
Alejandro González
SIII. C6
Alejandro Navarro- Galiano
SVII.C3
Alejandro Peñín
SVI. p5
Alejandro Vega-Vaquero
SI. p6
Alexander Weinhold
SIX. P1
Alfonso Albacete
SI. P1
Alfonso Azorín
SIV. p19
Alfonso González
SIII. C4
Beatriz Sánchez-Calvo
SVI. p10 SVI. p13
Beatriz Sánchez-Parra
SV. p7
Begonya Vicedo
SIV. p14
Begoña Benito
SVII.p30 SIX. p5
Begoña Orozco- Navarrete
SII. p3
Begoña Renau-Morata
SVII.p18
Belén Méndez-Vigo
SII. p2 SV. p3
Bérangère Lombard
SV. C1
Bert De Rybel
SVI. p7
Beverley Glover
SIX. p19
Blanca Gonsalvez
SII. p12
Blanca Sabarit
SIX. p1
Blanca San Segundo
SIX. p9
Bo Zhang
SIV. p17
Bob B. Buchanan
SVIII. p8
Borja Belda-Palazon
SVII.C1 SVII.p24
Borja Diego
SIII. C1
Bran López
SIX. p20
Brecht Wybouw
SVI. p7
Břetislav Brzobohatý
SV. P2
Briardo Llorente
SVIII. p12
Brigitte Poppenberger
SVIII. p10
Bruce McDonald
SIX. C1
Bruno Gugi
SIV. p15
Bruno Mezzetti
SII. p13
Camilla Molinari
SIV. p5
Candela Cuesta
SIV. p24
Capilla Mata-Pérez
SVI. p10 SVI. p13
Carla Inês
SIV. p13
Carla Méndez
SIV. p23
Carla Navarro-Quiles
SIV. p6
Carlos Alonso-Blanco
SII. p2 SV. p3 SVII. p 33
Carlos del Pozo
SV. p4 SVII.p31
Carlos García-Almodovar
SII. p12
Carlos Hernández- Cortés
SVI. p1 SVI. p5
Carlos Perea
SVI. p8 SVII.C2 SVII.p6
Carlos Rivero
SVII.p27
Carlos Sanz
SVI. p7 SVIII. p7
Carlos Tarancón
SI. P2
Carlos Vargas-Chávez
SI. p2
Carmen Castresana
SIX. p3 SIX. p20
Carmen Fenoll
SII. p1 SII. P9 SIX. C2 SIX. p4
Carmen González Bosch
SIX. p6
Carmen González-Murua
SVIII. p5
Carmen Martín-Pizarro
SIV. P1 SIV. p7 SIV. p8 SIV. p12
Carmen Pérez-Delgado
SVIII. p5
Andrea Sánchez-Vallet
SIX. C1 SIX. p8
Angel Chávez
SVIII. C1
Ángel Del-Espino Pérez
SIX. p7
Ángel Matilla
SIV. p29
Ángela María Sánchez- López
SII. P2 SVIII. p1 SVIII. p2 SVIII. p4
Ángela Martínez-Gómez
SIX. p4
Angela Saez
SVII.P2 SVII.p31
Ángeles Gómez- Zambrano
SV. P1 SIV. p18
Anja Krieger-Liszkay
SVII.p20
Anna Stepanova
SIV. p16
Anne-Flore Deton- Cabanillas
SV. C1
Annika Haugeneder
SII. p13
Antonella Locascio
SV. P2 SV. p2
Antonio Arjona
SII. p13
Antonio Cano
SII. p11 SIV. p19
Antonio Costa de Oliveira
SIV. p15
Antonio Granell
SII. P1 SIII. C1
Antonio J. Matas-Arroyo
SIV. p11
Antonio Leyva
SII. p2 SVII.p5 SVII.p 33
Antonio M. Corderio
SIV. p13
Antonio Molina
SIX. p8 SIX. p14 SIX. p17
Antonio Muñoz-Barrios
SIX. p17
Antonio Rodríguez Franco
SI. p1 SI. p4 SIV. p11 SIV. p30
Antonio Serrano Mislata
SIV. p3 SIV. p10
Araceli Castillo
SVII.C2 SVII.p3
Araceli Díaz Perales
SII. p3
Aránzazu Gómez-Garay
SII. p6 SIV. p36
Armando Albert
SVII.p4 SVII.p14 SVII.p17
Arnald Marcer
SV. p3
Arnaldo Schapire
SVII.C2
Arnau Rovira
SVI. p2
Arthur G. Hunt
SVII.p31
Åsa Strand
SIV. p33
Ascensión Corrales
SVII.p20
Asier Briones-Moreno
SI. p2 SIV. p9 SVI. C2
Auguste Genovesio
SV. C1
Aurélie Petit
SII. p13
Aurora Alaguero- Cordovilla
SIV. p4 SIV. p19
Avital Beeri
SIX. p10
Azeddine Driouich
SIV. p15
Barbara Telléz-Robledo
SVII.p31
Béatrice Denoyes
SII. p13
Beatriz Briegas
SIV. p13
Beatriz García
SIII. C4
Beatriz Pintos
SII. p6 SIV. p36
David Lara-Astiaso
