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Libro de resúmenes

xiiirbmp.uniovi.es

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i b r o d e r e s ú m e n e s xiiirbmp.uniovi.es

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Reunión de Biología Molecular de Plantas

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XIII R BM P XIII R BM P Índice Índice i Comité Organizador y Científico iii

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Comité Organizador y Científico

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Carta de Bienvenida

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Programa XIII RBMP

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Conferencias Plenarias

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Comunicaciónes Sesión I

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Comunicaciónes Sesión II

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Comunicaciónes Sesión III

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Comunicaciónes Sesión IV

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Comunicaciónes Sesión V

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Comunicaciónes Sesión VI

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Comunicaciónes Sesión VII

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Índice de Autores

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Comité Organizador

XIII R BM P Comité Organizador María Jesús Cañal Villanueva Ricardo Ordás Fernández Mónica Meijón Vidal

María Jesús Cañal Villanueva Ricardo Ordás Fernández Mónica Meijón Vidal Luis Valledor González Elena María Fernández González

Comité Científico

Metabolismo y aplicaciones biotecnológicas

Albert Boronat Margosa

Departamento de Bioquímica y Biología Molecular de Plantas Universidad de Barcelona

David González Ballester

Departamento de Bioquímica y Biología Molecular de Plantas Universidad de Córdoba

Mecanismos moleculares de desarrollo

Óscar Lorenzo Sánchez

Departamento de Fisiología Vegetal. Universidad de Salamanca

Miguel Ángel Moreno Risueño

Centro de Biotecnología y Genómica de Plantas (CBGP-UPM-INIA)

Ambiente, desarrollo y plasticidad fenotípica

Carlos Alonso Blanco

Centro Nacional de Biotecnología (CNB-CSIC)

Ignacio Rubio Somoza

Center for Research in Agricultural Genomics (CRAG)

Vías de señalización

Salomé Prat Monguio

Centro Nacional de Biotecnología (CNB-CSIC)

Javier Agustí Feliu

Instituto de Biología Molecular y Celular de Plantas (IBMCP-UPV)

Estrés abiótico

Luisa María Sandalio

Estación Experimental del Zaidín (EZ-CSIC)

Mar Castellano Moreno

Centro de Biotecnología y Genómica de Plantas (CBGP-UPM-INIA)

Estrés biótico e interacción planta- microorganismo

Isabel Díaz Rodríguez

Centro de Biotecnología y Genómica de Plantas (CBGP-UPM-INIA)

Julio Rodríguez Romero

Centro de Biotecnología y Genómica de Plantas (CBGP-UPM-INIA)

Temas y técnicas emergentes

Jesús Jorrín Novo

Departamento de Bioquímica y Biología Molecular Universidad de Córdoba

Sonia Osorio Algar

Departamento de Biología Molecular y Bioquímica Universidad de Málaga

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Bienvenida

La Reunión bianual de Biología Molecular de Plantas es el evento nacional de mayor relevancia para los investigadores de diferentes Sociedades Científicas y procedencias que utilizamos la biología molecular con distintas aproximaciones a diferentes problemas científicos, compartiendo un material de estudio común: las plantas. Por ello, es el foro más idóneo para compartir experiencias y resultados, renovar o iniciar colaboraciones o abrir nuevos horizontes e iniciar nuevas aventuras científicas.

En esta edición, dedicada especialmente a las jóvenes promesas científicas, contamos con excelentes ponencias, ampliándose el número de comunicaciones orales por sesión,

la participación de investigadores senior de gran prestigio internacional que nos impartirán tres conferencias plenarias muy interesantes.

y

El

Comité Organizador os da la bienvenida a la Reunión, agradece vuestra participación

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espera que el programa científico y otras actividades organizadas sean de vuestro

agrado, solicitando vuestra colaboración para el buen desarrollo de la misma.

También os damos la bienvenida a la ciudad de Oviedo, cuyo origen se remonta a la alta edad media, con monumentos únicos y muy bellos como son los templos pre-románicos, magníficos museos, espectaculares piezas de arquitectura contemporánea, gente muy acogedora y una magnífica gastronomía, todo ello enmarcado por el característico color verde del paraíso natural que es Asturias.

Esperando que la Reunión sea un éxito, recibid un cordial saludo.

María Jesús Cañal Villanueva

Presidenta del Comité Organizador

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Programa XIII RBMP

XIII Reunión de Biología Molecular de Plantas

Oviedo 22-24 de junio de 2016

Miércoles 22 de junio

13:00-15:00……

Registro y recogida de documentación

15:00-15:15……

Acto de apertura

15:15-16:00……

Plenaria I: Professor Philip Benfey (Trinity College of Arts and Science, Duke

University, USA): “Getting to the root of plant development”

Sesión I. Metabolismo y aplicaciones biotecnológicas Moderadores: Albert Boronat Margosa (Universidad de Barcelona)/ David González Ballester (Universidad de Córdoba)

16:00-16:30 David González Ballester (Universidad de Córdoba): “Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures”

16:30-16:45 Edurne Baroja Fernández (Instituto de Agrobiotecnología, CSIC/UPNA/ Gobierno de Navarra): “Isotope ratio mass spectrometric and genetic evidence for the occurrence of starch degradation and cycling in illuminated Arabidopsis leaves”

16:45-17:00 Juan Manuel Pérez-Ruiz (Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla): “2-Cys Peroxiredoxins control redox regulation of photosynthetic metabolism”

17:00-17:15 Mª Belén Pascual (Universidad de Málaga): “Transcriptional regulation of phenylalanine biosynthesis and utilization”

17:15-17:45……

Café y posters

Sesión BIOVEGEN: Empresas agroalimentarias con intereses en I+D

Moderador: Gonzaga Ruíz de Gauna (BIOVEGEN)

17:45-18:45 Presentaciones cortas de empresas (actividad de la empresa, áreas y demandas tecnológicas de interés, posibilidades de colaboración…etc.)

- IDEN BIOTECHNOLOGY: Pedro Molina

- AGRICOLA 2000: Giacomo Scatolino

- SAIONAMER: Joseba Sánchez

- BIONATUR ROSES: Héctor Sánchez

19:00……

Visita guiada por el centro histórico de Oviedo

20:30……

Cóctel de Bienvenida. Hotel La Reconquista

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Jueves 23 junio

09:00-09:45……….Plenaria II (Patronicado por UO-LIBERBANK): Dr. Wolfgang Busch (Gregor Mendel Institute of Plant Molecular Biology, Austria): “From genotype to phenotype: the systems genetics of root growth”

Sesión II. Mecanismos moleculares de desarrollo Moderadores: Óscar Lorenzo Sánchez (Universidad de Salamanca)/ Miguel Ángel Moreno Risueño (CBGP-UPM-INIA)

09:45-10:15 Miguel Ángel Moreno Risueño (CBGP-UPM-INIA, Madrid): “Building a root postembryonically: new factors integrate cell identity and auxin signalling”

10:15-10:30 Zaida Vergara (CSIC-UAM, Madrid) “Uncovering the role of Arabidopsis ORC1 during root organogenesis”

10:30-10:45 José Luis Micol (Universidad Miguel Hernández, Elche): “Role of DESIGUAL1 and auxin in bilateral symmetry of Arabidopsis leaves”

10:45-11:00 Tamara Lechón (CIALE-Universidad de Salamanca): “PROHIBITIN3 and NOA1 participate in the maintenance of the root stem cell niche in Arabidopsis thaliana”

11:00-11:15 Paula Suárez López (CRAG, CSIC-IRTA-UAB-UB): “Regulation of developmental timing by TEMPRANILLO through the age-dependent pathway”

11:15-11:45……

Café y posters

Sesión III. Ambiente, desarrollo y plasticidad fenotípica Moderadores: Carlos Alonso Blanco (CNB-CSIC)/ Javier Agustí Feliu (IBMCP-UPV)

11:45-12:15 Javier Agustí Feliu (IBMCP-UPV): “Using natural variation to understand lateral growth in plants”

12:15-12:30 Daniel Conde (CBGP, UPM-INIA, Madrid): “DNA demethylases control growth-dormancy transitions in Poplar”

12:30-12:45 Irma Roig Villanova (CRAG, CSIC-IRTA-UAB-UB): “Deciphering how plant density affects seed yield in Arabidopsis thaliana”

12:45-13:00 Adrián Valli (University of Cambridge-UK, CNB-CSIC Madrid): “Most microRNAs in the single-cell alga Chlamydomonas reinhardtii are produced by DCL3-mediated cleavage of introns and UTRs of coding RNAs”

13:00-13:15 Mª Luz Annacondia (Universidad de Oviedo): “Identification of novel epigenetically regulated genes involved in root development in Arabidopsis thaliana”

13:15-15:30……

Comida y posters

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Sesión IV. Vías de señalización Moderadores: Ricardo Ordás Fernández (Universidad de Oviedo)/ Ignacio Rubio Somoza (CRAG)

15:30-16:00 Ignacio Rubio Somoza (CRAG): “miRNA networks and their central role in molecular reprogramming”

16:00-16:15 Catharina Merchante (Universidad de Málaga): “Hormone-Mediated Gene- Specific Translation Regulation”

16:15-16:30 Emilio Gutiérrez-Beltrán (Upsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology): “Molecular composition of stress granules in Arabidopsis”

16:30-16:45 Sandra Fonseca (CNB-CSIC, Madrid): “New links between chromatin remodelling and photomorphogenesis in Arabidopsis”

16:45-17:00 Eduardo Bueso (IBMCP, Valencia): “Arabidopsis COGWHEEL1 links light perception and gibberellins with seed longevity”

17:00-17:30……

Café y posters

Sesión V. Estrés abiótico Moderadoras: Luisa María Sandalio González (EEZ-CSIC)/ Mar Castellano Moreno (CBGP-UPM- INIA)

17:30-18:00 Mar Castellano Moreno (CBGP-UPM-INIA, Madrid): “The At3P protein family plays an essential role in response to different abiotic stresses”

18:00-18:15 Jessica Pérez Sancho (Universidad de Málaga-CSIC): “Plasma membrane lipid remodeling during cold acclimation is mediated by the ER-PM contact sites- localized synaptotagmins 1 and 3”

18:15-18:30 Mónica Escandón (Universidad de Oviedo): Systems biology approach of heat-induced thermotolerance in Pinus radiate”.

18:30-18:45 Gaetano Bissoli (IBMCP-UPV-CSIC, Valencia): “Pivotal role of subtilisin SBT4.13 in pH homeostasis, oxidative stress and jasmonic acid response”

18:45-19:00 M Carmen Romero Puertas (EEZ-CSIC, Granada): “Insights into the ROS- dependent cell response to the herbicide 2,4-D in plants”

20:30………………. Cena en el Llagar Quelo (Tiñana)

Viernes 24 junio

09:00-09:45……….Plenaria III (Patronicado por UO-LIBERBANK): Professor Tamas Dalmay (School of Biological Science, University of East Anglia, UK): “Profiling microRNAs and their targets”

Sesión VI. Estrés biótico e interacción planta-microorganismo Moderadores: Isabel Díaz Rodríguez (CBGP, UPM-INIA)/ Julio Rodríguez Romero (CBGP, UPM- INIA)

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09:45-10:15 Julio Rodríguez Romero (CBGP, UPM-INIA): “Interconnections between mRNA processing, TOR pathway and plant pathogenesis in the rice blast fungus”

10:15-10:30 Araíz Gallo (CNB-CSIC, Madrid): “The Helper Component Proteinase and viral replication: unexpected requirements for the proper yield of virions in Plum pox potyvirus”

10:30-10:45 Sonia Campo (DD Plant Science Center, USA; CRAG, CSIC-IRTA-UAB-UB):

Small RNA-based antiviral defense in the phytopathogenic fungus Colletotrichum higginsianum”

10:45-11:00 Mª Estrella Santamaría (CBGP, UPM-INIA): “MATI, a novel protein involved in plant defence against spider mites”

11:00-11:15 Mayte Castellano (IBMCP, CSIC-UPV): “Epigenetic reprogramming of the host repetitive DNA induced by a pathogenic long noncoding RNA during infection”

11:15-11:45……

Café y posters

Sesión VII. Temas y técnicas emergentes Moderadores: Jesús Jorrín Novo (Universidad de Córdoba)/ Sonia Osorio Algar (Universidad de Málaga)

11:45-12:15 Sonia Osorio Algar (Universidad de Málaga): “Sugars plays an important role in cuticle metabolism and cell wall architecture or tomato and affects shelf-life softenin”

12:15-12:30 Luis Valledor (Universidad de Oviedo): “The integration of physiological, proteomic, and metabolomic levels reveals new adaptive and stress-responsive mechanisms in Pinus”

12:30-12:45 Marcos Egea Cortines (Instituto de Biología Vegetal, UP Cartagena):

Development of artificial vision systems for automatic phenotyping”

12:45-13:00 David Wilson Sánchez (Universidad Miguel Hernández, Elche): “Next- generation forward genetic screens using mapping-by-sequencing”

13:00-13:15 Alberto Carbonell (IBMCP, CSIC-Universidad Politécnica de Valencia):

Genome-wide identification of ARGONAUTE-bound target RNAs in Arabidopsis”

13:15-13:30………………. Clausura

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Getting to the root of plant development

Philip N. Benfey

Biology Department and HHMI, Duke University, Durham, NC USA. Hi Fidelity Genetics, Durham, NC USA

To understand the progression from stem cells to differentiated tissues we are exploiting the simplifying aspects of root development. We have developed new experimental, analytical and imaging methods to identify networks functioning within different cell types and developmental stages of the root. We are particularly interested in a subnetwork that regulates a key asymmetric cell division of a stem cell and the regulatory networks that control differentiation of the stem cell’s progeny. These networks are partially dependent on cell-to-cell signaling through movement of transcription factors. To quantify dynamic aspects of these networks, we are employing light-sheet microscopy to image accumulation of their different components. To find additional signaling molecules we performed ribosome profiling and identified putative peptide ligands. We have also uncovered a clock-like process responsible for the positioning of lateral roots along the root primary axis. Two sets of genes were identified that oscillate in opposite phases at the root tip and are involved in the production of prebranch sites, locations of future lateral roots. A derivative of the carotenoid biosynthesis pathway appears to act as a new mobile signal regulating root architecture.

This work is supported by grants from the NIH, NSF and the Gordon and Betty Moore Foundation.

