Libro de resúmenes - XIII Reunión de Biología Molecular de Plantas

Transcripción

Libro de resúmenes
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xiiirbmp.uniovi.es
Reunión de Biología Molecular de Plantas
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Índice
Índice ..................................................... i
Comité Organizador y Científico ........ iii
Carta de Bienvenida ............................. v
Programa XIII RBMP ............................ vii
Conferencias Plenarias ....................... 11
Comunicaciónes Sesión I .................... 17
Comunicaciónes Sesión II ................... 35
Comunicaciónes Sesión III .................. 77
Comunicaciónes Sesión IV ................. 95
Comunicaciónes Sesión V .................. 117
Comunicaciónes Sesión VI ................. 139
Comunicaciónes Sesión VII ................ 165
Índice de Autores ................................. 185
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Comité Organizador
María Jesús Cañal Villanueva
Ricardo Ordás Fernández
Mónica Meijón Vidal
Luis Valledor González
Elena María Fernández González
Comité Científico
Metabolismo y aplicaciones
biotecnológicas
Estrés abiótico
Albert Boronat Margosa
Luisa María Sandalio
Departamento de Bioquímica y Biología Molecular de
Plantas Universidad de Barcelona
Estación Experimental del Zaidín
(EZ-CSIC)
David González Ballester
Mar Castellano Moreno
Departamento de Bioquímica y Biología Molecular de
Plantas Universidad de Córdoba
Centro de Biotecnología y Genómica de Plantas
(CBGP-UPM-INIA)
Mecanismos moleculares de
desarrollo
Estrés biótico e interacción plantamicroorganismo
Óscar Lorenzo Sánchez
Isabel Díaz Rodríguez
Departamento de Fisiología Vegetal.
Universidad de Salamanca
(CBGP-UPM-INIA)
Miguel Ángel Moreno Risueño
Julio Rodríguez Romero
(CBGP-UPM-INIA)
(CBGP-UPM-INIA)
Ambiente, desarrollo y plasticidad
fenotípica
Temas y técnicas emergentes
Jesús Jorrín Novo
Carlos Alonso Blanco
Departamento de Bioquímica y Biología Molecular
Universidad de Córdoba
Centro Nacional de Biotecnología
(CNB-CSIC)
Sonia Osorio Algar
Ignacio Rubio Somoza
Departamento de Biología Molecular y Bioquímica
Universidad de Málaga
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)
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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,
y la participación de investigadores senior de gran prestigio internacional que nos
impartirán tres conferencias plenarias muy interesantes.
El Comité Organizador os da la bienvenida a la Reunión, agradece vuestra participación
y 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”
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 GeneSpecific 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”
Sesión V. Estrés abiótico
Moderadoras: Luisa María Sandalio González (EEZ-CSIC)/ Mar Castellano Moreno (CBGP-UPMINIA)
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 siteslocalized 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 ROSdependent 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, UPMINIA)
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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”
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): “Nextgeneration 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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Conferencias Plenarias
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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 Dalmay1
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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Sesión I: Metabolismo y Aplicaciones Biotecnológicas
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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-Ballester1, Jose Luis Jurado-Oller1, Alexandra Dubini1, Aurora Galván1,
Emilio Fernández1
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 Baslam1, Edurne Baroja-Fernández1, Ángela María Sánchez-López1, Iker
Aranjuelo1, Adriana Ricarte-Bermejo1, Abdellatif Bahaji1, Francisco José Muñoz1,
Goizeder Almagro1, Pablo Pujol2, Regina Galarza2, Pilar Teixidor3, and Javier PozuetaRomero1
1
Instituto de Agrobiotecnología (CSIC/UPNA/Gobierno de Navarra). Iruñako etorbidea 123,
31192 Mutiloabeti, Nafarroa, Spain. 2Servicio de Apoyo a la Investigación, Universidad Pública
de Navarra, Campus de Arrosadia, 31006 Iruña, Nafarroa, Spain. 3Serveis 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 14CO2 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 13CO2 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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Comunicación Oral 2 / SI CO2
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 secondarywall 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 Dubini1, Marko Boehm2, Erin Peden2, Wenqiang Yang3, Arthur Grossman3,
Maria Ghirardi2
1
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de
2
Córdoba, Córdoba, Spain. National Renewable Energy Laboratory, 15013 Denver West,
3
Parkway, Mail Box 3313, Golden, CO 80401, USA. 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.
23
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RBMP
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.
24
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RBMP
Poster 03 / SI P03
QTL mapping for primary metabolites responsible of organoleptic and
nutritional characteristics of strawberry (Fragaria x ananassa)
Delphine Pott1, José G. Vallarino1, Juan Jesús Medina2, Alisdair R. Fernie3, Iraida
Amaya4, Sonia Osorio1
1
Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, University of MalagaCSIC, Department of Molecular Biology and Biochemistry, 29071 Málaga, Spain. 2Instituto
Andaluz de Investigación y Formación Agraria y Pesquera (IFAPA), Julio Caro Baroja s/n,
Huelva, Spain. 3MaxPlanck-Institute für Molekulare Planzenphysiologie, Am Mühlenberg 1,
14476 Golm, Germany.4IFAPA, 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
F1 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.
25
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RBMP
Poster 04 / SI P04
Steryl ester metabolism in tomato
Alejandro Lara1, Karla Ramírez-Estrada1, Alma Burciaga1, María Martín1, Montserrat
Arró1,2 Albert Boronat1,3, Teresa Altabella1,4, Albert Ferrer1,2
1
Center for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Campus UAB, Bellaterra,
Barcelona España; 2Dpto de Bioquímica y Fisiología, UB, Barcelona, España, 3Dpto de
Bioquímica y Biomedicina Molecular, UB, Barcelona, España. , 4Dpto 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 AGL201313522-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
26
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RBMP
Poster 05 / SI P05
Lysophosphatidylcholine acyltransferase genes from sunflower
(Helianthus annuus L.)
Ana Mapelli-Brahm1, J.J. Salas1, Enrique Martínez-Force1, Rafael Garcés1, Mónica
Venegas-Calerón1
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.
27
XIII
RBMP
Poster 06 / SI P06
A mitochondrial lipoyltransferase from Helianthus annuus
Raquel Martins-Noguerol1, Rafael Garcés1, J.J. Salas1, Enrique Martínez-Force1
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), branchedchain 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, CO2 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.
28
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RBMP
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-López1, Abdellatif Bahaji1, Nuria De Diego2, Marouane Baslam1,
Jun Li1, Francisco José Muñoz1, Goizeder Almagro1, Pablo García-Gómez1, Kinia
Ameztoy1, Adriana Ricarte-Bermejo1, Ondřej Novák3, Jan F. Humplík2, Lukáš Spíchal2,
Karel Doležal2, Sergio Ciordia4, Maria del Carmen Mena4, Rosana Navajas4, Edurne
Baroja-Fernández1, and Javier Pozueta-Romero1
1
31192 Mutiloabeti, Nafarroa, Spain. 2Department of Chemical Biology and Genetics, Centre of
the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký
University, Olomouc, CZ-78371, Czech Republic. 3Laboratory of Growth Regulators, Centre of
the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký
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)
29
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RBMP
Poster 08 / SI P08
Coexpression networks as tool to identify novel elements in starch
metabolism regulation
M.Teresa Ruiz1, María Garcia1, M. Isabel Ortíz-Marchena1, Karen Chacón1, Francisco J.
Romero-Campero2, Federico Valverde1, José M. Romero1
1
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Seville, Spain.
Department of Computer Science and Artificial Intelligence, Universidad de Sevilla, Seville,
Spain.
2
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.
30
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RBMP
Poster 09 / SI P09
Complex floral scent profiles under selection pressure show
Mendelian based genetic structure and evolve via transposon activity
Victoria Ruiz-Hernández1, Julia Weiss1, Benjamin Hermans1, Joëlle K. Mühlemann,2,
Natalia Dudareva,3 and Marcos Egea-Cortines1
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
31
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RBMP
Poster 10 / SI P10
Castor plant (Ricinus communis): A biological model for production of
unusual and added-valued industrial oil.
Alfonso Sánchez1, Enrique Martínez-Force1, Rafael Garcés1, E. Guzmán2, Noemí RuízLópez1, Joaquín J. Salas
1
Department of biochemistry and molecular biology of plants products,
Instituto de la Grasa (CSIC), Sevilla, Spain. 2Department 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.
32
XIII
RBMP
Poster 11 / SI P11
Genome-wide description and functional characterization of SNF1related kinases in Chlamydomonas reinhardtii.
Francisco J Colina1, Joana Amaral2, Gloria Pinto2, María Carbó1,
María Jesús Cañal1, Luis Valledor1
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 (Calciumdependent 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.
33
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34
Sesión II: Mecanismos Moleculares de Desarrollo
XIII
RBMP
Comunicaciones Sesión II.
Mecanismos Moleculares de Desarrollo
35
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RBMP
36
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RBMP
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 position1.
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 cells2. 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
2
Moreno-Risueno, MA, et al. (2015) Science, 350(6259):426-30.
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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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
mutants1. 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
regulator1,2.
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 1naphthaleneacetic 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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PROHIBITIN3 and NOA1 participate in the maintenance of the
root stem cell niche in Arabidopsis thaliana
Tamara Lechón1, Noel Blanco-Touriñán2, Virginia Palomares1, Miguel A. Blázquez2, Ivett
Bárány3, Miguel A. Moreno-Risueño4, Mari C. Risueño3, Pilar S. Testillano3, Óscar
Lorenzo1, Luis Sanz1
1
Dpto. Botánica y Fisiología Vegetal, Instituto Hispano-Luso de Investigaciones Agrarias (CIALE)
– Universidad de Salamanca (USAL), Salamanca, Spain, 2Instituto de Biología Molecular y
Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Científicas (CSIC) –
Universidad Politécnica de Valencia (UPV), Valencia, Spain, 3Centro de Investigaciones
Biológicas (CIB), Madrid, Spain, 4Centro 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 pathways1,2. 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 function3,4. 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 mice5.
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: (BIO2014-57107R), 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.
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.
2
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Regulation of developmental timing by TEMPRANILLO through the
age-dependent pathway
Andrea E. Aguilar Jaramillo1, Esther Marín González1, Luis Matías Hernández1, Soraya
Pelaz1,2 and Paula Suárez López1
1
Centre de Recerca en Agrigenòmica, CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra
(Cerdanyola del Vallés), Spain, 2ICREA (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 agedependent developmental pathway.
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Poster 01 / SII P01
Genetic dissection of adventitious root formation in tomato hypocotyls
after wounding
Aurora Alaguero1, Joan Villanova1, Ana Belén Sánchez-García1, Antonio Cano2,
Manuel Acosta2, José Manuel Pérez-Pérez1
1
Instituto de Bioingeniería, Universidad Miguel Hernández, Avda. de la Universidad s/n, 03202
Elche. 2Departamento 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
Recent research on the reversible methylation of adenosine residues at their N6 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 N6-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, highthroughput 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 Fernández1, Ana Elisa Valdés3, María Jesús Cañal , Valeria
Gagliardini2, Alejandro Rivera1, Ueli Grossniklauss2
1
Area of Plant Physiology, Dept. BOS, University of Oviedo, Spain. 2Institute of Plant Biology &
Zurich-Basel Plant Science Center, University of Zurich, Switzerland, 3Physiological 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 onedimensional 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-Marcos1, Bénédicte Desvoyes1, Concepción Manzano2, Louisa M.
Liberman3, Philip N. Benfey3, Juan C. del Pozo2, Crisanto Gutierrez1
1
Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain, 2Centro de
Biotecnología y Genómica de Plantas, INIA, Madrid, Spain, 3Howard 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 domeshaped 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 H2O2 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
Recent studies have shown that the YTH domain is an RNA binding domain that mediates
the interaction with N6-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 Gatewaycompatible 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-Zambrano2, Yue Zhou1, Francisco Romero-Campero2, Wiam Merini2, ,
Franziska Turck1, Myriam Calonje2
1
Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research,
Carl-von-Linné-Weg 10, 50829 Köln, Germany. 2Institute 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 ChIPseq 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.
49
XIII
RBMP
Poster 09 / SII P09
A pollen caleosin with peroxygenase activity is critical for fertilization
María José Jiménez-Quesada1, Krzysztof Zienkiewicz1, José Feijó2, Agnieszka
Zienkiewicz1, Juan de Dios Alché1, Antonio Jesús Castro1
1
Plant Reproductive Biology Laboratory, Estación Experimental del Zaidín (CSIC), Granada,
Spain, 2Instituto 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).
50
XIII
RBMP
Poster 10 / SII P10
Gibberellin signaling in the endodermis modulates the hypocotyl
gravitropic response
Rodrigo Marí-Ordóñez1, Alberto Fuster1, Jana Crespo-Trives1, David Alabadí1, Miguel
Ángel Blázquez1, Eugenio G. Minguet1
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 (GallegoBartolome 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
51
XIII
RBMP
Poster 11 / SII P11
Arabidopsis CUPULIFORMIS genes are new players on the chromatin
remodeling scene
Eduardo Mateo-Bonmatí, Lucía Juan-Vicente, José Luis Micol
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 mechanism1,2. 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/Fe2+-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 LEAF3, 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 upregulated 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 icu111 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.
52
XIII
RBMP
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 (CelluSpotstm 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.
53
XIII
RBMP
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, BIO201233952 and PPII10-0194-4164. AA was supported by a predoctoral grant from JCCM
54
XIII
RBMP
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-Martín1, Fernando J. Yuste-Lisbona1, Benito Pineda2, Begoña GarcíaSogo2, Juan F. Campos3, Estela Giménez1, Teresa Antón2, Iván del Olmo4, Manuel A.
Piñeiro4, José A. Jarillo4, M. Carmen Bolarin3, Vicente Moreno2, Trinidad Angosto1, Juan
Capel1, Rafael Lozano1
1
Centro de Investigación en Biotecnología Agroalimentaria (BITAL). Universidad de Almería.
04120 Almería, Spain; 2Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC),
Universidad Politécnica de Valencia. 46022 Valencia, Spain; 3Centro de Edafología y Biología
Aplicada del Segura-CSIC. 30100 Murcia, Spain; 4Centro 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.
55
XIII
RBMP
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 (WilsonSánchez 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
56
XIII
RBMP
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-Rodriguez1, Miguel A. Moreno-Risueno1
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.
57
XIII
RBMP
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
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.
58
XIII
RBMP
Poster 18 / SII P18
MYB36 regulates root cell elongation by modulating auxin response in
Arabidopsis thaliana.
Paula Ragel1, Javier Pérez-Hormaeche1, Beatriz Cubero1, and José Manuel Pardo1
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.
59
XIII
RBMP
Poster 19 / SII P19
Hydra, a sporocyteless/noozle homologue, is required for
sporogenesis and controls fruit set in tomato
Pilar Rojas-Gracia1, Edelín Roque1, Mónica Medina1, Maricruz Rochina1, Rim Hamza1,
María Pilar Angarita-Díaz1, Vicente Moreno1, Fernando Pérez-Martín2, Rafael Lozano2,
Luis Cañas, José Pío Beltán1, Concha Gómez-Mena1
1
2
Instituto de Biología Molecular y Celular de Plantas (IBMCP) CSIC-UPV, Valencia, Spain
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
60
XIII
RBMP
Poster 20 / SII P20
Understanding the cues regulating morphogenesis of meristematic cells during
lateral root formation in Arabidopsis thaliana
Álvaro Sánchez-Corrionero1, Juan Perianez-Rodríguez1, Miguel Ángel Moreno-Risueno1.
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
pSKP2B0.5:ER::3xmcherry, b) pericycle cells undergoing reprogramming through the line
carrying DR5:D2eGFP::eGFP and pSKP2B0.5:NLS::3xmcherry, c) founder cells using the
marker linepWOX5:FP pSKP2B0.5: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.
2.
3.
4.
Brady S.M., et al (2007). Science, 318: 801–806
Casimiro, I., et al (2001). The Plant Cell, 13(4), 843-852.
Moreno-Risueno, M.A., et al (2010). Science, 1306–1311.
Manzano, C., et al (2012). Plant physiology, 160(2):749-62.
61
XIII
RBMP
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
Although the reversible methylation of adenosine residues at the N6 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 N6methyladenosine “writers” in mRNA molecules) and demethylation (i.e. demethylases that
function as N6-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-offunction 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 N6-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.].
62
XIII
RBMP
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-Cazorla1, Samanta Ortuño1, Till Kash1, Juan-José Ripoll1,
Antonio Martínez-Laborda1, Antonio Vera1
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
63
XIII
RBMP
Poster 23 / SII P23
The microRNA pathway regulates cuticle formation
Raquel Sarmiento Mañús, Sara Jover-Gil, Rosa Micol-Ponce, María Rosa Ponce
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 RNAs1. 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 laboratory2: 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
accumulation3. 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
64
XIII
RBMP
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.
65
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RBMP
Poster 25 / SII P25
ore functions of the AtBMI1 proteins as part of different PRC1s in
Arabidopsis
Wiam Merini1, Francisco J Romero-Campero2, Myriam Calonje1
1
2
Institute of Plant Biochemistry and Photosynthesis (IBVF-CSIC-University of Seville), Seville,
Higher Technical School of Computer Engineering, Faculty of Mathematics, University of Seville,
Spain
PcG regulation in Arabidopsis is required to maintain the differentiated state of the cells
and to allow developmental phase transitions. These tasks are performed by three different
PRC2 complexes, which act at different developmental stages, and by the activity of an
additional complex, the PRC1. Although several PRC1 subunits have been identified and
some data suggested the existence of different functional PRC1 variants (Yang et al.,
2013; Calonje et al., 2014; Merini and Calonje 2015), little is known about their composition
and function; furthermore, the functional relationship of some PRC1 components has been
controversial. Among the PRC1 components, the AtBMI1 proteins has been shown to
participate in the repression of seed maturation genes after germination and EMF1 in the
suppression of the reproductive program during vegetative development; however, it is not
known how PRC1 cooperates with PRC2 in regulating other developmental processes.
Here, by analyzing the transcriptome of single, double and triple atbmi1 mutants we have
defined genome-wide the genes whose expression is regulated by the AtBMI1 proteins,
providing a more comprehensive picture of the developmental processes that they
regulate. Our results indicate that the AtBMI1s not only are required to switch off seed
maturation program after germination, but also to promote the transition from one
developmental phase to the next one throughout development. In addition, our data
highlighted their crucial role in the control of cell proliferation during organ growth and
development. Furthermore, by comparing these datasets with previously published data,
we have determined their interplay with other PcG related proteins. Our analyses strongly
suggest that AtBMI1 and VAL proteins act together only in the regulation of seed
maturation genes. Conversely, AtBMI1 and EMF1 co-regulate a considerable number of
genes involved in different developmental processes, although the loss of function of their
respective activities has different impact on the repression of these genes. Nevertheless,
AtBMI1 and EMF1 have also a subset of specific target genes, supporting the existence
of different PRC1 variants with unique molecular functions.
This work is supported by Marie Curie CIG Grant ID 333748 and BIO2013-44078-P Grant from the
Spanish Ministry of Economy and Competitiveness (MINECO).
References:
Merini W & Calonje M. (2015). Plant J, 83(1):110-20.
Calonje M (2014). Mol Plant, (3):459-71.
Yang Cet al. (2013). Curr Biol, 23(14):1324-9.
66
XIII
RBMP
Poster 26 / SII P26
Identification of a novel protein that interacts with different factors of
the translation initiation complex
Ana B. Castro-Sanz*, Fco. René Toribio*, Alfonso Muñoz*, José M. García-Reyes, Elena
Ramírez-Parra, Juan Carlos del Pozo and M. Mar Castellano
1
Centro de Biotecnología y Genómica de Plantas, INIA, Pozuelo de Alarcón, Spain.
Plant’s architecture and development are orchestrated by multiple regulation mechanisms
that integrate both external and internal stimuli. Regulation of gene expression is tightly
coordinated by control checkpoints set at transcriptional, post-transcriptional, translational
and post-translational levels.To date, most efforts have focused on the transcriptional
regulation of gene expression, but nowadays the role of the translational control is
acquiring more importance, although the mechanisms involved in selective mRNA
translation are far less understood.
In other eukaryotes the main checkpoint on translational regulation is the initiation step,
mainly through the regulation of the availability of the translation initiation factor 4E
(eIF4E). eIF4E belongs, along with a number of well-known proteins, to the so called
translation initiation complex.
In our lab we are characterizing a novel protein that interacts with different components of
the translation initiation complex. According to our results, this protein is mainly expressed
in the root meristem, where the hormonal influence plays an essential role by determining
root architecture and lateral root disposition. Based on the pattern of expression and the
different evidences that demonstrate that this protein forms part of the initiation complex,
we hypothesize that this novel protein could have a role on the selection of mRNAs to be
translated in the root meristem.
67
XIII
RBMP
Poster 27 / SII P27
The DnaJ-like ANGULATA7 protein is required for plastid gene
expression and thylakoidal membrane organization in Arabidopsis
Tamara Muñoz-Nortes, Tamara González-Costa, María Rosa Ponce, Héctor Candela, José Luis
Micol
The characterization of mutants with altered leaf shape and pigmentation has previously
allowed the identification of nuclear genes that encode plastid-localized proteins that
perform essential functions in leaf growth and development1. A large-scale screen
previously allowed us to isolate ethyl methanesulfonate (EMS) induced mutants with small
rosettes and pale green leaves with prominent marginal teeth, which were assigned to a
phenotypic class that we dubbed Angulata2. The molecular characterization of the twelve
genes assigned to this phenotypic class should help us to advance our understanding of
the still poorly understood relationship between chloroplast biogenesis and leaf
morphogenesis3.
Here we report the phenotypic and molecular characterization of the angulata7-1 (anu7-1)
mutant of Arabidopsis, which we found to carry a novel hypomorphic allele of the EMB2737
gene, which was previously known only for its embryonic lethal mutations. ANU7 encodes
a plant-specific protein containing a domain with conserved cysteine and glycine residues
that is similar to an incomplete central cysteine-rich domain, which accounts for the
disulfide isomerase activity of DnaJ proteins. DnaJ proteins normally function as
chaperones, either alone or in combination with heat-shock protein 70, and have been
proposed to participate in the folding, unfolding, assembly and degradation of proteins,
maintaining protein homeostasis under normal or stress conditions4.
Although the complete loss of ANU7 function causes embryonic lethality, our EMS-induced
alleles are hypomorphic and viable, and have allowed us to demonstrate that ANU7 is
required for the accumulation of photosynthetic pigments and the correct organization of
the thylakoid membrane system. The observed genetic interaction of anu7-1 with a lossof-function allele of GENOMES UNCOUPLED15 suggests that the anu7-1 mutation
triggers a retrograde signal that is at least in part responsible for the observed phenotypic
defects. Our microarray expression studies show that many genes that normally function
in the chloroplasts are upregulated in anu7-1 rosettes, with a significant overrepresentation
of those required for the expression plastid genome genes.