SIV. p20
David Posé
SIV. P1 SIV. p7 SIV. p8 SIV. p12
David San León
SVII. p33
David Stroebel
SV. C1
David Wilson-Sánchez
SIII. C2
Deborah Goffner
SIX. p8
Delphine Pott
SI. p3 SII. p3 SII. p10
Detlef Weigel
SI. C4
Diana Leibman
SII. C2
Diego López-Márquez
SIX. p7 SIX. p13
Diego Orzaez
SIII. C1
Dietmar Geiger
SVII.p27
Dmitry Kremnev
SIV. p33
Dolores Garrido
SII. C2 SVII.p7 SVII.p12
Domenique André
SIV. p17
Dominique Eeckhout
SV. C1
Dorota Komar
SIV. p21
Edgar A. Rodríguez- Negrete
SIX. p7
Eduardo Berenguer
SIII. C5 SVII.p8 SVII.p15
Eduardo Bueso Ródenas
SVII.p16
Eduardo Burillo
SVI. p5
Eduardo Cruz-Rus
SII. p8
Eduardo González
SI. P2
Eduardo Mateo-Bonmatí
SIV. C5 SIV. p6
Eduardo R Bejarano
SIX. C4 SIX. p1 SIX. p7
Edurne Baroja- Fernández
SII. P2 SVIII. p1SVIII. p2 SVIII. p4
Edward Zurawicz
SII. p13
Eiji Nambara
SVI. p8
Elena Carneros
SII. p6 SIV. p36
Elena Minina
SVII.p8
Elena Monte
SV. C3 SVI. p2
Elena Pastor-Mora
SIV. C4
Elena Perpiñan
SIV. p2
Elena Ramírez-Parra
SVII.p9
Elena Sánchez Martín- Fontecha
SVI. p4 SVI. p15
Elisa Iniesto
SV. C1 SV. P2
Elisabetta Caporali
SVIII. p4
Elison B. Blancaflor
SV. p1
Elsa Martínez-Ferri
SII. p10
Emilio A. Cano
SII. p11
Emilio Gutierrez-Beltran
SVII.p10
Emilio Marcos-Barbero
SVII.p11
Emma Fernández- Crespo
SIV. p14
Encarnación Zambrana
SVII.p9
Enrique Martínez-Force
SVIII. p11
Enrique Rojo
SIV. C2
Carmen R. Beuzon
SIX. p7 SIX. p13
Carmen Soria
SII. p8
Carole Dubreuil
SIV. p33
Carolina Camacho- Fernández
SVIII. p1
Carolina Escobar
SIX. C2 SIX. p4
Catharina Merchante
SII. p3 SIV. p7
Cécile Breyton
SV. C1
Cecilia Gotor
SVI. C3
Charlotte Gommers
SV. C3
Chris Bowler
SV. C1
Chris Dervinis
SVII.p28
Christian Göschl
SIX. p2
Christoph Weiste
SVI. p11
Christophe Rothan
SIV. p19
Clara Bourbousse
SV. C1 SV. C3 SV. p9
Claudia Bou
SV. p2
Claudia Köhler
SIV. C3 SIV. p18
Claudio Novella-Rausell
SVII.p6
Clémence Plissonneau
SIX. C1
Concepción Almoguera
SI. p5
Concepción Ávila
SII. C1
Concepcion Manzano
SV. p4 SVII.p31
Coral Del Val
SVI. p12 SVII. P32
Cristian Carrasco-López
SVII.p6
Cristian Mateo
SII. p2 SVII.p5
Cristina Caballo
SIV. p25
Cristina Castillejo
SII. C3
Cristina Ferrándiz
SIV. P2 SIV. p2
Cristina López-Hidalgo
SI. C3 SVIII. p3
Cristina Marí-Carmona
SVI. p3
Cristina Martínez
SVI. P2
Cristina Martínez- Andújar
SI. P1
Cristina Navarro
SVII.p5
Cristina Peris-Peris
SIX. p9
D Tieman
SII. P1
Damarys Loew
SV. C1
Daniel Alique
SV. C2
Daniel Conde
SVII.p28
Daniel Croll
SIX. C1
Daniel Marino
SVII.p18 SVIII. C2 SVIII. P5 SVIII. p6
Daniela Barro-Trastoy
SIV. p9
Daniela Goretti
SIV. p23
Danuše Tarkowská
SVIII. p4
David Alabadí SI. p2 SIV. p3 SIV. p10 SV. P2 SV. p9 SVI. p3 SVII.p6
David Blasco-Escámez
SI. P1 SIII. C6
David Esteve-Bruna
SIV. p10 SV. p9 SVI. p3 SVII.p6
Fuqi Liao
SV. p1
Gabriel Sánchez- Martínez
SIV. p26
Geert de Jaeger
SV. C1
Gema Martín Corredoira
SII. p8
Gemma Camañes
SIV. p14
Gemma López
SIX. p8
George Bassel
SIV. p28
Gerald Zabulon
SV. C1
Gerardo Carrera- Castaño
SVI. p11
German Martinez
SIV. C3
Giancarlo Bonora