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From Genotype to Phenotype: The Systems Genetics of Root Growth

Wolfgang Busch

Gregor Mendel Institute, Vienna, Austria

To grow and survive plants need access to nutrients and water: resources that are not evenly distributed in the soil environment. Consequently, the ability of roots to acquire such resources largely determines the ability of a plant to grow. To efficiently explore and forage the soil to acquire these resources, plants have evolved complex, often highly branched root systems. A multitude of environmental factors, such as the distribution of moisture and nutrients, the presence of toxic minerals, soil compaction, and microbiome composition, are important constraints for efficient distribution of roots in the soil as plant root systems need to adjust their growth in response to these and many other environmental cues. Despite this tremendous impact of the environment on root growth, different species, as well as different natural strains within a species, exhibit remarkably different root systems, clearly demonstrating that root growth is genetically determined. Key to this genetically determined responsiveness to the environment is that different environmental cues are differently prioritized by different genotypes. However, very little is known about the genetic and molecular mechanisms that are responsible for this. To understand the genotypic and molecular bases for different root phenotypes and their responses to environmental cues, we use the root of Arabidopsis thaliana as a model. Here, the notable natural genetic variation of different Arabidopsis accessions gives rise to broad phenotypic variation of root growth. Due to the small dimension of the Arabidopsis root, we can observe and quantitatively describe processes ranging from the cellular bases of root growth variation such as cell divisions and cell elongation to organ level traits such as root growth rate, root growth direction, and branching pattern. High throughput phenotyping allows us to observe hundreds of different accessions in a multitude of different growth environments. Moreover, large systems-type data sets such as cell-type specific transcriptome, proteome, and metabolomics data sets are available for the Arabidopsis root. Using all these advantages, we conduct a systems genetics approach, a combination of phenomics, systems biology, and quantitative genetics, to study how genetic information is translated by molecular, cellular, and physiological networks in order to shape complex root phenotypes. We have recently identified key genes and their variants that shape complex phenotypes in roots. In this lecture, I will highlight two of our recently discovered genetic and molecular mechanisms that shape root growth in response to the environment. The first mechanism involves an EXOCYST subunit encoding gene that regulates whether the root system is shallow or deep by modulating the auxin pathway and has a potential adaptive role in areas with variable rainfall patterns. The second component is a regulatory module of genes encoding for LEUCINE-RICH RECEPTOR-LIKE-KINASES that regulate root growth in response to iron limitation, a major growth constraint in soils.

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Identifying microRNAs and their targets

Tamas Dalmay 1

1 School of Biological Sciences, University of East Anglia, Norwich, UK

MicroRNAs (miRNAs) are small non-coding RNA molecules regulating the expression of protein coding genes. The talk will describe how to use next-generation sequencing (NGS) to profile miRNA expression, identify new miRNAs and also their targets. Using these approaches we investigated the correlation between miRNA and target mRNA accumulation and found that contrary to expectation it is not always negative. We also identified an RNA ligation step during the NGS library preparation that introduce a bias into the protocol and an approach will be presented to reduce this bias (Sorefan et al 2012). A similar approach will be presented for target identification.

References:

Lopez-Gomollon S, et al. (2012) Planta, 236(6):1875-87. Sorefan K, et al. (2012) Silence, 3(1):4.

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Comunicaciones Sesión I. Metabolismo y Aplicaciones Biotecnológicas

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Ponencia Invitada / SI PI

Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed Chlamydomonas cultures

David González-Ballester 1 , Jose Luis Jurado-Oller 1 , Alexandra Dubini 1 , Aurora Galván 1 , Emilio Fernández 1

Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Córdoba, Córdoba, Spain

Currently, hydrogen fuel is derived mainly from fossil fuels, but there is an increasing interest in clean and sustainable technologies for hydrogen production. In this context, the ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen is a promising alternative for renewable, clean-energy production. Among a diverse array of photosynthetic microorganisms able to produce hydrogen, the green algae Chlamydomonas reinhardtii is the model organism widely used to study hydrogen production. Despite the well-known fact that acetate-containing medium enhances hydrogen production in this algae, little is known about the precise role of acetate during this process. We have examined several physiological aspects related to acetate assimilation in the context of hydrogen-production metabolism. We show that mixotrophic nutrient-replete cultures under low light can be an alternative for the simultaneous production of hydrogen and biomass. Measurements of oxygen and CO2 levels, acetate uptake, and starch accumulation were performed under different light conditions, and oxygenic regimes. Our data suggest that acetate plays an important role in the hydrogen-production process, during non-stressed conditions, other than establishing anaerobiosis, and independent of starch accumulation. We show that oxygen and light intensity control acetate assimilation and modulate hydrogen production. Low levels of oxygen allow for low acetate uptake rates, and paradoxically, lead to efficient and sustained production of hydrogen. Moreover, we highlight the importance of releasing the hydrogen partial pressure to avoid an inherent inhibitory factor on the hydrogen production. We also demonstrate that the determination of the contribution of the PSII-dependent hydrogen production pathway in mixotrophic cultures, using the photosynthetic inhibitor DCMU, can lead to dissimilar results when used under various oxygenic regimes. The level of inhibition of DCMU in hydrogen production under low light seems to be linked to the acetate uptake rates. Potential metabolic pathways involved in hydrogen production in mixotrophic cultures are discussed.

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Comunicación Oral1 / SI CO1

Isotope ratio mass spectrometric and genetic evidence for the occurrence of starch degradation and cycling in illuminated Arabidopsis leaves

Marouane Baslam 1 , Edurne Baroja-Fernández 1 , Ángela María Sánchez-López 1 , Iker Aranjuelo 1 , Adriana Ricarte-Bermejo 1 , Abdellatif Bahaji 1 , Francisco José Muñoz 1 , Goizeder Almagro 1 , Pablo Pujol 2 , Regina Galarza 2 , Pilar Teixidor 3 , and Javier Pozueta- Romero 1

1 Instituto de Agrobiotecnología (CSIC/UPNA/Gobierno de Navarra). Iruñako etorbidea 123, 31192 Mutiloabeti, Nafarroa, Spain. 2 Servicio de Apoyo a la Investigación, Universidad Pública de Navarra, Campus de Arrosadia, 31006 Iruña, Nafarroa, Spain. 3 Serveis Científico-Tècnics Universitat de Barcelona, C/ Lluís Solé I Sabarís 1-3, 08028 Barcelona, Spain.

Substrate or “futile” cycles are metabolic cycles of synthesis and degradation of a compound resulting in ATP consumption and dissipation of energy. Although there is a great wealth of data supporting the occurrence of storage carbohydrate cycling in many organisms, previous 14 CO 2 pulse-chase studies indicated that starch degradation and cycling do not operate in illuminated Arabidopsis leaves. In this work we show that leaves of different starch breakdown mutants cultured under continuous light conditions accumulate higher levels of starch than WT leaves, which shows that starch degradation operates during illumination. To investigate whether starch breakdown products can be recycled back to starch during illumination through a mechanism involving ADP-glucose pyrophosphorylase (AGP) we conducted time-course analyses of the stable isotope carbon composition ( δ13C) of starch in leaves of 13 CO 2 pulsed-chased wild type (WT) and AGP lacking aps1 plants. Maximum δ13C values of starch in aps1 leaves reached at the end of the pulse were exceedingly higher than those of WT leaves. Furthermore, δ13C reduction in starch of aps1 leaves during the chase was much more rapid than that of WT leaves. Notably, aps1/mex1 leaves impaired in the export of maltose derived from starch breakdown displayed a high-maltose phenotype. Results presented in this work provide strong evidence for the occurrence of simultaneous synthesis and breakdown of starch and the operation of starch cycling through a mechanism involving AGP in illuminated Arabidopsis leaves.

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2-Cys Peroxiredoxins control redox regulation of photosynthetic metabolism

Juan Manuel Pérez-Ruiz, Belén Naranjo, Valle Ojeda and Francisco Javier Cejudo

Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla and CSIC, Avda Américo Vespucio 49, 41092-Sevilla, Spain

Chloroplast metabolism needs to rapidly respond to ever changing environmental conditions, a key component of this response being redox regulation of enzyme activity. This regulatory mechanism, based in the disulphide-dithiol interchange of regulatory cysteines, relies on a complex set of thioredoxins (Trxs), which uses photosynthetically reduced ferredoxin thus linking redox regulation in this organelle to light. In addition, chloroplasts contain an NADPH-dependent Trx reductase (NTR) with a joint Trx domain at the C-terminus, NTRC (Serrato et al. 2004). This enzyme allows the use of NADPH for redox regulation of several enzymes previously known to be Trx-regulated, suggesting that both redox systems act concertedly to regulate chloroplast processes. Photosynthesis, the primary source of biomass and oxygen into the biosphere, inevitably produces reactive oxygen species (ROS), which can cause oxidative damage but have also signalling function. To balance the toxic and signalling activities of ROS, chloroplasts harbour different antioxidant systems including 2-Cys peroxiredoxins (Prxs), which are efficiently reduced by NTRC and, to a lesser extent, by Trxs (Perez-Ruiz et al. 2006; Pulido et al. 2010). In this work, we have analysed the genetic interaction of NTRC and 2-Cys Prxs by the study of Arabidopsis thaliana mutants simultaneously deficient in both enzymes. Strikingly, the deficiency of 2-Cys Prxs had a suppressor effect on the phenotype caused by the lack of NTRC. Moreover, overexpression of 2-Cys Prx A or B in the ntrc mutant background aggravated the phenotype of this mutant, indicating a dose-dependent suppressor effect of 2-Cys Prxs. The simultaneous deficiencies of NTRC and Trx f, the ntrc-trxf1f2 triple mutant, causes a severe impairment of chloroplast redox homeostasis and a dramatic growth inhibition phenotype, which were suppressed by decreased levels of 2-Cys Prxs, in the ntrc-trxf1f2- 2cp quintuple mutant. Overall, our results uncover the key function of 2-Cys Prxs modulating chloroplast redox homeostasis and plant growth.

References:

Perez-Ruiz JM, et al. (2006). Plant Cell 18: 2356-2368 Pulido P, et al. (2010). J Exp Bot 61: 4043-4054 Serrato AJ, et al. (2004). J Biol Chem 279: 43821-43827

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Comunicación Oral 3 / SI CO3

Transcriptional regulation of phenylalanine biosynthesis and utilization

Mª Belén Pascual, Rafael A. Cañas, Blanca Craven-Bartle, Francisco M. Cánovas, Concepción Ávila

Departamento de Biología Molecular y Bioquímica. Facultad de Ciencias. Universidad de Málaga. Campus de Teatinos s/n, Málaga, Spain.

Conifer trees divert large quantities of carbon into the biosynthesis of phenylpropanoids, particularly to generate lignin, an important constituent of wood. Since phenylalanine is the precursor for phenylpropanoid biosynthesis, the precise regulation of phenylalanine synthesis and utilization should occur simultaneously. This crucial pathway is finely regulated primarily at the transcriptional level. Transcriptome analyses indicate that the transcription factors (TFs) preferentially expressed during wood formation in plants belong to the MYB and NAC families. Craven-Bartle et al. (2013) have shown in conifers that Myb8 is a candidate regulator of key genes in phenylalanine biosynthesis involved in the supply of the phenylpropane carbon skeleton necessary for lignin biosynthesis. This TF is able to bind AC elements present in the promoter regions of these genes to activate transcription. Constitutive overexpression of Myb8 in white spruce increased secondary- wall thickening and led to ectopic lignin deposition (Bomal et al. 2008). In Arabidopsis, the transcriptional network controlling secondary cell wall involves NAC-domain regulators operating upstream Myb transcription factors. Functional orthologues of members of this network described have been identified in poplar and eucalyptus, but in conifers functional evidence had only been obtained for MYBs. We have identified in the P. pinaster genome 37 genes encoding NAC proteins, which 3 NAC proteins could be potential candidates to be involved in vascular development (Pascual et al. 2015). The understanding of the transcriptional regulatory network associated to phenylpropanoids and lignin biosynthesis in conifers is crucial for future applications in tree improvement and sustainable forest management.

This work is supported by the projects BIO2012-33797, BIO2015-69285-R and BIO-474

References:

Bomal C, et al. (2008) J Exp Bot. 59: 3925-3939. Craven-Bartle B, et al. (2013). Plant J, 74: 755-766. Pascual MB, et al. (2015). BMC Plant Biol, 15: 254.

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Poster 01 / SI P01

Ferredoxin-mediated hydrogen production

Alexandra Dubini 1 , Marko Boehm 2 , Erin Peden 2 , Wenqiang Yang 3 , Arthur Grossman 3 , Maria Ghirardi 2

1 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Córdoba, Córdoba, Spain. 2 National Renewable Energy Laboratory, 15013 Denver West, Parkway, Mail Box 3313, Golden, CO 80401, USA. 3 Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305, USA

Ferredoxins (FDXs) are small proteins that can distribute electrons originating from either photosynthesis, fermentation, or other reductant-generating pathways to specific redox enzymes in different organisms. Chlamydomonas reinhardtii contains multiple isoforms that are not fully characterized in terms of their biological function. However, we recently provided experimental evidence that FDX1 serves as the primary electron donor to two important biological pathways: NADPH and H2 photo-production. On the other hand, FDX2 is capable of driving these two reactions at less than half of the rate observed for FDX1. We have characterized the FDX2 crystal structure, which helped highlighting the sequence differences between FDX2 and FDX1. These differences directly affect both the midpoint potentials and their ability to catalyze NADPH and H2 production. The differences in the ability of catalyzing NADPH and H2 are possibly due to an altered binding capacity to interact with FNR and HYDA, respectively. We have also characterized FDX5 and showed that this protein has a more positive midpoint potential than FDX1 and FDX2. FDX5 interact with some common partners of FDX1 and 2, but also has specific ones. A mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised while growth in the light was unaffected. Under sulfur deprivation, the mutant strain shows also differences in term of photosynthetic activity and H2 production when compared to the wild type. The results suggest that in photosynthetic organisms, redox reactions are being tailored by specific electron carriers such as ferredoxins to unique intracellular metabolic circuits under distinct redox conditions. These ferredoxins are specific and potentially interchangeable depending on the condition while always impacting hydrogen production to some degree.

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Poster 02 / SI P02

Studying the role of the strawberry Fra protein family in the flavonoid metabolism during fruit ripening

Begoña Orozco-Navarrete, Araceli G. Castillo, Ana Casañal, Victoriano Valpuesta, and Catharina Merchante

Instituto de Hortofruticultura Subtropical y Mediterránea, Universidad de Málaga-CSIC, Spain.

Strawberry fruits are highly appreciated worldwide due to their pleasant flavor and aroma and to the health benefits associated to their consumption. An important part of these properties is due to their content in secondary metabolites, especially phenolic compounds, of which flavonoids are the most abundant in the strawberry fruit. Although the flavonoid biosynthesis pathway is uncovered, little is known about its regulation. The strawberry Fra a (Fra) genes constitute a large family of homologs of the major birch pollen allergen Bet v 1 and for which no equivalents exist in Arabidopsis. Our group has shown that Fra proteins are involved in the formation of colored compounds in strawberries (Muñoz et al., 2010), which mainly depends on the production of certain flavonoids; that they are structurally homologs to the PYR/PYL/RCAR Arabidopsis ABA receptor, and that they are able to bind flavonoids (Casañal et al., 2013). With these previous results, our working hypothesis is that the Fra proteins are involved in the regulation of the flavonoids pathway. They would mechanistically act as the ABA receptor, binding a protein interactor and a ligand to regulate a signaling cascade and/or act as molecular carriers. The main objective of this research is to characterize the Fra family in strawberry and gain insight into their role in the flavonoid metabolism. By RNAseq expression analysis in ripening fruits we have identified transcripts for 10 members of the Fra family. Although expressed in all tissues analyzed, each family member presents a unique pattern of expression, which suggests functional specialization for each Fra protein. Then, our next approach was to identify the proteins that interact with Fras and their ligands to gain knowledge on the role that these proteins play in the flavonoids pathway. To identify the interacting partners of Fras we have performed a yeast two hybrid (Y2H) screening against cDNA libraries of strawberry fruits at the green and red stages. A protein that shares a 95% homology to the Heat stress transcription factor A-4-C like of Fragaria vesca (HSA4C) interacts specifically with Fra1 and not with other family members, which suggests functional diversification of Fra proteins in specific signaling pathways. The Y2H screening is not yet saturated, so characterization of other interacting proteins with other members of the Fra family will shed light on the functional diversity within this gene family. This research will contribute to gain knowledge on how the flavonoid pathway, and hence, the fruit ripening, is regulated in strawberry; an economically important crop but for which basic research is still very limited.