References:
1.- Micol, J.L. (2009). Current Opinion in Plant Biology 12, 9-16.
2.- Berná, G., et al (1999). Genetics 152, 729-742.
3.- Casanova-Sáez, R., et al. (2014). Journal of Experimental Botany 65, 2391-2404.
4.- Craig, E.A., et al. (2006). Reviews of Physiology, Biochemistry and Pharmacology 156, 1-21.
5.- Cottage, A., et al. (2010). Journal of Experimental Botany 61, 3773-3786
68
XIII
RBMP
Poster 28 / SII P28
Arabidopsis ABCE proteins function in leaf development and venation
patterning
Carla Navarro-Quiles, Miquel Sendra-Ortolà, Eduardo Mateo-Bonmatí, José Luis Micol
Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche,
Spain.
In a large-scale screen for ethyl methanesulfonate-induced Arabidopsis mutants that are
altered in leaf development1, we isolated the apiculata7-1 (api7-1) mutant, which has
small, indented and pointed leaves, with aberrant venation pattern. Several of these
phenotypes occur in many mutants with defects in the translation machinery. We combined
map-based cloning and genome-wide sequencing to identify the causal mutation and
found that the api7-1 mutation is a hypomorphic allele of RNASE L INHIBITOR2 (RLI2),
which encodes one of the two Arabidopsis ABCE proteins. We also found that the null
insertional allele api7-2 is recessive lethal. The very large ABC protein superfamily is
present in organisms ranging from bacteria to humans. Plant ABC proteins fall into nine
subfamilies: ABCA to ABCI2, which include the lesser-studied, soluble ABCE and ABCF
proteins.
Human and Arabidopsis ABCE proteins participate in RNA silencing
suppression3,4_ENREF_3 and in ribosome biogenesis and recycling5. Consistent with its
relationship to ribosome function, we observed synergistic phenotypes in double mutants
combining api7-1 and loss-of-function alleles of the ASYMMETRIC LEAVES1 (AS1) and
AS2 genes, which encode transcription factors known to play a role in leaf dorsoventral
patterning and polarity. We constructed an API7pro:API7:GFP translational fusion, which
allowed us to demonstrate that RLI2/API7 is a cytoplasmic protein, consistent with the
absence of predicted transmembrane domains in ABCE proteins. The api7-1 mutation is
the first viable mutant allele of the RLI2/API7 gene and our examination of this allele
revealed an unexpected role for ABCE proteins in whole-leaf development and venation
patterning.
References:
1.- Berná, G., et al. (1999). Genetics 152, 729-742.
2.- Verrier, P.J., et al. (2008). Trends in Plant Science 13, 151-159.
3.- Braz, A.S., et al. (2004). Journal of Molecular Evolution 59, 20-30.
4.- Sarmiento, C., et al. (2006). Plant Molecular Biology 61, 153-163.
5.- Pisarev, A.V., et al. (2010). Molecular Cell 37 196-210
69
XIII
RBMP
Poster 29 / SII P29
Transcriptional regulation of the Arabidopsis ARGONAUTE1 gene
Adrián Cabezas-Fuster, Rosa Micol-Ponce, Raquel Sarmiento-Mañús, María Rosa
Ponce
The Arabidopsis ARGONAUTE1 (AGO1) ribonuclease is the main effector of
posttranscriptional gene silencing pathways that are mediated by small interfering RNAs,
including microRNAs (miRNAs)1. Also, a miRNA targets AGO1 transcripts. Hypomorphic
ago1 alleles disrupt many developmental pathways and responses to biotic and abiotic
stress factors; null ago1 alleles are lethal. Expression of AGO1 is widespread and virtually
constitutive under normal conditions; also AGO1 expression increases under certain
environmental conditions, such as viral infection. We have a wealth of information on the
mechanisms by which AGO1 post-transcriptionally regulates expression of other genes,
but we lack information on the transcriptional regulation of AGO1 itself.
To analyse the transcriptional regulation of AGO1, we generated an AGO1pro:GUS
transcriptional fusion, which carries a putative full-length AGO1 promoter. Plants
homozygous for the AGO1pro:GUS transgene were grown on media with different
concentrations of NaCl, abscisic acid (ABA), or sucrose, or exposed to different light
intensities and dark periods. ABA treatments and dark exposure significantly altered GUS
activity, but sucrose and NaCl did not affect GUS activity.
Among other subcellular locations, Arabidopsis AGO1 occurs in the endoplasmic
reticulum, where it represses translation of target mRNAs2. An in silico analysis of the
promoter of AGO1 allowed us to identify two regulatory elements associated with the
unfolded protein response (UPR) in the endoplasmic reticulum, which we also found in the
promoters of the AGO1 orthologs of rice and Brassica rapa; these sequences seem to be
relevant for the regulation of AGO1 expression.
To identify the key regulatory AGO1 promoter sequences, we constructed four
transcriptional GUS fusions driven by different segments of the AGO1 promoter, lacking
one or both UPR elements. We are examining these fragments by mobility shift assays
with nuclear protein extracts. In addition, we are analysing the effects of each mutant
promoter variant on the expression of the GUS reporter gene in plants grown in the
different culture conditions mentioned above, and in the presence of tunicamycin or
dithiothreitol, which induce UPR.
References:
1. Kidner, C.A., et al. (2005). Current Opinion in Plant Biology 8, 38-44.
2. Li, S., et al. (2013). Cell 153, 562-574.
70
XIII
RBMP
Poster 30 / SII P30
Arabidopsis CUPULIFORMIS genes are new players on the chromatin
remodeling scene
Eduardo Mateo-Bonmatí, Lucía Juan-Vicente, José Luis Micol
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 mechanism1,2. 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/Fe2+-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 LEAF3; 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 upregulated in a RNA-seq analysis of icu11-1 leaves, including members of the MADS-box
family of transcription factors. 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 the SEP3 gene in the icu11-1 mutant. Taken together, our results
reveal 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.
71
XIII
RBMP
Poster 31 / SII P31
Role of DESIGUAL1 and auxin in bilateral symmetry of Arabidopsis
leaves
David Wilson-Sánchez, Sebastián Martínez-López, Sara Jover-Gil, José Luis Micol
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
mutants1. Only one of these mutants exhibited defects in bilateral symmetry; we named
this mutant desigual1-1 (deal1-1).
Arabidopsis has bilaterally symmetric 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
regulator2,3.
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 1naphthaleneacetic 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, D., et al. (2014). Plant Journal 79, 878-891.
2.- Bilsborough, G.D., et al. (2011). PNAS USA 108, 3424-3429.
3.- Kasprzewska, A., et al. (2015). Plant Journal 83, 705-718.
72
XIII
RBMP
Poster 32 / SII P32
Ribosome biogenesis requires RRP7 and NOP53 in Arabidopsis
Rosa Micol-Ponce, Raquel Sarmiento-Mañús, Alejandro Ruiz-Bayón, Sara FontcubertaCervera, Jorge Ruiz-Ramírez, María Rosa Ponce
We identified mas2 (morphology of argonaute1-52 suppressed2) alleles as informational
suppressors of ago1-52, a hypomorphic allele of Arabidopsis AGO1 (ARGONAUTE1)1.
Positional cloning and sequence analysis showed that MAS2 encodes the Arabidopsis
ortholog of NKAP (NF-kappa B activating protein)2, a protein conserved in most eukaryotes
and involved in transcriptional repression in animals. A yeast two-hybrid assay with MAS2
as bait identified 14 interactors, including two putative orthologues of proteins that
participate in ribosome biogenesis in Saccharomyces cerevisiae: Ribosomal RNA
Processing Protein 7 (RRP7) and Nucleolar Protein 53 (NOP53). Ribosome biogenesis
requires stoichiometric amounts of ribosomal proteins and ribosomal RNAs (rRNAs).
Although rRNA biogenesis consumes most of the transcriptional activity of eukaryotic cells,
its regulation remains largely unclear in plants.
We obtained rrp7-1, rrp7-2, nop53-1, and nop53-2 insertional mutants from public
collections; these mutants exhibited pointed and reticulate leaves, similar to many mutants
defective in ribosome biogenesis. We constructed NOP53pro:NOP53:GFP and
RRP7pro:RRP7:GFP translational fusions; examination of transgenic plants showed that
RRP7 is nucleolar, and NOP53 both nucleolar and nucleoplasmic.
The yeast orthologs of NOP53 and RRP7 participate in the control of 45S rDNA
transcription and transcript processing, processes that we found to be defective in
Arabidopsis nop53 and rrp7 mutants. All rrp7 and nop53 mutations synergistically
interacted in double mutants with parallel1 (parl1), a loss-of-function allele of Arabidopsis
NUCL1 isolated in a screen for mutants with altered venation patterning3. NUCL1 encodes
the nucleolar protein NUCLEOLIN1, which participates in the epigenetic control of 45S
rDNA expression4. Morphometry of the vasculature of cotyledons, leaves, and petals
showed strong reductions in venation length, density and number of bifurcations in rrp7-1
and parl1 mutants, but not in nop53-1. Since altered abscisic acid (ABA) responses have
been described for several mutations in genes involved in different pathways of RNA
metabolism, seeds of rrp7-1, nop53-1 and parl-1 were sown in medium supplemented with
3 µm ABA. Their germination rates were undistinguishable from wild type, but rrp7-1 and
parl1 showed a strongly reduced cotyledon expansion and greening.
We also found synergistic phenotypes in double mutant combinations of nop53 or rrp7 with
mutations in genes encoding microRNA machinery components. Taken together, our
results suggest a functional relationship between the miRNA pathway and ribosome
biogenesis.
References:
1. Micol-Ponce, R et al. (2014). Scientific Reports 4, 5533.
2. Sánchez-García, A.B. et al. (2015). Plant Cell 27, 1999-2015.
3. Petricka, J.J., et al. (2007). Plant Physiology 144, 173-186.
4. Pontvianne, F., et al. Molecular Biology of the Cell 18, 369-379
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Poster 33 / SII P33
Regulation of the Arabidopsis DESIGUAL1 gene and identification of
DESIGUAL1 interactors
Sergio Navarro-Cartagena, David Wilson-Sánchez, Sebastián Martínez-López, José Luis
Micol
Bilateral symmetry is a striking property of many plants and animals. In bilateral organs
such as plant leaves, acquisition of symmetry requires properly regulated development on
both sides of the midplane. However, how this occurs remains unclear at the molecular
level. We are studying the Arabidopsis DESIGUAL (DEAL) gene family, whose members
seem to be required for bilateral symmetry at very early stages of leaf organogenesis.
To study the developmental networks that provide positional information to leaf cells, we
identified the DEAL1 gene, which is expressed during early leaf development. The DEAL1
protein resides at the membrane of a sub-compartment of the endoplasmic reticulum and
a split-ubiquitin membrane-based yeast two-hybrid screen for DEAL1 interactors identified,
among other proteins, several components of the Very-Long-Chain Fatty Acid (VLCFA)
elongation complex. VLCFA lipids are known to negatively regulate leaf cell proliferation
through cytokinin signaling1.
To examine DEAL1 function further, we are using yeast one-hybrid (Y1H) screens and
other approaches to identify additional components of the auxin- and cytokinin-mediated
regulatory networks that mediate symmetry in leaves. To design the Y1H baits we used
the mVISTA program2 to search in silico for conserved regulatory sequences in the
promoter of DEAL1. This analysis identified two regions conserved in the Brassicaceae
family. Constructs including only one or both of these regions were cloned in the pTUY1H
plasmid and transformed into Saccharomyces cerevisiae. A screen was performed with
these baits and a prey library of 1400 Arabidopsis transcription factor genes. We observed
only one strong interaction, corresponding to the ACTIVATOR OF SPOMIN::LUC2
(ASML2) protein, a known transcriptional activator that regulates the expression of several
sugar-inducible genes3.
References:
1.- Nobusawa, T., et al. (2013). PLoS Biology 11, e1001531.
2.- Frazer, K.A., et al. (2004). Nucleic Acids Research 32, W273-W279.
3.- Masaki, T., et al. (2005). Plant Journal 43, 142-152.
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Poster 34 / SII P34
Arabidopsis MAS2 interactors identified in a yeast two-hybrid screen
Alejandro Ruiz Bayón, Raquel Sarmiento Mañús, Rosa Micol-Ponce, María Rosa Ponce
Arabidopsis MAS2 is an essential gene that encodes a homolog of animal NF-kappa B
Activating Protein and seems to be a key player in the regulation of rRNA synthesis in
plants1. Fluorescence in situ hybridization showed that MAS2 colocalizes with the 45S
rDNA in the nucleolar organizer regions. To better understand the function of MAS2, we
screened for interactors in a yeast two-hybrid (Y2H) assay. Two Arabidopsis cDNA
libraries obtained from whole Arabidopsis plants, totalling 21 million prey clones, were
used in the screening. The bait contained the full-length coding region of MAS2. The
screen identified 91 prey clones, representing 14 different genes, and these clones were
confirmed by directed Y2H assays.
The most represented interactor in the Y2H-based screen, in 23 of the 55 clones, was
CAX INTERACTING PROTEIN4 (CXIP4), a protein of unknown function that was
previously identified as interacting with the high-affinity vacuolar calcium antiporter CAX1
and found in the nucleus and cytoplasm2. CXIP4 occurs exclusively in plants and 30 amino
acids of its N-terminal region show 70% similarity to the mammalian splicing factor SREK1interacting protein 1. Three MAS2 interactors were related to ribosome biogenesis,
including RPS24B, the second most represented interactor, which is one of the two
Arabidopsis S24-type proteins in the 40S ribosomal subunit.
CXIP4 is encoded by AT2G28910, a plant-specific, single-copy gene. The CXIP4 Nterminal region contains a conserved CysX2CysX4HisX4Cys (CCHC)-type zinc finger
domain, termed a zinc knuckle. Selfing of plants heterozygous for the cxip4-1 insertional
allele did not produce homozygous mutant progeny, the only exception being a few
extremely dwarf, morphologically aberrant escapers, whose growth arrested several days
after germination. We constructed two artificial microRNAs designed to target CXIP4, to
circumvent the lethality associated with the lack of CXIP4 function. Additional transgenes
were obtained to complement the mutant phenotype of homozygous cxip4-1 plants, and
to visualize the spatial pattern of expression of CXIP4 and the subcellular localization of
the CXIP4 protein. We are also studying the genetic interactions of CXIP4 and RPS24B,
with a particular focus on MAS2 viable alleles and alleles of genes encoding components
of the microRNA pathway.
References:
1. Sánchez-García, A.B. et al. (2015). Plant Cell 27, 1999-2015.
2. Cheng, N.H., et al. (2004). FEBS Letters 559, 99-106
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Poster 35 / SII P35
A transcriptional repressor complex regulates asymmetric divisions in
Arabidopsis roots
Mar Martín-Trillo*2, Marta Boter*1, Regla Bustos1, Liam Dolan3, Monica Pernas1
1
Centro de Biotecnología y Genómica de Plantas (CBGP, INIA-UPM). Instituto Nacional de
Investigación y Tecnología Agraria y Alimentaria–Universidad Politécnica de Madrid, Madrid.
2
Dpto. de CC. Ambientales-Área de Fisiología Vegetal, Universidad de Castilla-La Mancha,
Toledo. 3Deparment of Plant Sciences, University of Oxford, Oxford, UK.
* These authors contributed equally to this work
The formation of the plant body requires the assembly of different tissue systems in an
organized pattern to make organs. The formation of tissue systems in all multicellular
organisms including plants depends on the activity and development of a group of
undifferentiated cells called stem cells. Plant cells are surrounded by rigid cell walls that
prevent cell movement therefore stem cells need to follow a strict pattern of cell division
and acquisition of specific cell identities to ensure the maintenance of tissue organization
during the life cycle of the plant. The cortex and endodermal cell layers, collectively known
as ground tissue, arise from two successive asymmetric cell divisions of the
cortex/endodermis stem cell initial (CEI). Ground tissue formation involves a complex
interplay of transcription factors. SCHIZORIZA (SCZ) is a heat shock transcription factor
(HSFB4) that regulates asymmetric divisions of the stem cell initials in the Arabidopsis
root. Thus, in the scz-1 mutant the CEI divides periclinally to form two endodermis layers
instead of the usual cortex-endodermis ground tissue layers. The aim of our work is to
address how SCZ regulates asymmetric cell division in the root meristem. SCZ lacks an
activation domain, and carries a functional repression domain and an ethylene response
factor–associated amphiphilic repression (EAR) motif at their C-terminal suggesting that
SCZ acts as a transcriptional repressor. Using yeast two hybrid assays, we have found
that SCZ specifically interacts with the transcriptional co-repressor TOPLESS (TPL). TPL
comprise a conserved family of plant transcriptional corepressors related to Groucho/Tup1
corepressor family that regulate different developmental processes through interaction
with EAR motifs. Consequently, mutations in the SCZ EAR motif completely abolish this
interaction in yeast, confirming that this motif mediates the interaction of SCZ with TPL.
Furthermore, defective asymmetric divisions similar to scz-1 in the ground tissue of tpl1-1
mutant and the additive phenotype of the scz tpl double mutant suggest a genetic
interaction of SCZ with TPL. Additionally, although the role of SCZ in asymmetric cell
division is well stablished, its downstream targets remain unknown. The analysis of the
genes differentially expressed in the root meristem of scz-1 mutant compare to wild type
plants by RNAseq further supports a role of SCZ as part of a repressor complex and shed
light on how SCZ could regulates asymmetric stem cell divisions in the root meristem.
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Sesión III: Ambiente, Desarrollo y Plasticidad
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Comunicaciones Sesión III.
Ambiente, Desarrollo y Plasticidad
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Ponencia Invitada / SIII PI
Using natural variation to understand lateral growth in plants
Javier Agusti
Instituto de Biología Molecular y Celular de Plantas (IBMCP) “Eduardo Primo Yúfera”. (CSICUPV). Carrer de l’enginyer Fausto Elio, s/n. 46011. Valencia (Spain).
Multicellular organisms develop by integrating and coordinating growth programs. In plants,
the lateral growth (thickening) of stems and roots is a central developmental process that
provides the indispensable mechanical support and stability that plants need to expand their
growth and sustain their structures. In addition, lateral growth is the direct source of large
amounts of extremely useful plant biomass for food and energy security: lateral growth
brings about wood in trees and regulates the yield of fundamental crops for food security
like cassava or sweet potato.
Lateral growth is mediated by a specialized group of stem cells called cambium. Remarkably
for such a crucial cell-type for plant development and plant biomass production, very little is
known about the genetic regulation of cambium.
We have used a strategy that combines natural variation and Genome-Wide Association
Studies (GWAS) in the model system Arabidopsis thaliana to identify new cambium
regulators.
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Comunicación Oral 1 / SIII CO1
DNA demethylases control growth-dormancy transitions in Poplar
Daniel Conde1, Alicia Moreno-Cortés1, Anne-Laure Le Gac2, Christopher Dervinis3, José
M. Ramos-Sánchez 1, Matias Kirst3-4, Stéphane Maury2-5, Mariano Perales1, Pablo
González-Melendi1 and Isabel Allona1
1
Departamento de Biotecnología-Biología Vegetal, Centro de Biotecnología y Genómica de
Plantas UPM-INIA, Universidad Politécnica de Madrid (UPM), Campus de Montegancedo, E28223 Pozuelo de Alarcón (Madrid), Spain, 2Université d’Orléans, Faculté des Sciences,
Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), UPRES EA 1207, 45067
Orléans, France, 3School of Forest Resources and Conservation, University of Florida, Florida
32611,USA, 4University of Florida Genetics Institute, University of Florida, FL 32611,USA, 5INRA,
USC1328 Arbres et Réponses aux Contraintes Hydriques et Environnementales (ARCHE),
45067 Orléans, France.
Coordinating growth and reproduction with the environment is essential for the survival of
trees in temperate and boreal latitudes. In these regions, deciduous and periodic growth
habits evolved into a single trait known as winter dormancy. This trait consists of growth
cessation through the arrest of meristem activity and consequent transformation of the
apical meristem into a dormant winter bud. For many trees and perennial plants, the
perception of short days (SD) is sufficient to promote cessation of growth and bud
formation. Restoration of meristem activity and bud break during the spring are trigged by
the environmental favourable conditions (long days and warm temperatures) once the
chilling requirement is fulfilled.
Active chromatin rearrangement has been proposed as key steps in growth-dormancy
transitions in trees, but yet functional studies are needed to support this hypothesis. In this
work we investigated the role of DNA demethylases during dormancy. Thought
phylogenetic and protein sequence analyses we firstly identified poplar and chestnut
Demeter-like genes (DMLs). Expression studies showed that CsDML and its closer
homolog gene PtaDML6, are induced during dormancy entrance. Overexpression of
CsDML accelerated short day-induced bud formation in poplar. Comparative
transcriptional profiling revealed that the overexpression of this 5mC DNA demethylase
promoted the molecular changes previously observed under short-day conditions. In the
other hand, two poplar DMLs genes, PtaDML8 and PtaDML10, were induced during
dormancy exit. The phenological assays showed that PtaDML8-10 knockdown plants (KD)
have a delayed bud break. Transcriptome analyses revealed that KD plants have altered
biological processes such as cellular metabolic process, photosynthesis, ribosome
biogenesis and response to light and temperature stimulus. Whole Genome Bisulphite
Sequencing let us to identify the DMR (differentially methylated region) in KDs vs. WT at
bud break, indicating that transcriptomic changes could be associated to the changes in
DNA methylation status. Taken all together, we demonstrate for the first time that
chromatin rearrangement leading by active DNA demethylation control dormancy entrance
and exit in poplar.
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Deciphering how plant density affects seed yield in Arabidopsis
thaliana
Irma Roig-Villanova1, Jaime F. Martínez-García1,2
1
Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Edifici CRAG,
Campus UAB, Bellaterra, 08193 - Barcelona, Spain;
2
Institució Catalana de Recerca i Estudis Avançats (ICREA), Ps. Lluís Companys 10, 08010 Barcelona, Spain.
Fruit production has huge agronomical and economical interest, being the yield and quality
associated with fruits two traits of key importance to agricultural production. Yield refers to
the final amount of seeds generated by the plant. In the model plant Arabidopsis thaliana,
the final number of mature seeds produced is determined by key environmental and
genetic factors affecting plant architecture and the ovule and seed development.