SI. p6
Gianfranco Diretto
SVI. p2
Goizeder Almagro
SII. P2 SVIII. p1SVIII. p2 SVIII. p4
Guillermo García- Martínez
SV. p2
Harrold A. van den Burg
SIX. p1
Héctor Candela
SVI. p1 SVI. p5 SVI. p14 SIX. p10
Helena Ruiz-Cano
SIV. p31
Henning Frerigmann
SV. P2
HJ Klee
SII. P1
Hua Jiang
SIV. C3
Hugo Mélida
SIX. p8 SIX. p14 SIX. p17
Ignacio Ezquer
SVIII. p4 SIV. p15
Ignacio Rubio Somoza
SI. C4 SII. p7 SIV. p10 SIX. p7 SIX. p15
Ikhlak Ahmed
SV. C1
Ilse Kranner
SVI. p8 SVII.p29
Inés Sánchez
SIV. p9
Inmaculada Coleto
SVIII. C2 SVIII. p6
Inmaculada Garrido
SVII.p2
Inmaculada Moreno
SVI. C3
Inmaculada Sánchez Vicente
SVI. P7 SIV. p5 SVII.p20
Iraida Amaya
SII. C3 SII. p8 SII. p13
Iraide Bejarano
SVIII. C2 SVIII. p6
Irene García
SVI. C3
Irene Garcia-Maquilon
SVII.p17 SVII.p14
Irene Martínez Almonacid
SVII.p16
Irene Martínez- Fernández
SIV. P2
Iris Martínez
SV. p8
Isabel Allona
SV. C2 SVII.p28
Isabel Betegón-Putze
SIII. C6
Isabel Diaz SIII. C5 SV. p7 SVII.p13 SIXP2
Isabel Feito
SI. p8 SIV. p24
Isabel Lopez-Diaz
SIV. p9
Isabel Manrique Gil
SIX. p11
Isabel Martínez
SIX. p4
Isabel Mateos
SVI. p7 SVI. p8 SVII.p23
Enriqueta Moyano
SI. p1 SIV. p11 SIV. p30
Erica Gil Velasco
SVII. p33
Ernesto Escoms
SIV. p9
Ernesto Pérez Benito
SIX. p12
Erundina Ruiz
SVI. p1 SVI. p5
Erwann Arc
SVI. p8
Estefanía Berrio
SVI. p9
Esther Carrera
SIV. p9
Esther Lechner
SVII.C1
Eugenio G. Minguet
SV. P2
Eugenio Llorens
SIV. p14
Eva Lucas-Reina
SIV. P1 SIV. p12
Eva Miedes
SIX. p8 SIX. p14
Eva Núñez-Delegido
SIV. p26 SIV. p34
Eva Rodríguez-Alcocer
SVI. p1 SVI. p5 SVI. p14
F. Xavier Picó
SV. p3
Fabio Pasin
SIII. C4
Fabiola Ruiz-Ramón
SII. p12
Fanny Hartmann
SIX. C1
Fátima Carvajal
SII. C2 SVII.p7 SVII.p12
Federico Valverde
SI. C2 SVIII. p9
Felipe Gómez del Castillo
SVI. p14
Félix Juan Martínez- Rivas
SI. p1 SI. p4 SIV.p11 SIV. p30
Fernando E. Díaz- Manzano
SIX. p4
Fernando Pérez-Sanz
SIV. p22 SV. p5 SV. p6 SIX. p19
Ferran Sánchez-Sanuy
SIX. p9
Fidel Lozano-Elena
SI. P1
Florine Dupeux
SII. p3
Fran Romero
SI. P2
Francesca Maio
SIX. p1
Francisca Blanco- Herrera
SV. p7
Francisco Amil-Ruiz
SI. p9
Francisco Espinosa
SVII.p2
Francisco J Colina
SII. p4 SII. p5
Francisco J. Romero
SI. C2 SIV. p35
Francisco Javier Cejudo
SVIII. P1
Francisco Javier Gran
SIV. p4 SIV. p19
Francisco Javier Molina Hidalgo
SI. p1 SI. p4 SIV. p11 SIV. p30
Francisco José Muñoz
SII. P2 SVIII. p1 SVIII. p2 SVIII. p4
Francisco M. Cánovas
SII. C1
Francisco Madueño
SIV. p23 SIV. p25 SIV. p27
Francisco Palma
SII. C2 SVII.p7 SVII.p12
Francisco Pérez Alfocea
SI. P1
Francisco Rubio
SVII.C5 SVII.p1 SVII.p22
Francisco Vera-Sirera
SIV. p9
Fredy Barneche
SV. C1 SV. C3 SV. p9
Jordi Moreno-Romero
SIV. C3 SIV. p18 SV. p11
Jorge Corbacho
SIV. p13
Jorge Fung-Uceda
SIV. C1
Jorge Hernández-García
SI. p2 SVI. C2
Jorge J. Casal
SV. P2
Jorge Lozano-Juste SVII.C1 SVII.p4 SVII.p14 SVII.p17 SVII.p24
Jorge Morales
SIX. p17
José A. Fernández
SVII.p21
José A. Gómez
SII. p13
José A. Jarillo
SIV. p18 SIV. p21 SV. p10 SVII.P1
José A. Márquez
SII. p3
José Alfredo Zambrano