References:

Muñoz, C, et al. (2010). Molecular Plant, 3(1): 113–124. Casañal, A, et al (2013). Journal of Biological Chemistry, 288(49): 35322–35332.

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Poster 03 / SI P03

QTL mapping for primary metabolites responsible of organoleptic and nutritional characteristics of strawberry (Fragaria x ananassa)

Delphine Pott 1 , José G. Vallarino 1 , Juan Jesús Medina 2 , Alisdair R. Fernie 3 , Iraida Amaya 4 , Sonia Osorio 1

1 Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, University of Malaga- CSIC, Department of Molecular Biology and Biochemistry, 29071 Málaga, Spain. 2 Instituto Andaluz de Investigación y Formación Agraria y Pesquera (IFAPA), Julio Caro Baroja s/n, Huelva, Spain. 3 MaxPlanck-Institute für Molekulare Planzenphysiologie, Am Mühlenberg 1, 14476 Golm, Germany. 4 IFAPA, Centro de Churriana, Cortijo de la Cruz S/N, Churriana, 29140 Malaga, Spain.

The cultivated strawberry (Fragaria x ananassa) is the berry fruit most consumed worldwide and is well-known for its delicate flavour and nutritional properties. However, fruit quality attributes have been lost or reduced after years of traditional breeding focusing mainly on agronomical traits. To face the obstacles encountered in the improvement of cultivated crops, new technological tools, such as genomics and high throughput metabolomics, are becoming essential for the identification of genetic factors responsible of organoleptic and nutritive traits. Integration of “omics” data will allow a better understanding of the molecular and genetic mechanisms underlying the accumulation of metabolites involved in the flavour and nutritional value of the fruit. To identify genetic components affecting/controlling? fruit metabolic composition, here we present a quantitative trait loci (QTL) analysis using a 95 F 1 segregating population derived from genotypes ‘1392’, selected for its superior flavour, and ‘232’ selected based in high yield (Zorrilla-Fontanesi et al., 2011; Zorrilla-Fontanesi et al., 2012). Metabolite profiling was performed on red stage strawberry fruits using gas chromatography hyphenated to time-of-flight mass spectrometry, which is a rapid and highly sensitive approach, allowing a good coverage of the central pathways of primary metabolism. Around 50 primary metabolites, including sugars, sugars derivatives, amino and organic acids, were detected and quantified after analysis in each individual of the population. QTL mapping was performed on the ‘232’ x ‘1392’ population separately over two successive years, based on the integrated linkage map (Sánchez-Sevilla et al., 2015). First, significant associations between metabolite content and molecular markers were identified by the non-parametric test of Kruskal-Wallis. Then, interval mapping (IM), as well as the multiple QTL method (MQM) allowed the identification of QTLs in octoploid strawberry. A permutation test established LOD thresholds for each metabolite and year. A total of 132 QTLs were detected in all the linkage groups over the two years for 42 metabolites out of 50. Among them, 4 (9.8%) QTLs for sugars, 9 (25%) for acids and 7 (12.7%) for amino acids were stable and detected in the two successive years. We are now studying the QTLs regions in order to find candidate genes to explain differences in metabolite content in the different individuals of the population, and we expect to identify associations between genes and metabolites which will help us to understand their role in quality traits of strawberry fruit.

References:

Zorrilla-Fontanesi, et al. (2011) Theor Appl Genet, 123: 755-778; Zorrilla-Fontanesi, et al (2012) Plant Physiol, 159: 851-870; J.F. Sánchez-Sevilla, et al. (2015) PloS one. 10, e0144960.

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Poster 04 / SI P04

Steryl ester metabolism in tomato

Alejandro Lara 1 , Karla Ramírez-Estrada 1 , Alma Burciaga 1 , María Martín 1 , Montserrat Arró 1,2 Albert Boronat 1,3 , Teresa Altabella 1,4 , Albert Ferrer 1,2

1 Center for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Campus UAB, Bellaterra, Barcelona España; 2 Dpto de Bioquímica y Fisiología, UB, Barcelona, España, 3 Dpto de Bioquímica y Biomedicina Molecular, UB, Barcelona, España. , 4 Dpto de Biología, Sanidad y Medio Ambiente, UB, Barcelona, España.

Phytosterols are integral components of plant membranes that modulate their fluidity and permeability. Recent studies have shown that sterols play also essential roles not only in plant growth and development but also in mediating their responses to a variety of stress conditions. Each plant species has its own qualitative and quantitative profile of sterols, with the three most common being sitosterol, campesterol and stigmasterol. Plant sterols are found in free form (FE) and conjugated as esters (SE), glycosides (SG) and acylglycosides (ASG). Tomato, along with other species of the Solanaceae family, displays an atypical profile of conjugated sterols, which shows marked changes during the fruit ripening process (Duperon et al., 1984; Whitaker, 1988). However, the biological and evolutionary significance of this peculiar sterol composition is currently unknown and the knowledge about the enzymes involved in the metabolism of conjugated sterols is still very limited. These include, among others, phospholipid sterol acyltransferases (PSAT) and acyl-CoA sterol acyltransferases (ASAT) responsible for SE biosynthesis. Sterol acylation is considered an essential process for maintaining sterol homeostasis in cell membranes, and the level of SEs has been reported to increase in plants exposed to different stresses. In tomato (Solanum lycopersicum cv. Micro-Tom), we have identified a single gene coding for PSAT (SlPSAT), whose functional identity has been demonstrated by complementation of the A. thaliana null mutant psat 1-1 (Banas et al. 2005), and 8 genes coding for putative ASATs (SlASAT1-8). These genes are differentially expressed in different tomato organs and during fruit ripening, as well as in response to several exogenous stimuli (abscisic acid, salycilic acid, methyl jasmonate and flagellin). The cDNAs corresponding to the four most highly expressed genes (SlASAT1, SlASAT2, SlASAT5 and SlASAT8) have been cloned. SlASAT1 encodes a plasma membrane protein whose functional identity has been demonstrated by complementation of the A. thaliana null mutant asat1-1 (Bouvier-Navé et al. 2010) while SlASAT2, SlASAT5 and SlASAT8 encode proteins located in the endoplasmic reticulum, whose functionality is being studied. Data will also be presented on the possible involvement of the different tomato sterol acyltransferases in plant response to stress. These data will set the basis for further studies aimed at understanding the role of SE in tomato plant growth and development, fruit ripening and their response to biotic and abiotic stress.

This work was financed by the Spanish Ministerio de Economia y Competitividad (grant number AGL2013- 13522-R) and the Generalitat de Catalunya (grant number 2014SGR 1434). A.L. and A.B. are recipients of predoctoral fellowships from the CONACYT (México). K.R-E. is recipient of a postdoctoral fellowship from the CONACYT (México).

References:

Banas et al., (2005). J. Biol. Chem. 280: 34626–34634 Bouvier-Navé et al., (2010). Plant Physiol. 152: 107-119

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Poster 05 / SI P05

Lysophosphatidylcholine acyltransferase genes from sunflower (Helianthus annuus L.)

Ana Mapelli-Brahm 1 , J.J. Salas 1 , Enrique Martínez-Force 1 , Rafael Garcés 1 , Mónica Venegas-Calerón 1

1 Group of Genetics and Biochemistry of Seed Lipids, Department of Biochemistry and Molecular Biology of Plant Products, Instituto de la Grasa (CSIC), Seville, Spain.

Oil crops and other oleaginous plants accumulate triacylglycerols (TAGs) in seeds as the stock of carbon and reducing equivalents necessary to feed the embryo. TAGs in oil crops are an important source of food and provides with highly reduced carbon chains for biofuel and oleochemical industries. The fatty acids composition and their distribution within the TAG molecules determine the quality and property of oils. The main TAG biosynthesis pathway is the Kennedy pathway, which occurs at the endoplasmic reticulum by successive acylation of glycerol-3-phosphate with acyl-CoA derivatives producing lysophosphatidic acid, phosphatidic acid and diacylglycerol as intermediates. In addition to the Kennedy pathway, the phosphatidylcholine (PC) acyl-editing pathway or Lands cycle is important for TAG synthesis in plants. In this regard, PC plays an important role because esterified fatty acids in the position sn-2 of this lipid can suffer different modifications like desaturation, hydroxylation or epoxidation. In this pathway there are a continuous acyl exchange between PC and acyl CoA pools, involving the production of lysophosphatidylcholine (LPC). The re-acylation of LPC to yield PC is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT), which leads to an enrichment of polyunsaturated or unusual fatty acids synthesized in PC in the acyl-CoA pool. Thus, these modified fatty acids are available for their incorporation into TAG. In this work, we report the isolation of two LPCAT genes from sunflower (Helianthus annuus L.), HaLPCAT1 and HaLPCAT2. Both HaLPCATs are members of the membrane bound O-acyltransferases (MBOAT) family and homologous to previously described LPCAT genes in Arabidopsis thaliana, flax (Linum usitatissimum L.), castor (Ricinus communis L.) and rapeseed (Brassica napus L.). Expression levels of both LPCATs genes from sunflower have been studied revealing distinct tissue-specific expression patterns. Sequence analysis and functional characterization of these genes are also reported. Results in this research could contribute to a better understanding of the synthesis of TAGs in oil crops, and to find novel paths for the production of oils with new compositions and applications.

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Poster 06 / SI P06

A mitochondrial lipoyltransferase from Helianthus annuus

Raquel Martins-Noguerol 1 , Rafael Garcés 1 , J.J. Salas 1 , Enrique Martínez-Force 1

1 Group of Genetics and Biochemistry of Seed Lipids, Department of Biochemistry and Molecular Biology of Plants Products, Instituto de la Grasa (CSIC). Sevilla, Spain.

Lipoic acid is a sulfur containing coenzyme found in most bacteria and eukaryotic organisms, as well as some archaea. This cofactor is essential for the functionality of several key enzymes involved in oxidative and single carbon metabolism including pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase (2-OGDH), branched- chain 2-oxoacid dehydrogenase, acetoin dehydrogenase and the glycine cleavage system. All these complexes require lipoic acid molecules covalently bound to at least one of their protein components to be functional. Lipoic acid is essentially a modified form of the short-chain fatty acid octanoate with two thiol substituents at C6 and C8. In its oxidized form these thiols give place to a disulphide bond forming a 1,2-dithiolane ring. Pyruvate dehydrogenase complex (PDH) is a large multienzyme structure catalyzing the oxidative decarboxylation of pyruvate to produce acetyl-CoA, CO 2 and NADH. Plants contains two distinct spatially separated PDH complexes, one within mitochondrial matrix, where it serves as a primary entry point for carbon into the citric acid cycle, and the other in the plastid stroma, providing acetyl-coA for fatty acid biosynthesis. A specific lysine residue of the E2 subunit of PDH is covalently bound via an amide linkage to the carboxy group of lipoic acid. This cofactor is synthesized from octanoyl-acyl carrier protein (ACP) through a reaction catalyzed by lipoic acid synthase (LS). Then a lipoyltransferase (LT) transfers the lipoyl group from lipoyl-ACP to apoproteins such as E2-PDH. Therefore, lipoic acid biosynthesis and supply is essential for PDH complex to be functional. Despite the importance of the lipoyl prosthetic group in the function of enzyme complexes involved in central metabolism, little is known about its synthesis and the enzymes responsible for its incorporation into proteins in higher plants. In the present work, a mitochondrial lipoyltransferase from sunflower (HaLIP2m) was identified, sequenced and cloned in a heterologous production system (Escherichia coli). Also we studied the expression of this enzyme in different vegetal tissues. The contribution of this cofactor to sunflower plant development and oil synthesis was investigated.

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Poster 07 / SI P07

Arabidopsis is capable of responding to volatile phytostimulants emitted by phytopathogenic microorganisms by triggering plastidic phosphoglucose isomerase independent mechanisms

Ángela María Sánchez-López 1 , Abdellatif Bahaji 1 , Nuria De Diego 2 , Marouane Baslam 1 , Jun Li 1 , Francisco José Muñoz 1 , Goizeder Almagro 1 , Pablo García-Gómez 1 , Kinia Ameztoy 1 , Adriana Ricarte-Bermejo 1 , Ond ř ej Novák 3 , Jan F. Humplík 2 , Luká š Spíchal 2 , Karel Dole ž al 2 , Sergio Ciordia 4 , Maria del Carmen Mena 4 , Rosana Navajas 4 , Edurne Baroja-Fernández 1 , and Javier Pozueta-Romero 1

1 Instituto de Agrobiotecnología (CSIC/UPNA/Gobierno de Navarra). Iruñako etorbidea 123, 31192 Mutiloabeti, Nafarroa, Spain. 2 Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palack ý University, Olomouc, CZ-78371, Czech Republic. 3 Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palack ý University and Institute of Experimental Botany ASCR, Olomouc, CZ-78371, Czech Republic. 4 Unidad de Proteómica Centro Nacional de Biotecnología, CSIC, Darwin 3, Campus de Cantoblanco, Madrid 28049, Spain

Volatile compounds (VOCs) emitted by phylogenetically diverse microorganisms (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote photosynthesis, growth and accumulation of exceptionally high levels of starch in leaves through cytokinin (CK) regulated processes (Sánchez-López et al. 2016). In VOCs non-treated Arabidopsis plants, plastidic phosphoglucose isomerase (pPGI) plays an important role in photosynthesis and growth likely as a consequence of its involvement in the synthesis of plastidic CKs and/or its participation in the glycolytic and oxidative pentose phosphate pathways. Moreover, this enzyme plays an important role in connecting the Calvin Benson cycle with the starch biosynthetic pathway in leaves. To better elucidate mechanisms involved in the plants´ responses to microbial VOCs, and to investigate the extent to which pPGI is involved in this phenomenon, in this work we characterized pPGI null pgi1-2 Arabidopsis plants cultured in the presence or absence of VOCs emitted by Alternaria alternata. We found that volatile emissions from this fungal phytopathogen promoted growth, photosynthesis and accumulation of plastidic CKs in pgi1-2 plants. Contrary to expectations, mesophyll cells of exposed pgi1-2 leaves accumulated exceptionally high levels of starch. Isobaric labeling based differential proteomic analyses revealed that VOCs promote global changes in the expression of proteins involved in photosynthesis, starch metabolism and growth that can account for the observed responses in pgi1-2 plants. The overall data show that Arabidopsis plants are capable of responding to volatile phytostimulants emitted by microorganisms by triggering pPGI independent mechanisms.

References:

Sánchez-López, A.M., et al. (2016). Plant Cell Environ. (in press)

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Poster 08 / SI P08

Coexpression networks as tool to identify novel elements in starch metabolism regulation

M.Teresa Ruiz 1 , María Garcia 1 , M. Isabel Ortíz-Marchena 1 , Karen Chacón 1 , Francisco J. Romero-Campero 2 , Federico Valverde 1 , José M. Romero 1

1 Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Seville, Spain. 2 Department of Computer Science and Artificial Intelligence, Universidad de Sevilla, Seville, Spain.