In the close presence of neighbouring vegetation, such as in a crop field, plants sense the
proximity of competing vegetation as a change in light quality, i.e., a reduced red (R) to
far-red (FR) light ratio (R:FR). Upon this signal, many plants display “shade avoidance
responses” that comprehend various developmental changes intended to overgrow or
survive neighbouring plants. In seedlings, these responses include increased elongation
of the hypocotyl, which has been extensively studied by several laboratories. In adult
plants, responses include decreased branching and accelerated flowering, that are also
associated with an altered seed set and production. However, little is known about how
plant proximity affects and controls the molecular network regulating seed production once
the architecture of the adult plant is defined and flowering is induced. In our laboratory we
are characterizing the specific changes occurring in the reproductive tissues of
Arabidopsis plants in response to shade related to ovule and seed development, i.e., the
establishment of a specific number of ovule primordia, the correct formation of the ovule,
the successful double fertilization, and the right maturation of the seed. Moreover, we are
investigating the molecular mechanisms (i.e. identification of genes) that participate in this
regulatory network. So far we have discovered that some of the factors that control the
shade avoidance responses in seedlings also play a role in controlling these responses in
the reproductive tissues, whereas other factors are specific of this later developmental
phase. The last advances will be presented in this meeting.
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Most microRNAs in the single-cell alga Chlamydomonas reinhardtii are
produced by DCL3-mediated cleavage of introns and UTRs of coding
RNAs
Adrian Valli1,2, Bruno Santos1, Claire Agius1, Silvia Hnatova1, David Baulcombe1
1
Department of Plant Sciences, University of Cambridge, United Kingdom, 2Department of Plant
Molecular Genetics, Spanish National Center of Biotechnology (CNB-CSIC), Madrid, Spain.
Most of our knowledge regarding miRNA-mediated regulation of gene expression derives
from studies in multicellular organisms – in the case of plants, it mostly comes from studies
in Arabidopsis thaliana. We also know that miRNAs are present in unicellular organisms,
such as the green alga Chlamydomonas reinhartii (Molnár et al., 2007). Until now, the
biogenesis, mode of action, and biological function of miRNAs in this organism were
unknown.
Chlamydomonas reinhardtii has been used as a biological model to study several aspects
of plant biology for decades. The availability of its genome sequence and its short
generation time also makes this alga an ideal tool for genetic analyses. We have recently
developed a forward genetic approach to identify factors involved in the miRNA-mediated
silencing pathway in Chlamydomonas, and among the isolated lines we identified three
independent mutants that were unable to produce miRNAs and transposon-derived
sRNAs. A PCR-based mapping technique located the mutagenic insertions into the Dicerlike protein (DCL) 3 coding gene. To further characterize the effect of these mutations at
the molecular level, sRNA and mRNA high-throughput sequencing analysis of the dcl3
mutant and parental lines were carried out. These analyses helped us to identify and
classify not only the whole population of mature miRNAs (and many others DCL3dependent sRNAs), but also their corresponding precursors. Results indicate that, in many
respects, miRNA precursors in Chlamydomonas resemble those from animals rather than
miRNA precursors from higher plants, and revealed a further difference from higher plants
in that the sRNAs are rarely negative switches of mRNA accumulation. The few transcripts
that were more abundant in dcl3 mutant lines than in wild type cells were not due to sRNAtargeted RNA degradation but to direct DCL3 cleavage of miRNA and siRNA precursor
structures embedded in both the untranslated and translated regions of the mRNAs.
This analysis reveals that miRNA-mediated RNA silencing in C. reinhardtii differs from that
of higher plants, and informs about the evolution and function of this pathway in
eukaryotes.
Our current work on the characterization of DCL3-dependent control of transcriptional
silencing will be presented and discussed at the meeting.
References:
Molnar A, et al. (2007) Nature, 447(7148):1126-1129.
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Comunicción Oral 4 / SIII CO4
Identification of novel epigenetically regulated genes involved in root
development in Arabidopsis thaliana
María Luz Annacondia1, Jesús Pascual1, Luis Valledor1, Agustín Fernández2, Mario F
Fraga2, María Jesús Cañal1, Mónica Meijón1
1
Plant Physiology, Department of Organisms and Systems Biology, University of Oviedo,
Spain. 2Cancer Epigenetics Laboratory, Institute of Oncology of Asturias (IUOPA-HUCA),
Spain.
The root of Arabidopsis thaliana can be used as a model system to study epigenetic
regulation in plants and adaptive processes to changing environmental conditions. In
recent years, genome wide association studies (GWAS) have been positioning in plant
species research as a simple and inexpensive tool allowing to associate specific genes
with certain processes (Meijón et al., 2014), opening the possibility of studying the
epigenetic regulation of root development using quantitative genetics. In this context, a
novel approach combining the use of epigenetic drugs and GWAS was employed to
unmasked novel genes involved in this process. Root development traits were quantified
by BRAT software (Slovak et al., 2014) in 160 different natural accessions, thereby
sampling the worldwide distribution of Arabidopsis, subjected to the action of genistein
(DNA demethylating agent). After GWAS analysis, the use of the most significant SNPs
on nearby genes allowed the identification of several high confidence genes potentially
mediating in root development that have been unmasked by epigenetic drugs. Notably,
polymorphisms related to CG islands in the promotor and the 5’ UTR of a ROOT HAIR
gene were significantly associated with the variation in the tortuosity of roots. In Col-0
plants, genistein affects the tortuosity of roots, specifically causing its loss. In this way,
Col-0 plants grown in MS-genistein had straight roots, in contrast to the twisted roots they
had in MS.
Two independent T-DNA insertion lines in CDS region of this gene showed a strongly
twisted root phenotype compared to wild type, both in MS and MS-genistein culture media,
affecting not only macro-morphological traits but also cell orientation. Moreover, the
extreme accessions in relation to tortuosity trait, Wilcox and EM-183, were grown in MS
and MS-genistein culture media to unveil the effects of genistein at a cellular level. Wilcox
is an accession with highly twisted roots and not responsive to genistein, while EM-183
root twist was strongly reduced in the same conditions. Interestingly, in the case of Wilcox,
the most significant SNP associated to this gene was changed from C to A, altering a
potential DNA-methylation-related regulatory locus. qPCR analyses demonstrated that the
expression of this gene is increased when C-Allele accessions were grown in MSgenistein, being its expression associated to straighter roots and lower twist degree.
Bisulfite pyrosequencing analysis of DNA methylation confirmed the specific methylation
differences between treatments and accessions.
In summary, these data reveal that the epigenetic mechanisms of gene regulation and
gravitropism traits, such as, root tortuosity, are key elements for adaptation across
worldwide in Arabidopsis thaliana.
References:
Meijón, M., et al (2014) Nature Genetics 46 (1): 77-81
Slovak, R., et al (2014) The Plant Cell 26 (6): 2390-2403
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Poster 01 / SIII P01
Development of a regional platform for GWAS in Arabidopsis from the
Iberian Peninsula
Belén Mendez-Vigo1, Noelia Arteaga1, Daniel Tabas-Madrid1, Mercedes Ramiro1, Marija
Savic1, F. Xavier Picó2, Carlos Alonso-Blanco1
1
Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas
(CSIC), Madrid, Spain, 2Deparmento de Ecología Integrativa, Estación Biológica de Doñana
(EBD), Consejo Superior de Investigaciones Científicas (CSIC), Sevilla, Spain,
In the past decade, Arabidopsis thaliana has become a model species not only for plant
molecular biology but also for ecological and evolutionary genetics (Alonso-Blanco and
Mendez-Vigo, 2014). This has been achieved thanks to the unique resources that have
been developed for the analysis of its natural variation, such as the availability of more
than 6000 wild genotypes that span A. thaliana world distribution in Eurasia, North Africa
and North America. In addition, the genome sequence of a large number of world-wide
accessions has become available (Cao et al., 2011). Both resources have opened
Genome-Wide Association Analysis (GWAS) as a general approach to dissect the natural
variation in A. thaliana, although GWAS statistical power is limited by the frequency of
segregating mutations (Horton et al., 2012). Analyses of world-wide collections have
shown that most of the natural mutations altering gene functions (functional mutations) in
A. thaliana display very low frequency and strong geographic structure. Therefore, GWAS
using world-wide collections is mainly focused in the small proportion of widely spread
mutations (Horton et al., 2012).
To increase the frequency of natural functional mutations of A. thaliana, in the past ten
years we have developed a new regional collection of wild accessions exclusively from the
Iberian Peninsula (Mendez-Vigo et al., 2011; Manzano-Piedras et al., 2014). This region
provides an ideal scenario to study A. thaliana adaptation because it is part of the species
native range, it spans a large diversity of climates, altitudes (0-2600 m) and ecological
habitats, and it has been shown to contain the largest amount of genetic variation of A.
thaliana in Eurasia (Picó et al. 2008; Cao et al. 2011). As part of the Arabidopsis 1001
Genomes Project, we have obtained nearly-complete genomes from 180 Iberian
accessions and we have analysed these sequences to stablish a platform for GWAS in
the Iberian Peninsula. Currently, the Iberian GWAS platform contains more than two million
segregating SNPs that have been used to identify new genomic regions contributing to the
natural variation for two important adaptive traits: flowering time and trichome density in
the leaves.
References:
Alonso-Blanco and Mendez-Vigo,(2014) Current Op Plant Biol 18:37-43
Cao et al. (2011) Nature Genetics 43:956-963
Manzano-Piedras et al., 2014. PLOS One 9: e87836
Mendez-Vigo et al., 2011. Plant Physiology 157:1942-1955
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Securing yield stability of Brassica crops in changing climate
conditions
Iván del Olmo, Marta Boter, Begoña Prieto, Manuel Piñeiro, José A. Jarillo and Mónica
Pernas
Montegancedo, 28223 Madrid
European farmers are currently facing the crucial challenge of securing crop yield by
adapting agricultural practices and crop varieties to climate change. Oilseed rape
(Brassica napus) is considered one of the main sources of high-quality vegetable oil for
human nutrition and biofuels worldwide. Crops respond to environmental variation (high
temperature, drought, etc…) resulting from global climate change with alterations in
developmental traits such as flowering time and root system architecture (RSA) that cause
important production losses. One way to minimize the negative impact of rising
temperatures on yield is by fine-tuning the floral transition to optimize the production of
seeds and by modulating the root architecture in the soil to improve water and nutrient
uptake.
In the framework of the SYBRACLIM (Securing yield stability of Brassica crops in changing
climate conditions, FACCE-JPI-ERA-NET+ CLIMATE SMART AGRICULTURE) project
we are performing a detailed evaluation of the impact that warm temperatures have on
flowering time and root development traits in a genetically diverse panel of spring oilseed
rape genotypes. Using controlled greenhouse conditions at 21°C and 28°C, we have
observed differential responses, accelerated or delayed flowering time in the warmth, for
the spring varieties assessed in these temperature assays. The oilseed rape genotypes
showing differential responses to warm temperatures have been chosen to perform
expression analysis of master genes involved in flowering time control. A time lapse
analysis of different tissues in the spring oilseed rape varieties has shown differential
transcriptional behaviours regarding the expression of a number of putative Brassica
orthologous genes involved in the regulation of the floral transition and floral meristem
development.
On the other hand, root growth tends to increase with warmer temperatures until an
optimum is reached above which root growth is decreased. The roots of oilseed rape
seedlings grown in vitro at 21 and 28ºC have been screened for morphological and
dynamic parameters such as primary root length, lateral root number and root growth
rates. The analysis of these parameters in early development stages has uncovered the
natural variation among oilseed rape genotypes. Root phenotyping of the same panel of
genotypes has also been performed in soil grown plants under controlled greenhouse
conditions to establish whether the uncovered RSA variability is maintained under
physiological conditions.
Phenotypic data on flowering time and RSA will be presented and discussed in the context
of understanding how these developmental traits affect yield. Furthermore, the use of
genome-wide (RNA-seq) approaches in SYBRACLIM will allow us to uncover the genetic
determinants and the transcriptional networks involved in the adaptability of oilseed rape
to warm temperatures.
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Flavonols mediate root phototropism and zonation establishment
Javier Silva-Navas1,2, Miguel A. Moreno-Risueno3, Concepción Manzano1; Bárbara
Téllez-Robledo1, Sara Navarro-Neila1, Víctor Carrasco3, Stephan Pollmann3 and F.
Javier Gallego2, Juan C. del Pozo1,
1.
Tecnología Agraria y Alimentaria. Campus de Montegancedo, Pozuelo de Alarcón, 28223
Madrid, Spain. 2. Dpto. de Genética, Facultad de Biología, Universidad Complutense de Madrid,
Madrid 28040 Spain 3. Centro de Biotecnología y Genómica de Plantas (CBGP). Universidad
Politécnica de Madrid. Campus de Montegancedo, Pozuelo de Alarcón, 28223 Madrid, Spain.
In nature, roots grow in darkness, but, under certain circumstances, they may be exposed
to light. After perceiving light focus, they react showing negative phototropism, also called
root light avoidance. In this response, roots bend to escape from light, and in addition they
reduce their growth. Despite this response is known for decades, the molecular
mechanisms that govern this negative growth response are poorly understood. Genetic
and physiological experiments showed that root negative phototropism is induced by blue
light signaling and the activity of PHOT blue-light receptors. In roots, PHOT1 is mainly
expressed in the transition zone, where dividing cells start to differentiate and elongate.
Tropic responses, which involved organ curvature, occur due to asymmetric sides growth
that normally correlates with an asymmetric distribution of the plant hormone auxin.
However, it should be highlighted that the majority of these studies have been performed
with etiolated seedling or using seedling which roots have grown in presence of light.
Recently, we have designed a novel system to grow Arabidopsis plants with the root
rowing in darkness and the shoot in presence of light. Using this system we used light as
a growth-conditional cue that allow us to show that flavonols regulate root growth
responses to light through repression of cell proliferation and promotion of cell
differentiation. Unilateral illumination of roots induces accumulation of flavonols at the
meristem side closer to light, promoting local cell differentiation and growth re-orientation
to avoid light. Genetic and chemical analyses show that flavonol accumulation in the root
transition zone is triggered by pathways promoting differentiation (cytokinin and hydrogen
peroxide), and that these pathways are required for root light avoidance. Flavonol levels
are low when roots grow in darkness, but they strongly increase upon illumination. This
higher content of flavonols establishes a new developmental zonation through reduction
of auxin signaling, the PLETHORA gradient, and superoxide radical content, and
subsequently reduces root growth.
Flavonols, therefore, function as positional signals, integrating hormonal and ROS
pathways to regulate organ growth in response to light.
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Functional analysis of the mTERF5 and mTERF9 genes of Arabidopsis
thaliana
Almudena Ferrández-Ayela, Pedro Robles Ramos y Víctor Quesada Pérez
Instituto de Bioingeniería. Universidad Miguel Hernández, 03202, Elche, Alicante, Spain
Using a reverse genetics approach, we previously identified and characterized the
Arabidopsis mda1 and mterf9 insertional mutants affected in the chloroplast-localized
mitochondrial transcription termination factors mTERF5 and mTERF9, respectively1,2. In
order to shed light into the molecular and developmental functions of both genes, we are
carrying out different experimental approaches. We have studied by qRT-PCR the
expression patterns of mTERF5 and mTERF9 along development and detected transcripts
of both genes in all the stages analysed, reaching their highest levels at the earliest time
point studied (7 days after stratification). To investigate the effect that defective chloroplast
biogenesis might have on mTERF5 and mTERF9 activity, we analysed their expression in
the scabra3-2 (sca3-2) mutant, affected in the plastid-RNA polymerase RpoTp and
severely impaired in chloroplast development. mTERF5 transcript levels were similar in
sca3-2 and the wild-type (Col-0), whereas mTERF9 was significantly upregulated. We
obtained the mda1-1 sca3-2 and mterf9 sca3-2 double mutants and found that sca3-2 is
epistatic on mda1-1, while mterf9 and sca3-2 synergistically interact. This suggests that
the affected genes participate in the same genetic pathway required for accurate
chloroplast development.
In metazoans, the MTERF3 and MTERF4 proteins are involved in the biogenesis of
mitochondrial ribosomes. Besides, our previous results point to a functional relationship
between mTERF5 and some nuclear genes encoding plastid ribosomal proteins2. We have
quantified the levels of the different plastid rRNA species in the mda1 and mter9 mutants
given that rRNA abundance is commonly used as a proxy for the levels of 50S and 30S
ribosomal subunits. Our results revealed a differential accumulation of some plastid rRNAs
in mda1 and mterf9 mutants compared to Col-0, which would be consistent with a defect
in chloroplast ribosomal assembly in the mutants. Currently, we are generating different
constructs to overexpress mTERF5 and mTERF9 and study their effect on Arabidopsis
development in wild-type and different mterf defective mutants.
References:
1
2
Robles, P., et al. (2012). PLoS One. 7: e42924.
Robles, P., et al. (2015). Physiol. Plant. 154: 297–313.
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Role of two subunits of the Arabidopsis NuA4 complex in the
regulation of flowering time
Alfonso Mouriz, María M. Martín Trillo, Iván del Olmo, Begoña Prieto, Mónica Pernas,
José A. Jarillo and Manuel Piñeiro
Montegancedo, 28223 Madrid, Spain
Chromatin remodelling plays a crucial role in the establishment and maintenance of gene
expression patterns that drive developmental transitions in plants, and it is of utmost
importance in the control of master regulators of the floral transition, including the floral
repressor FLOWERING LOCUS C (FLC). In particular, the exchange of histone H2A by
the histone variant H2A.Z in the FLC chromatin mediated by the SWR1 complex (SWR1C) is required for the transcriptional activation of this floral repressor. In yeast, the SWR1C shares four subunits with the histone acetyltransferase complex NuA4 (NuA4-C), and a
close functional relationship between these complexes in the control of gene expression
has been described. Most of the 13 subunits of the Saccharomyces cerevisiae NuA4-C
are conserved in Arabidopsis, suggesting that interplay between these two complexes
could exist in Arabidopsis, and that histone acetylation mediated by this putative NuA4-C
may also have a role in the regulation of flowering time.
The Arabidopsis homologues of the yeast NuA4-C subunits Esa1 (AtHAM1 and AtHAM2),
Yaf9 (AtYAF9a) and Eaf1 (AtEAF1) subunits regulate H4 acetylation levels in the
chromatin of the FLC locus. In this work, we have characterized the functions of some of
the Arabidopsis NuA4-C subunits, paying special attention to their roles in flowering time.
We have shown that ING1 and ING2, the Arabidopsis homologues of Yng2, play opposite
roles in the regulation of flowering. Both proteins bind the chromatin of FLC and are
required to maintain proper H4 acetylation levels in this locus. Interestingly, ING2 also
binds the chromatin of FT. Their ability to physically interact with NuA4-C components
suggests that ING1 and ING2 regulate the acetylation status of their target genes in the
context of this multiprotein complex. Transcriptomic data regarding ing1 and ing2 mutants
indicate that ING1 and ING2 have independent and redundant functions in the control of
gene expression in Arabidopsis, and suggest key roles for these PHD-containing proteins
as central regulators of several biological processes.
Furthermore, ING proteins are also conserved in other pant species. To understand the
role of these chromatin remodeling factors in flowering control in crop species and explore
the possible use of these members of histone acetylation complexes as tools for the fine
modulation of the floral transition, we are characterizing Brassica napus homologues of
ING genes. Progress in the use of transgenic approaches to study the involvement of
NuA4-C and SWR1-C in the control of flowering time and thermosensory responses in this
crop will be presented.
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Role of sugars in the posttranslational regulation of FRI protein in the
temperature pathway to flowering in Arabidopsis
Mercedes Pallero-Baena, M. Isabel Ortiz-Marchena, Marina Alexandra Ribeiro-Pedro,
José M. Romero, Federico Valverde
Institute of Plant Biochemistry and Photosynthesis, CSIC-University of Sevilla, Sevilla, Spain.
The floral transition is a crucial process in plant development as it determines the
reproductive success of the species. The vernalization signal implies the exposure to low
winter temperatures during several weeks before flowering (winter annuals). Vernalization
depends mainly on the action of two loci in Arabidopsis that show a high degree of allelic
variation in natural populations. The FRIGIDA (FRI) gene codes a coiled-coil domaincontaining protein that activates the expression of the transcriptional repressor
FLOWERING LOCUS C (FLC), a MADS-box transcription factor that strongly represses
the flowering pathway by inhibiting the expression of the floral integrators FT and SOC1.
FLC is regulated at chromatin level by epigenetic signals that are influenced by low
temperatures through vernalization. An alternative gene activated by FRI is FLOWERING
LOCUS M (FLM). FLM (also known as MAF1) belongs to a family of MADS-box genes
similar to FLC, localized in a tandem repeat region of the genome (MAF1-5) and also code
for flowering repressors. Although the effect of photoperiod or vernalization over flowering
time is well documented in the literature, the effect of sugars and temperature and overall,
how these signals are integrated into a single common decision, remains obscure.
Using proteomics we were able to isolate the truncated protein encoded by the FRI allele
from Col-0 seedling in a differential screening experiment with or without the addition of
external sucrose to the growing media on plates. The addition of sugars enhanced FRI
protein stability in Arabidopsis due to the inhibition of its proteasoma-dependent
degradation. This caused the accumulation of high levels of FLC mRNA and late flowering.
In this case, glucose and not trehalose, seemed to have the strongest effect on FLC
expression. This result may explain why adding sugars early in plant development
produces a delay in flowering time. Later, we overexpressed FRI:GFP protein fusion under
a 35S promoter. 35S:FRI:GFP plants were not only late flowering in Col-0, that has an
active FLC allele, but also in Ler background that is a natural flc mutant, identifying a role
for several MAF genes in flower retardation. Therefore, sugars interfere with the
temperature-dependent pathway by affecting the stability and activity of FRI protein.
Results will be shown on the possible regulatory mechanism controlling flowering time in
mutants and FRI overexpressing plants.
This work was funded by projects BIO2011-28847-C00 and BIO2014-52452-P (MINECO) to FV and JMR
and PAI BIO-281 (Junta de Andalucía).
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The Arabidopsis mTERF6 gene is required for organelle gene
expression, development and response to abiotic stress
Víctor Quesada Pérez, Almudena Ferrández-Ayela, Sergio Navarro Cartagena y Pedro
Robles Ramos
Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche,
Alicante.
The molecular mechanisms that control gene expression in chloroplasts are far from fully
understood and the results hitherto obtained show that this regulation is much more
complex than initially thought. In recent years, the characterization of different mutants in
Arabidopsis thaliana and maize, assigns an increasingly prominent role to the family of the
mitochondrial transcription termination factors (mTERF) in the control of organelle gene
expression1. Plant genomes harbor a considerably larger number of mTERFs than
animals. However, very little is known about its function in plants. To advance the
understanding of the roles of plant mTERFs, we have performed a reverse genetic
approach in Arabidopsis, to identify and characterize mutants affected in these genes.