SIV. p17
Jose Alonso
SIV. p16
Jose Antonio Duarte- Conde
SIV. p8
José Antonio Fernández
SV. p2
José Domínguez- Figueroa
SI. p7 SVII.p18
José F. Sánchez Sevilla
SII. C3 SII. p8 SII. p13
José G. Vallarino
SI. p3 SII.C3 SII. p10 SIV. p7
Jose Gadea Vacas
SVII.p16 SVII.p29
Jose Julián
SVII.C1 SVII.p24
José Luis Caballero
SI. p1 SIV. p11 SIV. p30
José Luis Micol
SIII. C2 SIV. p6 SIV. p31 SIV. C5
José Luis Riechmann
SI. P1
José M Jiménez-Gómez
SVII.P1
José M. Alonso
SIV. p9
José M. Colmenero- Flores
SVII.p27
José M. Franco-Zorrilla
SIV. p18 SVI. P2 SVI. p8
Jose M. Garcia-Mina
SVI. P1 SVII.P2
José M. Martínez-Rivas
SVII.p19 SVIII. C3
José M. Romero
SI. C2 SVIII. p9
Jose Mª Estavillo
SVIII. p5
José Manuel Álvarez
SIV. p24
José Manuel Franco
SVII.p5
Jose Manuel Pérez- Pérez
SII. p11 SIV. p4 SIV. p19 SIV. p31
Jose Manuel Ramos- Sánchez
SV. C2
Jose María Seguí- Simarro
SVIII. p1
José Miguel Mulet
SV. p2
José Pruneda-Paz
SVII.p5
Jose S. Rufián
SIX. p13
José-Antonio Daròs
SIII. C3
Josefa Rubio
SIV. p25
Jose-Juan Sánchez- Serrano
SIV. C2
José-Luis Crespo
SIII. C5
Juan A. López
SIV. p18 SVI. p9
Juan Alejandro Lara
SVIII. C1
Isabel Monte
SVI. P1
Isabel Torres-Quezada
SIX. p12
Iván del Olmo
SV. p10 SIV. p20
Ivan Lebovka
SIV. p10
Ivett Bárány SIII. C5 SIV. p36 SVI. C1 SVII.p8 SVII.p15
Izargi Vega-Más
SVIII. p5
J. Alan Sparks
SV. p1
J.J. Salas
SVIII. p11
Jaime F Martínez-García
SV. p11
Jaime López Cruz
SIX. p6
James Giovannoni
SIV. p7
Javier Brumos
SIV. p16
Javier Cabrera
SIX. C2 SIX. p4
Javier Canales
SVII.p18
Javier Iserte
SVII.p6
Javier Paz-Ares
SVII.p5 SVII.p33
Javier Pozueta-Romero
SII. P2 SVIII. p1 SVIII. p2 SVIII. p4
Javier Rivero Bravo
SIX. P1
Javier Rueda-Blanco
SIX. p13
Javier Ruiz –Albert
SIX. p1 SIX. p13 SIX. p7
Javier Silva-Navas
SVII.P2 SVII.p31
Jenifer Pozas
SIV. p20 SV. p10
Jerald D. Noble
SVII.p28
Jeremy Pillet
SIV. p7
Jessica Pérez-Sancho
SVII.C2
Jesús Agüero
SII. p12
Jesus Amo
SVII.C5 SVII.p22
Jesús Praena
SIV. p27
Jesús V. Jorrín-Novo
SI. C3 SI. p9 SVIII. p3
Jesús Vázquez
SIV. p18
Jesús Vicente-Carbajosa
SI. p7 SIV. C4 SVII.p18
Jimena Solana
SIV. p32
JJ Ruiz
SII. P1
JL Rambla
SII. P1
Joan Bernabé
SIII. C1
Joan Renard Meseguer
SVII.p16
Joan Sánchez-Pascual
SIV. p4
Joan Villanova
SII. p11
Joanne Chory
SVII.p5
Joaquin Ariño
SIX. p9
Joaquín Medina
SI. p7 SVII.p18
Johanna Trinkl
SII. p13
Jonatan Illescas
SII. P9 SII. p1
Joost Stassen
SIX. p3
Jordi Díaz-García
SVII.p21
Jordi Gamir
SVI. p9
Leonardo Furci
SIX. p3
Lidia Jiménez
SII. p10 SII. p13
Lieven De Veylder
SIV. C1
Liudmilla Rubbi
SI. p6
Lize Jourbert
SIX. p19
Loredana Scalschi
SIV. p14
Lorena Aranda-Caño
SVI. p10 SVI. p13
Lorena Blázquez
SVII.p30
Lorena Sánchez Giménez
SIX. p6
Lothar Willmitzer
SI. p3 SVII.C2
Lourdes García-Vico
SVIII. p7
Lourdes Infantes
SVII.p4
Lourdes Rubio
SVII.p21
Luca Mazzoni
SII. p13
Lucía Arenas-Alfonseca
SVI. C3
Lucia Colombo
SIV. p15
Lucía de la Rosa
SVII.p9
Lucía Jordá
SIX. p17
Lucía Juan-Vicente
SIV. C5
Lucio López
SIII. C3
Luis C. Romero
SVI. C3
Luis Gómez
SIV. p28 SVI. p11