Molecular System Biology has proven a powerful tool to unveil novel elements in known biological processes such as starch biosynthesis (Romero-Campero et al., 2013). Starch is essential not only in the long-term nutrient storage or the daily supply of carbon to the plant, but also in the events that lead to flowering in Arabidopsis (Ortiz et al., 2014). In order to find genes involved in the regulation of starch metabolism, we constructed a gene co-expression network for A. thaliana Col-0 using data available in GEO (Gene Expression Omnibus), corresponding to 31 microarray experiments comprising 134 conditions in which starch metabolism was altered. The application of clustering algorithms combined with gene ontology analysis identified in the network five clusters with defined functional enrichment. APS1 and APL1 are the two major isoforms of ADP-Glucose pyrophosphorylase (ADPGase) in leaves (Ventriglia et al., 2008), responsible for the synthesis of ADP-Glu, the monomer from which starch is synthetized. The APS1 and APL1 genes were found in a central position in the gene co-expression network, in a cluster containing genes involved in macromolecules biosynthesis, as well as carbon and phosphorous metabolism. Surprisingly, they were directly connected to two genes of unknown function. However, some structural and functional features [one of them presented an oligonucleotide/oligosaccharide binding motive (OB) and the other a FHA domain (SMAD)], suggested that they may act as transcriptional regulators. To investigate the role of these genes in starch metabolism and the processes controlled by these two genes, T-DNA insertion mutant lines for both genes were selected and analysed. Preliminary results show that these lines present and altered expression of APS1 and APL1 genes, as well as a delay in flowering time and reduced levels of starch and sugar accumulation. Lines overexpressing the two genes, both in their native form or fused to GFP for subcellular localization, are also being generated. A role for these transcriptional regulators in the control of the expression of the ADPGase gene cluster, which has not been described to date, will be presented.

This work was funded by project BIO2014-52452-P (MINECO) to FV and JMR and PAI BIO-281 (Junta de Andalucía).

References:

Romero-Campero FJ, et al (2013) Front. Plant Sci., 4:291. Ventriglia T, et al (2008). Plant Physiol. 148: 65-76. Ortiz-Marchena MI, et al. (2014). Plant Cell, 26: 565-584.

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Poster 09 / SI P09

Complex floral scent profiles under selection pressure show Mendelian based genetic structure and evolve via transposon activity

Victoria Ruiz-Hernández 1 , Julia Weiss 1 , Benjamin Hermans 1 , Joëlle K. Mühlemann, 2, Natalia Dudareva, 3 and Marcos Egea-Cortines 1

1 Genética Molecular, Instituto de Biotecnología Vegetal, Universidad Politécnica de Cartagena 30202 Cartagena, Spain, 2 Department of Biology, Wake Forest University, North Carolina 27109, USA, 3 Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA

Scent and volatiles are an interface driving interaction between plants and a large array of organisms such as bacteria, fungi, hervibores or pollinators. Floral scent is formed by combinations of Volatile Organic Compounds. We have used the Antirrhinum genus as a model to study the genetic structure and evolution of scent as it comprises over 25 species with a history of intercrossing and evolution back to a parental type. Analysis of floral scent in eight Antirrhinum wild species and two A. majus lines identified 130 compounds such as phenylpropanoids, benzenoids, mono- and sesquiterpenes, nitrogen-containing compounds and aliphatic alcohols. Using the volatile profiles we were able to construct the phylogenetic subsections of the genus. Cluster analysis showed that scent is probably selected as a combination of components in most cases but single pathways may also be a target of selection. Despite the complexity of the scent profiles, a cross of A. majus x A. linkianum differing in methyl benzoate, methyl cinnamate, acetophenone and ocimene showed Mendelian segregations of these volatiles. The null A. linkianum BENZOIC ACID CARBOXYMETHYL TRANSFERASE showed multiple polymorphisms in the 5’ promoter region including an IDLE MITE transposon insertion. The strong match between scent profiles and phylogeny, the complexity of blends based on combination of single genes coding for enzymes or regulatory elements coupled to transposon activity may allow rapid changes of scent profiles, which seem to be under strong evolutionary pressure.

This work is part of Project BFU-2013-45148-R. VRH is recipient of a FPU fellowship

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Poster 10 / SI P10

Castor plant (Ricinus communis): A biological model for production of unusual and added-valued industrial oil.

Alfonso Sánchez 1 , Enrique Martínez-Force 1 , Rafael Garcés 1 , E. Guzmán 2 , Noemí Ruíz- López 1 , Joaquín J. Salas

1 Department of biochemistry and molecular biology of plants products, Instituto de la Grasa (CSIC), Sevilla, Spain. 2 Department of vegetal Biology. Facutad de Ciencias.Universidad de Málaga.

Unusual fatty acids, such as epoxy and hydroxylated ones, have many uses as industrial feedstock for polymers, lubricants, and synthetic chemistry. Plants that accumulate these unusual fatty acids in their seed oils typically have poor agronomic performance. Castor plant (Ricinus communis) is an important non-edible oilseed crop widely cultivated in tropical-sub-tropical and temperate countries. Castor oil compromises up to 50-60% of the seed weight of this plant, which reaches productivities up to 3000 kg oil/ha. This oil contain high amount of ricinoleic acid because it has special enzymatic machinery that channel this fatty acid into TAGs. Therefore, castor plant looks like an ideal platform to produce and accumulate unusual fatty acids via genetic engineering. Unfortunately, in addition to large amounts of oil, castor seed also contain concentrated amounts of cytotoxic lectins, ricin and agglutinin, potent ribosome inactivating proteins that makes castor seeds and extracted meal highly toxic. In the present work we are studying unusual fatty acid accumulation (other than ricinoleic acid) by castor seeds. First of all we have designed interference RNA constructs to silence ricin gene expression and studied the metabolism of unusual fatty acid accumulation in this specie. In this regard, one of the main objectives is to modify the special TAG accumulation enzymatic machinery of castor plant by means of genetic transformation. At the present moment we are testing sonication-assisted Agrobacterium-mediated transformation as a transient and stable castor seed transformation method. Regeneration of transgenic explants is the main obstacle to develop transgenic castor plants due the low reproducibility of the methods previously reported. In the present work the settlement of a robust method of stable transformation and regeneration of castor will be pursued.

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Poster 11 / SI P11

Genome-wide description and functional characterization of SNF1- related kinases in Chlamydomonas reinhardtii.

Francisco J Colina 1 , Joana Amaral 2 , Gloria Pinto 2 , María Carbó 1 , María Jesús Cañal 1 , Luis Valledor 1

1 Plant Physiology, Department B.O.S., Faculty of Biology, University of Oviedo, Oviedo, Asturias, Spain. 2 Department of Biology and CESAM, University of Aveiro, Aveiro, Portugal.

The sustainability and long-term profitability of microalgae-based production of biomolecules relies on increasing biomass production while reducing the production costs.

A determinant proportion of these expenses is related to the process of stressing the

cultures, a needed step for triggering the accumulation of the biomolecules of interest. In

consequence, advancing in the characterization of stress-responsive mechanisms and regulatory pathways is paramount both for industry and stress-biology research. Among regulatory protein families, Sucrose non fermenting related kinases (SnRK) stands as a key stress-metabolism regulatory hub in plants and animals, controlling entire metabolic pathways related to energy deficit and abiotic stress response by interaction with bZIP, TOR and other factors (Baena-Gonzalez and Sheen 2008).

In Chlamydomonas the first description of this family, which is known to respond to nutrient

deprivation and abiotic stress, was reported considering 8 sequences (Gonzalez-Ballester

et al. 2008). In this work we completed the picture of this family employing genome-wide

characterization tools. Employing both known sequences in this species and in Arabidopsis (Coello et al. 2011) we searched for homology into the entire genome of Chlamydomonas. Afterwards we performed a search based on domain structure that confirmed the previous sequences identified by homology, provided new members of this family, and served as a filter for removing elements of the close family CDPK (Calcium- dependent protein kinases). Resulting sequences were grouped by domain content and sequence similarity through trees and Clustal alignments into SnRK1, SnRK1 regulatory subunits and SnRK2 subfamilies. Interestingly, sequences homologous to Arabidopsis SnRK3 subfamily were not found. All of the members of this family were functionally characterized by qPCR, evaluating its expression change over 9 different abiotic stresses (high and low temperature, S and N starvation, P and C limitation, salt, osmotic and UV stress). All of the members of this family were proved to be stress-responsive being some of them, like SnRK9 (expressed only under heat stress), markers of specific stress situation. This work increases our microalgae stress biology knowledge, opening new possibilities for biotechnological improvement of algae, focusing on the regulation of the members of this family, which are linked to the accumulation of biomolecules of interest.

References:

Coello P, et al. (2011). Journal of Experimental Botany 62: 883-893. Gonzalez-Baena and Sheen (2008). Trends in Plant Science 13: 474-482.

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Ponencia Invitada / SII PI

Building a root postembryonically: new factors integrate cell identity and auxin signaling

Miguel A. Moreno-Risueno + , Juan Perianez-Rodriguez + , Alvaro Sanchez-Corrionero + , Javier Silva-Navas ¤

+ Center for Plant Biotechnology and Genomics (CBGP) UPM - INIA. Department of Biotechnology. Universidad Politécnica de Madrid. Pozuelo de Alarcon (Madrid), Spain; ¤ CBGP UPM-INIA. Instituto Nacional de Investigaciones Agrarias, Madrid, Spain

Plants develop continuously through the formation and growth of organs. In roots, growth requires the activity of stem cells localized in a specific micro-environment known as the root stem cell niche. Stem cells generate tissue lineages and cell position is crucial for assignment of identity. After ablation, a cell in a specific location can be replaced by a cell generated from a contiguous tissue, and thus position has been assigned a predominant role in cell fate specification. Cell lineages were interpreted as the activity of positional signaling along the root longitudinal axis. In Arabidopsis thaliana, we have recently demonstrated that cells in the position of the ground tissue require ground tissue factors to interpret positional signaling; being the ground tissue lineage pre-set prior position 1 . Tissue specification emerges as a result of the combined activity of lineage determinants and positional signaling. We have also found that three of these factors: SCARECROW (SCR), JACKDOW (JKD) and BLUEJAY (BLJ) have a role in specification of the stem cell niche. Regulation of stem cell niche maintenance by these factors occurs through integration of the parallel pathways PLETHORA, downstream of auxin signaling, and SHORT-ROOT, through a non-autonomous mechanism. In blj jkd scr triple mutants QC cells are rapidly lost in the course of development. This phenomenon is accompanied by reduction in cell numbers within every tissue and correlates with poor replenishment of tissue stem cells by the QC. Eventually, blj jkd scr roots contain very few cells (~4-5) that differentiate. In blj jkd scr roots, formation of postembryonic roots such as lateral roots is also affected. Postembryonic organogenesis occurs through the specification of organ founder cells (FCs) and tissue stem cells 2 . Our data are consistent with the early establishment of a stem cell organizer, and its activity being required for the construction of the new organ. To gain further insight into this morphogenetic mechanism we have generated a number of plants carrying specific cell-type markers and we will profile the transcriptome of FCs and the newly formed cells with stem cell organizer properties. Out of a mutagenesis screen, we have identified a novel mutation with altered postembryonic organogenesis, which we named potent. In potent, many pericycle cells change their identity becoming FCs, which results in overproduction of lateral roots. Our results indicate that POTENT integrates auxin signaling with factors with oscillating expression, which are required to pre-pattern the sites that will give rise to new lateral roots. The outcome of this interaction is the specification of FCs at specific locations of the longitudinal axis following a temporal pattern. In addition, POTENT specifically regulates auxin signaling required for asymmetric divisions of FCs to generate tissue stem cells.

References:

1 Moreno-Risueno, MA, et al. (2015) Science, 350(6259):426-30. 2 Perianez-Rodriguez, J, et al. (2014) Front Plant Sci, 5:219.1-219.11

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Comunicación Oral 1 / SII CO1

Uncovering the role of Arabidopsis ORC1 during root organogenesis

Zaida Vergara, Joana Sequeira-Mendes and Crisanto Gutierrez

Department of Genome Dynamics and Function – DNA Replication, Chromatin and Cell Division Laboratory, Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.

Plant stem cells are organized into meristems that will actively divide throughout the lifespan of the plant to produce new organs. When cells stop proliferating they frequently enter the endocycle program, linked to the differentiation process. Reliable genome duplication is a key step towards maintaining genomic stability of meristematic and endoreplicating cells. DNA replication starts at discrete sites called origins of DNA replication (ORIs), marked by a set of proteins that form the pre-replication complex, namely, the heterohexamer origin recognition complex (ORC), CDC6, CDT1 and the helicase MCM. ORC1, the largest subunit of ORC, is one of the major components regulated in higher eukaryotes to assure a complete genome replication (Marks et al., 2016). Arabidopsis ORC1 is encoded by two genes, which were suggested to have different roles during development (Diaz-Trivino et al., 2005). The aim of our study is to understand the role of the two ORC1 genes from a genome replication and developmental perspective. To assess ORC1 dynamics during Arabidopsis root organogenesis, the native expression of both ORC1a and ORC1b proteins fused to a fluorescent tag was followed by live imaging and differential labelling of cells in S-phase, G2 or mitosis. While ORC1a is only present at the endoreplication domain, ORC1b appears in both proliferating and endocycling cells. The two are absent along the elongation and mature zones, except for the lateral root primordia (LRP), where ORC1b is detected as soon as LRP founder cells are activated for proliferation. We found that ORC1b is quickly degraded upon G1/S transition through the proteasome pathway. Experiments to identify the E3 ligase involved are in progress. A small fraction of the protein is already synthesized in G2 and remains bound to the chromatin throughout mitosis and in G1, the rest of ORC1b is slowly loaded onto chromatin. In the endocycle both proteins are degraded prior to DNA replication and synthesized again along the G-phase. Analysis of orc1 mutants reveals a delay in S-phase progression and also a failure to rescue dormant ORIs under replication stress conditions. These studies led us to propose that ORC1 is crucial for the selection of ORIs. ORC1 may have additional roles since the proteins are accumulated at chromocenters. We are using mutants compromised in chromocenter compaction to elucidate the role of ORC1 in heterochromatin dynamics.

References:

Marks, A et al. (2016) Curr. Opin. Genet. Dev. 37: 67-75. Diaz-Trivino, S et al. (2005) Nucleic Acids Res. 33(17): 5404-5414.