One of the mutants identified in our screen proved to be a new allele not yet described of
the recently characterized mTERF6 gene2, that we named mterf6-5. The mterf6-5 mutant
exhibited markedly reduced growth and developmental retardation, pale cotyledons,
leaves, stems and sepals. The insertion of the T-DNA caused a strong reduction in
mTERF6 transcript levels in mterf6-5 plants. We found that mTERF6 is a plant-conserved
protein expressed in different organs and developmental stages, which was reported to be
dually targeted to chloroplasts and mitochondria2. We studied by qRT-PCR the steadystate levels of several characteristic plastid and mitochondrial genes in the mterf6-5 mutant
and found that all of them were misregulated. Interestingly, the expression of several
nuclear genes was also altered in mterf6-5 indicating that defective chloroplast and/or
mitochondrial function is signalled to the nucleus. Bioanalyzer profiles showed abnormal
ratios of different plastid rRNAs in mterf6-5 suggesting that the accumulation of plastid
ribosomal subunits was likely altered in this mutant. Our double mutant analysis revealed
that mterf6-5 synergistically interacts with other mterf-deficient mutants as well as with
sca3-2, affected in the plastid RpoTp RNA polymerase. These results point to a functional
relationship between mTERF6, other mTERF genes and RpoTp/SCA3.
We have studied the response of the mterf6-5 mutant to different abiotic stresses and
found that the mterf6-5 mutation enhanced salt, osmotic and ABA stress sensitivity during
germination and early growth. Our results highlight the important role of mTERF6 in
organelle gene expression and reveal new functions not yet described for this gene in
development and response to stress in plants.
References:
1
2
Romani, I. et al. (2015). Plant Physiol. 169(1): 627-246.
Quesada, V. (2016). Physiol. Plant. doi: 10.1111/ppl.12416.
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Functional characterization of Arabidopsis CRD genes
Pedro Robles Ramos, Almudena Ferrández Ayela, Eva Núñez Delegido, Moisés
Cabanes Martínez, Víctor Quesada Pérez
Instituto de Bioingeniería. Universidad Miguel Hernández, 03202, Elche, Alicante, Spain
The crd (chloroplast ribosome defective) mutants of Arabidopsis thaliana were previously
isolated in our laboratory in a search for T-DNA mutants presumably affected in the flux of
genetic information in the chloroplasts. All the crd mutants are loss-of-function alleles of
four nuclear genes encoding different chloroplast ribosomal proteins: three of the small
subunit (30S) and one of the large one (50S). The crd mutants share some common
phenotypic traits such as impaired growth, pale leaves, stems and sepals as well as
abnormal chloroplasts. In addition, two of them, crd2 and crd4 synergistically interact with
the mda1-1 mutant affected in the mTERF5 gene encoding a mitochondrial transcription
termination factor chloroplast-localized1. We have confirmed that increased paleness of
crd leaves correlates with reduced levels of chlorophylls. We checked by qRT-PCR
transcription of CRD genes at different time points in whole plants, finding that their
expression decrease 20 days after stratification. In order to investigate, as it may be
assumed, if chloroplast function was perturbed in the crd mutants, we are conducting
molecular analyses at different levels. A quantification of the ribosomal RNA species
present in the mutants may indicate specific assembly problems of one or both subunits
of the chloro-ribosome and hence altered assembly and/or stability of this organelle2. We
found that the crd mutants showed changes in the 30S:50S ratio: three of them being lower
and one higher than the wild type, which is consistent with the physical locations of the
affected proteins in each ribosomal subunit. We have also studied by qRT-PCR whether,
in addition to potential defects in chloroplast translation, the crd mutants showed altered
steady-state levels of plastid gene transcripts. Our results revealed that most of the studied
genes displayed transcripts levels significantly higher than the wild type, including those
of genes involved in photosynthesis, translation or transcription in the organelle. Finally,
as part of the functional characterization, we want to study the effects of CRD gain of
function in wild-type and mutant genetic backgrounds. For this purpose, we are obtaining
CRD overexpression lines in which the transcription of the CRD genes is constitutively
driven by the CaMV 35S promoter.
References:
1
2
Robles, P., et al. (2015). Physiol. Plant. 154: 297–313.
Tiller, N., et al. (2012). Plant J. 69: 302-316.
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Unraveling phenology in Picea abies: epigenetic memory laid down
during embryogenesis adjusts timing of bud set and bud burst
Marcos Viejo1, Elena Carneros2, Hugh Cross2, YeonKyeon Lee1, Igor Yakovlev2, Carl
Gunnar Fossdal2, Jorunn E. Olsen1
1
Department of Plant Sciences, Norwegian University of Life Sciences (NMBU), Ås, Norway,
2
Norwegian Institute of Bioeconomy Research (NIBIO), Ås, Norway
Timing of bud set and bud burst in Picea abies is of capital importance for the survival of
meristems during winter and reflects local adaptations depending on the latitude in which
the individuals live. The plasticity involved in this cyclic behaviour is a consequence of the
response to seasonal abiotic stimuli but also relies on an epigenetic memory whose
establishment depends on the temperature during zygotic embryogenesis (Øystein et al.,
2005). Similarly, the in vitro development of somatic embryos at different temperatures not
only gives rise to epitypes resembling the situation observed in plants originating from
zygotic embryos (Kvaalen and Johnsen, 2008) but also is associated with profound
epigenetic changes on the chromatin and at the transcriptional level (Yakovlev et al.,
2016).
In order to unravel the epigenetic mechanisms involved in this behaviour, 12 year-old
clonal individuals from two epitypes generated in vitro at 18 and 28°C are being
characterized from several angles: (1) phenology is being studied by following the spring
bud burst to assess the differential performance of the epitypes. (2) Transcriptomic study
using RNA-seq data to identify differentially transcribed genes (focusing on epigenetic
machinery and bud burst-related genes) in different cell types. The transcriptomic data will
also help to (3) identify candidate genes and study their specific methylation through
bisulfite sequencing; (4) analysis of the expression patterns of genes with different
methylation between epitypes during dormancy release and bud burst. Finally, (5) the
study of epigenetic changes during bud burst via the identification of differential DNA
methylation and various posttranslational modifications of histones within the meristems.
The first results of transcriptomic studies in different cell tissues of contrast epitypes will
be presented and discussed.
References:
Kvaalen and Johnsen (2008) New Phytol. 177: 49-59.
Yakovlev I, et al. (2016). Planta. 243 (5): 1237-1249.
Øystein J, et al. (2005). Plant Cell Environ. 28: 1090-1102.
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Epigenomic landscapes of DNA replication orgins in a live organism
Joana Sequeira-Mendes1, Irene Aragüez1, Christopher Hale2, Celina Costas1, Ugo
Bastolla1, Steven E. Jacobsen2, Crisanto Gutierrez1
1
Centro de Biologia Molecular Severo Ochoa, CSIC-UAM, Nicolas Cabrera 1, Cantoblanco,
28049 Madrid. 2 Department of Molecular, Cell and Developmental Biology, Howard Hughes
Medical Institute, University of California, Los Angeles, California, USA
Replication origins (ORIs) are the genomic sites where DNA replication initiates.
Determining how ORI specification is linked to transcriptional activity and epigenetic
landscape is crucial to unravel their regulation. Prior studies in our lab identified and
thoroughly characterised a collection of genome-wide ORIs in a synchronised Arabidopsis
cell line(1).
Still, studies on the replication field have focused on cultured cells, loosing the possibility
to evaluate factors beyond the cellular level, e.g. developmental signals. Therefore, we are
filling this gap by identifying ORIs directly in the developing organism, by next generation
sequencing of short nascent DNA strands (SNS) contained in the replication bubbles. We
are currently characterising the originome in all the proliferating cell types of the seedling,
at different developmental stages. We have performed high resolution analysis of the
epigenetic features in the vicinities of these ORIs based on the chromatin states described
recently by our group(2). Surprisingly, initiation sites are not associated with a single
chromatin signature common to all of them. Instead, various signatures are associated
with ORI specification. Such observation discloses a significant plasticity of ORIs regarding
their chromatin context. Even so, from the possible combinations of epigenetic marks,
some chromatin states are preferred over others.
The implications of our results in the study of replication control at the organismal level will
be discussed.
References:
(1) Costas et al., 2011. Nat Struct Mol Biol, 18, 395-400
(2) Sequeira-Mendes et al., 2014. Plant Cell, 26, 2351-2366
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Comunicaciones Sesión IV.
Vías de Señalización
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Ponencia Invitada / SIV PI
miRNA networks and their central role in molecular reprogramming
Ignacio Rubio Somoza1
1
Molecular Reprogramming and Evolution (MoRE) Laboratory, CRAG, Barcelona, Spain.
Plant smallRNAs (sRNAs) regulate from genome stability, preventing re-activation and
invasion of transposable elements, to the expression of their mRNA targets. sRNAs come
in different flavors depending on the RNA source they are produced from. microRNAs
(miRNAs), a sub-class of sRNAs, mainly target mRNAs coding for transcription factors
(TFs). Thus, in the model plant Arabidopsis thaliana 20% of the miRNA families regulate
3% of the total repertoire of TFs found in its genome. miRNA-targeted TFs regulate
common sets of downstream targets through binding to their regulatory regions therefore
forming regulatory circuits. Those unrelated TFs tend to engage in different protein
regulatory complexes that are pivotal for the control of several developmental programs
and their transition throughout the plant life cycle1,2,3. Interestingly, the combination of
different TFs in those regulatory complexes determines different developmental outputs3.
To obtain a global view of those miRNA-TF regulatory circuits, we have leveraged ChIPseq, RNA-seq and protein-protein interaction techniques along with functional studies. Our
results assessing the interaction among 5 unrelated miRNAs and 10 of their TF targets
and their implications in molecular reprogramming will be discussed.
References:
1- Rubio-Somoza, I and Weigel, D. (2011). Trends Plant Sci, 16:258-264.
2- Rubio-Somoza, I and Weigel, D. (2013). PLoS Genet, 9: e1003374.
3- Rubio-Somoza, I, et al. (2014). Curr Biol, 24: 2714-2719.
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Comunicación Oral 1 / SIV CO1
Hormone-Mediated Gene-Specific Translation Regulation
Catharina Merchante1,2, Javier Brumós2, Jeonga Yun2, Anna Stepanova2, José Alonso2
1
Departmento de Biología Molecular y Bioquímica, UMA, Málaga, Spain. 2Department of Plant
and Microbial Biology, NCSU, Raleigh, USA
The central role of translation regulation in the control of critical cellular processes has
long been recognized. Yet the systematic exploration of quantitative changes in translation
at a genome-wide scale in response to specific stimuli has only recently become
technically feasible. Using a genetic approach, we have identified new Arabidopsis weakethylene insensitive mutants that also display defects in translation, which suggested the
existence of a previously unknown molecular module involved in ethylene-mediated
translation regulation of components of this signaling pathway. To explore this link in detail,
we implemented for Arabidopsis the ribosome-footprinting technology, which enables the
study of translation at a whole-genome level at single codon resolution[1]. Using ribosomefootprinting we examined the effects of short exposure to ethylene on the Arabidopsis
translatome looking for ethylene-triggered changes in translation rates that could not be
explained by changes in transcript levels. The results of this research, in combination with
the characterization of a subset of the aforementioned weak-ethylene insensitive mutants
that are defective in the UPF genes (core-components of the nonsense-mediated mRNA
decay machinery), uncovered a translation-based branch of the ethylene signaling
pathway[2]. In the presence of ethylene, translation of a negative regulator of ethylene
signaling EBF2 is repressed, despite induced transcription of this gene. These
translational effects of ethylene require the long 3ÚTR of EBF2 (3ÉBF2), which is
recognized by the C-terminal end of the key ethylene-signaling protein EIN2 (EIN2C) in
the cytoplasm once EIN2C is released from the ER-membrane by proteolytic cleavage.
EIN2C binds the 3ÉBF2, recruits the UPF proteins and moves to P-bodies, where the
translation of EBF2 in inhibited despite its mRNA accumulation. Once the ethylene signal
is withdrawn, the translation of the stored EBF2 mRNAs is resumed, thus rapidly
dampening the ethylene response. These findings represent a mechanistic paradigm of
gene-specific regulation of translation in response to a key growth regulator.
Translation regulatory elements can be located in both 3′ and 5′ UTRs. We are now
focusing on the ead1 and ead2 mutants, another set of ethylene-signaling mutants
defective in translational regulation. Ribosome-footprinting on the ead1 mutant revealed
an accumulation of translating ribosomes in the 5ÚTRs of uORF-containing genes and
reduction in the levels of ribosomes in the main ORF. The mutant is also impaired in the
translation of GFP when this reporter is fused to WT 5ÚTR of potential EAD1 targets but
not when GFP is fused to the uORF-less versions of the same 5ÚTRs. Our hypothesis is
that EAD1/2 work as a complex that is required for the efficient translation of mRNAs that
have common structural (complex 5ÚTR with uORFs) and functional (regulation of key
cellular processes) features. We are working towards the identification of the conditions
where the EAD1 regulation of translation is required.
References:
[1] Ingolia, N. et al. (2009) Science, 324; 218-222
[2] Merchante, C. et al. (2015) Cell, 163(3): 684-697
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Molecular composition of stress granules in Arabidopsis
Emilio Gutierrez-Beltran1, Panagiotis N. Moschou2, Andrei P. Smertenko3, Peter V.
Bozhkov1
1
Department of Chemistry and Biotechnology, Uppsala BioCenter, Swedish University of
Agricultural Sciences and Linnean Center for Plant Biology, PO Box 7015, SE-75007 Uppsala,
Sweden. 2Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural
Sciences and Linnean Center for Plant Biology, PO Box 7080, SE-75007 Uppsala, Sweden.
3
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA
Efficient adaptation to stress depends on the availability of energy resources. Stress drives
cells to an energy crisis whereupon they have to reduce energy expenditure in order to
survive. To this end, eukaryotic cells compartmentalize specific mRNAs and proteins in
cytoplasmic ribonucleoprotein complexes known as stress granules (SGs) 1. In these
structures mRNA molecules are stored, degraded or kept silent in order to prevent energy
expenditure on producing useless, surplus or even harmful proteins under stress
conditions 2. Molecular composition, structure, and function of SGs in plants are largely
unknown. Recently, we have revealed that Tudor staphylococcal nuclease (TSN) is
essential for the integrity and function of SGs in Arabidopsis thaliana 3. Yet, TSN is stably
associated with SGs, suggesting that it may serve scaffolding role to recruit other proteins
to the mRNP complexes. Therefore we used TSN as bait in tandem affinity purification of
SGs-associated proteins. Localization of identified proteins to SGs in vivo has been further
verified by live imaging techniques. We have finally obtained a list of SGs-associated
proteins.
References:
1. Muench, et al. (2012) Wiley Interdiscip Rev RNA. 3(2): p. 178-94.
2. Anderson, et al. (2014) Biochim Biophys Acta.
3. Gutierrez-Beltran, E., et al. (2015). Plant Cell.
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Comunición Oral 3 / SIV CO3
New links between chromatin remodelling and photomorphogenesis in
Arabidopsis
Amr Nassrallah1, Sandra Fonseca1, Vicente Rubio1
1
Centro Nacional de Biotecnología- Consejo Superior de Investigaciones Científicas, Madrid,
Spain
Photomorphogenic plant growth while responding to environmental stimuli is a complex
process integrated through highly structured intracellular networks. These rely on tight
transcription coordination, achieved by chromatin remodelling synchronized with
transcription factor binding to DNA and their ubiquitination and proteolysis. The last step
of protein-targeted proteolysis is mediated, among others, by Cullin 4 RING Ligases
(CRL4s) associated to COP10-DDB1-DET1-DDA1 (CDDD) complexes. Recently we have
characterized DDA1 as a novel type of substrate adaptor for CRL4-CDDD (Irigoyen et al.,
2014). In an yeast two hybrid screening we found SGF11 as a DDA1 interactor. SGF11 is
a member of the SAGA (SPT-ADA-GCN5-Acetyltransferase) complex deubiquitination
module (DUBm). The SAGA complex provides a paradigm for multisubunit histone
modifying complexes because it combines two-histone-modifying activities: of
acetyltransferase and of ubiquitin protease. In yeast and animals the role of SAGA DUBm
on H2Bub (ubiquitinated histone 2B) deubiquitination to promote transcription is well
described. However, in plants, the function of SAGA DUBm remains unexplored.
Arabidopsis SAGA DUBm is composed of three subunits: SGF11, ENY2 and UBP22, with
UBP22 having the predicted catalytic function on H2B deubiquitination.
SGF11 is a nuclear protein and we could confirm its interaction in vivo with DDA1. By
performing TAP assays with tagged SGF11, we could recover ENY2 and UBP22,
suggesting that as well as in animals and yeast, the SAGA DUBm in Arabidopsis might
have a functional significance as a whole. Therefore we tested SGF11 on H2B
deubiquitination. As expected sgf11 mutant displays increased H2B monoubiquitination
levels compared to wild-type, while SGF11 overexpression complements sgf11 mutant
defects.
Previous reports showed that GCN5, a subunit of SAGA histone acetylation module acts
on light-responsive chromatin regions overlapping with HY5 binding sites (Barneche et al.,
2014). According to our results, the DUBm also plays a role in the control of plant
responses to light. Thus, under low light conditions, sgf11 displays lower hypocotyl length
than wt plants. In addition, sgf11 mutation enhanced the detiolated phenotype of det1-1
plants under dark conditions, whereas SGF11 overexpression ameliorated det1-1
photomorphogenic defects These phenotypes correlated with increased and reduced
accumulation of HY5 protein, respectively. Altogether our findings, indicate that SGF11
acts as a negative regulator of photomorphogenesis, and point to a role for H2B
deubiquitination in this process. We propose a model in which de DUBm facilitates
recruitment of CRL4-CDDD complexes to light-responsive promoter regions in order to
increase recognition and targeted degradation of HY5 to repress photomorphogenic
development.
References:
Irigoyen I, et al. (2014). Plant Cell 26, 712-728.
Barneche F, et al. (2014). J Exp Bot 65, 2895-2913.
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Arabidopsis COGWHEEL1 links light perception and gibberellins with
seed longevity
Eduardo Bueso, Jesús Muñoz, Gaetano Bissoli, Irene Martinez and Ramón Serrano.
Estrés abiótico, Ibmcp, Valencia, Spain
Light is a major regulator of plant growth and development by antagonizing gibberellins
(GA) and we provide evidence for a role of light perception and GA in seed coat formation
and seed longevity. We have identified two activation-tagging mutants of Arabidopsis
thaliana (cog1-2D and cdf4-1D) with improved seed longevity linked to increased
expression of COG1/DOF1.5 and CDF4/DOF2.3 respectively. These encode two highly
homologous DOF (“DNA-binding with One Finger”) transcription factors, with COG1 most
expressed in seeds. Resistance to seed deterioration was reproduced in transgenic plants
over-expressing these genes, and loss-of-function by RNA interference resulted in
opposite phenotypes. Over-expressions of COG1 and CDF4 have been described to
attenuate various light responses mediated by phytochromes and we found that phyA and
phyB mutants exhibit increased seed longevity. The additional seed longevity conferred
by gain-of-function of COG1 and by loss-of-function of phytochromes is of maternal origin
and correlates with a seed coat with increased suberin and reduced permeability. In
developing siliques of the cog1-2D mutant expression of the GA biosynthetic gene
GA3OX3 and levels of GA1 are higher than in wild type. These results underscore the
important role of GA in the reinforcement of the seed coat and explain the antagonism
between phytochromes and COG1 in terms of inhibition and activation, respectively, of GA
action.
101
XIII
RBMP
Poster 01 / SIV P01
Identification and characterization of the WUSCHEL-RELATED
HOMEOBOX gene family in Pinus pinaster
José M. Álvarez1, Natalia Bueno1, Rafael A. Cañas2, Concepción Ávila2, Francisco M.
Cánovas2, Ricardo J. Ordás1
1
Departamento de Biología de Organismos y Sistemas, Universidad de Oviedo, Spain.
2
Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, Spain.
In angiosperms, members of the WUSCHEL-RELATED HOMEOBOX (WOX) gene family
play an important role in key developmental processes, such as embryonic patterning,
stem-cell maintenance and organ formation. The genome of Arabidopsis (Arabidopsis
thaliana) contains 15 WOX genes. Phylogenetic analyses have identified three major
clades in the WOX gene family (van der Graaff et al., 2009): the modern clade/WUS clade
(AtWUS and AtWOX1-7), specific to seed plants; the intermediate clade (AtWOX8, 9, 11
and 12), present in vascular plants; and the ancient clade (AtWOX10, 13, and 14), with
representatives in the earliest diverging green plants and therefore probably representing
an ancestral WOX gene. The role of the WOX genes during plant development has been
studied to some detail in some angiosperms, but little information is available in conifers.
Recent works in Picea abies have shown functional conservation of some WOX genes in
conifers (Zhu et al., 2014; 2016; Alvarez et al., 2015). In this work, 13 members of the
WOX gene family, spanning all the major clades, have been identified in Pinus pinaster.
The expression pattern for each of the 13 WOX genes was studied during embryo
development and in different plant parts (shoot tip, root tip, cotyledons, and hypocotyl) by
quantitative real-time PCR or RNAseq. Similar to that described in angiosperms, members
of the ancient clade (PipsWOX13 and PipsWOXA) are expressed in most tissues and
developmental stages. PipsWOXB, PipsWOXC, PipsWOXD, PipsWOXE and PipsWOXF
(belonging to the intermediate), and PipsWOX2 (belonging to the modern clade) are
preferentially expressed during embryo development. In Arabidopsis, AtWOX8, AtWOX9
and AtWOX2 play important roles during the patterning and morphogenesis of the early
embryo. In angiosperms, some members of the modern clade are involved in stem-cell
regulation. WUS and WOX5 are involved in the maintenance of the shoot apical meristem
and root apical meristem, respectively. WOX4 is involved in the cambial meristem, while
WOX3 is involved in lateral organ development through recruiting organ founder cells
forming the lateral domain. Their homologues in P. pinaster PipsWUS, PipsWOX5,
PipsWOX4, and PipsWOX3 are preferentially expressed in shoot tip, root tip, vascular
cambium, and shoot tip respectively, suggesting that their functions could be conserved.
However, the identification of a new member of the modern clade, PipsWOXX, with no
homologues in angiosperms suggests a divergence of the WOX gene family after the split
between angiosperm and gymnosperms.
References:
Alvarez, JM, et al. (2015) New Phytol., 208(4): 1078-1088
van der Graaff, E, et al. (2009) Genome Biol., 10: 248-256
Zhu, T, et al. (2014). J. Exp. Bot., 65(22): 6543-6552
Zhu, T, et al. (2016). BMC Plant Biol., 16: 19-32
102
XIII
RBMP
Poster 02 / SIV P02
The signaling role of cyanide in root hair formation in Arabidopsis
thaliana
Lucía Arenas-Alfonseca, Cecilia Gotor, Luis C. Romero and Irene García
Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas,
Sevilla, España
In non-cyanogenic plants, cyanide is a co-product of ethylene and camalexin biosynthesis.
To maintain cyanide at non-toxic levels, Arabidopsis plants express the mitochondrial ßcyanoalanine synthase CAS-C1. CAS-C1 knockout is not toxic for the plant and leads to
an increased level of cyanide in the roots and leaves, a severe defect in root hair
morphogenesis (1) and an alteration on the plant immune response (2), suggesting that
cyanide acts as a signaling factor in these processes (3).