Luis Miranda
SII. p13
Luis Oñate-Sánchez
SIV. p28 SVI. p11
Luis Quintero
SVII.p23
Luis Rodríguez-Moreno
SII. p12
Luis Sanz
SVI. C1
Luis Valledor SI. p8 SII. p4 SII. p5 SV. p8 SVII.C4 SVIII. p3
Luis Villar-Martin
SII. p7 SIX. p15
Luisa M. Sandalio
SVI. p12 SVII. P32 SIX. P2
Lukas Meile
SIX. C1
Lukás Spíchal
SII. P2 SVIII. p2 SVIII. p4
Lydia Ugena
SVIII. p2
Lynne Yenush
SV. p2
M Hamberg
SVI. P1
M José Domenech
SIV. p25
M Perez-Hedo
SII. P1
M. Águila Ruiz-Sola
SVIII. P2
M. Ángeles Botella
SVII.p1
M. Carmen Sampedro
SVIII. p4
M. Dolores Sicardo
SVII.p19 SVIII. C3
M. Luisa Hernández
SVII.p19 SVIII. C3
M. Teresa Ruiz
SVIII. p9
M. Victoria Barja
SVIII. P2
M.T. Solís
SIV. p37
Mª Begoña González- Moro
SVIII. p5
Juan Antonio García
SIII. C4 SIX. p16 SIX. P21
Juan B. Arellano
SVII.p11 SVII.p20
Juan B. Barroso
SVI. p7 SVI. p10 SVI. p13
Juan C. del Pozo
SVII.P2
Juan C. Triviño
SII. C3
Juan Carlos Begara- Morales
SVI. p13 SVI. p10 SVI. p7
Juan D. Franco-Navarro
SVII.p27
Juan Gil
SIV. p25
Juan Jordano
SI. p5
Juan José López-Moya
SIX. C3
Juan José Ripoll
SIV. p9
Juan Luis Benavente
SVII.p4
Juan Majada
SI. p8
Juan Manuel Pérez Ruiz
SVIII. P1
Juan Muñoz Blanco
SI. p1 SI.p4 SIV. p11 SIV. p30
Juan Santos-González
SIV. C3 SIV. p18
Juana Labrador
SIV. p13
Jules Beekwilder
SVIII. P2
Julia Kehr
SV. p7
Julia Weiss
SIV. p22 SV. p5 SV. p6 SIX. p19
Julián Calleja-Cabrera
SVII.p6
Julian Lehmann
SVII.p27
Julie Caruana
SII. C3
Julio Salinas
SVI. p8 SVII.P1SVII.C2 SVII.p6
Jun Li
SVIII. p1
Jurriaan Ton
SIX. p3
Kaisa Kajala
SV. p4
Karel Dolezal
SII. P2 SVIII. p2 SVIII. p4
Kazumi Nakabayashi
SVI. p8
Kerstin Dalman
SVII.p10
Kinia Ameztoy
SII. P2 SVIII. p2
Kiyoshi Tatematsu
SVI. p8
Klaas Vandepoele
SI. C4
Klaus Olbricht
SII. p13
Kyounghee Lee
SIV. C1
Lara García
SV. p8
Laura Bacete
SIX. p8 SIX. p14
Laura Bouza-Morcillo
SIV. p28
Laura C. Terrón-Camero
SVI. p12 SVII.p32
Laura Carrillo
SVII.p18
Laura Castro-Labrador
SIV. p20
Laura de Lorenzo
SVII.p31
Laura García-Abad
SIV. p26
Laura Jaén
SIX. p5
Laura Lamelas
SVII.C4
Laura Poza-Viejo
SIV. p20 SV. p10
Maria Lopez de la Calle
SVII.p25
Maria Luisa Domingo- Calap
SIX. C3
María N. Padilla
SVI. p10 SVI. p13
María Ribaya
SIII. C4
María Rosa Ponce
SIV. p1
María Salud Justamante
SII. p11 SIV. p31
María Teresa Escribano
SIX. p12
María Victoria Díaz- Galián
SV. p5 SV. p6 SIV. p22
María-Dolores Rey
SI. C3 SI. p9
Mariana Bustamante
SI. P1
Mariano Perales
SV. C2 SVII.p28
María-Teresa Solís SIII. C5 SIV.p36 SVII.p8 SVII.p15
Marie Pierre Rivieré
SIX. p8
Marie-Louise Körner
SIV. p22
Marina Moreno
SII. P9
Marina Rueda-López
SII. C1
Marina Silvestre
SIV. p23
Mario Fenech-Torre
SVII.p26
Mariona Estapé
SIX. C3
Markus Schmid
SIV. p23
Marouane Baslam
SII. P2 SVIII. p2
Marta Adelina Mendes
SVIII. p4
Marta Barcala
SIX. p4
Marta Godoy
SVI. p8
Marta Hervás
SIX. p16 SIX.p21
Marta Isabel Terry
SV. p5
Marta Vázquez
SIII. C1
Marta-Marina Pérez Alonso
SV. p7
Martin Černý
SV. P2
Martin F. Yanofsky
SIV. p9
Martín Ramos-Alvelo
SIV. p12
Martin Rougée
SV. C1
Martin Weih