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Comunicación Oral 2 / SII CO2

Role of DESIGUAL1 and auxin in bilateral symmetry of Arabidopsis leaves

David Wilson-Sánchez, Sebastián Martínez-López, Sara Jover-Gil, José Luis Micol

Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Spain

Most living beings exhibit some form of symmetry; however, there is a dearth of mutations affecting bilateral symmetry in all biological systems. This lack of mutations has hampered genetic analysis of bilateral symmetry in multicellular organisms, particularly plants. To examine the regulation of symmetry and other facets of leaf development, we screened 19,850 Arabidopsis lines from the Salk homozygous T-DNA collection, and found 706 leaf mutants 1 . Only one of these mutants exhibited defects in bilateral symmetry; we named this mutant desigual1-1 (deal1-1). Arabidopsis has bilaterally symmetrical leaves with interspersed marginal lobes and indentations along the margin. Several overlapping regulatory pathways establish these marginal features; these pathways involve feedback loops of auxin, the PIN-FORMED1 (PIN1) auxin efflux carrier, and the CUP-SHAPED COTYLEDON2 (CUC2) transcriptional regulator 1,2 . The deal1 mutants have randomly asymmetric leaves that fail to acquire symmetry in the early stages of leaf primordium development, but instead form ectopic lobes and sinuses. In the leaves of deal1 mutants, improper regulation of cell division (simultaneous over- and under-proliferation) along the organ margins alters bilateral symmetry during the primordium stage. Auxin maxima are mislocalized at the margins of expanding deal1 leaves and this asymmetry can be enhanced by treatment with the polar auxin transport inhibitor 1-N-naphthylphthalamic acid or alleviated by treatment with the synthetic auxin 1- naphthaleneacetic acid. Among other defects, deal1 mutants show aberrant recruitment of marginal cells expressing properly polarized PIN1, resulting in misplaced auxin maxima. Normal PIN1 polarization requires CUC2 expression and CUC2 genetically interacts with DEAL1; DEAL1 also affects CUC2 expression in the leaf primordium margin. DEAL1, a protein of unknown molecular function, localizes to the endoplasmic reticulum membrane and functions in the leaf, acting partially redundantly with its two closest paralogs. DEAL1 also participates in flower development, revealing that this gene has diverse functions in plant morphogenesis.

References:

1.- Wilson-Sánchez et al. (2014). Plant Journal 79, 878-891. 2.- Bilsborough, G.D., et al. (2011). Proc Natl Acad Sci USA, 108, 3424-3429. 3.- Kasprzewska, A., et al. (2015). Plant Journal 83, 705-718.

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Comunicación Oral 3 / SII CO3

PROHIBITIN3 and NOA1 participate in the maintenance of the root stem cell niche in Arabidopsis thaliana

Tamara Lechón 1 , Noel Blanco-Touriñán 2 , Virginia Palomares 1 , Miguel A. Blázquez 2 , Ivett Bárány 3 , Miguel A. Moreno-Risueño 4 , Mari C. Risueño 3 , Pilar S. Testillano 3 , Óscar Lorenzo 1 , Luis Sanz 1

1 Dpto. Botánica y Fisiología Vegetal, Instituto Hispano-Luso de Investigaciones Agrarias (CIALE) – Universidad de Salamanca (USAL), Salamanca, Spain, 2 Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC) – Universidad Politécnica de Valencia (UPV), Valencia, Spain, 3 Centro de Investigaciones Biológicas (CIB), Madrid, Spain, 4 Centro de Biotecnología y Genómica de Plantas (CBGP), Universidad Politécnica de Madrid, Madrid, Spain.

Compelling evidence demonstrates a key role of nitric oxide (NO) on primary root growth

in Arabidopsis, suggesting a link between NO and auxin signalling pathways 1,2 . Prohibitins

are a group of highly conserved proteins across different taxa that form multimeric complexes in the inner mitochondrial membrane. They have been linked to regulation of

NO homeostasis, cell cycle, protein folding and mitochondrial function 3,4 . We now attempt

to further understand the role of additional NO molecular players in the stem cell niche. To

this end, we have characterized the role of PHB3 on root development and stem cell niche homeostasis through its functional interaction with AtNOA1, since both have been shown

to physically interact in mice 5 .

A transcriptomic metanalysis revealed that 26% of genes misregulated in phb3 are also

misregulated in the same direction in noa1, and many of them are involved in root development. The double phb3 noa1 mutant shows inhibited primary root elongation, abnormal cell divisions and differentiation in the root meristem and severe alterations in the root cap. Ultrastructural analyses in this area showed that mitochondria have less cristae and are less dense to electrons, a morphology similar to those of mitochondria present in the stem cell niche. These changes correlate with a progressive expansion in WOX5 expression pattern in phb3 which is further exacerbated in phb3 noa1. Taken together, these results suggest that PHB3 and NOA1 could represent a means to maintain root growth patterns through a mechanism involving mitochondrial retrograde signaling upon WOX5 expression, although further analyses are necessary to test this possibility.

This work is financed by grants: ERC.KBBE.2012.1.1-01 (EcoSeed-311840), MINECO: (BIO2014-57107- R), CONSOLIDER (CSD2007-00057), Junta de Castilla y León (SA239U13).

References:

1 Fernández-Marcos M, et al. (2011) Proc Natl Acad Sci USA, 108(45): 18506-11. 2 Sanz L, et al. (2014) Plant Physiol, 166(4): 1972-84. 3 Van Aken O, et al. (2007) Plant J, 52(5): 850-64. 4 Wang Y, et al. (2010) Plant Cell, 22(1): 249-59. 5 Heidler J, et al. (2011) J Biol Chem, 286(37): 32086-93.

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Comunicación Oral 4 / SII CO4

Regulation of developmental timing by TEMPRANILLO through the age-dependent pathway

Andrea E. Aguilar Jaramillo 1 , Esther Marín González 1 , Luis Matías Hernández 1 , Soraya Pelaz 1,2 and Paula Suárez López 1

1 Centre de Recerca en Agrigenòmica, CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra (Cerdanyola del Vallés), Spain, 2 ICREA (Institució Catalana de Recerca i Estudis Avançats), Barcelona, Spain

During their life cycle, plants undergo several developmental transitions. The timing of these transitions is essential for proper development and adjustment of growth to environmental conditions. In Arabidopsis thaliana, the microRNA 156 (miR156) controls plant developmental timing by negatively regulating several SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, which promote the juvenile-to-adult and the floral transition in part through up-regulation of miR172. This developmental pathway is known as the age-dependent pathway. TEMPRANILLO1 (TEM1) and TEM2 are transcriptional repressors that delay flowering. TEM and miR156 levels are high early in development and then decrease gradually, allowing progression from one developmental phase to another. Given the similarities in expression patterns and functions, we hypothesized that TEMs and miR156 may act through a common genetic pathway. We have found that the miR156/SPL module affects mainly the juvenile-to-adult transition, with a smaller effect on the floral transition. Conversely, TEMs play a minor role in the juvenile-to-adult transition and a major role in the floral transition. TEMs have a small effect on the levels of miR156 and regulate the abundance of several SPL mRNAs and miR172. TEM1 binds to SPL9 chromatin, suggesting that TEM1 regulates SPL9 through both transcriptional repression and miR156-mediated post-transcriptional control. TEM1 also binds to MIR172 chromatin, suggesting that the regulation of miR172 is both direct and mediated by SPL9. Genetic analyses show that TEM2 affects the juvenile-to-adult and the floral transition through miR156-dependent and independent pathways, consistent with the miR156-dependent and independent regulation of SPL9 and miR172 by TEMs. We are currently analysing the genetic interactions between TEMs, SPL9 and miR172. Overall our results indicate that TEMs regulate the timing of the juvenile-to-adult and floral transitions through the age- dependent developmental pathway.

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Poster 01 / SII P01

Genetic dissection of adventitious root formation in tomato hypocotyls after wounding

Aurora Alaguero 1 , Joan Villanova 1 , Ana Belén Sánchez-García 1 , Antonio Cano 2 , Manuel Acosta 2 , José Manuel Pérez-Pérez 1

1 Instituto de Bioingeniería, Universidad Miguel Hernández, Avda. de la Universidad s/n, 03202 Elche. 2 Departamento de Biología Vegetal (Fisiología Vegetal), Universidad de Murcia, 30100 Murcia

Adventitious roots (ARs) are formed from non-root tissues, such as stems or leaves, in response to some stresses (i.e. flooding) or after wounding (Pacurar et al. 2014). Tomato is an attractive model to study the genetic basis of de novo adventitious organ formation, since there is a considerable natural genetic variation for this trait among wild relatives (Arikita et al. 2013). To identify the genetic determinants contributing to AR formation in this species, we are using young hypocotyl explants of S. lycopersicum cv. Micro-Tom grown in vitro. Our results indicate that active polar auxin transport through the hypocotyl leads to a localized auxin gradient required for AR formation in the hypocotyl base. Quantitative histology allowed us to define the cellular dynamics during the early stages of AR initiation. Gene expression profiling at different phases of AR formation have been analysed. AR formation has been studied on a number of tomato mutants affected in hormonal signalling and a model for wound-induced organ regeneration from hypocotyl explants in this species will be presented. The identification of the genetic networks involved in AR formation will contribute to our basic understanding of the molecular events leading to this complex developmental response.

Work funded by MINECO/FEDER (AGL2012-33610 and BIO2015-64255)

References:

Arikita FN, et al. (2013). Plant Sci., 199-200: 121-130 Pacurar DI, et al. (2014) Physiol. Plant., 151: 83-96

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Poster 02 / SII P02

Elucidating the interaction networks at work in the methyladenosine epitranscriptome

Eva Rodríguez-Alcocer, Natalia Gómez-Peral, Carlos Hernández-Cortés, Sara Jover-Gil, Héctor Candela

Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Spain

Recent research on the reversible methylation of adenosine residues at their N 6 position, i.e. the most abundant internal modification present in the messenger RNA (mRNA) of eukaryotes, has led to the establishment of an entirely new field of study called epitranscriptomics. Despite this post-transcriptional modification has been known for about 4 decades, the study of adenosine methylation has emerged as a hot research topic only in the past three years, coinciding with the implementation phase of our BFU2012-31719 grant, placing us in a privileged position to address frontier research questions in this field using Arabidopsis thaliana as a model organism. Some key advances in this field have been: (i) the characterization of the multisubunit complex that methylates adenosine residues (“writer” proteins), (ii) the identification of enzymes with demethylase activity, such as the one encoded by the FTO gene, whose polymorphisms have been associated to obesity in humans (“eraser” proteins), and (iii) the identification of the YTH domain as the N 6 -methyladenosine (m6A) binding domain of some RNA binding proteins (“reader” proteins). As a continuation of our former project, we have already performed several yeast two-hybrid screens using proteins of the three functional categories (writers, erasers and readers) as baits, which have yielded some promising protein-protein interactions. We have also implemented a novel, high- throughput RNA tagging protocol that should allow us to identify specific RNA molecules targeted by proteins from the three functional categories. Our ultimate goal 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 was initiated with funds from Spain's Ministry of Economy and Competitiveness and the European Regional Development Fund (ERDF) (‘Una manera de hacer Europa') [BFU2012-31719 grant to H.C.]

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Poster 03 / SII P03

Central components of the DNA replication machinery interact with chromatin remodelling complexes to mediate epigenetic gene silencing

Iván del Olmo, Manuel Piñeiro and José A. Jarillo

Centro de Biotecnología y Genómica de Plantas (CBGP). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria – Universidad Politécnica de Madrid (INIA-UPM). Campus de Montegancedo, Pozuelo de Alarcón 28223 Madrid

In eukaryotic organisms chromatin is duplicated during cell division to ensure faithful transmission of both genetic and epigenetic information, maintaining in the daughter cells the memory of the chromatin status of their progenitors. The epigenetic inheritance during DNA replication is crucial to maintain cellular identity following cell division. The role of POLYCOMB REPRESSIVE COMPLEXES 1 and 2 (PRC1 and PRC2) is essential for the temporal regulation of gene repression involved in different developmental processes, but how these complexes may interact with the DNA replication machinery to contribute to the mitotic inheritance of cellular identity in the daughter cells remains unknown. The Arabidopsis ESD7 locus encodes the catalytic subunit of the DNA Polymerase (Pol) ε complex that is involved in the synthesis of the DNA leading strand and is essential for embryo viability. The esd7-1 mutant is a viable hypomorphic allele but displays a number of pleiotropic alterations including an acceleration of flowering time. Furthermore, Pol ε is involved in the epigenetic silencing of the floral integrator genes FT and SOC1, but the molecular nature of the transcriptional gene silencing mechanisms involved remains elusive. We have revealed that ESD7 interacts with components of PRC2 such as CURLY LEAF, EMBRYONIC FLOWER 2 and MULTICOPY SUPPRESSOR OF IRA 1, and that mutations in ESD7 cause a decrease in the levels of the H3K27me3 mark present in the chromatin of FT and SOC1 [1]. We have also demonstrated that a domain of the C-terminal region of the DNA polymerase ε catalytic subunit mediates the binding to the different PRC2 components. In addition, we have showed that this interaction with ESD7 is necessary for the proper recruitment of PRC2 to FT and SOC1 chromatin. Using the well characterized flowering time regulatory network we have unveiled the existence of interplay between the DNA replication machinery and the Polycomb Group chromatin remodelling complexes in epigenetic transcriptional silencing. Altogether these observations provide an insight into the mechanisms ensuring that the epigenetic code at pivotal loci in developmental control is faithfully transmitted to the progeny of eukaryotic cells and might help to explain how these complexes preserve chromatin modification states during DNA replication.

References:

[1] Del Olmo et al. 2016. Nucleic Acids Research DOI: 10.1093/nar/gkw156.

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Poster 04 / SII P04

Differential gene expression by RNA sequencing between one- and two-dimensional apogamous gametophytes of Dryopteris affinis ssp. affinis

Stefan Wyder 2 , Helena Fernández 1 , Ana Elisa Valdés 3 , María Jesús Cañal , Valeria Gagliardini 2 , Alejandro Rivera 1 , Ueli Grossniklauss 2

1 Area of Plant Physiology, Dept. BOS, University of Oviedo, Spain. 2 Institute of Plant Biology & Zurich-Basel Plant Science Center, University of Zurich, Switzerland, 3 Physiological Botany, Uppsala BioCenter, Uppsala University, Sweden

Performing proteomic studies on non-model organisms with little or no genomic information is still difficult. However, many specific processes and biochemical pathways occur only in species that are poorly characterized at the genomic level. For example, many plants can reproduce both sexually and asexually, the first one allowing the generation of new genotypes and the latter their fixation. Thus, both modes of reproduction are of great agronomic value. However, the molecular basis of asexual reproduction is not understood in any plant. In ferns, it combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells (apogamy). Processes such as apogamy and apomixis share molecular aspects, although their genetic landscape remains undeciphered. Hence, the study of the apogamy commitment in naturally apogamous species, such as ferns, might shed some light for the understanding of the apomixis process in angiosperms. In this study an RNAseq approach was used to disentangle dynamic changes in gene expression leading to the development of an apogamous gametophyte by comparing one- and two-dimensional gametophytes of the apogamous fern Dryopteris affinis ssp. affinis. Our data show a total of 10679 genes differentially expressed between filamentous and prothallial architectures. Ca. 6110 genes were up-regulated and 4570 were down-regulated in two-dimensional relative to one- dimensional gametophytes. Regulation of meristem growth, auxin signaling, reproduction and sucrose metabolism are biological functions enriched in the two-dimensional gametophytes, while response to stimulus, and defense are overrepresented in the filamentous gametophytes. In addition, protein domain annotations related to epigenetic regulation and ubiquitin degradation were emphasized. Our results supply a reference dataset for the free-living gametophyte development, focusing on filamentous-to-prothallus transition requirements, and provide a rich apogamous library, useful for further investigation on embryo development by asexual means.

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Poster 05 / SII P05

MYB36 modulates redox balance and defines boundaries of arabidopsis lateral roots

María Fernández-Marcos 1 , Bénédicte Desvoyes 1 , Concepción Manzano 2 , Louisa M. Liberman 3 , Philip N. Benfey 3 , Juan C. del Pozo 2 , Crisanto Gutierrez 1

1 Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain, 2 Centro de Biotecnología y Genómica de Plantas, INIA, Madrid, Spain, 3 Howard Hughes Medical Institute and Department of Biology, Duke University, Durham, USA.