This work aims to clarify the CAS-C1 and cyanide function in root hair development. Three
approaches are presented here. First, a genetic approach is being carried out to elucidate
the site of action of CAS-C1 in the signaling pathway leading to the root hair development.
For this, we have used mutants in SCN1, a regulator of the root hair initiation, and in the
NADPH oxidase RHD2, which produces the superoxide anions necessary for the root hair
elongation. The CAS-C1 expression is being analyzed in these mutants and the
expression of RHD2 and SCN1 is being analyzed in the cas-c1 mutant. On the other hand,
rhd2 and scn1 mutants have been crossed with the cas-c1 mutant to establish the genetic
relationships (epistasy) between CAS-C1 and genes involved in the regulation of root hair
formation.
A second approach will analyze the spatiotemporal expression of CAS-C1 in the process
of root hair formation. For this, we have fused the CAS-C1 promoter with or without the
CAS-C1 ORF to the GFP reporter gene. These constructs have been introduced in plants
and the expression of the reporter gene driven by the CAS-C1 promoter observed by
fluorescence/confocal microscopy during the root hair development.
Finally, a proteomic approach is being performed in order to investigate post-translational
modifications of proteins in roots or isolated root mitochondria, produced directly or
indirectly by cyanide. This will establish a mode of action for this molecule.
Supported by grant no. BIO2013–44648–P.
References:
1. García I, et al. (2010) Plant Cell 22(10):3268-3279.
2. García I et al (2013). Plant Physiol. 162: 2015-27.
3. García I, et al (2014) Plant Signal. Behav. 7:12.
103
XIII
RBMP
Poster 03 / SIV P03
The ESCRT component FYVE1/FREE1 interacts with the PYL4 ABA
receptor and mediates its delivery to the vacuolar degradation
pathway
Borja Belda-Palazon1, Lesia Rodriguez1, Maria A. Fernandez1, Mari-Cruz Castillo1, Caiji
Gao2, Miguel Gonzalez-Guzman1, Geert de Jaeger3, Liwen Jiang2, José León1 and
Pedro L. Rodriguez1
1
Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones
Científicas-Universidad Politécnica de Valencia, 46022 Valencia, Spain 2Centre for Cell and
Developmental Biology and State Key Laboratory of Agrobiotechnology, School of Life Sciences,
The Chinese University of Hong Kong, China 3Department of Plant Systems Biology, Vlaams
Instituut voor Biotechnologie, Ghent University, Ghent, Belgium.
Removal of signaling/transport components from the plasma membrane and their delivery
to endosomal compartments is crucial for the regulation of cellular homeostasis.
Ubiquitylation of membrane proteins is a trigger for endocytosis and cargo trafficking to
the vacuole. Recently we described the ubiquitylation of PYL4 and PYR1 by the RING E3
ubiquitin ligase RSL1 in plasma membrane, which suggested that ubiquitylated ABA
receptors might be targeted to the vacuolar degradation pathway. In this study, we have
found that FYVE1/FREE1, which is a recently described component of the ESCRT
machinery, interacts with RSL1-PYL4 or RSL1-PYR1 complexes and recruits ABA
receptors to endosomal compartments. The biological relevance of the ESCRT pathway
for the modulation of ABA signaling is illustrated by the ABA-hypersensitive phenotype of
knock-down fyve1 alleles. fyve1 mutants are impaired in the endosomal transit/vacuolar
degradation of ABA receptors, which leads to higher accumulation of PYL4 and enhanced
response to ABA. Thus, pharmacological and genetic approaches reveal a dynamic
turnover of ABA receptors from plasma membrane to the endosomal/vacuolar degradation
pathway mediated by FYVE1 and dependent on RSL1 as well as the trafficking of PYL4
through the ESCRT pathway, which serves to regulate the turnover of ABA receptors and
to attenuate ABA signaling.
104
XIII
RBMP
Poster 04 / SIV P04
The R domain, a novel and unknown motif of TCP proteins
Isabel Domínguez1, Florian Chevalier1 and Pilar Cubas1
1
Centro Nacional de Biotecnología (CNB-CSIC) Dpto. de Genética Molecular de Plantas. c\
Darwin. 3 Campus Universidad Autónoma de Madrid Cantoblanco 28049 Madrid, Spain.
The BRANCHED1 (BRC1) gene encodes a transcription factor of the TCP family. The
BRC1 protein has two conserved domains typical of class II TCP transcriptional
regulators1: the TCP and the R domains. The R domain is rich in arginine (Arg, R) residues,
and is predicted to form an α-helix. To date, nothing is known about the function of this
domain.
To investigate this, we generated mutant proteins (BRC1*) with alterations in this motif: we
made C-t deletions lacking the R domain (BRC1∆R), point mutations in Serine282, a residue
potentially phosphorylable (BRC1S282D, and BRC1S282R) and a triple point mutant,
BRC1RRK290 IGE, in three highly conserved residues of the domain. In addition, we are
generating mutants in the R domain using the CRISPR/Cas9 system. The goal is to create
small in-frame deletions that remove a part of this domain but leave intact the rest of the
protein and the gene is expressed in its wild type domains of expression.
We are now analyzing the activity of these mutant proteins in transient assays in Nicotiana
and in Arabidopsis transgenic lines. In Nicotiana, we are testing how the mutations affect
BRC1 transcriptional activity using a promoter:LUC reporter assays combined with
estradiol-inducible (pMDC7:GFP:BRC1*) constructs. We are also assaying protein-protein
interactions using 35S:BRC1*:GFP and confirmed BRC1 protein partners. In estradiolinducible pMDC7:GFP:BRC1 transgenic lines we are testing BRC1 stability and nuclear
location. Finally we are comparing the phenotypes of 35S:BRC1 vs 35S:BRC1* lines and
pMDC7:GFP:BRC1 vs pMDC7:GFP:BRC1* to determine whether loss of the R domain
leads to a loss or a gain of BRC1 function. In our CRISPR/Cas9 mutagenesis we have
isolated a plant with a single nucleotide insertion in the R domain that generates a frameshift and consequently a truncated protein, but we are currently searching for additional
point mutations in the T2 generation.
Preliminary evidence indicates that the R domain may play a important role in the negative
control of the BRC1 protein. Thus, mutations in the R domain would generate BRC1 gainof-function mutants of great interest to increase lateral branch suppression in many crop
species.
References:
Cubas, P., et al. (1999) Plant Journal 18(2), 215-222.
105
XIII
RBMP
Poster 05 / SIV P05
Regulation of SAM activity by DELLAs through their interaction with
CIN-TCPs and KNAT1
Felipo-Benavent, Amelia1; Blanco-Touriñan, Noel1; Rubio-Somoza, Ignacio2; Blázquez,
Miguel A1; Alabadí, David1.
1
Instituto de Biología Molecular y Celular de Plantas (CSIC-Universidad Politécnica de
Valencia), Ingeniero Fausto Elio, s/n 46022 Valencia, Spain.2Centre for Research in
Agricultural Genomics (CRAG), c/ de la Vall Moronta, edifici CRAG, Bellaterra 08193
Barcelona, Spain.
In plants, new aerial organs are continuously generated by cells in the shoot apical
meristem (SAM). Coordination of cell division and differentiation in this context is essential
for plant development. KNOX genes are required for meristem maintenance and their
expression is accordingly confined to these meristematic cells, while their ectopic
expression favours the appearance of ectopic meristems (Hay et al., 2002). Therefore,
there are several mechanisms by which plants down-regulate KNOX expression in organ
primordia. A very important one is the repression exerted by ASYMMETRIC LEAVES1
(AS1), which is in turn activated by CIN-TCP transcription factors (Koyama et al., 2010;
Guo et al., 2008).
KNOX transcription factors have been proposed to maintain meristem function by both
preventing gibberellin biosynthesis and promoting gibberellin inactivation and therefore
DELLA accumulation. Here we describe two mechanisms by which DELLA proteins exert
a positive feedback loop on the KNOX gene KNAT1/BREVIPEDICELLUS (BP): First,
DELLA proteins interact physically with virtually all protein members of the CIN-TCP family
and impair their transcriptional activity, probably by a sequestration mechanism. As a
consequence, higher DELLA levels lead to a decreased AS1 and an increased KNAT1/BP
gene expression. And second, DELLA and KNAT1/BP proteins also interact, and RNAseq experiments indicate that they might regulate jointly the expression of a set of genes.
These results suggest that gibberellins are involved in the homeostasis of KNOX activity
at the shoot apical meristem, and probably coordinates the production of undifferentiated
meristem cells with the production of new organs.
References:
1. Hay A et al .(2002) Current Biology 12:1557–65.
2. Koyama et al .(2010) The Plant cell 22(11):3574-88.
3. Guo M et al (2008) The Plant cell 20(1):48-58.
106
XIII
RBMP
Poster 06 / SIV P06
bZIP transcription factors as central nitric oxide sensors
María Guadalupe Fernández-Espinosa1, Inmaculada Sánchez-Vicente1, Isabel Mateos1,
Oscar Lorenzo1
1
Dpto. de Botánica y Fisiología vegetal, Instituto Hispano-Luso de Investigaciones Agrarias
(CIALE), Facultad de Biología, Universidad de Salamanca, 37185 Salamanca, Spain.
Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of many
different processes throughout plant development, comprising seed dormancy, seed
germination, postgerminative vegetative growth, flowering, stomatal aperture and
response to pathogens, among others. Plants have developed mechanisms for NO
sensing to trigger a variety of specific responses in their life cycle1. Remarkably, Snitrosylation has been suggested as one of the most relevant posttranslational
modifications regulated by NO through which plants are able to sense this gaseous signal.
In this way, there are NO-dependent protein alterations which have been described as
modifying protein function or stability.
Arabidopsis thaliana bZIP (basic region/leucine zipper) transcription factors regulate
diverse biological processes such as pathogen defense, light and stress signaling, seed
maturation and flower development. There are ten defined groups of bZIP proteins based
on sequence similarity of the basic region and the presence of additional conserved motifs.
Among them, ABI5 (ABA-INSENSITIVE 5) and TGA1 (TGACG motif binding factor 1) have
been suggested to be regulated by NO. ABI5 S-nitrosylation promotes the interaction with
CUL4-based and KEG E3 ligases and consequently, ABI5 is rapidly degraded by the
proteasome during seed germination2. TGA1 is also S-nitrosylated in vitro after Snitrosoglutathione (GSNO) treatment. The GSNO-dependent modifications probably result
in conformational changes of TGA1, which allow a more effective TGA1–NPR1 interaction
and enhanced DNA-binding of TGA1, emphasizing the role of NO in the plant defense
response3.
To deepen the hypothesis that members of the bZIP family are key to the sensing of NO
within the cell, in this work we make an in silico study to analyze if essential cysteines
residues of TGA1 are conserved in other bZIP members and if these cysteines could be
potentially S-nitrosylated. We present data about the characterization of another bZIP and
their relationship with NO in different developmental processes, studying the phenotypes
when loss-of-function and gain-of-function bZIP lines grow in the presence of the NO
scavenger, cPTIO (2- (4-carboxyphenyl) -4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide) or
the NO donor, SNAP (S-Nitroso-N-Acetyl-D,L-Penicillamine). In addition, we have studied
how bZIP transcriptional levels are modified during treatment with NO pharmacological
compounds. Finally, insights in the potential S-nitrosylation of this bZIP in vitro after GSNO
treatment will be presented.
This work is financed by grants: ERC.KBBE.2012.1.1-01 (EcoSeed-311840). MINECO: (BIO2014-57107R), CONSOLIDER (CSD2007-00057). Junta de Castilla y León (SA239U13).
References:
1
Gibbs D.J, et al. (2014) Mol. Cell, 53; 369-379.
Albertos P, et al. (2015) Nature Commun, 23; 6:8669.
3
Lindermayr C, et al. (2010) Plant Cell, 22. (8): 2894:2907.
2
107
XIII
RBMP
Poster 07 / SIV P07
Negative regulation of autophagy by sulfide in Arabidopsis thaliana is
independent of reactive oxygen species
Ana M. Laureano, Inmaculada Moreno, Luis C. Romero, Cecilia Gotor
Sevilla, España
Accumulating experimental evidence in mammalian, and recently plant, systems has led
to a change in our understanding of the role played by hydrogen sulfide in life processes.
In plants, hydrogen sulfide mitigates stress and regulates important plant processes such
as photosynthesis, stomatal movement, and autophagy, although the underlying
mechanism is not well known. In this study, we provide new experimental evidence that,
together with our previous findings (Álvarez et al, 2012), demonstrates the role of hydrogen
sulfide in regulating autophagy. We used green fluorescent protein fluorescence
associated with autophagic bodies and immunoblot analysis of the ATG8 protein to show
that sulfide (and no other molecules such as sulfur-containing molecules or ammonium)
was able to inhibit the autophagy induced in Arabidopsis thaliana roots under nitrogen
deprivation. Our results showed that sulfide was unable to scavenge reactive oxygen
species generated by nitrogen limitation, in contrast to well-established reducers. In
addition, reducers were unable to inhibit the accumulation of autophagic bodies and ATG8
protein forms to the same extent as sulfide (Laureano et al, 2016). Therefore, we conclude
that sulfide represses autophagy via a mechanism that is independent of redox conditions.
References:
Laureano-Marín A. M, et al. (2016) Plant Physiol. doi:10.1104/pp.16.00110
Álvarez C. et al. (2012). Plant Cell.24: 4621-4634.
108
XIII
RBMP
Poster 08 / SIV P08
ANAC089, a new componente of ABA/NO signalling involved in seed
germination and stress
Isabel Mateos1, Pablo Albertos1, Kiyoshi Tatematsu2, Alejandro Fernández-Arbaizar1,
Kazumi Nakabayashi3, Eiji Nambara4, Marta Godoy5, José M. Franco5, Roberto Solano5,
Carlos Perea6, Julio Salinas6, Thomas Roach7, Erwann Arc7, Ilse Kranner7 and Oscar
Lorenzo1
1
Dpto. de Botánica y Fisiología Vegetal, CIALE, Universidad de Salamanca, Salamanca, Spain,
2
Laboratory of Plant Organ Development, National Institute for Basic Biology, Okazaki, Japan,
3
School of Biological Sciences, University of London, Egham, United Kingdom, 4Department of
Cell & Systems Biology, University of Toronto, Toronto, Canada, 5Dpto. de Genética Molecular
de Plantas. CNB-CSIC, Madrid, Spain, 6Dpto. de Biología Medioambiental. CIB-CSIC, Madrid.
Spain,7Institut für Botanik, Innsbruck, Austria
Seed dormancy and germination are complex traits regulated by the interaction of different
signalling molecules such as abscisic acid (ABA) and nitric oxide (NO)1. In order to
elucidate their crosstalk, a genetic screening in presence of (+)-S-ABA coupled to NO
scavenger (cPTIO) was performed. In this screening we identified two mutants gap1 and
gap2 (germination in ABA and cPTIO1 and 2)2, that show ABA and cPTIO-insensitive
phenotypes in the transition from dormancy to germination. After characterization and
positional cloning of both of them, we found that GAP2 encodes ABI5 and GAP1 the
ANAC089 transcription factor. anac089 mutants lack the critical transmembrane domain
of ANAC089 protein that confers the mutated proteins constitutive nuclear localization.
Interestingly, activity of ANAC089 is regulated by mono-ubiquitinatination and this
modification has been suggested to regulate its traffic to the nucleus. We also
demonstrated that mutants lacking the membrane-related domain of ANAC089 displayed
ABA, salt osmotic and cold stress insensitivity revealing a repressor function of ABA and
abiotic stresses responses. Furthermore, mutants exhibited higher endogenous NO levels
avoiding the effect of NO-depletion during seed germination. In addition, whole-genome
transcriptional profiling indicated the existence of different groups of ABA- and redoxrelated genes that are differentially regulated by ANAC089. According with this idea, a thiol
and disulphide analysis revealed that mutants presented differences in the ratio of
GSH/GSSG, compared to the corresponding parental lines. This transcription factor can
specifically bind to the core cis-regulatory element GCGTCAGC harbour in the promoters
of ANAC089 regulated genes. Consistently, translocation of ANAC089 protein to the
nucleus was directed by changes in cell redox status after NO- and redox-related
compound treatments. Collectively, our results indicate that ANAC089 transcription factor
integrates ABA signalling with NO levels to modulate redox homeostasis as a novel master
regulator during seed germination and abiotic stresses in Arabidopsis.
Supported by Fundación memoria de Don Manuel Solorzano Barruso
(FS/8-2015), USAL.
ERC.KBBE.2012.1.1-01 (EcoSeed-311840). MINECO: (BIO2014-57107-R), CONSOLIDER (CSD200700057). Junta de Castilla y León (SA239U13)
References:
1
2
Sanz, l, et al. (2015) J Exp Bot. 66(10): 2857-68.
Albertos, P, et al. (2015) Nat. Commun , DOI: 10.1038/ncomms9669.
109
XIII
RBMP
Poster 09 / SIV P09
Strigolactones are involved in Pinus pinaster branching and apical
dominance signaling
Álvaro Calderón1, Isabel Feito2, Carolina DelaTorre2, Jesús Pascual1, Francisco J.
Colina1, Luis Valledor1, Juan Majada3, Ana Rodríguez1, María Jesús Cañal1, Mónica
Meijón1
1
Plant Physiology, Faculty of Biology, University of Oviedo, Asturias, Spain. 2Regional Institute
for Research and Agro-Food Development in Asturias (SERIDA), Experimental Station “La
Mata”, Asturias, Spain. 3CETEMAS, Asturias, Spain.
Polycyclism is the manifestation of more than a growth cycle in the same annual period,
increasing the number of branches within a tree. This behavior occurs in some forest
species such as Pinus pinaster, reducing the wood quality and having a high impact over
timber industry. The branching stem is controlled by various plant regulators. Among them,
a newly described group of plant hormones, the strigolactones, seem to have a key role in
the control of apical dominance and branching. Recently, new genes involved in
strigolactones signaling in Arabidopsis have been described (Beveridge et al., 2010);
however, there is scarce information about the physiological and molecular implications of
these hormones in forest species. In this work, a brief phylogenetic study including the
major genes in strigolactone signaling pathway (AXR1, MAX1, MAX2, MAX3 and MAX4)
was performed in order to determine the most conserved gene sequence between species
(11 species were studied) for later analyses. Through designing of degenerated primers
considering Arabidopsis thaliana, Populus trichocarpa and Pinus taeda sequences,
putative sequence of P. pinaster MAX1 and AXR1 were obtained. Additionally, two-yearsold P. pinaster seedlings were treated in dominant and/or lateral buds with a synthetic
strigolactone (GR-24) and/or auxin transport inhibitor (NPA) in different combinations.
Elongation of dominant and lateral shoots were measured 30 days after the treatment,
showing these data that only when GR-24 was applied over the lateral bud and NPA was
not used, dominant shoot significantly increased elongation, while lateral shoot almost
stopped to growth. The expressions of PpMAX1 and PpAXR1 were quantified by qPCR in
apical and lateral bud of all the treatment applied, showing both genes high expression
levels in lateral bud. Moreover, the quantification of the expression levels of these genes
in field samples from different provenances of P. pinaster that show different degrees of
polycyclism, validated the use of these genes as biomarkers of wood quality. Those
provenances with high frequency of polycyclism growth (this mean high branching)
showed low level of expression of these genes, while provenances with low polycyclism
(low branching) showed high expression levels of PpAXR1 and PpMAX1. Furthermore, by
fluorescence in situ hybridization analyses it was identified the exact location of the
expression of both genes in lateral bud tissues.
This work demonstrates a relation between the strigolactones, auxins and the degree of
branching and apical dominance in P. pinaster. Also, it is a first molecular approach to the
determination of the apical dominance and branching signaling in P. pinaster, providing a
knowledgebase that will benefit not only forest developmental biology, but also tree
breeding programs since branching is one of the current main challenges in forest
management.
Supported by funds from INIA and FEDER co-funding through the project RTA 2013-00048-C03-02.
References:
Beveridge CA, et al (2010). Curr Opin Plant Biol. 13, 34-39.
110
XIII
RBMP
Poster 10 / SIV P10
CSK regulates the SIG5-mediated expression of PSII genes and
promotes UV stress resistance
Jesús Pascual1, Mónica Meijón1, Moona Rahikainen2, Mónica Escandón1, Saijaliisa
Kangasjärvi2, María Jesús Cañal1, Luis Valledor1
1
Plant Physiology Lab, Organisms and Systems Biology, Faculty of Biology, University of Oviedo,
Oviedo, Asturias, Spain, 2 Department of Biochemistry, Molecular Plant Biology, University of
Turku, FI-20014 Turku, Finland
Chloroplasts and photosynthetic machinery act as sensors of the physiological state of the
cell. Under an environmental stress, an imbalance in the photosynthetic electron transport
chain is generated, leading to a chloroplastic oxidative burst that triggers the stress
responsisve mechanisms. In this context, the regulation of photosynthesis stoichiometry
plays a paramount role. In fact, it is one of the main mechanisms involved in the adaptation
to environmental stresses. The CHLOROPLASTIC SENSOR KINASE (CSK) is the main
regulator of this process, regulating the expression of PSI genes in a redox-dependent
manner through the interaction with the SIGMA FACTOR 1 (SIG1). CSK and the regulation
of the photosynthesis were revealed as key for UV stress response in an integrated
physiological, proteomic and metabolomic analysis of UV stress in Pinus radiata (Pascual
et al., 2016a; Pascual et al., 2016b). However, the performance of mutant-based studies
in pine have several drawbacks regarding the generation times and the inexistance of
collections of mutants. In consequence, it was decided to take advantage of the mutants
available for the model plant Arabidopsis and use it as a platform for the functional studies
about CSK. So, in this work, we performed UV and oxidative stress sensitivity assays using
a partial silencing and an overexpressing Arabidopsis CSK mutants obtained from SALK
colection. Moreover, we characterized their photosystems by lpBN-PAGE and 2-DE and
tested the interactions of CSK with the 6 SIG factors encoded in the Arabidopsis genome.
Preliminary results support the involvement of CSK in UV stress, and the interaction of
CSK with SIG5, previously unreported, along with the results of the 2-DE, a role of CSK in
regulating PSII genes and a more complex and relevant role in chloroplast signalling and
phosphorylation networks than previously expected and reported. Mutant rescue assays
with PrCSK will validate the obtained results for radiata pine and will open the door to the
use of CSK as a marker for the selction of trees more resistant to UV stress.
References:
Pascual J et al. (2016a). J Proteomics (in press)
Pascual J et al. (2016b). Under review
111
XIII
RBMP
Poster 11 / SIV P11
Functional characterization of Arabidopsis AMIDASE1 (AMI1) in IAA
biosynthesis
Beatriz Sánchez-Parra1, Stephan Pollmann1.