SVII.p18
Martina Legris
SV. P2
Mary-Paz González- García
SIV. C2
Matias Kirst
SVII.p28
Mats Hamberg
SIX. p20
Matteo Pellegrini
SI. p6
Meike Burow
SVIII. C2
Melania Ghita
SIV. p28
Mercedes Gallardo
SIV. p13
Mercedes Ramiro
SII. p2 SV. p3
Micaela Navarro
SVII.p5
Michael Hahn
SIX. p8
Michael Holdsworth
SIV. p28
Mª Belén Pascual
SII. C1
Mª Carmen Marqués
SV. p2
Mª Estrella Santamaria SIII. C5 SV. p7 SVII.p13 SIX. P2
Mª Mar Castellanos
SVII.P1
Macarena Menéndez- García
SVIII. p3
Macedonia Trigueros
SVIII. p3
Magdalena Delgado
SIX. p17
Maite Sanmartín
SIV. C2
Maite Saura-Sánchez
SIV. p9
Manuel Acosta
SII. p11 SIV. p19
Manuel Arroyo-Mateos
SIX. p1
Manuel Guinea Diaz
SIV. p33
Manuel Jamilena
SII. C2 SVII.p7 SVII.p12
Manuel Martinez
SVII.p13 SIX. P2
Manuel Nieves-Cordones
SVII.C5 SVII.p1 SVII.p22
Manuel Pacín
SV. P2
Manuel Piñeiro
SIV. p18 SIV. p21 SV. p10 SVII.P1
Manuel Rodriguez- Concepcion
SVIII. p12 SVIII. P2
Mar González
SVII.p18
Marc Valls
SIX. p2
Marcel Prins
SIX. p1
Marcello Zala
SIX. C1
Marcelo J Yanovsky
SVII.p6
Marco Morselli
SI. p6
Marcos Egea-Cortines
SIV. p22 SV. p5 SV. p6 SIX. p19
María A. Fernández
SVII.p24
Maria Ajenjo
SIII. C1
María Ángeles Fernández-López
SIV. p31
Maria Angels De Luis Balaguer
SI. C1 SVI. p6SIV. p12
Maria C. Camarero
SIV. p13
Maria C. Gomez- Jimenez
SIV. p13
María C. Risueño SII. p6 SIII. C5 SIV. p36 SVI. C1 SVII.p8 SVII.p15
Maria C. Romero- Puertas
SVI. p12 SVII. P32 SIX. P2
María Carbó
SII. p4 SII. p5
María del Carmen Rodríguez-Gacio
SIV.p29
María Dolores Gomez
SIV. p9
María Dolores Rodríguez
SVII.p23
Maria Fe Andrés
SIX. C2
María Garrido-Arandia
SII. p3
María Guadalupe Fernández-Espinosa
SIV. p32 SVI. p6
María I. Vaquero-Sedas
SI. p6
Maria J. Hernaiz
SVII.P2
Maria J. Pozo
SVI. p9 SIX. P1
María Jesús Cañal
SII. p4 SII. p5 SV. p8 SVII.C4
Pablo Albertos
SVI. p7 SVI. p8 SVIII. p10
Pablo Benito
SIX. p20
Pablo Bielza
SIX. p19
Pablo Díaz-Rueda
SVII.p27
Pablo García-Gómez
SII. P2
Pablo Gonzalez-Melendi
SVII.p13 SVII.p28 SIX. P2
Pablo Leivar
SVI. p2
Pablo Perez-Garcia
SI. C1
Pablo Ric-Varas
SIV. p11
Paloma Cubero-Font
SVII.p27
Paloma Mas
SIV. C1
Panagiotis N. Moschou
SVII.p10
Paolo M. Triozzi
SV. C2 SVII.p28
Parvathy Krishnan
SIX. C1
Pascal Genschik
SVII.C1
Pascual Rodríguez- Sepúlveda
SII. p12
Patricia Fresnillo
SIX. p10
Patricia M. Arjona
SVII.p19 SVIII. C3
Patrick Brunner
SIX. C1
Pau Bretó
SII. p12
Paula Jadczak
SIV. p19
Pawel Roszak
SIV. p18
Paxti San Martín-Uriz
SIV. p20
Pedro Crevillén
SIV. p18 SIV. p20 SV. p10 SVII.P1
Pedro De Los Reyes
SI. P2 SI. C2
Pedro J. Navarro
SIV. p22 SV. p5 SV. p6
Pedro L. Rodriguez SVII.p4 SVII.p17 SVII.C1 SVII.p14 SVII.p24
Pedro Pastor-Andreu
SV. p11
Pedro Robles
SIV. p26 SIV. p34
Pepe Cana-Quijada
SIX. C4
Pernilla H. Elander
SVII.p10
Peter Bozhkov
SVII.p8 SVII.p10
Peter Schürmann
SVIII. p8
Petra Stamm
SIV. p28
Philip Wigge
SV. C2
Philippe Chartier
SII. p13
Philippe Ranocha
SIX. p8
Pil Joon Seo
SIV. C1
Pilar Carbonero
SIV. C4
Pilar Cubas
SI. P2 SVI. p4 SVI. p15
Pilar García-Agustín
SIV. p14
Pilar Hoyos