Root branching in plants relies on the continuous de novo formation of lateral roots (LR). LRs initiate from founder cells that generate lateral root primordia (LRP) by formative divisions and eventually emerge from the primary root (PR). To identify novel components controlling root architecture we screened the TRANSPLANTA collection of Arabidopsis lines expressing individual transcription factors (Coego et al., 2014). Here we focus on MYB36, recently known to participate in the Casparian strip formation and the proliferation to differentiation transition (Kamiya et al., 2015; Liberman et al., 2015). We found that myb36 mutants had a delayed and reduced LR emergence. MYB36 is expressed from stage V of LR development, and is restricted to the LRP boundary (LRPB) cells that surround LRP. Quantification of developmental stages in the mutants indicates an over-abundance of stage IV LRP, revealing a defect in the transition from flat- to dome- shaped LRP. This is when the growing LRP emerges through the outer cell layers, concomitant with the cessation of cell divisions in the pericycle. Accordingly the myb36 mutants contain more cells at the base of the LRP, suggesting that MYB36 in the LRPB cells control the LRP width. We also found that PER9 and PER64 were drastically reduced in the LRP of myb36 mutants. Importantly, reducing the H 2 O 2 levels in myb36 mutants significantly reverts the mutant LR phenotype. Our data are consistent with a role of MYB36 at stages V-VI of LRP development by affecting the balance of reactive oxygen species that contribute to setting the outer boundary of the growing LRP and the transition from a flat to dome LRP.

References:

Coego A, et al. (2014) Plant J, 77 (6): 944-953. Kamiya T, et al. (2015) PNAS, 112(33): 10533–10538. Liberman L M, et al. (2015) PNAS, 112(39): 12099-12104.

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Poster 06 / SII P06

Understanding the function of the YTH-domain proteins of Arabidopsis thaliana

Natalia Gómez-Peral, Eva Rodríguez-Alcocer, Erundina Ruiz, Sara Jover-Gil, Héctor Candela

Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Spain

Recent studies have shown that the YTH domain is an RNA binding domain that mediates the interaction with N 6 -methyladenosine (m6A) residues present in messenger RNA molecules. The genome of Arabidopsis thaliana has previously been reported to contain as many as thirteen RNA binding proteins containing a YTH domain (Li et al., 2014), but the function of these proteins remains largely 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.

As a first step, we are characterizing transgenic lines carrying T-DNA insertions residing at or near the coding regions of these genes. Our preliminary results show that most of the examined lines lack a mutant phenotype on their own, suggesting extensive functional redundancy among the members of this protein family. To overcome this problem, we have initiated the isolation of double mutants involving loss-of-function alleles of the phylogenetically closest gene pairs. In addition to this, we have prepared Gateway- compatible constructs containing the full-length cDNAs (with and without stop codons) as well as the promoter regions of most of these genes. These constructs are systematically being transferred to Arabidopsis plants and should inform us on the consequences of overexpressing the YTH genes of Arabidopsis thaliana and their normal expression patterns.

This work received support from Spain's Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund (ERDF) (‘Una manera de hacer Europa') [BFU2012-31719 grant to H.C.].

References:

Li D, et al. (2014). Plant Mol. Biol. Rep. 32: 1169-1186

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Poster 07 / SII P07

Unveiling the impact of PRC1 function on PRC2 mediated H3K27me3 marking in Arabidopsis

Ángeles Gómez-Zambrano 2 , Yue Zhou 1 , Francisco Romero-Campero 2 , Wiam Merini 2 , , Franziska Turck 1 , Myriam Calonje 2

1 Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Köln, Germany. 2 Institute of PlantBiochemistry and Photosynthesis, Instituto de Bioquímica Vegetal y Fotosíntesis-Consejo Superior de Investigaciones Científicas-University of Seville, Isla de La Cartuja, 41092 Seville, Spain

Polycomb group (PcG)-mediated repression constitutes a major epigenetic mechanism for controlling gene expression throughout the plant life. However, the mechanism by which the PcG machinery mediates gene repression is still largely unknown in plants. As far as it is known, PcG proteins associate in two multi-protein complexes in Arabidopsis:

Polycomb Repressive Complex 1 (PRC1) and PRC2 that mediate Histone 2A monoubiquitination and Histone 3 Lysine 27 trimethylation, respectively (Förderer et al., 2016; Merini and Calonje, 2015). Interestingly, recent data indicated that the binding and activity of PRC1 is required for H3K27me3 marking at some target genes (Yang et al., 2013; Merini and Calonje 2015), which challenges the classical hierarchical model for recruitment of PcG complexes; however, it is not known to which extent this is a general mechanism.To investigate this, we analyzed the localization of H3K27me3 marks by ChIP- seq in wild type Col and the strong PRC1 mutant atbmi1a/b/c at different stages of seedling development. Our data confirmed a requirement of PRC1 for H3K27me3 marking of a subset of targets, but also unveiled that the loss of PRC1 function may affect the maintenance of H3K27me3 marks at a different subset of genes, as suggested the progressive loss of H3K27me3 marks detected at these genes.

References:

Merini W. and Calonje M. (2015). Plant J, 83: 110–120. Yang C., et al (2013). Curr. Biol. 23, 1324–1329. Förderer A., et al(2016). CurrOpin Plant Biol.29:169-78

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Poster 08 / SII P08

Gibberellins, a new player in the determination of ovule initiation

Maria Dolores Gomez, Esther Carrera, Isabel Lopez-Diaz, Miguel A Perez-Amador

Instituto Biiología Molecular y Celular de Plantas (IBMCP), CSIC-UPV, Valencia, Spain

The formation of ovules and seeds is an essential process in the life cycle of plants as it ensures proper reproduction, and has great economic importance by its direct impact on crop yields. Several key components of the genetic and hormonal network controlling the initiation and development of ovule primordia are known. So far, the GAs have not been involved in this process, despite being implicated in a plethora of developmental processes. Results from our group suggest that GAs have a key role in controlling the development of ovules of Arabidopsis and tomato. The DELLA activity favors the initiation of ovules while its absence results in a decreased number of ovules and altered morphology. In Arabidopsis, null mutants of the GA receptor GID1s or dominant mutations of DELLA proteins result in an increase in ovule number. Conversely, null DELLA mutants reduce the number of ovules and alter the ovule and seed shape. Genetic analysis of multiple null mutant combinations of the five DELLAs of Arabidopsis revealed that the DELLAs GAI, RGA and RGL2 have a function in ovule initiation, being RGL1 and RGL3 not involved. In contrast, ovule development is controlled by GAI, RGA RGL1 and RGL2, by a molecular mechanism that relies in the interaction of DELLAs with the transcriptional factor ATS. In tomato, inhibition of GA synthesis with PCB or the null DELLA mutation procera increase or decrease ovule number, respectively. Recent data on the interaction of GAs and other hormones, mainly brassinosteroids and cytokinins are presented.

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Poster 09 / SII P09

A pollen caleosin with peroxygenase activity is critical for fertilization

María José Jiménez-Quesada 1 , Krzysztof Zienkiewicz 1 , José Feijó 2 , Agnieszka Zienkiewicz 1 , Juan de Dios Alché 1 , Antonio Jesús Castro 1

1 Plant Reproductive Biology Laboratory, Estación Experimental del Zaidín (CSIC), Granada, Spain, 2 Instituto Gulbenkian de Ciência, Oeiras, Portugal.

Caleosins are lipid body intrinsic proteins involved in a plethora of functions including storage lipid mobilization during seed germination and defence against biotic and abiotic stress. Yet, the biological role of pollen caleosins remains unknown. In this work, a gene encoding a 239-aa caleosin with a predicted molecular mass of 26.67 kDa was functionally characterized in Lilium longiflorum (Eastern lily). Expression analysis showed that the caleosin gene is transcribed not only in pollen but also in the vegetative tissues. During pollen germination, both mRNA and protein levels gradually decreased, indicating that there was neither net gene transcription nor net protein synthesis. Fluorescent immunolabeling using an anti-caleosin antibody combined with simultaneous Nile red staining of neutral lipids showed a good co-localization of lily pollen caleosin with lipid bodies in elongating pollen tubes. At ultrastructural level, gold labeling mainly appeared attached to the surface of lipid bodies randomly distributed in the pollen tube cytoplasm or in the surrounding RER membranes. The plasma membrane and vacuoles containing membrane-like structures were immunostained, whereas the vegetative nucleus and other cytoplasmic organelles showed no labelling. Small secretion vesicles at the clear zone also displayed a significant immunolabelling. To further characterize the protein at molecular level, a fusion recombinant caleosin was produced in Escherichia coli. In Western blot experiments, the specific anti-caleosin antibody was able to bind to the ~40 kDa fusion protein. Under non-reducing conditions, the antibody also detected a second band with an apparent molecular weight of ~80 kDa. We further confirmed the identity of the antibody-bound monomeric and dimeric proteins by immunoprecipitation and MS analysis. The recombinant caleosin was also capable to bind calcium ions in vitro. Purified lipid bodies isolated from lily pollen tubes were able to perform hydroxylation of aniline, a cooxidation reaction known to be catalyzed by peroxygenases. The recombinant caleosin, either alone or on reconstituted artificial lipid bodies also catalysed cooxidation of aniline, thus probing that the lily pollen caleosin is a peroxygenase. The study of the biological function of the pollen caleosin was achieved by microinjecting the anti-caleosin antibody in growing pollen tubes. Antibody microinjection causes cytoplasmic streaming cessation and the loss of the clear zone at the pollen tube tip, leading to permanent apical growth arrest within a few min after loading. In parallel, oil body mobilization was blocked in injected pollen tubes, leading to characteristic accumulation patterns. These data suggest that the pollen caleosin is a key regulator of pollen tube tip growth and, consequently, is critical for pollen to achieve successful fertilization.

This work was supported by ERDF-cofinanced grants AGL2013-43042-P (MICINN) and P10-CVI-5767 (Junta de Andalucía).

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Poster 10 / SII P10

Gibberellin signaling in the endodermis modulates the hypocotyl gravitropic response

Rodrigo Marí-Ordóñez 1 , Alberto Fuster 1 , Jana Crespo-Trives 1 , David Alabadí 1 , Miguel Ángel Blázquez 1 , Eugenio G. Minguet 1

1 Laboratory2.07, Instituto de Biología Molecular y Celular de Plantas, CSIC-UPV, Valencia, Spain.

Plants, as sessile organisms, adapt their growth in response to environmental changes to optimize their survival. Gravitropism is one of these mechanisms allowing optimal orientation of aerial and radicular growth in the direction of gravity vector. Starch-loaded amyloplasts have been shown to be an integral part of the mechanism that allows gravity perception (Kiss et al., 1989; Boonsirichai et al., 2003; Hashiguchi et al., 2013; Sato et al., 2015). Cells accumulating amyloplasts are located in the tip of the roots and in the endodermis of aerial tissues, such as the hypocotyl. Gravistimulation leads to the generation of an auxin gradient that causes differential growth, and the extent and response to this gradient depend in turn on the presence of other environmental cues, such as light. It has been proposed that gibberellins (GAs) attenuate auxin response to provide flexibility in situations under which plants face competing tropic signals (Gallego- Bartolome et al., 2011). A critical issue in the control of the gravitropic response is the spatial localization of the machinery that perceives gravity and directs reorientation. The presence of amyloplasts in the endodermis suggest a critical role of this tissue in gravity perception but it raises the question whether the coordination is also commanded from endodermis in the hypocotyl. We have addressed this issue by blocking GA signaling in different cell types and examining the reorientation capacity of hypocotyls subject to gravistimulation. The accumulation of the DELLA proteins, negative regulators of GA signalling, either by treatment with the inhibitor of GAs synthesis paclobutrazol (PAC) or in the GAs insensitive dominant gai-1D mutant, enhance the gravitropic response (Gallego-Bartolome et al., 2011). We have used several cell-type specific promoters for expressing gai-1D in Arabidopsis thaliana: pML1 (epidermis), pRbcS (green tissue, not epidermis, specially induced by light), pSUC2 (phloem companion cells) or pSCR (endodermis). After gravistimulation by 90 degrees rotation with respect to the gravity vector, 3 day-old seedlings of gai-1D reorient faster than wild type. This behavior was only phenocopied when gai-1D was expressed in the endodermis. These results suggest that the endodermis is not only the main aerial tissue responsible for gravity perception but it is also the main tissue involved in the integration of other signaling cues that modulate the gravitropic response.

References:

Boonsirichai K, et al. (2003). Plant Cell 15: 2612-2625. Gallego-Bartolome J, et al. (2011) Plant Physiol 156: 1819-1825. Hashiguchi Y, et al. (2013) Am J Bot 100: 91-100. Kiss JZ, et al. (1989) Planta 177: 198-206 Sato EM, et al. (2015) J Exp Bot 66: 2155-2165

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Poster 11 / SII P11

Arabidopsis CUPULIFORMIS genes are new players on the chromatin remodeling scene

Eduardo Mateo-Bonmatí, Lucía Juan-Vicente, José Luis Micol

Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Spain

We conducted forward and reverse genetic screens for Arabidopsis mutants with abnormally shaped or sized leaves. In these screens, gene-morphology relationships among mutants were reproducible and in not few cases predictable: mutations classified together based on morphological phenotype actually affect genes involved in a single pathway or molecular mechanism 1,2 . One of the most represented phenotypic classes was that of incurvata (icu) mutants, with incurved, hyponastic leaves. Several icu mutants had defects in chromatin remodeling, an essential process for all eukaryotes that impacts growth and development. We are studying a family of five Arabidopsis proteins that present the PF03171 domain, with putative 2-oxoglutarate/Fe 2+ -dependent dioxygenase activity. We dubbed CP this gene family because its founding member, ICU11, was identified in the icu11-1 mutant, which was initially named cp (cupuliformis). The effects of loss of ICU11 function on the morphological and molecular phenotypes are similar to those of two other genes with epigenetic activity that we previously studied, CLF (CURLY LEAF 3 , we initially named this gene ICU1) and ICU2 (INCURVATA2) 4 , with which ICU11 synergistically interacts. CLF is a component of the Polycomb Repressive Complex 2, which functions as an H3K27me3 histone methyltransferase. ICU2 is the catalytic subunit of DNA polymerase alpha, and plays a role in the maintenance of repressive epigenetic marks. The CP family includes redundant and essential genes in Arabidopsis, as shown by lethality of the icu11 cp2 and cp3 cp4 double mutants. In addition, we found the ICU11 and CP2 proteins solely localized at the cell nucleus. Hundreds of genes were found up- regulated in a RNA-seq analysis of icu11-1 leaves, including members of the MADS-box family. Double mutants combining icu11 alleles with alleles of genes known to participate in chromatin remodeling exhibit synergistic phenotypes. The leaf phenotype of the icu11- 1 mutant is caused by over-expression of the SEPALLATA3 (SEP3) MADS-box gene; the phenotype is suppressed by a microRNA designed against SEP3 mRNA. Chromatin immunoprecipitation assays revealed altered patterns of H3K27me3 deposition in SEP3. Taken together, our results indicate that ICU11 and other CP genes are new players on the chromatin remodeling scene.

References:

1.- Pérez-Pérez, J.M et al (2011). Plant, Cell and Environment 34, 2200-2211. 2.- Wilson-Sánchez, D., et al (2014). Plant Journal 79, 878-891. 3.- Goodrich, J., et al. (1997). Nature 386: 44–51. 4.- Barrero, J.M., et al (2007). Plant Cell 19, 2822-2838.