1
Centre for Plant Biotechnology and Genomics (CBGP) U.P.M., Madrid, Spain.
Plant growth and development is regulated in great parts by phytohormones, which are
small signalling molecules of various chemical natures, acting at sub-micromolar
concentrations. One of them is the plant hormone indole-3-acetic acid (IAA), considered
as the most abundant naturally occurring auxin. IAA is controlling many aspects of plant
development, e.g. cell division, apical dominance, senescence, etc. (Davies, 2004). Much
is known about the physiology of auxin, however, how IAA is biosynthesized remains, at
least in parts, unclear. There is evidence for different pathways involved in IAA production
that work either in parallel or in a redundant manner. They are principally classified in two
groups: Tryptophan independent- and dependent-pathways. In the latter group, we can
find a number of different pathways. One of them, designated by its hallmark-intermediate,
is called the IAM-pathway. Herein, indole-3-acetamide (IAM) is converted to IAA by the
virtue of AMIDASE1 (AMI1) (Pollmann et al., 2003). However, so far, this conversion has
only been demonstrated in vitro.
Here, we provide in vivo-evidence that AMI1 is capable of influencing auxin contents by
analysing different knock out (k.o.) mutants as well as inducible over-expressor lines for
AMI1.
Moreover, we investigated the distribution and the enzymatic properties of AMI1-like
proteins in the plant kingdom, disclosing a widespread occurrence of AMI1-like proteins
and very similar characteristics for the amidases studied. These results suggest a
conserved and important role of this enzyme in plant development.
In addition, comprehensive whole-genome wide transcriptomics analyses were used to
characterize AMI1 functionally, comparing wild-type Arabidopsis, with ami1-2 and AMI1ind
lines. In these enrichment assays, we found groups of genes involved in plant defense
responses towards pathogen attacks and genes participating in related phytohormonessignalling pathways [e.g. the jasmonic acid (JA)], being overrepresented. For instance, in
the ami1-2 mutant that contains increased IAM- and decreased IAA contents, we
discovered an overexpression of several JA biosynthesis-associated genes. This induction
of JA biosynthesis does not occur in AMI1ind, where IAM is effectively converted into IAA.
This suggests an important role for AMI1 in maintaining the balance between IAM and IAA
in plants.
References:
Davies, PJ (2004). Kluwer Academic Publishers, Dordrecht, Boston, London.
Pollmann, S, et al. (2003). Phytochemistry. 62(3):293-300.
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Poster 12 / SIV P12
SUMO modulates senescence through the control of the ethylenesignaling pathway in Arabidopsis
Arnaldo L. Schapire1, Abraham Mas1, and L. Maria Lois1
1
Center for Research in Agricultural Genomics-CRAG, Edifici CRAG-Campus UAB, Bellaterra
(Cerdanyola del Vallés), 08193 Barcelona, Spain
In plants, senescence represents the final stage of leaf development. Several endogenous
and environmental signals control the intricate and highly orchestrated process of leaf
senescence. All major plant hormones have been reported to affect leaf senescence, with
ethylene, jasmonic acid, salicylic acid, abscisic acid, and brassinosteroids as inducers and
with cytokinins, gibberellic acid, and auxin as inhibitors (Jibran et al, 2013).
Ethylene is a volatile hormone that regulates many physiological processes including
induction of leaf senescence. The EIN3 transcription factor positively regulates leaf
senescence by activating at least two key regulators of leaf senescence, ORE1 and AtNAP
(Kim et al, 2014). Knowledge of ethylene signaling pathway has evolved from an initial
linear transduction cascade to a more complex regulatory network including feedback
regulations, multiple levels of protein stability control, and broad existence of signaling
interplay and integration (Zhao and Guo, 2011).
SUMO (small ubiquitin-related modifier) conjugation (i.e., sumoylation) to protein
substrates is a reversible posttranslational modification that regulates protein function
(Park and Yun, 2013). In this work, we present evidence that SUMOylation negatively
regulates ethylene signaling, which represents a novel regulatory mechanism to be added
to the highly complex ethylene signaling network. Among the potential SUMO targets in
the ethylene signaling pathway, we will show a major role of SUMO conjugation regulating
the key transcription factor EIN3. We will present and discuss data related to the molecular
consequences of SUMOyaltion in EIN3 activity and its implications in leaf senescence.
References:
Jibran R, et al. (2013) Plant. Mol. Biol., 82(6): 547-561.
Kim HJ, et al. (2014). J. Exp. Bot., 65(14): 4023-4036.
Park HJ, et al. (2013) Int. Rev. Cell. Mol. Biol., 300: 161-209.
Zhao Q, et al. (2011) Paradigms and paradox
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Poster 13 / SIV P13
Organ-specific evolution and subfunctionalization of circadian clock in
Petunia
Marta Isabel Terry López1, Claudio Brandoli1, Marta Carrera-Alesina1, Marcos EgeaCortines1, Julia Weiss1
1
Genética Molecular, Instituto de Biotecnología Vegetal, Universidad Politécnica de Cartagena
30202 Cartagena, Spain
The plant circadian clock is comprised by a set of genes that form the so-called loops.
These loops are molecular and genetic interactions of activation and repression, and are
classified as morning, central and evening loops based on the expression of their
components in Arabidopsis leaves. We have performed a comprehensive analysis of the
clock components in Petunia1 and found that PRR7, PRR5, GI, ELF3 and ELF4 show gene
copy numbers ranging from 2 to 4. In contrast, PRR9, PRR3, TOC1, ZTL and LHY are
single copy gene while CCA1 is absent. This suggests possible subfunctionalizations of
different paralogs. We investigated the transcription of circadian clock genes in leaves and
petals of Petunia and found highly significant difference in gene expression in single copy
genes such as PRR9 and ZTL. The differences identified included changes in their
circadian expression pattern and level of expression. The paralogs PRR7a and PRR7b
showed differences of expression between leaves and flowers in terms of daily expression
profiles and expression levels. A similar effect was seen in PRR5a and PRR5b as PRR5a
was 159 fold higher in petals than in leaves. Our results shows that the plant circadian
clock is different in petals and leaves, and there is a possible subfunctionalization of
paralogs that become specific for the leaf or petal.
This work was developed under projects Fundación Séneca 11895/PI/09, 19398/PI/14 and BFU-201345148-R
References:
1.
Bombarely, A. et al. Nat. Plants In press, (2016).
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Poster 14 / SIV P14
Seed-specific transcription factor HSFA9 links embryogenesis and
photomorphogenesis
Pilar Prieto-Dapena1, Concepción Almoguera1, José-María Personat1, Francisco
Merchan2 and Juan Jordano1
1
Departamento de Biotecnología Vegetal, Instituto de Recursos Naturales y Agrobiología de
Sevilla. Consejo Superior de Investigaciones Científicas (CSIC). 41012 Seville, Spain.
2
Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla,
41012 Seville, Spain.
HSFA9 is a seed-specific transcription factor involved in desiccation tolerance and seed
longevity (Prieto-Dapena et al., 2006; 2008). In sunflower plants, HSFA9 disappears a few
days after seed germination (Almoguera et al., 2002). Here we uncover a connection
between HSFA9 and the initial acquisition of photosynthetic competence, which occurs
following seed germination and seedling emergence. The constitutive overexpression of
HSFA9 in the 35S:A9 tobacco seedlings enhanced expression of genes that are relevant
for early light signaling (as the phytochromes PhyA and PhyB), and for light-dependent
synthesis of chlorophylls [for example, a NADPH:protochlorophyllide oxidoreductase
(POR, EC 1.3.1.33)]. The 35S:A9 seedlings also showed reduced expression of a crucial
photomorphogenesis repressor (CONSTITUTIVE PHOTOMORPHOGENIC 1, COP1).
Similar effects were observed in dark-imbibed seeds of DS10:A9 lines, where the
overexpression of HSFA9 occurs in a time window similar to that for the expression of
HSFA9 in sunflower. In the 35S:A9 seedlings, HSFA9 enhanced PhyA- and PhyBdependent light signaling, as shown by intensified hypocotyl length reduction under
continuous far-red and red light, respectively. Following exposure to white light, HSFA9
accelerated the initial photosynthetic development of the 35S:A9: seedlings. This occurred
by augmenting the accumulation of chlorophyllide and chlorophyll, leading to earlier
unfolding of the cotyledons. Converse effects on gene expression, greening, and
cotyledon unfolding were observed using a dominant-negative form of HSFA9 expressed
within the same time window as for DS10:A9. Our results demonstrate a transcriptional
link between late embryogenesis and early photomorphogenesis that involves HSFA9, a
transcription factor acting below the top hierarchical regulators of embryogenesis. We
conclude that, in developing seeds and before the first exposure to light, HSFA9 enhances
gene expression relevant for photomorphogenesis. HSFA9 subsequently boosts lightresponses that promote early greening. HSFA9 would thus help facilitating quick seedling
establishment after seedlings emerge from the soil and are exposed to light.
References:
Almoguera, et al. (2002) J. Biol. Chem. 277(46): 43866-43872.
Prieto-Dapena, et al. (2006). Plant Physiol. 142(3): 1102-1112.
Prieto-Dapena, et al. (2008). Plant J. 54(6): 1004-1014.
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Poster 15 / SIV P15
Nitric oxide (NO) sensing in seeds
Inmaculada Sánchez-Vicente1, Pablo Albertos1, Isabel Mateos1, María Romero-Puertas2,
Kiyoshi Tatematsu3, Eiji Nambara4, Óscar Lorenzo1
1
Dpto. de Botánica y Fisiología Vegetal, Instituto Hispano-Luso de Investigaciones Agrarias
(CIALE), , Universidad de Salamanca, Salamanca, Spain, 2Dpto. de Bioquímica, Biología Celular
y Molecular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones
Científicas, Granada, Spain, 3Laboratory of Plant Organ Development, National Institute for Basic
Biology, Okazaki, Japan, 4Department of Cell and Systems Biology, University of Toronto,
Toronto, Canada
Seed germination and nitric oxide sensing are two concepts of key relevance in our
research context. Seeds constitute a vital stage along the plant life cycle, leading the adult
plant to transmit it genetic background to the next generation. Seed germination (sensu
stricto) can be defined as a set of physiological and molecular events initiated by the first
water uptake in non-dormant seeds, finishing with the radical protrusion after the rupture
of the external envelopes. Nitric oxide (NO) constitutes a signalling molecule implicated in
the regulation of a plethora of different processes, beginning at the first developmental
stages: seed maturation, dormancy and germination and early seedling growth (1,2). Our
recent research is focused on how NO regulates seed germination in a complex signalling
pathway through the posttranslational modification of specific proteins by S-nitrosylation
and how this tight checkpoint influences the interaction with other plant growth regulators
(i.e. abscisic acid, ABA). To this purpose, we examine the fundamental role of different
basic region/leucine zipper motif (bZIP) transcription factors, which have been reported to
be of great relevance along the plant life cycle regulation. The identification of these bZIPs
as key NO targets in plant growth and development processes help us to understand the
function of NO in plant signal transduction, establishing a molecular framework during seed
development and germination. We found that ABI5 plays an important role, in the ABA-NO
crosstalk during seed germination (3). Whereas ABA induces ABI5, NO promotes its
degradation in a proteasome dependent manner. By using pharmacological and genetic
approaches, we describe how the posttranslational modification of ABI5 protein in specific
Cys residue can modulate its stability, leading to the promotion of seed germination. The
molecular pathways by which NO selectively modify specific Cys residues impact on
protein function, stability or location, but the real potential of all these changes remain
poorly understood. Thus, we analyze the Cys conservation along the bZIP transcription
factor family, and focus on another bZIP, which presents the ABI5 key Cys residue. By
means of phenotypic and molecular characterization we explore the functional features of
NO posttranslational modification inside this protein and the physiological effects during
seed maturation.
Grants: BIO2014-57107-R and CSD2007-00057 (TRANSPLANTA) from the MINECO (Spain), Impacts
‘EcoSeed-311840’ ERC.KBBE.2012.1.1-01 and SA239U13 from Junta de Castilla y León
References
1. Fernández-Marcos, M, et al.(2011) Proc. Nat. Acad. Sci. USA, 108:18506-18511
2. Sanz, L,et al. (2014) Plant Physiol. 166(4):1972-84
3. Albertos, P, et al. Nature Commun. 23; 6:8669
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Comunicaciones Sesión V.
Estrés Abiótico
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Ponencia Invitada / SV PI
The At3P protein family plays an essential role in response to different
abiotic stresses
Nuria Fernández*, Lourdes Fernández*, Alfonso Muñoz and M. Mar Castellano
Centro de Biotecnología y Genómica de Plantas, INIA-UPM, Pozuelo de Alarcón, España
Plants as sessile organisms are constantly exposed to a wide spectrum of stress
conditions, and so, they have developed sophisticated mechanisms to cope with different
environmental challenges.
In this study, we have characterized the function of the different members of the At3P
family in the response to different abiotic stresses. This family has 3 members in
Arabidopsis that are characterized by the presence in their sequences of three TPR
domains. These domains have been involved across eukaryotes in protein-protein
interactions. Despite some homologs in other species are involved in aging and in the
protection from prion diseases, the role of the 3P family in the plant response to abiotic
stresses has not been addressed before.
During this research, we have found that 3P3 plays an essential role during the unfolded
protein response (UPR). Our analysis demonstrates that this protein is induced by different
UPR inducer agents and it is partially localized in the endoplasmic reticulum (ER).
Furthermore, different T-DNA insertion mutants with reduced expression of At3P3 show a
marked hypersensitive phenotype to UPR inducers. This phenotype is alleviated in the
presence of a chemical chaperone, suggesting a main role of 3P3 in assisting protein
folding during UPR.
Despite 3P3 is the only member of the family highly induced by heat, the analysis of the
3p1/3p2/3p3 triple mutants demonstrates that the three members of the family act together
to promote long term acquired thermotolerance in plants. This function seems to be
partially linked to the possible role of 3Ps in protein folding through their interactions with
different cytosolic chaperones. Apart from their role in the maintenance of protein
homeostasis, a large fraction of the bulk of these proteins is also localized at the nucleus
under heat challenge. This change in their subcellular localization suggests that they could
also have a different role in transcriptional regulation, an aspect that we are currently
exploring.
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Comunicación Oral 1 / SV CO1
Plasma membrane lipid remodeling during cold acclimation is
mediated by the ER-PM contact sites-localized synaptotagmins 1 & 3
Jessica Pérez-Sancho1, Abel Rosado2, Arnaldo L. Schapire1, Noemí Ruiz-López1, Sonia
Osorio1 Steffen Vanneste3, Lothar Willtmizer4, Carlos Perea5, Julio Salinas5, Miguel
Ángel Botella1
1
Departamento de Biología Molecular y Bioquímica, IHSM (Universidad de Málaga-CSIC),
Málaga, Spain, 2 Department of Botany, Faculty of Sciences, University of British Columbia,
Vancouver, Canada, 3Department of Plant Systems Biology, VIB-Ghent University, Ghent,
Belgium, 4Central Metabolism Group, Max Planck Institute of Molecular Plant Physiology,
Potsdam-Golm, Germany, 4Departamento de Biología Medioambiental, Centro de
Investigaciones Biológicas (CSIC), Madrid, Spain.
Cold acclimation is the capacity of certain plants to increase their freezing tolerance in
response to a period of low non-freezing temperatures. Cold acclimation involves a series
of biochemical and physiological adaptations, including a deep transcriptional
reprogramming and drastic changes in the lipid composition of cellular membranes in order
to prevent the freeze-induced damage (1). While a profound knowledge has been acquired
on the regulation of gene expression triggered by cold-acclimation, very little is known
about the mechanisms governing the cold-induced changes in membranes’ lipid
composition.
In this study we report that in Arabidopsis, the constitutively expressed Synaptotagmin 1
(SYT1) and the cold-induced homolog Synaptotagmin 3 (SYT3) are essential for coldacclimated freezing tolerance and for the lipid remodelling of the plasma membrane during
cold-acclimation. SYT1 and SYT3 are phospholipid-binding proteins located in
Endoplasmic Reticulum-Plasma Membrane contact sites (ER-PMcs), conserved
structures defined as regions of the cortical ER in close apposition to the PM (2). ER-PMcs
facilitate the non-vesicular lipid transport between ER and PM in yeast and mammals, and
are essential for lipid homeostasis (3). In contrast to the high and ubiquitous SYT1
expression, SYT3 expression is low and mainly restricted to meristemoids, young stomata,
and old primary root. TIRF microscopy analyses show that during cold acclimation there
is an increase of SYT1::SYT1:GFP and SYT3::SYT3:GFP signals as spots at the PM.
High-resolution lipidome analyses show the over-accumulation of phosphatidylinositols
phosphate (PIPs) and glycerolipids in vivo in syt1 and specially syt1/syt3 mutant plants
compared to WT in one-week cold-acclimated plants. Interestingly, protein-lipid overlay
assays (membrane-strips and PIP-strips) reveal PIPs and glycerolipids as major
interactors for both, SYT1 and SYT3. Here we show that 1) Arabidopsis SYT1 and SYT3
are induced by cold, 2) SYT1 and SYT3 localize to ER-PMcs, 3) the specific lipids that
directly interact with SYT1 and SYT3 accumulate in syt1/syt3 mutant after cold acclimation,
and 4) syt1/syt3 show reduced cold acclimated freezing tolerance. We propose that SYT1
and SYT3 have essential roles in ER-PMcs mediated lipid remodelling during cold
acclimation, which in turn leads to freezing tolerance.
References:
(1)
(2)
(3)
Degenkolbe T. et al (2012) The Plant Journal. 72: 972–982).
Pérez-Sancho J., et al. (2015) Plant Physiol. 168: 132–143.
Prinz, W.A. (2014). J. Cell Biol. 205: 759–769.
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Systems biology approach of heat-induced thermotolerance in Pinus
radiata
Mónica Escandón1, Luis Valledor1, Jesús Pascual1,Gloria Pinto2, Mónica Meijón1, María
Jesús Cañal1
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.
Studying the tolerance mechanisms of trees to heat stress is essential to preserve forest
productivity and quality in the currently global warning. Pinus radiata is the most widely
planted pine species in the world and studying thermotolerance processes would be the
key to unravel conifer stress response and improve their breeding programs.
Pinus radiata plants were exposed to 40 °C during 5 days and sampling was performed
at: 3 hours after 40 ºC were reached on day 1 (T1/2) and the end of the 6 h heat treatment
on day 1 (T1), day 2 (T2), day 3 (T3), and day 5 (T5). Control plants was collected at 1
day and recovery plants (R) of each exposure after 1 month in control conditions.
Metabolites, proteins and RNA were extracted from the same sample according Valledor
et al., (2014).
Metabolomics response was analysed in all treatments using complementary mass
spectrometry techniques (GC-MS and LC-Orbitrap-MS). These allowed the reliable
quantification of 2286 metabolites. Multivariable analysis clustered two types of heat stress
response in P radiata: immediate (T1/2, T1, T2 and T3) and delayed (T5 and R) responses
according to previous studies related to physiological and hormonal parameters
(Escandón et al. 2015). Fatty acids metabolism is showed as the most relevant pathway
in both stress responses. However, the major number of pathways were identified in shortterm treatments
Given the relevance for the plant survival, shorter-term response was analysed in details
by the analysis of the proteome and transcriptome in Control, T1 and T3 treatments.
Proteins were identified by GeLC-Orbitrap-MS which allowed the quantification of 848
proteins. Additionally, 15 candidate genes, getting from integrative analysis, were studies
by qPCR.
In order to reduce the dimensionality of the results and integrate metabolomics, proteomics
and physiological parameters, sPLS analysis was used. Multivariable ordination provided
an overall picture of the -omics response condition of P. radiata plants during the
experimental setup. Three main networks were revealed in relation to immediate
thermotolerance response: 1) Heat shock network with several proteins that contain the
dominion of HSP20 family, the most all of them overexpressed in T1 and T3 treatments
according qPCR analysis; 2) Sugar network with sucrose synthase candidate where fatty
acids and hormones (like abscisic acid) have an key role; and 3) Dehydrogenase network
with several dehydrogenase proteins, include alcohol dehydrogenase candidate, which
reduces their presence in the first impact to heat stress.
In summary, this integrated approach pinpointed the basic mechanisms that underlying
immediate physiological response in P. radiata during heat-induced thermotolerance.
References:
Valledor L, et al. (2014) Plant J., 79(1), 173-80
Escandón M, et al. (2016) Tree Physiol., 36, 63-77
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Pivotal role of subtilisin SBT4.13 in pH homeostasis, oxidative stress
and jasmonic acid response
Gaetano Bissoli1, Eduardo Bueso1, Jesús Muñoz-Bertomeu2, Lourdes Rubio3, José
Antonio Fernández3, Ramón Serrano1
1
IBMCP-UPV-CSIC, Valencia, Spain, 2 Departament de Biologia Vegetal, Facultat de Farmàcia,
Universitat de València , Spain, 3 Departamento de Biología Vegetal, Universidad de Málaga,
Spain
In order to uncover novel determinants of intracellular pH homeostasis, we are screening
activation tagging lines of Arabidopsis selecting for tolerance to intracellular acidification
caused by weak organic acids (acetic, propionic, sorbic). Previously we have identified the
prolyl-isomerase ROF2 (Bissoli et al., 2012) and the β-adaptin WAT1 (Niñoles et al., 2013)
and the mechanism in both cases there is indirect activation of proton efflux by increasing
K+ uptake and anion efflux. We report here the isolation of subtilisin SBT4.13 as another
determinant of pH homeostasis. Subtilisin proteins are serine proteases located in the
secretory pathway or secreted to the apoplast and with relevant roles in plant defence
against pathogens and in plant development. Their known targets are peptide hormones
or transcription factors. Over-expression of SBT4.13 conferred at the seedling
establishment stage tolerance to: a) intracellular acidification generated by weak organic
acids; b) toxic cations (lithium, norspermidine and hygromycin); and c) oxidative stress
(hydrogen peroxide and paraquat). On the other hand, loss of function in plants expressing
artificial micro RNAs (ami-sbt4.13) resulted in sensitivity to all these three kinds of stress.
We detected in roots of 35S::SBT4.13 over-expressing plants a higher rate of proton
extrusion than wild type in long term experiments (12-16 h) in plates containing
bromocresol purple. This can explain the observed tolerance to weak organic acids. In
electrophysiological measurements these over-expressing lines exhibited a plasma
membrane depolarization (-90 versus -135 mV in wild type) that can explain tolerance to
toxic cations. This depolarization could result from either increased K+ uptake or increased
anion (malate) efflux. Transcriptomic analysis indicates regulation by over-expression of
SBT4.13 of 550 genes.
References:
Bissoli G, et al. (2012) Plant J., 70 (4): 704-716.
Niñoles R, et al. (2013) J., 74 (4): 557-568.