SVII.P2
Pilar Lasierra
SVII.C3
Pilar Martínez Hidalgo
SIX. p11
Pilar Muñoz del Rio
SII. p8 SII. p13 SII. C3
Pilar Pérez
SVII.p11
Miguel A. Aranda
SII. p12
Miguel A. Blázquez SI. p2 SIV. p3 SIV. p10 SV. P2 SV. p9 SVI. C1 SVI. p3 SVI. C2 SVII.p6
Miguel A. Moreno- Risueño
SI. C1 SVI. C1SIX. C2
Miguel A. Paredes
SIV. p13
Miguel A. Perez-Amador
SIV. p9
Miguel A. Rosales
SVII.p27
Miguel A. Vega-Palas
SI. p6
Miguel Angel Botella SIV. p7 SIV.p12 SVII.C2 SVII.p3 SVII.p21 SVII.p26 SIX. C4
Miguel Ángel Delgado- Gutierrez
SI. p7 SIV. C4
Miguel Angel Torres
SVII.p28 SIX. p17
Miguel de Lucas
SV. p9 SVI. P2
Miguel Miñambres
SVII. p33
Miguel Sánchez-Durán
SIX. p1
Miroslav Ovecka
SVIII. p1
Mónica Balsera
SVIII. p8
Mónica Escamilla Aguilar
SIX. p6
Mónica Lanza
SIX. p5
Mónica Meijón
SI. p8 SII. p4SV. p8 SVII.C4
Monserrat Arró
SVIII. C1
Montaña Mena
SII. p1 SII. p9
Mounira Chaki
SVI. p7 SVI. p10 SVI. p13
Myriam Calonje
SIV. p35 SV. P1
Natalia Bueno
SIV. p24
Natalia Gómez-Peral
SVI. p1 SVI. p5
Nerea Valdebenito
SIV. p10
Néstor Carrillo-Barral
SIV.p29
Nicholas Smirnoff
SVII.p26
Nicolas Denance
SIX. p8
Nicolás Oiza
SII. C3
Nicole M. van Dam
SIX. P1
Nieves López-Pagan
SIX. p7
Niko Geldner
SV. p4
Noel Blanco-Touriñán SV. P2 SIV. p10 SV. p9 SVI. C1 SVI. p3 SVII.p6
Noelia Arteaga
SV. p3
Noemí Ruiz-López
SVII.C2
Norma Fàbregas
SI. P1
Nuria De Diego
SII. P2 SVIII. p2 SVIII. p4
Nùria Real
SIX. p18
Núria S. Coll
SIX. p2
Odette Deen
SII. p2
Óscar Crespo Salvador
SIX. p6
Oscar Lorenzo
SIV. p5 SIV. p32 SVI. C1 SVI. p6 SVI. p7 SVI. p8 SVII.p20 SVII.p23 SIX. p11 SIX. p12
Ouardia Ait-Mohammed
SV. C1
Ove Nilsson
SIV. p17
P Reymond
SVI. P1
Rosa Morcuende
SVII.p11
Rosana Navajas
SIX.p21
Rosangela Sozzani
SI. C1 SIV. p12 SVI. p6
Rosario Alonso
SIV. C4
Rosario Blanco Portales
SI. p1 SI. p4 SIV. p11 SIV. p30
Rosario Haro
SIX. p5
Rosario Sánchez
SVIII. p7
Rosa-V Molina
SVII.p18
Rubén M. Buey
SVIII. p8
Rubén Mateos Fernández
SVII.p16
Rumyana Karlova
SVIII. P2
Ruth Marcos
SIX. p20
S Garcia-Martinez
SII. P1
S Lassueur
SVI. P1
S Weiss
SVI. P1
Sabrina Iñigo
SVII.p24
Salomé Prat
SVI. P2 SVII.C3
Salva Duran-Nebreda
SIV. p28
Samuel Daniel Lup
SIII. C2
Samuel F. Brockington
SIII. C4
Samuel Gámez-Arcas
SVIII. p4
Sandra Fonseca
SV. C1
Sandra Martínez-Turiño
SIX. p16 SIX.p 21
Sanjay Swami
SIX. p17
Santosh B Satbhai
SIX. p2
Sara Aljama
SV. p2
Sara Guerrero
SVII.C4
Sara Jover-Gil
SVI. p1 SVI. p5 SIX. p10
Sara Martín
SV. p8
Sara Navarro-Neila
SVII.P2 SVII.p31
Sara Selma
SIII. C1
Sara Sopeña-Torres
SIX. p17
Satish Kulasekaran
SIX. p20
Sergio Andreu-Sánchez
SIII. C2
Sergio Ciordia
SIX.p21
Sergio Diaz
SVII. p33
Sergio Ibáñez
SIV. p19 SIV. p31
Sharareh Ghasemi
SI. p7 SVII.p18
Sherezade Ortíz-Villajos Cano
SIV. p3
Shubhada Kulkarni
SI. C4
Silke Lesemann
SII. p13
Silvia Jurado
SIV. p18
Silvio Collani
SIV. p23
Siobhan M.Brady
SV. p4
Sonia Campo
SIX. p9
Sonia Osorio SI. P1 SI. p3 SII. C3 SII. p10 SII. p13 SIV. p7 SVII.C2
Pilar Prieto-Dapena
SI. p5
Pilar S. Testillano SII. p6 SIII. C5 SIV. p36 SVI. C1 SVII.p8 SVII.p15