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Poster 12 / SII P12

Role of two putative histone lysine methyltransferases during Arabidopsis organogenesis

Carla-Dianela Méndez, Joana Sequeira-Mendes, Crisanto Gutiérrez

Department of Genome Dynamics and Function, Laboratory of DNA replication, chromatin and cell division, Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain

There are 40 proteins in Arabidopsis that contain a SET domain, a conserved amino acid region that confers methyltransferase activity at lysine (K) residues. ASHH3 (ASH1 HOMOLOG 3) and ASHH4 belong to a group of proteins closely related to Drosophila and yeast SET domain containing histone lysine methyltransferases (KMTases). Our work is aimed at deciphering the molecular mechanisms by which these ASHH family members act during Arabidopsis development. Native expression of ASHH3 and ASHH4 fused to different reporter genes (GUS, eCFP and mRFP) has allowed us to carry out localization studies in a variety of tissues. In the case ASHH3, its localization is restricted to the root apical meristem (RAM), lateral root primordia (LRP), shoot apical meristem (SAM), leaves, gynoecium and embryo development. ASHH4 has two major splice variants, ASHH4-s and ASHH4-l (shorter and longer peptides) that differ in their expression pattern; ASHH4-s localizes to the RAM, LRP, trichomes, stamens and ovules while ASHH4-l is confined to stamens. Phenotypic studies of knockout ashh3-1 and ashh4-2 mutants revealed a smaller root area coverage in 10 day-old seedlings. Experiments to determine effects during cell cycle progression and cell cycle gene expression are in progress and the developmental defects of ashh3-1 and ashh4-2 mutants. Based on the nuclear localization of both proteins and the presence of a SET domain we investigated whether they interact with histones and possess KMTase activity in vitro. Combinatorial peptide binding assays (CelluSpots tm MODified Histone Array, Active Motif) revealed that purified ASHH3-His6 preferentially binds to H3K9me2 and H3K27me1 in a context where H3R8/R17/R26 residues are dimethylated and histones H2A, H2B and H4 are acetylated. Purified ASHH4s-His6 showed preferential interaction with H4K20me1/me2 combined with the recognition of H3K27me2, me3 marks. Further binding and KMTase assays are under way to link the activity of ASHH3 and ASHH4 on the chromatin landscape with the specific spatial and temporal expression patterns.

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Poster 13 / SII P13

Functional analysis of stomatal b-HLHs from crop species in arabidopsis

Alfonso Ortega, Alberto de Marcos, Mar Martín, Carmen Fenoll and Montaña Mena

Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-la Mancha, Toledo 45071, Spain

The gene networks controlling the development of stomata from protodermal cells in aerial

organs have been studied mostly in Arabidopsis. In this model, the process is regulated by and interplay of positive, stomata-promoting factors, and negative regulators that inhibit stomatal fate in those cells in contact with stomata or stomata precursors. The activity of all these factors determine how abundant are stomata in a mature organ, a parameter known to influence the maximum stomatal pore area available for gas exchange between the plant and the atmosphere and, therefore plant performance under different growth conditions. For instance, higher stomatal abundance is related to higher transpiration and photosynthesis, improving cooling and productivity under heat in irrigated crops; lower stomatal abundance, in contrast, optimizes water use efficiency during water shortage. In crops, alleles for these regulators presenting a modified activity have an interesting potential to modify stomatal numbers and thus potentials for photosynthesis and transpiration. The key positive regulators of stomata development are three related bHLH-type transcription factors (SPCH, MUTE and FAMA). The evolutionary footprint of the three Arabidopsis proteins has been tracked from Arabidopsis to mosses, and the partial conservation of their functions has been determined for the Physcomitrella patens and Oryza sativa putative orthologs. However, information on the genes that determine stomatal development and thence stomatal abundance and the related physiological traits

in crop species is lacking. Using the SGN network (http://www.solgenomics.net) and the

PLAZA database, we have identified the putative orthologues of the three Arabidopsis bHLH-coding genes in Solanum lycopersicum. We obtained the full length cDNAs for the SPCH, MUTE and FAMA putative orthologues from developing tomato cotyledons, and cloned them under the control of the corresponding Arabidopsis promoters as such or as C-terminal translational fusions to GFP. The constructs were mobilized to Arabidopsis plants carrying loss-of-function mutations in each of the three genes, double homozygous plants (for the transgene and the mutation) were identified and their phenotypes and accumulation pattern of the GFP fusions examined. We will show that the tomato genes can complement the loss of function of Arabidopsis SPCH and MUTE and present data regarding the two FAMA putative orthologues found. The cDNAs were also cloned in in a

system for β-estradiol inducible over-expression, and mobilized to Arabidopsis to determine the phenotypes resulting from their overexpression.

A similar approach has been started for Vitis vinifera. Since in Arabidopsis alleles with

partial loss of function show reduced stomatal abundance, finding the tomato orthologues for these positive stomatal development regulators is the first step towards Identifying mutant alleles for these genes (by TILLING or eco-TILLING) or designing specific variants with altered properties conferring beneficial physiological traits will contribute to crop

breeding for future climate scenarios. Work was funded by grants AGL2015-65053-R, BIO2012- 33952 and PPII10-0194-4164. AA was supported by a predoctoral grant from JCCM

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Poster 14 / SII P14

The MEDIATOR COMPLEX SUBUNIT 18 (MED18) encoded by the tomato POLLEN DEFICIENT1 (POD1) gene is essential for pollen ontogeny

Fernando Pérez-Martín 1 , Fernando J. Yuste-Lisbona 1 , Benito Pineda 2 , Begoña García- Sogo 2 , Juan F. Campos 3 , Estela Giménez 1 , Teresa Antón 2 , Iván del Olmo 4 , Manuel A. Piñeiro 4 , José A. Jarillo 4 , M. Carmen Bolarin 3 , Vicente Moreno 2 , Trinidad Angosto 1 , Juan Capel 1 , Rafael Lozano 1

1 Centro de Investigación en Biotecnología Agroalimentaria (BITAL). Universidad de Almería. 04120 Almería, Spain; 2 Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia. 46022 Valencia, Spain; 3 Centro de Edafología y Biología Aplicada del Segura-CSIC. 30100 Murcia, Spain; 4 Centro de Biotecnología y Genómica de Plantas, Instituto Nacional de Investigaciones Agrarias - Universidad Politécnica de Madrid. 28223 Madrid, Spain.

Pollen development and maturation depend on a coordinated spatio-temporal regulation of gene expression, which takes place at early stages of reproductive development. A suitable pollen formation is required not only for biological diversity maintenance but also for fruits and seed production in agronomical important crop species. Furthermore, in fleshy fruit plants like tomato (Solanum lycopersicum L.), defects in pollen ontogeny produces parthenocarpic (seedless) fruits, which are considered to be of great importance since they have a high commercial value. In this study, we described the tomato enhancer trap T-DNA mutant pollen deficient1 (pod1) that displayed abnormalities in pollen development, which leads to production of parthenocarpic fruits. Detailed histological study of anther development displayed that microspores were degenerated at the tetrad stage but tapetum development was not affected. Cloning of flanking sequences at T-DNA integration site showed that a single T-DNA copy was located in an intergenic region of chromosome 6 between ZINC FINGER HIT-type (ZF-HIT) and MEDIATOR COMPLEX SUBUNIT 18 (MED18) genes. Expression analysis and characterization of silencing lines revealed that the pod1 mutant phenotype relies on the tomato MED18 gene (POD1/SlMED18). Interestingly, POD1/SlMED18 is required for the proper pollen formation and fruit development, as indicated pollen marker gene analysis. As far as we know, most genes isolated so far regulating pollen development encode transcription factors or control different stages of meiotic cycle, whereas MED18 encodes a member of the Mediator multi-protein complex involved in the regulation of RNA polymerase II transcription (Bjorklund and Gustafsson, 2005). Additionally, we demonstrated that MED18 homologs share functional homology in Arabidopsis and tomato species as POD1/SlMED18 is able to rescue the flowering time and floral organ identity abnormalities of the Arabidopsis med18 mutant (Zheng et al., 2013). Nevertheless, our results indicated that SlMED18 has evolved to acquire a novel function in tomato, which is the genetic control pollen ontogeny.

This work was supported by grants of Junta de Andalucia (P12-AGR-1482) and Ministerio de Economía y Competitividad (AGL2015-64991-C3-1-R)

References:

Bjorklund, S. and Gustafsson, C.M. (2005). Trends Biochem Sci, 30: 240-244. Zheng, Z, et al. (2013) PLoS ONE, 8: e53924.

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Poster 15 / SII P15

A developmental framework for adventitious root development in Arabidopsis thaliana

María Ángeles Fernández-López, Sergio Ibáñez, Samuel Daniel Lup, José Luis Micol, José Manuel Pérez-Pérez

Instituto de Bioingeniería, Universidad Miguel Hernández, Avda. de la Universidad s/n, 03202 Elche, Spain

Adventitious roots (ARs) are ectopic roots that arise either naturally or in response to stress from various plant tissues, such as stems and leaves; they may also be induced by mechanical damage or following in vitro tissue culture regeneration. The formation of ARs is a complex genetic process regulated by both environmental and endogenous factors, among which the plant hormone auxin plays a central role (Bellini et al. 2014). Using Arabidopsis thaliana excised leaves as a model for de novo root organogenesis (Chen et al. 2014), we characterized both at the histological and molecular level the different stages during AR formation. Our results indicate that, shortly after excision, a localized auxin maximum is established on a subset of vascular cells near the wound. Then, cytokinin-dependent cell proliferation leads to callus formation in this region which will later acquire root identity markers. To identify additional gene functions required for AR development, we previously screened the Arabidopsis thaliana unimutant collection with a visible leaf phenotype (Wilson- Sánchez et al. 2014). Here, we present new data on a subset of these mutants selected on the basis of their defective AR formation from excised leaves.

Work funded by MINECO/FEDER (AGL2012-33610 and BIO2015-64255)

References:

Bellini C, et al. (2014) Annu. Rev. Plant Biol., 65: 639-66 Chen X, et al. (2014) Front. Plant Sci., 5: 208 Wilson-Sánchez D, et al. (2014) Plant J., 79: 878-91

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Poster 16 / SII P16

A novel auxin signaling factor regulates root periodic branching through the specification of root organ founder cells and its patterning

Juan Perianez-Rodriguez 1 , Miguel A. Moreno-Risueno 1

1 Center for Plant Biotechnology and Genomics, Universidad Politécnica de Madrid, Pozuelo de Alarcón, Madrid, Spain.

Plants have a postembryonic mode of development forming and growing new organs continuously. Plant postembryonic organogenesis requires new organs to be positioned and formed through the specification of organ founder cells and their subsequent development to generate new tissues. In Arabidopsis thaliana has been described that lateral root positioning is dependent on oscillating gene expression. Gene expression oscillations can be followed with the marker DR5, subsequently a static site of expression will be form, this marks the location where a new lateral root will be form (Moreno-Risueno et al., 2010) through the specification of a root organ founder cell. Out of an ethyl methanesulfonate screen, we identified a heritable mutation with altered postembryonic organogenesis, which we named potent. Stem cell and tissue specification is not affected in the main root of potent but the mutant does not produce lateral roots. We use lineage analyses and stem cell specification markers and found that new tissues are not specified in potent. To investigate defects in founder cell specification we focused on pericycle tissue because it is the tissue that is reprogrammed to generate lateral roots. In potent many pericycle cells present an altered identity, they do not present the marker J2661 that marks all pericycle cells, but however, they present the founder cell marker SKP2B. The founder cells in our mutant, although are normally arrested in subsequent development, can be stimulated to undergo organogenesis by auxin treatment, at a low auxin concentration that only induces the formation of lateral root from founder cells. We found that potent overproduces lateral roots. We mapped the mutation by next generation sequencing and we identify the affected gene, an Aux/IAA factor. The mutation is located in the domain that is involved in degradation of the protein upon auxin perception. We checked gene expression oscillations in our mutant detecting non-oscillating, almost continuous expression that could explain the elevated number of founder cells. When we checked more deeply potent pericycle we found regions where cells divided. However, they appeared to be divided symmetrically. We checked the marker MAKR4 that is expressed in the anticlinal membrane between two founder cell prior the nuclear migration that precede the asymmetric cell division and in the next divisions. The result in our mutant was that this marker was expressed in the anticlinal membrane of each pericycle cell or was not polarized. In addition, we observed that some divisions appear to be morphologically asymmetric in potent, however new cells show abnormal morphologies and no change in cell fate. These results suggest that our mutant may be involved in the correct polarization of founder cell to make a correct asymmetric cell division.

References:

Moreno-Risueno, M, et al., (2010) Science; 329(5997):1306-11.

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Poster 17 / SII P17

Crosstalk between histone marks is involved in the control of multiple stages of reproductive development in arabidopsis

Dorota Komar, José A. Jarillo, and Manuel Piñeiro

1 Centro de Biotecnología y Genómica de Plantas (CBGP). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria – Universidad Politécnica de Madrid (INIA-UPM). Campus de Montegancedo, Pozuelo de Alarcón 28223 Madrid, Spain

Successful sexual reproduction of plants requires not only an appropriate timing of flowering but also a proper inflorescence growth and flower development to ensure an optimal balance between the number of flowers and production of resources through photosynthesis. The plant specific chromatin protein, EARLY BOLTING IN SHORT DAYS (EBS) is required for the proper regulation of flowering time through the repression of the floral integrator gene FT. EBS recognizes di- and trimethylated lysine 4 (K4) in histone H3 (H3K4me2/3), and binds regulatory regions of FT chromatin. EBS interacts with histone deacetylases such as HDA6 and mutations in the EBS gene cause an increase in the levels of histone H3 acetylation throughout the FT gene body [1]. Our recent studies have revealed a role for EBS in the control of additional developmental processes related to reproductive growth. Under short day conditions plants deficient in EBS function display multiple morphological alterations including reduced apical dominance and phyllotaxy abnormalities. The expression of class B, C and E floral identity genes as well as the genetic network controlling shoot apical meristem maturation is deregulated resulting in defects in flower development including the appearance of frequent floral reversion events. Most recent advances in our understanding of the molecular mechanisms underlying EBS activity in the chromatin-mediated modulation of different aspects of reproductive development will be presented.

References:

(1) López-González, et al. (2014). Plant Cell, 26: 3922-3938.

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Poster 18 / SII P18

MYB36 regulates root cell elongation by modulating auxin response in Arabidopsis thaliana.