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Insights into the ROS-dependent cell response to the herbicide
2,4-D in plants
Romero-Puertas M. C.1, Rodríguez-Serrano M.1, Bautista R.2, Pazmiño D. M.1, GómezCadenas A.3, Claros M. G.2, León J.4, Sandalio L. M.1
1
Departamento de Bioquímica, Biología Celular y Molecular de Plantas, EEZ, CSIC, C/ Prof.
Albareda, 18008 Granada, Spain, 2Departamento de Biología Molecular y Bioquímica, Ciencias,
Univ. de Málaga, Campus de Teatinos s/n, E-29071, Málaga, Spain. 3Department Ciències
Agràries i del Medi Natural, Universitat Jaume I, E-12071, Castelló de la Plana, Spain, 4Instituto
de Biología Molecular y Celular de Plantas (CSIC-Univ. Valencia), CPI Edificio 8E, Avda.
Ingeniero Fausto Elio s/n, 46022 Valencia, Spain
2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin specific for dicotyledons and is
considered to be among the most successful herbicides used in agriculture (Grossmann,
2010). One of the most characteristic effects of 2,4-D on sensitive plants is the
development of epinasty and stem curvature, as well as reduction of root and stem growth
(Grossmann, 2010; Pazmiño et al., 2011). The processes involved in signalling under 2,4D toxicity, specifically those involved in developing epinasty, are not well established
although ET-dependent and –independent pathways have been described.
Reactive oxygen species (ROS) play an important role in signalling by controlling the
response to biotic and abiotic stress; in addition, they regulate processes such as growth
and development, and participate in programmed cell death (Sandalio and RomeroPuertas, 2016). We found that ROS overproduction is a key point in the effect of high
concentrations of 2,4-D (Pazmiño et al. 2011) and that two peroxisomal enzymes (AcilCoA oxidase, ACX and xanthine sehydrogenase, XDH) may be involved in this
overproduction (Romero-Puertas et al., 2004). To gain further insights into the role or ROS
in the regulation of plant response to the herbicide 2,4-D, we analysed development of
epinasty, oxidative metabolism and transcriptome of WT and acx1 mutants.
Plants treated with 2,4-D showed a strong epinasty while in acx1 mutants this effect is
considerably reduced concomitantly with H2O2 production. Actually, WT plants showed a
strong reduction in actin bundling and polymerization due to actin modifications by
oxidation and S-nitrosylation, which affect the polymerization of F-actin (Sandalio and
Romero-Puertas, 2016). The transcriptome of WT leaves subjected to 2,4-D showed a
peroxisomal ROS-specific signature at the very early signalling and an increase was
detected in transcripts associated with mitochondrial and peroxisomal ROS stresses at
longer treatment. Differences in the transcriptome with acx1 mutant will be also discussed.
Supported by ERDF-Cofinanced grants BIO2012-36742 and BIO2015-67657 from MICINN and Junta de Andalucía
(BIO-337).
References:
Grossmann (2010) Pest Manag. Sci. 66, 113–120.
Pazmiño et al. (2011) Plant, Cell Environ. 34: 1874–1889
Romero-Puertas et al. (2004). Plant, Cell Environ. 27: 1135–1148
Sandalio and Romero-Puertas (2016). Annals Bot. 116(4):475-85
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Poster 01 / SV P01
Role of HKT1-like Na+ transporters in tomato salt tolerance*
Noelia Jaime-Pérez1, Benito Pineda2, Begoña García-Sogo2, Ana Pilar Ortega1, Raquel
Olías1, Alejandro Atares2, María José Asins3, Vicente Moreno2 and Andrés Belver1
1
Department of Biochemistry, Molecular and Cellular Biology of Plants, Estación Experimental
del Zaidín, (CSIC), Granada, Spain, 2Laboratory of Biotechnological Breeding, IBMCPUPV/CSIC, Valencia, Spain, 3Plant Protection and Biotechnology Center, IVIA, Moncada,
Valencia, Spain.
Excessive soil salinity causes abiotic stress and consequently diminishes crop yields. The
regulation of intracellular concentration of Na+ and K+ (homeostasis) in plant cells and
tissues is a key mechanism in saline stress tolerance. HKT1-like genes encode Na+
transporters which play an important role in Na+ and K+ homeostasis. These Na+
transporters, located at the plasma membrane of parenchyma cells surrounding the xylem
vessels, are responsible for unloading Na+ from the xylem, thus preventing Na+
accumulation in aerial parts and indirectly improving K+ homeostasis. In previous studies,
two closely linked tomato genes encoding HKT1-like transporters, HKT1;1 and HKT1;2,
were shown to be positional candidate genes for a major tomato QTL (lkc7.1) related to
shoot Na+/K+ homeostasis identified using two populations of recombinant inbred lines
(RILs) derived from Solanum cheesmaniae and S. pimpinellifolium as male parents (1,2).
However, using two tomato near-isogenic lines (NIL) differing in terms of the HKT1 gene
allele they contain (from S. lycopersicum or S. cheesmaniae) showed that the connection
between the allelic variants of tomato HKT1;1 and HKT1;2 and salt tolerance was unclear
and mostly depended on salt tolerance criteria used (1). In this study, different transgenic
lines derived from these NILs, in which each HKT1;1/HKT1;2 allelic variant was silenced
by stable gene transformation, were used. The phenotype of each genotype was
characterized in order to determine which HKT1 locus is responsible for lkc7.1 and plays
the most significant role in tomato salt tolerance, measured as tissue growth in plants
cultured in different media (Petri dishes, pots and hydroponics). Results obtained may be
the basis for future research in order to improve the tolerance of plant crops to salinity in
water and soils.
*Funded by ERDF-cofinanced grant AGL2013-41733-R (AB), AGL2015-64991-C3-3-R (VM) and FPI
fellowships from MINECO (NJP).
References:
1. Asins MJ et al. (2013) Plant Cell & Environm 36: 1171-1191.
2. Asins et al., (2015) Theor & App Gen128: 667-679,
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Poster 02 / SV P02
Arabidopsis Ubiquitin Ligases RGLG1 and RGLG5 Regulate Abscisic
Acid Signaling by Controlling the Turnover of PP2CA
Qian Wu1, Xu Zhang1, Borja Belda-Palazon2, Marta Peirats-Llobet2, Alberto Coego2,
Jose Julian2, Xiaofeng Wang1, Shao Cui1, Xiangchun Yu1, Chengcai An1 Pedro L.
Rodriguez2
1
The State Key Laboratory of Protein and Plant Gene research, College of life sciences, Peking
University, Beijing, China 2Instituto de Biología Molecular y Celular de Plantas, Consejo Superior
de Investigaciones Científicas-Universidad Politécnica de Valencia, Valencia, Spain
Abscisic acid (ABA) is an essential hormone for plant development and stress response.
ABA signaling is suppressed by clade A PP2Cs, which are key repressors of the pathway
through inhibition of ABA-activated SnRK2s. Upon ABA perception, the PYR/PYL/RCAR
ABA receptors bind to PP2Cs with high affinity and biochemically inhibit their activity.
Whereas this mechanism has been extensively studied, how PP2Cs are regulated at the
protein level is only starting to be explored. Arabidopsis RING DOMAIN LIGASE5
(RGLG5) belongs to a five-member E3 ubiquitin ligase family whose target proteins remain
unknown. We report RGLG5, together with RGLG1, releases PP2C blockade of ABA
signaling by mediating PP2CA protein degradation. ABA promotes the interaction of
PP2CA with both E3 ligases, which mediate ubiquitination of PP2CA and are required for
ABA-dependent PP2CA turnover. Down-regulation of RGLG1 and RGLG5 stabilizes
endogenous PP2CA, diminishes ABA-mediated responses and the reduced response to
ABA in germination assays is suppressed in the rglg1 amiRrglg5 pp2ca-1 triple mutant,
supporting a functional link among these loci. Overall, our data indicate RGLG1 and
RGLG5 are important modulators of ABA signaling, and further unveil a mechanism for
activation of the ABA pathway by controlling PP2C half-life.
125
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Poster 03 / SV P03
Identification and characterization of an lsm8 suppressor
Cristian Carrasco-López1, Carlos Perea-Resa1, José Manuel Jiménez-Gómez2, Rafael
Catalá1, and Julio Salinas1
1
Departamento de Biología Medioambiental, Centro de Investigaciones Biológicas, CSIC, 28040
Madrid, Spain. 2INRA, Institut Jean-Pierre Bourgin, UMR 1318, 78026 Versailles, France.
Recent studies show that a key regulatory step for proper gene expression is the removal
of introns from pre-mRNAs and the subsequent binding of exons, through a process known
as splicing. This process is catalyzed by the spliceosome, a large molecular weight
complex comprised of five small nuclear ribonucleoproteins (snRNPs) and hundreds of
additional proteins. The five snRNPs contain small nuclear uridine-rich RNAs (U1, U2, U4,
U5 and U6 snRNAs) and their associated proteins, namely the Sm for U1, U2, U4 and U5
snRNAs, and the LSM2-8 heteroheptameric complex for the U6 snRNA. Recently, we
reported the identification and characterization of two Arabidopsis LSM8 null mutants
(lsm8-1 and lsm8-2) that are deficient in LSM2-8 nuclear complex, and we demonstrated
that the LSM2-8 complex is required for pre-mRNA splicing through U6 snRNA
stabilization. More important, our results also revealed that LSM2-8 complex is necessary
for the correct splicing of several development-related mRNAs and, consequently, for
normal development of Arabidopsis. On the other hand, we have shown that LSM2-8
complex plays a critical role in plant tolerance to abiotic stresses. Our data indicate that
this complex negatively regulates the ability of Arabidopsis to cold acclimate but functions
as a positive regulator of salt tolerance. To better understand the genetic network
connecting U6 snRNP, post-transcriptional regulation, plant development, and abiotic
stress tolerance, and to identify the molecular mechanisms by which LSM2-8 complex
function in pre-mRNA splicing, we have performed a genetic screen for suppressors of the
lsm8-1 mutation. To this aim, EMS mutagenized M2 lsm8-1 seeds were first screened for
plants disclosing wild-type (WT) morphological phenotype. One of the plants identified
from the screening, we named sol8 (suppressor of lsm8-1), also showed WT levels of U6
snRNA, WT splicing patterns and WT abiotic stress tolerance phenotypes. The genetic
characterization of the sol8 mutant revealed that it is produced by a single recessive
mutation in a nuclear gene. We anticipate that the sol8 mutation should correspond to an
intermediate component of the regulatory networks governing the pre-mRNA splicing
function of LSM2-8 complex in Arabidopsis tolerance to freezing temperatures and salt
stress. Results regarding the identification and characterization of the sol8 mutation using
a mapping-by-sequencing approach will be presented and discussed.
126
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Poster 04 / SV P04
Functional characterization of two novel long non-coding RNAs
involved in abiotic stress response
Diego Gómez-Martínez1, Javier Barrero-Gil1, Mª Fernanda Ruiz1, Rafael Catalá1 and
Julio Salinas1
1
Departamento de Biología Medioambiental, Centro Investigaciones Biológicas, CSIC, Ramiro de
Maeztu 9, 28040 Madrid, Spain
In the past decade, next generation sequencing technologies have revealed the pervasive
transcription of stretches of DNA that carry little protein-coding capacity leading to the
identification of thousands of non-coding RNA molecules in a wide range of organisms
including plants. The largest class of this type of molecules has been named long noncoding RNAs (lncRNAs) as they typically are longer than 200 nucleotides and lack an open
reading frame. Rather than constituting transcriptional noise, lncRNAs are thought to
perform regulatory functions on gene expression at both transcriptional and posttranscriptional levels. We have performed a high-throughput RNA sequencing analysis in
Arabidopsis to detect lncRNAs that respond to low temperature. Two representative
lncRNAs, a cold-induced and a cold-repressed lncRNA, have been selected for further
study. The molecular characterization of their structure and expression pattern has
confirmed the existence of these lncRNAs and their ability to respond in a specific manner
to diverse abiotic stresses. Furthermore, preliminary genetic evidence suggests that these
lncRNAs play an important role in Arabidopsis tolerance to abiotic stress. Additional
experiments are in progress to characterize the function of these novel lncRNAs in
Arabidopsis response to abiotic stress.
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Poster 05 / SV P05
Unusual histones HTR6 and HTR14 as potential players during abiotic
stress response in Arabidopsis.
Sofia Madeira1, Bénédicte Desvoyes1, Sofia Otero1, José M. Franco-Zorrilla2, Crisanto
Gutierrez1
1
Department of Genome Dynamics and Function, Laboratory of DNA replication, chromatin and
cell division, Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain, 2Genomics
unit, Centro Nacional de Biotecnologia, CSIC, Madrid, Spain
Histones are packaged with DNA to form a dynamic structure named chromatin that
encompasses different states of complexity (Zhou et al.,2011; Sequeira-Mendes et al.,
2014). Understanding how histone composition influences chromatin architecture and
consequently regulates various processes during developmental transitions and stress
responses is of crucial importance. There are two main classes of histone H3, H3.1 and
H3.3, that differ in only 4 amino acids and which dynamics is of primary relevance for cell
differentiation during Arabidopsis development (Otero et al., 2014; Otero el al., 2016).
Additionally, two other H3 variants, HTR6 and HTR14 are known as ‘unusual’ and have
heterogeneous features sharing characteristics of both H3.1 and H3.3 proteins. Here we
show, by gene expression analysis, that HTR6 and HTR14 are induced by salt stress and
abscisic acid. We generated GFP-tagged versions of HTR6 and HTR14 and by live
imaging we observed that both proteins are expressed in a subset of epidermal cells. The
rapid induction of these genes under drought conditions suggests a role as part of the
responses to stress. Thus, we are studying some regulatory mechanisms to understand
their dynamics and how chromatin states could influence the response to abiotic stress in
Arabidopsis.
References:
Otero, S., et al. (2014). Cytogenet genome Res. 143:114-124.
Otero, S., et al. (2016) Plant Cell. (under second review).
Sequeira-Mendes et al. (2014). Plant Cell. 26:2351-2366.
Zhou, V., et al. (2011). Nat Rev Genet.12: 7-18.
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Poster 06 / SV P06
Cytokinin determines thiol-mediated arsenic tolerance and
accumulation in Arabidopsis thaliana
Thotegowdanapalya C. Mohan1, Gabriel Castrillo1, Cristina Navarro1, Sonia ZarcoFernández2, Eswarayya Ramireddy3, Cristian Mateo1, Angel Mª Zamarreño4, Javier PazAres1, Riansares Muñoz2, Jose Mª García-Mina4, Luis E. Hernández5, Thomas
Schmülling3, Antonio Leyva1
1
Department of Plant Molecular Genetics, Centro Nacional de Biotecnología (CSIC), Madrid,
Spain, 2Department of Analytical Chemistry, School of Chemical Sciences, Universidad
Complutense de Madrid, Madrid, Spain, 3Institute of Biology/Applied Genetics, Dahlem Centre of
Plant Sciences, Freie Universität Berlin, Berlin, Germany, 4Department of Environmental Biology
(Agricultural Chemistry and Biology Group), Faculty of Sciences, University of Navarra,
Pamplona, Spain, 5Departamento de Biología, Universidad Autónoma de Madrid, Edif. de
Biológicas BS13, Madrid, Spain
The presence of arsenic in soil and water is a constant threat to plant growth in many
regions of the world. Arsenate [As(V)] is the most prevalent arsenic chemical species in
nature. Due to its structural similarity to phosphate (Pi), it is easily incorporated into plants
and other organisms through phosphate transporters. Once taken up by the cell, As(V) is
rapidly reduced to arsenite (AsIII) by arsenate reductases. As(III) is either extruded from
the cytoplasm or is sequestered by phytochelatins (PC) and other related thiol-containing
compounds and is compartmentalized into vacuoles. When As(V) is perceived, Pi
transporters are rapidly downregulated and thiol compound accumulation increases
concomitantly to cope with the metalloid. These two responses are key As(V) tolerance
strategies for natural plant populations. Phytohormones act in the integration of growth
control and stress response, but their role in plant responses to arsenic remains to be
elucidated. Here we show that As(V) exposure causes severe depletion of endogenous
cytokinins (CK) in the model plant Arabidopsis thaliana. We found that CK signaling
mutants and transgenic plants with reduced endogenous CK levels showed an As(V)tolerant phenotype. Our data indicate that in CK-depleted plants exposed to As(V),
transcript levels of As(V)/phosphate-transporters were similar or even higher than in wild
type plants. In contrast, CK depletion provoked the coordinated activation of As(V)
tolerance mechanisms, leading to the accumulation of thiol compounds such as
phytochelatins and glutathione, which are essential for arsenic sequestration.
Endogenous CK depletion in response to As(V) also affects expression of the arsenate
reductase gene AtARQ1 (Sánchez-Bermejo et al., 2014). Transgenic CK-deficient A.
thaliana and tobacco lines show a marked increase in arsenic accumulation. Our findings
indicate that cytokinin is an important regulatory factor in plant adaptation to arsenic stress.
References:
Mohan TC, et al. (2016) Plant Physiol (in press). doi: http://dx.doi.org/10.1104/pp.16.00372
Sanchez-Bermejo E, et al. (2014) Nat Commun 5: 4617
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Poster 07 / SV P07
Remodeling ATPase BRAHMA via Core ABA Signaling Pathway
Components
Marta Peirats-Llobet1, Soon-Ki Han2, Miguel Gonzalez-Guzman1, Cheol Woong Jeong2,
Lesia Rodriguez1, Borja Belda-Palazon1, Doris Wagner2, and Pedro L. Rodriguez1
1
Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones
Científicas-Universidad Politécnica de Valencia, Valencia, Spain 2Department of Biology,
University of Pennsylvania, Philadelphia, USA
Optimal response to drought is critical for plant survival and will affect biodiversity and crop
performance during climate change. Mitotically heritable epigenetic or dynamic chromatin
state changes have been implicated in the plant response to the drought stress hormone
abscisic acid (ABA). The Arabidopsis SWI/SNF chromatin-remodeling ATPase BRAHMA
(BRM) modulates response to ABA by preventing premature activation of stress response
pathways during germination. We show that core ABA signaling pathway components
physically interact with BRM and post-translationally modify BRM by phosphorylation/dephosphorylation. Genetic evidence suggests that BRM acts downstream of
SnRK2.2/2.3 kinases, and biochemical studies identified phosphorylation sites in the Cterminal region of BRM at SnRK2 target sites that are evolutionarily conserved. Finally,
the phosphomimetic BRMS1760D S1762D mutant displays ABA hypersensitivity. Prior studies
showed that BRM resides at target loci in the ABA pathway in the presence and absence
of the stimulus, but is only active in the absence of ABA. Our data suggest that SnRK2dependent phosphorylation of BRM leads to its inhibition, and PP2CA-mediated
dephosphorylation of BRM restores the ability of BRM to repress ABA response. These
findings point to the presence of a rapid phosphorylation-based switch to control BRM
activity; this property could be potentially harnessed to improve drought tolerance in plants.
130
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Poster 08 / SV P08
Vacuolar NHX antiporters: understanding structure-function
relationships and regulation.
Belén Rombolá-Caldentey1, Zaida Andrés1,2, Javier Pérez-Hormaeche1, Beatriz Cubero1,
José Manuel Pardo1,3
1
Instituto de Recursos Naturales y Agrobiologia de Sevilla (IRNAS-CSIC), Seville, Spain. 2Centre
for Organismal Studies, Heidelberg University, Heidelberg, Germany. 3 Instituto de Bioquimica
Vegetal y Fotosintesis (IBVF-CSIC), Seville, Spain.
Potassium (K) is an essential nutrient for plants and the most abundant cation in plant
cells, comprising up to 10% of plant dry weight. While cytosolic K is kept at homeostatic
concentrations close to 100 mM, surplus K is stored in vacuoles. The tonoplast-localized
K+,Na+/H+ exchangers NHX1 and NHX2 proteins of Arabidopsis mediate this K+
accumulation in the vacuole, thereby increasing the osmotic potential, water uptake and
the turgor pressure necessary for cell expansion and growth. Vacuolar remodeling during
stomatal movements also depends on these proteins.
Structural domains and essential amino acid residues putatively involved in ion transport,
cation coordination and pH sensing, have been identified by phylogenetic analysis and
computational modeling of the NHX1 protein. To determine the relevance of these residues
in the biochemical activity of NHX1, and its pH dependence, point-mutation alleles have
been generated. Mutant NHX1 proteins have been functionally tested in yeasts nhx1
mutants and in vitro ion transport assays. The presence of a calmodulin-binding domain
comprising amphipathic α-helices at the C-termini of NHX1 and NHX2 have also been
detected by computational and biochemical analyses. The importance of the putative
calmodulin-binding domain for NHX1 activity has been demonstrated by functional
analyses in yeast, whereas the interaction of NHX1 and NHX2 with CalModulin-Like18
(CML18) has been analyzed by BiFC and Y2H assays. Our results evidence the fine-tuning
of NHX1 and NHX2 activity in response to developmental and environmental cues. In
addition, we expect to unravel the biochemical mechanisms for pH sensing and regulation
of these critical K transporters of Arabidopsis.
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Poster 09 / SV P09
ROS metabolism and nutrient uptake are differentially regulated by
RBOH C, RBOH D and RBOH F under cadmium toxicity
L.M. Sandalio1, D.K. Gupta2, L.B. Pena 3, A. Hernández4, M. Inouhe5, M. SanzFernández1, C. Hafsi6, M.C. Romero-Puertas1
1
Department of Biochemistry and Cellular and Molecular Biology of Plants, Estación
Experimental del Zaidín, CSIC, Granada, Spain, 2 Institut für Radioökologie und Strahlenschutz,
Gottfried Wilhelm Leibniz Universität Hannover, Herrenhäuser Deparment-Laboratory, Hannover,
Germany, Institution, City, Country, 3Department of Biological Chemistry, Faculty of Pharmacy
and Biochemistry, University of Buenos Aires and IQUIFIB, CONICET, Argentina,4Postgrados de
Agronomía. Universidad Centroccidental Lisandro Alvarado. Apdo 400, Barquisimeto,
Venezuela,5 Department of Biology, Faculty of Science, Ehime University, Matsuyama Japan, 6
Laboratoire d’Adaptation des Plantes aux Stress Abiotiques, Centre de Biotechnologie à la
Technopole de Borj Cédria, Tunisia
Cadmium (Cd) is toxic for plants, animals and humans. In plants, Cd-specific transporters
have not been identified and Cd seems to be transported via several classes of Ca2+, Fe2+
and Zn2+ transporters, affecting their uptake and distribution and therefore inducing
deficiency of those elements (Clemens 2006). In plants Cd can cause disturbances in
photosynthesis and growth inhibition (Sandalio et al. 2001). Oxidative stress is one of the
primary effects of Cd exposure, although the sources of reactive oxygen species involved
are not well established. In this work, the role of NADPH oxidases under cadmium (Cd)
toxicity was studied using Arabidopsis thaliana mutants AtrbohC, AtrbohD and AtrbohF
which were grown under hydroponic conditions with 25 and 100 µM Cd for 1 and 5 days.