Priscilla Rossetto
SIV. p2
Purificación Castillo Martínez
SVI. p14
Qian Wu
SVII.C1
R Fernandez-Munoz
SII. P1
R Ortega
SIV. p25
Rafael Catalá
SVII.P1
Rafael Garcés
SVIII. p11
Rafael Martínez- Carrasco
SVII.p11
Ramon Contreras
SIV. C2
Ramón José Barrio
SVIII. p4
Ramón Serrano
SVII.p29
Ramón Serrano Salom
SVII.p16
Raquel Bertí
SVII.p29
Raquel Iglesias- Fernández
SI. p7 SIV. C4
Raquel Jiménez-Muñoz
SII. C2 SVII.p7 SVII.p12
Raquel Martins-Noguerol
SVIII. p11
Raquel N. Sempere
SII. p12
Raquel Rosales
SVII.p7
Raquel Valderrama
SVI. p10 SVI. p13
Raúl Huertas
SVII.P1
Raúl Rivas González
SIX. p11
Regina Niñoles
SVII.p29 SVII.p16
Renaud Dumas
SVI. C2
Reyes Benlloch
SIV. p27
Reyes Ródenas
SVII.C5 SVII.p22
Riad Nadi
SIV. C5
Ricardo Ordás
SIV. p24
Ricardo Parreño-Montoro
SVI. p14
Ritushree Jain
SIX. p3
Robert Sablowski
SIV. p3 SIV. p10
Robertas Ursache
SV. p4
Roberto Baigorri
SVII.P2 SVII.p31
Roberto Solano
SVI. P1 SVI. p8 SIV. p18
Rocío Álvarez-Aragón
SVII.p30
Rocío Olmo
SIX. C2 SIX. p4
Rocío Sánchez- Montesino
SIV. p28 SVI. p11
Rocio Torres-Vera
SVI. p9
Rodrigo García-del Campo
SVI. p15
Rodrigo Sagaceta
SVIII. p5
Ronald Pierik
SV. p7
Rosa Lozano-Durán
SIX. C4
Rosa M Rivero
SVII.p25 SVII.C5
Rosa Micol-Ponce
SIV. p1
Wilfried Schwab
SII. p13
Wolfgang Busch
SIX. p2
Wolfgang Dröge-Laser
SVI. p11
Xavier Bartel
SIX. p8
Xavier Sevillano
SIII. C6
Xu Zhang
SVII.C1
Y Tikunov
SII. P1
Yogev Burko
SVII.p5
Yolanda Leo-del Puerto
SII. p2 SVII.p5
Yolanda Pérez-Pérez SII. p6 SIII. C5 SIV. p36 SVII.p8 SVII.p15
Yovanny Izquierdo
SIX. p20
Yun Kang
SV. p1
Yves Gibon
SVII.p11
Yves Marco
SIX. p8
Yvon Jaillais
SVII.p3
Zhongchi Liu
SII. C3
Stefanie Polyn
SIV. C1
Steffen Vanneste
SVII.C2
Stella Bernardo-García
SVI. P2
Stephan Pollmann
SI. p7 SV. p7 SVII.p18
Stéphanie Drevensek
SV. C1
Sttefany Rosario
SVII.p17
Svenja Bomers
SVIII. p10
Tábata Rosas-Díaz
SIX. C4 SIX. p1
Takayuki Tohge
SI. P1
Tamara Hernández- Verdeja
SIV. p33
Tamara Lechón
SVI. C1
Teresa Altabella
SVIII. C1
Teresa Fuertes- Mendizábal
SVIII. p5
Teresa Millán
SIV. p25
Thomas Roach
SVI. p8 SVII.p29
Tim Crawford
SIV. p33
Timo Hytönen
SII. C3
Tinguaro Rodríguez
SIX. p8
Tobias Langenecker
SIV. p23
Tobias Maierhofer
SVII.p27
Tomás Albi
SVIII. p9
Tomás Cascón
SIX. p20
Toshiaki Mitsui
SII. P2
Trine Bundgaard Andersen
SVIII. p12
Valle Ojeda
SVIII. P1
Vanessa Masson
SV. C1
Verónica Parra-Vega
SVIII. p1
Vicente Balanzà
SIV. P2
Vicente Martínez
SVII.p1 SVII.p25 SVII.C5
Vicente Rubio
SV. P2 SV. C1
Víctor Carrasco Loba
SV. p7
Víctor M. Guerrero- Sánchez
SI. C3 SI. p9
Víctor Quesada
SIV. p26 SIV. p34
Víctor Roces
SVII.C4
Victoria Ruiz-Hernández
SV. p5 SIX. p19
Victoriano Meco
SIV. P1 SIV. p7
Victoriano Valpuesta SII. p3 SIV. p7 SIV. p12 SVII.p3 SVII.p26
Virginia Palomares
SVI. C1
Virginia Ruiz-Ferrer
SIX. p4
Vitor Amorim-Silva
SVII.C2 SVII.p3 SVII.p26 SIX. C4
Vívian Ebeling Viana
SIV. p15
Viviana Escudero
SIX. p17
Vojislava Grbic
SIX. P2
Walter Dewitt
SVI. C1
Wiam Merini
SIV. p35 SV. P1
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