Paula Ragel 1 , Javier Pérez-Hormaeche 1 , Beatriz Cubero 1 , and José Manuel Pardo 1

1 Instituto de Recursos Naturales y Agrobilogía de Sevilla (IRNAS), CSIC, Sevilla, Spain.

We and others [1,2] have studied the role of MYB36, an Arabidopsis R2R3-MYB class transcription factor (TF), as a master regulator of the differentiation of the endodermis during root development. Our results support that MYB36 regulates a developmental switch from proliferative to differentiated state that promotes the development of the Casparian band, in part by regulation of the expression of genes involved in the very localized lignin assembly and deposition in cells from root endodermis. The genetic and molecular mechanisms controlling the cell size in the root elongation zone and the regulators driving the coordinated arrest of cell elongation in the transition from elongation to differentiation zone, are still poorly understood. Here, we present the transcriptional and developmental outcome from MYB36 overexpression, which supports the idea that MYB36 is involved in this process by modulating auxin signalling/perception in the root. Arabidopsis transgenic lines overexpressing MYB36 have pleiotropic phenotypes in auxin-related growth and development, reduced sensitivity to exogenous auxin, and altered gene expression in response to auxin. Both the initiation and lateral root emergence were impaired when MYB36 was overexpressed, but the lateral root phenotype was partially rescued by auxin treatments. However, the expression of DR5:GUS and LAX3pro:YFP auxin-marker genes were not properly induced by auxin in the overexpressing line confirming that MYB36 function affects auxin responses. Moreover the transcriptome analysis of a line conditionally overexpressing MYB36 revealed a strong down-regulation of auxin signalling in shoots 24 hours after induction. These results would explain the drastic effect on cell elongation/expansion when MYB36 is expressed ectopically in Arabidopsis.

References:

1 Kamiya, T, et al. (2015). Proc Natl Acad Sci USA 112(33):10533–10538.

2 Liberman, LM, et al. (2015). Proc Natl Acad Sci USA 112(39):12099-104.

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Poster 19 / SII P19

Hydra, a sporocyteless/noozle homologue, is required for sporogenesis and controls fruit set in tomato

Pilar Rojas-Gracia 1 , Edelín Roque 1 , Mónica Medina 1 , Maricruz Rochina 1 , Rim Hamza 1 , María Pilar Angarita-Díaz 1 , Vicente Moreno 1 , Fernando Pérez-Martín 2 , Rafael Lozano 2 , Luis Cañas, José Pío Beltán 1 , Concha Gómez-Mena 1

1 Instituto de Biología Molecular y Celular de Plantas (IBMCP) CSIC-UPV, Valencia, Spain 2 Centro de Investigación en Biotecnología Agroalimentaria (BITAL), Universidad de Almería, Almería, Spain.

Fruit set is an essential process to ensure successful sexual plant reproduction. Pollination and fertilization are coordinated processes that stimulate the growth of the structures that will protect the developing seeds. However, some species develop seedless (parthenocarpic) fruits that overcome the standard restriction for the ovaries to growth in the absence of pollination and fertilization (Medina et al 2013). The study of parthenocarpic lines in tomato, a major crop plant and a model system for fleshy fruits, has been very useful to understand the genetic and molecular mechanisms associated to fruit set and development. We report here the identification of a new parthenocarpic mutant in tomato, the hydra mutant. Seedless fruit production in these plants is linked to the absence of both male and female sporocyte development. Using positional cloning, virus induced gene silencing and expression analysis we identitied the HYDRA gene and demonstrated that it encodes the tomato ortholog of SPOROCYTELESS/NOZZLE (Schiefthaler et al 1999; Yang et al 1999) of Arabidopsis thaliana. Despite SPL/NZZ and SlSPL/HYD proteins only showed high protein identity in the described functional domains, the tomato protein is able to replace function in the spl/nzz mutants. Remarkably SlSPL/HYDRA is the first SPL/NZZ ortholog characterized since the identification of the Arabidopsis spl/nzz mutants sixteen year ago. We have also analysed the hormonal basis of the parthenocarpy in hydra mutants and shown that precocious ovary growth is associated to changes in auxin distribution within the ovary. Our results showed that the tomato HYDRA gene is essential for gametophyte development and that hormonal signals generated during microgametogenesis must repress precocious ovary growth assuring coordinated pollination and fertilization and successful fruit set. This study supports the conservation of a genetic pathway and the critical role of SPL-like genes during plant reproductive development. Moreover, our data provided evidence of the pivotal role of male gametophyte development in the control of ovary growth and also revealed a new role for SPL-like genes in the control of fruit set in fleshy fruit plants.

References:

Medina, M, et al. (2013) Plant Biotech. J., 11. (6): 770-779. Schiefthaler U, et al. (1999) PNAS. 96: 11664-11669. Yang, WC, et al. (1999) Genes Dev. 13: 2108-2117

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Poster 20 / SII P20

Understanding the cues regulating morphogenesis of meristematic cells during lateral root formation in Arabidopsis thaliana

Álvaro Sánchez-Corrionero 1 , Juan Perianez-Rodríguez 1 , Miguel Ángel Moreno-Risueno 1 .

1 Center for Plant Biotechnology and Genomics CBGP, Department of Biotechnology, Universidad Politécnica de Madrid, Madrid, Spain

The pericycle tissue gives rise to lateral root founder cells (LRFC) through a reprogramming process, and subsequently, distinctive cell fates are specified through asymmetrical divisions. Morphogenesis of lateral roots initiates with the asymmetric division of LRFC to generate small and large cells. These divisions require external inputs (auxin hormone) and are driven by intrinsic cues (such as polarity and nuclear migration). The mechanism(s) regulating these developmental transitions and specifying different cell fates is not well understood. We hypothesize that self-organizing properties of founder cells are controlled by a regulatory network which incorporates external cues such as auxin. Trough double Fluorescent Activated Cell Sorting we will be able to know the expression levels of genes in pericycle, lateral root founder cells and its daughters. To these end, we have already generated a range of plants carrying cells markers, and based on our preliminary studies we can isolate the cell types of interest: a) pericycle cells capable of undergoing reprogramming will be isolated using the line carrying the markers J0121 and pSKP2B 0.5 :ER::3xmcherry, b) pericycle cells undergoing reprogramming through the line carrying DR5:D2eGFP::eGFP and pSKP2B 0.5 :NLS::3xmcherry, c) founder cells using the marker linepWOX5:FP pSKP2B 0.5 :NLS::3xmcherry, and LRFC daughter cells will be isolated using d) the line carrying the markers pWOX5:FP and pSCR:ER::3xmcherry for the small daughter cells), and e) the markers pWOX5:FP and pHB53-2K:ER::3xmcherry for the large daughter cells. This approach will define the regulatory program between crucial developmental states (pericycle, lateral root founder cells and its daughters) associated to root organ morphogenesis, and it will address how two distinct fates are specified from a single cell. We except that our approach provides novel relationships between pluripotency and cell identity.

References:

1. Brady S.M., et al (2007). Science, 318: 801–806

2. Casimiro, I., et al (2001). The Plant Cell, 13(4), 843-852.

3. Moreno-Risueno, M.A., et al (2010). Science, 1306–1311.

4. Manzano, C., et al (2012). Plant physiology, 160(2):749-62.

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Poster 21 / SII P21

Genetic and molecular analysis of mRNA adenosine methylation in Arabidopsis thaliana

Eva Rodríguez-Alcocer, Natalia Gómez-Peral, Daniel Blasco-Espada, Francisca María Lozano, Sara Jover-Gil, Héctor Candela

Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Spain

Although the reversible methylation of adenosine residues at the N 6 position seems to be universally present in the messenger RNAs (mRNAs) of all eukaryotes, we still know very little on the cellular and developmental functions played by this post-transcriptional modification. In an attempt to advance the knowledge of this methylation mark, we are systematically following a reverse genetics approach to identify genes encoding proteins that are likely to participate in the methylation (i.e. methyltransferases that function as N 6 - methyladenosine “writers” in mRNA molecules) and demethylation (i.e. demethylases that function as N 6 -methyladenosine “erasers”), using Arabidopsis thaliana as a model organism. We have selected five different genes for further functional studies, two encoding subunits of the methyltransferase complex and three encoding putative demethylases. To investigate the function of these five genes, we are characterizing plants carrying loss-of- function alleles (T-DNA insertion lines) as well as transgenic plants overexpressing their full-length coding sequences. Using the combinatorial power of the Gateway cloning technology, we have generated a large collection of constructs that should help us to address questions on the consequences of experimentally increasing or reducing the levels of N 6 -methyladenosine in the Arabidopsis transcriptome.

This work received support from Spain's Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund (ERDF) (‘Una manera de hacer Europa') [BFU2012-31719 grant to H.C.].

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Poster 22 / SII P22

The HUA-PEP nuclear ribonucleoproteins regulate ovule development in Arabidopsis via post-transcriptional control of D-function identity genes

Encarnación Rodríguez-Cazorla 1 , Samanta Ortuño 1 , Till Kash 1 , Juan-José Ripoll 1 , Antonio Martínez-Laborda 1 , Antonio Vera 1

1 Área de Genética, Universidad Miguel Hernández, Campus de Sant Joan d’Alacant, Sant Joan d’Alacant, Alicante, Spain,

Development of multicellular organisms encompasses a series of processes, among which correct specification of organ identity plays a fundamental role, and it is subject to tight genetic control at the transcriptional and post-transcriptional levels. Indeed, production of functional eukaryotic RNA is a very elaborate process that involves a complex interplay between transcription and RNA processing activities (Bentley, 2014). In the model plant Arabidopsis, a set of interacting ribonucleoproteins encoded by the so-termed HUA-PEP activity genes were previously demonstrated to regulate the MADS-box floral homeotic gene AGAMOUS (AG), thus affecting flower organ identity (stamens and carpels) and determinacy (Rodríguez-Cazorla et al., 2015). Inside carpels, ovules are critical structures for plant reproductive success that house the female gametophyte and give rise to the seeds after fertilization. Closely related to AG, the D-function genes SHATTERPROOF 1 (SHP1), SHP2, and SEEDSTICK (STK) redundantly confer ovule identity (Pinyopich et al.,

2003).

Here, we report that mutational perturbation of the HUA-PEP gene function leads to the homeotic transformation of developing ovules into ectopic flower organ-like structures. Correspondingly, hua-pep mutants displayed reduced expression of D-function genes along with the accumulation of aberrant transcripts that retain intronic sequences, strongly suggesting post-transcriptional misregulation of MADS-box ovule identity genes. In addition, unlike previous studies in which converted ovules were reported to resemble carpeloid structures (Pinyopich et al., 2003), our morphological and molecular studies showed that transformed ovules in hua-pep mutant backgrounds displayed obvious sepaloid features. This is most likely due to concomitant reduction of AG expression in our hua-pep mutant combinations (Rodríguez-Cazorla et al., 2015) together with D-function decline. Thus, ectopic expression of APETALA1 (AP1) protein was observed in ovules transformed into sepaloid organs. Remarkably, the loss of AP1 restored carpeloid traits in hua-pep transformed ovules, as did the increase of AG gene dosage. These findings suggest the interesting possibility that proper ovule development may require the exclusion of factors such as AP1 which might otherwise promote alternate cell fates. This scenario evokes mutual exclusion of A and C floral homeotic activities during flower development (Coen and Meyerowitz, 1991).

References:

Coen ES, Meyerowitz EM (1991). Bentley, D. L. (2014). Nat Rev Genet 15: 163-175. Pinyopich, A. et al. (2003). Nature 424, 85–88. Rodriguez-Cazorla, E. et al. (2015). PLoS Genet 11(2): e1004983

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Poster 23 / SII P23

The microRNA pathway regulates cuticle formation

Raquel Sarmiento Mañús, Sara Jover-Gil, Rosa Micol-Ponce, María Rosa Ponce

Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Spain

Genes encoding components of RNA metabolism often cause pleiotropic phenotypes when mutated, probably because their products have many targets. Pleiotropy is also exhibited by mutants affected in components of gene silencing pathways mediated by small RNAs 1 . For example, mutations in genes encoding components of the microRNA (miRNA)-silencing pathway often show drought resistance and hypersensitivity to abscisic acid (ABA); the processes underlying these mutant traits are unknown. Indeed, we found increased tolerance to water deprivation, as well as hypersensitivity to salt and ABA, in seven mutants carrying loss-of-function alleles of genes that encode components of the miRNA machinery, six of which had been isolated in our laboratory 2 : dcl1-9 (dicer-like1-9), hyl1-11 (hyponastic leaves1-11), hyl1-12, hen1-13 (hua enhancer1-13), hst-21 (hasty-21), ago1-51 (argonaute1-51) and ago1-52. DCL1 and HYL1 participate in miRNA biogenesis, HEN1 in miRNA stabilization, and HST in miRNA nuclear export. AGO1 is the core component of the miRNA-induced silencing complex. The aerial surfaces of land plants are covered by the cuticle, a hydrophobic layer composed of cutin and cuticular waxes, which acts as a protective barrier. We hypothesized that the mutants mentioned above have a less-permeable cuticle than that of wild-type plants, conferring drought resistance and hypersensitivity to salt and ABA. Indeed, we found that the dcl1, hyl1, hen1, hst, and ago1 mutants studied exhibit reduced water loss and cuticle permeability, which might be caused by the increased epicuticular wax deposition that we also observed. At least one mutant, hst-21, has a thicker cuticle than that of the wild type. WAX INDUCER1 (WIN1), also named SHINE1 (SHN1), encodes an ethylene-responsive transcription factor whose overexpression triggers the induction of several genes of the wax biosynthesis pathway, leading to an increase of epidermal cutin and wax accumulation 3 . Plants over-expressing SHN1 are drought-tolerant. We found the transcription factor SHN1 was upregulated in all the mutants studied, except ago1-51 and ago1-52. Mis-regulation of SHN1 could explain the decrease in permeability and water loss shown by the mutants. These results suggest that the microRNA pathway is involved in the regulation of waxes and cutin production mediated by SHN1.

References:

1. Jover-Gil, S., et al. (2005). 49, 733-744.

2. Jover-Gil, S., et al. (2012). Plant and Cell Physiology 53, 1322-1333.

3. Aharoni, A., et al. (2004). Plant Cell 16, 2463-248

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Poster 24 / SII P24

Alternative polyadenylation regulates plant development and response to stress

Téllez-Robledo, B.; Manzano, C.; Navarro, S.; Marconi, M.; Wilkinson, M; del Pozo, J.C.

Centro de Biotecnología y Genómica de Plantas (UPM-INIA) Dpto. de Biotecnología, Carretera de la Coruña Km. 7,800. 28040 Madrid, Spain. 2, Unidad de Química y Bioquímica. Dpto. de Biotecnología. E.T.S.I. Montes. U.P.M. 28040, Madrid.

In eukaryotes, polyadenylation process defines the end of messenger RNA in a highly regulated manner. Polyadenylation site election influences in RNA translocation, stability and protein translation. The use of different polyadenylation sites has been related to cell differentiation, division, plant growth and response to many stresses. Despite of its great importance, the polyadenylation process and site usage is poorly understood, likely because the majority of the mutants involved in this process are lethal. Here, we have identified a single nucleotide mutation in FIP1, one of the key proteins of the polyadenylation machinery, which leads to alternative polyadenylation (APA) of a large number of genes involved in the regulation of plant development and responses to different abiotic stresses. By bioinformatics means we have found that fip1-1 mutant prefers to use distal polyadenylation sites rather than proximal ones. The fip1-1 mutation alters a large number of biological processes, such as seed dormancy, lateral root formation, leaves growth and flower development among many other processes. In addition, we have found that fip1-1 affects plant responses to salt stress or nitrate starvation. In summary, fip1-1 mutation causes a severe pleiotropic phenotype, likely as consequence of the generation of new proteins isoforms, differential protein translation or RNA stability. In fact, RNAseq analysis shows that fip1-1 mutation affects transcript accumulation of a large number of genes that belongs to many different functional categories. Our results will contribute to understand the role of APA in plant development and also in responses to different abiotic stresses.

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