A cadmium-dependent reduction of growth was observed in WT, AtrbohC and D, but not
in AtrbohF. H2O2 and lipid peroxidation content increased with the time of Cd exposure in
all genotypes, with AtrbohC showing the smallest increase. An opposite behaviour was
observed with NO accumulation. Cadmium increased catalase activity in WT plants and
decreased it in all Atrbohs, while glutathione reductase and glycolate oxidase activities
increased in Atrboh mutants, and superoxide dismutases were down-regulated specifically
in AtrbohC. The redox couples GSH/GSSG and ASA/DHA differentially changed in
AtrbohC and AtrbohF, respectively. The translocation of Cd to the leaves was severely
reduced in Atrboh mutants mainly after 1 d of treatment. Similar results were observed for
S, P, Ca, Zn and Fe accumulation, while an opposite trend was observed for K
accumulation, except in AtrbohF. The regulation of several K and Fe transporters by RBOH
will be disused. In conclusion, under Cd stress RBOHs differentially regulate ROS
metabolism, redox homeostasis, and nutrient balance. This result could be of potential
interest in biotechnology for the phytoremediation of polluted soils.
This study was supported by ERDF co-financed grant BIO2012-36742 from MICINN, Ramon Areces
Foundation through the project CIVP16A1840 (http://www.fundacionareces.es) and Junta de Andalucía
(BIO-337 group) in Spain
References:
Clemens S. (2006) 88, 1707-1719.
Sandalio L.M et al. (2001) J Exp Bot 52, 2115-2126.
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Poster 10 / SV P10
Masking the reality: the negative effect of light on
root responses to phosphate starvation
Javier Silva-Navas, Clara Echevarría, Sara Navarro, Juan Carlos del Pozo
Centro de Biotecnología y Genómica de Plantas. Universidad Politécnica de Madrid (UPM) Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). Campus
Montegancedo UPM. 28223-Pozuelo de Alarcón (Madrid), Spain
Phosphorous (Pi) is an essential macronutrient for plant growth. Pi is needed for the
generation of ATP, nucleic acids, membrane phospholipids and, in addition, Pi is involved
in many metabolic and regulatory processes. Phosphorous starvation is one of the most
critical nutritional deficiencies that severely affects plant survival and reproduction. Pi
starvation alters many morphological and physiological parameters. Using in vitro
conditions, in which roots are normally grown under light conditions or submerged in liquid
medium, Pi starvation strongly reduces Arabidopsis root growth, while increases lateral
root density. Most current research in root biology, including Pi deficiency, has been
carried out with the root system grown in the presence of light. Recently, our group have
engineered the D-Root device that allows the in vitro cultivation of plants with the aerial
part exposed to normal photoperiodic conditions but the root system on darkness (SilvaNavas et al., 2015). Using the D-Root, we found that root system architecture under Pi
deficiency significantly differs from the phenotype observed in light grown conditions. For
example, reduction of primary root length was minor (only 30% less than high Pi medium).
Conversely to light grown roots, we found that Pi starvation decreases lateral root density
when grown in darkness. Further characterization of Pi starvation response in the D-root
system revealed that plants accumulated higher levels of phosphorous and less
anthocyanins compared to light grown-root plants. RNAseq analyses of dark-grown roots
grown with or without Pi identified more than 1000 transcripts that change their levels in
response to Pi starvation in roots. Importantly, over 30 % of them have not been described
previously in other Pi-deficiency experiments. Taken together, our data indicate that light
strongly influence Pi starvation response in roots. We have identified T-DNA mutants for
some of the deregulated genes in our condition. One of these mutants, m12, shows a
significant reduction in root and shoot growth while lateral root density is increased and
also that accumulates, pointing out a possible role in regulating root system architecture
under Pi starvation conditions. Ionomics analyses reveals that m12 accumulates about
30% less Pi than wt, although the levels of other ions are reduced, such as Ni. Further
analyses of m12 mutant will be presented at the meeting.
References:
Silva-Navas J, et al. (2015) Plant J. 84(1): 244-255.
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Poster 11 / SV P11
Arabidopsis PHOSPHATE STARVATION RESPONSE 1 acts via two cismotifs displaying different functional properties and links plant water
content with phosphate homeostasis
Laura de Lorenzo1, Mª Isabel Puga1, Nathalie Prat-Leonhard2, George Coupland3, Detlef
Weigel4 and Javier Paz-Ares1
1
2
Centro Nacional de Biotecnología-CISC, Darwin 3, Campus Cantoblanco,Madrid, Institut de
Biosciences et Biotechnology Aix-Marseille, CEA, Saint Paul Les Durance, Franc CEA,
3
Cadarache, Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, 50829
4
Köln, Germany, Max Planck Institute for Developmental Biology, Tübingen, Germany
Plants evolved a complex array of responses to cope with growth under low Phosphate
(Pi) regimens, involving morphological and metabolic/biochemical changes. PHOSPHATE
STARVATION RESPONSE1 (PHR1) and related transcription factors are key regulators
of these responses. Here, we determined the in vivo binding sites of PHR1 and found it
binds more than 2600 sites, out of which more than 550 correspond to Pi responsive
genes (cut off 2x). These targets are enriched in two motifs, the already known P1BSI and
P1BSII, which display high and low intrinsic binding affinity and are bound by PHR1 as
dimer and monomer, respectively. Results using fusions of multimers of P1BSI and P1BSII
with a minimal promoter showed that both motifs are bona fide Pi starvation response
element, although of different strength. P1BSI and P1BSII containing targets display only
minor differences in P1BS unrelated motifs, out of which bZIP and bHLH binding sites are
most prominently enriched, but show significant differences in ontology term enrichment
and capacity to produce a transcriptional effect. These two sites display different
evolutionary constraints, but in both cases site conservation in part depends on
occupancy, independent of transcriptional output. This finding raises the possibility of
functionality of PHR1 binding beyond its effect on transcription. We also found a link
between phosphate starvation and drought/osmotic stress, indicated by the fact that Pi
starvation responsive genes are overrepresented in drought/osmotic stress responsive
genes and that plants with impaired PHR1(like) activity displayed decreased osmotic
stress induction of PHR1 targets. In addition, we found that plants display reduced water
content during Pi starvation, an effect dependent on PHR1(like) activity. These findings
underline the physiological importance of the link between water content and Pi
homeostasis
134
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Poster 12 / SV P12
Implication of 3P proteins in the heat stress response in plants
Transcriptional and subcellular localization changes of 3P proteins
during the heat stress response
Lourdes Fernández-Calvino, Nuria Fernández-Bautista, M. Mar Castellano
Department of Biotechnology, CBGP (INIA), Pozuelo de Alarcón (Madrid), Spain.
Climate change is one of the most important environmental problems of our society. One
of the consequences of climate change is the global warming. In Spain, the global
temperature increase is a great problem for agriculture since heat stress is one of the
abiotic stresses that affect more severely crop production. Therefore, the study of the
molecular mechanisms involved in plant acclimation to heat is a key issue for the
agriculture future.
During the last years our lab has carried out different -omics approaches, analyzing
transcriptional and translational changes during the survival process of plants to the
increase of temperatures. This research has allowed the identification of a group of three
proteins whose functions in heat stress response had not been studied before, the 3P
protein family.
Transcriptional expression analyses of these genes during the heat stress response (HSR)
show that 3P3 is the only member whose expression is highly increased during the heat
challenge, and actively degraded during the recovery from the stress. Despite this fact, the
three members act redundantly during the acclimation of plants to high temperatures.
The transcriptional and subcellular localization changes for these three 3P members
during the heat shock treatment are shown and compared with other already known
proteins involved in heat stress response. Their function during the acclimation process of
the plants to heat is also discussed.
Homologous genes of the 3P family have been previously studied in other organisms and
their functions described to be involved in aging and in the protection from prion diseases,
but this is the first time that their function is related to abiotic stress responses.
135
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Poster 13 / SV P13
Role of At3P in assisting protein folding under stress conditions
Nuria Fernández-Bautista, , Alfonso Muñoz, Lourdes Fernández-Calvino and M. Mar
Castellano
Centro de Biotecnología y genómica de plantas, INIA-UPM, Pozuelo de Alarcón, España
Proteins are translated by ribosomes as unfolded polypeptide chains that need to be folded
and assembled into functional proteins. This process is assisted by molecular chaperones,
a group of proteins that facilitate the correct folding of proteins in order to avoid
inappropriate interactions and aggregations under physiological and stress conditions.
Different abiotic stresses as heat stress, anoxia or chemical agents induce proteotoxic
conditions that course with an accumulation of missfolded proteins in the cell.
We have characterized the role of the 3P protein family in response to different abiotic
stresses in Arabidopsis. These proteins are characterized by the presence within their
sequence of three TPR domains. These domains have been involved in different proteinprotein interactions in other species.
In order to analyze their function in plants, we have carried out immunoprecipations of the
At3P3 protein from Arabidopsis extracts and we have identified different proteins as 3P3
interactors. Independent analyses corroborate that 3P3 interacts with cytosolic HSP90 and
HSP70, suggesting a role of 3P3 in protein folding in the cytoplasm. Phenotypic analyses
demonstrate that the lack of expression of the At3P members does not constrain plant
growth or development under control conditions; however, their expression is essential for
the proper acquisition of long term thermotolerance in plants.
In addition to the interaction with these cytosolic chaperones, At3P3 also interacts in vivo
with the main ER-resident chaperone BiP, suggesting that At3P3 could also play a main
role in protein folding in the ER. According to this possible role, 3p3 mutants show a high
hypersensitivity to ER inducer agents and this phenotype is reverted when the assays are
carried out in the presence of a chemical chaperone.
In this presentation, we will provide a model suggesting the role of the 3P proteins in
protein folding during different stress conditions, a function that seems to be linked to their
association to different chaperone complexes.
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Poster 14 / SV P14
Unraveling of the main physiological processes affected by the Na+, K+
transporters NHX1 and NHX2 of Arabidopsis thaliana
Beatriz Cubero1, Javier Pérez-Hormaeche1, Belén Rombolá1, José Manuel Pardo2
1
Instituto de Recursos Naturales y Agrobiología, Consejo Superior de Investigaciones
Científicas, Sevilla, Spain. 2Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de
Investigaciones Científicas, Sevilla, Spain
The Arabidopsis thaliana NHX1 and NHX2 are the two major tonoplast-localized isoforms
of Na+,K+/H+ antiporters. NHX1 and NHX2 have similar expression patterns and identical
biochemical activity, and together they account for a significant amount of the Na+,K+/H+
antiport activity in tonoplast vesicles. Double mutants nhx1 nhx2 have reduced ability to
create the vacuolar K+ pool, show high K+ retention in the cytosol, impaired
osmoregulation, and compromised turgor generation for cell expansion. Moreover NHX1
and NHX2 exchangers are pivotal in the vacuolar accumulation of K+ of guard cells as the
nhx1 nhx2 mutant lines are dysfunctional in stomatal regulation, showing impaired
stomatal opening and closure due to the abrogation of K+ accumulation in the guard cells.
Other phenotypic traits of the double mutant are poor growth, hypersensitivity to osmotic
stress and very low fertility. Some of these phenotypic traits can be suppressed by the
addition of moderate amounts of NaCl in the substrate. To determine what is the precise
physiological function critically affected by nhx1 nhx2 that accounts for most of the mutant
phenotype, we have analyzed the root and aerial part functions of NHX proteins by
micrografting of wild-type and mutant roots and shoots, and by guard cell specific
expression of NHX1 and NHX2 proteins in a double mutant genetic background. We have
evaluated the growth, fertility and stomatal function of these chimaeric plants. The results
indicate that defective osmotic regulation is the main cause of the pleiotropic phenotype of
mutants impaired in NHX function.
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138
Sesión VI: Estrés Biótico e Interacción Planta-Microorganismo
XIII
RBMP
Comunicaciones Sesión VI.
Estrés Biótico e Interacción PlantaMicroorganismo
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Ponencia Invitada / SVI PI
Interconnections between 3’-UTR mRNA processing, TOR pathway and
plant pathogenesis in the rice blast fungus
Julio Rodríguez-Romero 1, Marco Marconi 1, Mark Wilkinson1 and Ane Sesma1
1
Centro de Biotecnología y Genómica de Plantas. Universidad Politécnica de Madrid (UPM) Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Campus
Montegancedo UPM28223-Pozuelo de Alarcón (Madrid), Spain.
Rice is the most widely distributed dietary staple in the world. It also represents a
significant percentage of global farm land reaching up to 160 million hectares each year.
One of the most devastating rice pathogen is the blast fungus, Magnaporthe oryzae. Yield
losses caused by blast disease oscillate between 10-30 % per annum, which, even at the
most conservative estimate, are sufficient to feed 60 billion people.
The polyadenylation of mRNAs is a two-step process. Pre-mRNAs are first cleaved at their
3' end and then, the poly (A) tail is added by RNA polymerases during 3’ end formation.
Presence of multiple 3’ end cleavage sites is common in eukaryotic genes, and the
selection of a proper cleavage site represents an important step of regulation of gene
expression. Several proteins of the polyadenylation machinery have been shown to
regulate alternative polyadenylation (APA), including Rbp35/CfI25 complex in
Magnaporthe oryzae and Hrp1 in yeast. The M. oryzae Rbp35/Cfi25 complex regulates
the length of 3’UTRs of transcripts with developmental and virulence-associated functions
(Rodriguez-Romero et al., 2014).
In M. oryzae, Rbp35 regulates APA in ~30% of genes, and nearly 75% of then show a
preference for proximal poly(A) sites. The Δrbp35 mutant lacks precision in the cleavage
and shows an increase of proximal cut sites in pre-mRNAs. In addition, we have observed
that APA is involved in regulating M. oryzae gene expression in response to nutritional
fluctuations.
Significantly, Rbp35 regulates APA predominantly in genes related with signaling. These
include regulatory proteins such as 14-3-3 and several genes related with Target of
Rapamycin (TOR) pathway, which is the most severely affected signaling pathway in
Δrbp35, with at least eighteen genes of the pathway presenting altered 3’UTRs in carbon
depleted cells. TOR is a conserved serine/threonine kinase present in all eukaryotes from
fungi to humans. It is also a key component of the most central nutrient-sensing signal
transduction pathways in eukaryotic cells.
A prominent feature of the 14-3-3 proteins is their ability to bind a multitude of functionally
diverse signaling proteins, including kinases and phosphatases. M. oryzae has two
proteins 14-3-3 (MGG_01588 and MGG_13806) and both mRNAs contain several
polyadenylation sites. Both genes are required for full infection of leaves and roots. The
3’-UTRs of 14-3-3A and 14-3-3B shows a shortening during infection. In addition, delta
mutants of 14-3-3A and 14-3-3B shows altered response under osmotic stress, possibly
connected to MoMsn2 and MoOsm1 (Hog1) pathway. The identification of Rbp35/CfI25
as a component of the alternative polyadenylation machinery and its interconnections with
protein kinase signaling are important step to unravel post-transcriptional networks that
regulate M.oryzae plant colonization.
References:
Rodriguez-Romero, et al. (2014). Nucleic Acid Res 43, 179–95.
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Comunicación Oral 1 / SVI CO1
The Helper Component Proteinase and viral replication: unexpected
requirements for the proper yield of virions in Plum pox potyvirus
Araíz Gallo1, Adrian Valli1, Juan Antonio García1
1
Plant Molecular Genetics, Spanish National Centre for Biotechnology-CSIC, Madrid, Spain.
The helper component proteinase (HCPro) of potyviruses is a multifunctional protein that
is involved in diverse steps of the viral infection, such as aphid transmission, polyprotein
processing and suppression of host antiviral RNA silencing. Recently, it has been
described a new function of HCPro by which this viral factor enhances the yield of Plum
pox virus (PPV) particles and, as a consequence, the stability of its cognate capsid protein
(CP) (Valli et al., 2014). This new function is highly specific and cannot be fulfilled by either
the HCPro from other potyvirus or heterologous silencing suppressors. In the absence of
HCPro the virions of PPV are not well formed, meaning low production of unstable viral
particles. In more recent studies, we found that even when the presence of HCPro was
indeed necessary for the proper virion production, there seemed to be additional
requirements. We carried out several experiments to test the putative involvement of other
viral proteins, including the RNA viral genome, to find that none of the tested factors are
necessary to support the proper virion production. Interestingly, these experiments also
showed a correlation between viral replication and encapsidation: only replication-capable
virus variants supported correct virion formation. To formally probe this hypothesis, we
expressed a PPV mutant (PPV-CIKS91,92AA) unable to replicate and we found that this PPV
variant is unable to form normal viral particles even in the presence of functional HCPro.
Altogether, we conclude that only replicating viruses that express their cognate HCPro can
properly encapsidate their RNA genome.
References:
Valli A, et al. (2014) J. Virol., 88(17): 9808-9818.
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Coumnicación Oral 2 / SVI CO2
Small RNA-based antiviral defense in the phytopathogenic fungus
Colletotrichum higginsianum
Sonia Campo1,2, Kerrigan B. Gilbert1, James C. Carrington1
1
Donald Danforth Plant Science Center (DDPSC), St. Louis, Missouri, USA, 2 Center for
Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Campus UAB, Bellaterra,
Barcelona, Spain
The genus Colletotrichum comprises an important number of fungal species that cause
disease in over 3,000 species of plants, including nearly every crop grown. C.
higginsianum infects many members of the Brassicaceae, including Arabidopsis, offering
the opportunity to study and manipulate both host and pathogen. In this work, components
of the RNAi machinery were identified in C. higginsianum and knock-out mutants were
created. High-throughput sequencing of mRNA and small RNA populations from each
mutant genotype, as well as small RNA populations from an AGO1 immunoprecipitation,
was used to identify small RNA-producing loci in C. higginsianum mycelial tissue. Analyses
revealed the presence of an uncharacterized dsRNA virus within C. higginsianum that was
de-regulated in the Δdcl1 and Δago1 strains. No effect was observed on vegetative growth
in the RNAi mutants when grown on synthetic media. However conidiation and conidia
morphology were negatively impacted in the fungal Δdcl1 and Δago1 strains. Generation
of viral-free fungal strains indicated that virus proliferation in the ∆dcl1 mutant was the
major contributing factor to the severe conidiation defect observed. C. higginisianum
parental strains containing the virus showed no negative effects in growth, conidiation and
germination when grown in vitro. Conversely, they were slightly less pathogenic in
Arabidopsis than those cured of the virus. Both strains activated the expression of plant
defense genes at the same level, suggesting that viral-induced decrease in pathogenesis
was no related to suppression of plant host immunity. Future applications from these
research activities should finally help to define new strategies to improve disease
resistance in plants that will also benefit breeding programs for crop protection
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MATI, a novel protein involved in plant defence against spider mites
Mª Estrella Santamaria1, Manuel Martinez1, Ana Arnaiz1, Mercedes Diaz-Mendoza1,
David Perez-Herguedas1, Vojislava Grbic2, Felix Ortego3, Isabel Diaz1
1
Centro de Biotecnología y Genómica de Plantas (UPM-INIA), Madrid, Spain, 2Deparment of
Biology, Biological & Geological Sciences, University of Western Ontario, London, Canadá,
3
Deparmento de Biología Medioambiental, Centro de Investigaciones Biológicas (CSIC), Madrid,
Spain.
The two-spotted spider mite, Tetranychus urticae, is an ubiquitous polyphagous arthropod
that feeds on a remarkably array of plant species, including many important cultivars. In
addition T. urticae has the tendency to develop resistance to a wide battery of acaricides
which makes this pest a major challenge in agriculture. Spider mite has been considered
a model species because of its rapid life cycle, the genome availability and the fact they
can be easily rear in the laboratory. Furthermore, all prediction studies conclude that with
the climate change T. urticae will multiply even faster and there are no crop cultivars
resistant to spider mites which made spider mite a significant threat to cause severe
reduction in crop yields. The main aim of our research is to understand plant responses at
the different levels (perception, signalling and final defence molecules) to confer resistance
to spider mite feeding and to apply the knowledge obtained through basic science as a
potential new avenue for spider mite-pest control. The general goal of this project is being
developed by an international consortium, GAP-M (Genomics in Agricultural Pest
Management, http://www.spidermite.org/gapm/?page_id=2), in which different members
are focused on partial and complementary objectives. The natural variation in susceptibility
to spider mite damage among Arabidopsis (Zhurov et al. 2014) and tomato (Martel et al.
2015) accessions and the use of functional genomics techniques enabled us to select
several candidate genes putatively involved in plant defence against spider mites. Using
transgenic plants we identified an unknown gene, among others, that confers resistance
to spider mite attack. Molecular characterization suggests that this unknown protein, now
named MATI (Mite Attack Trigger Immunity), provides control plants higher SA levels. On
the contrary, infestation of over-expressing MATI lines favour JA biosynthesis and
signalling under an optimal redox state without an energy cost which leads to high
tolerance to T.urticae. In addition, Spodoptera exigua experiments reveal the MATI effect
it is not specific against spider mites.
References:
Martel, C, et al. (2015). Mol Plant Microbe Interact vol. 28 (3): 343-361.
Zhurov, V, et al. (2014). Plant Phys vol. 164 :384-399.
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Epigenetic reprogramming of the host repetitive DNA induced by a
pathogenic long noncoding RNA during infection
Mayte Castellano, Vicente Pallas and Gustavo Gómez
Instituto de Biología Molecular y Celular de Plantas (IBMCP), Consejo Superior de
Investigaciones Científicas (CSIC)-UPV, CPI, Edificio 8 E, Av. de los Naranjos s/n, 46022
Valencia, Spain.
Organisms exposed to adverse conditions are impelled to favor a certain degree of
transcriptional plasticity to cope with stress. Epigenetic regulation of the genome is a key
regulatory mechanism allowing dynamic changes of the transcriptional status in plantresponse to stress. Viroids, ancient plant-pathogenic long noncoding-RNAs (lncRNAs),
have developed a singular evolutionary strategy based on reprogramming specific phases
of host-metabolism to ensure that their infection cycle can be completed in infected-cells.
However, the molecular aspects governing this trans-regulatory phenomenon remain
elusive.
We observed that cucumber (Cucumis sativus) plants infected with the Hop stunt viroid
(HSVd) accumulate high levels of sRNAs derived from ribosomal RNA and transcripts of
Transposable Elements (TEs). This effect correlated with a transcriptional reactivation of
rRNAs and TEs during infection and with a significant modification of their DNA methylation
pattern in symmetric (CG and CHG) sequence context revealing that some rRNA-genes
and TEs are hypomethylated and transcriptionally reactivated during infection. These
results support that HSVd impairs the epigenetic control of rRNA genes and TEs in
cucumber, a phenomenon thus far unknown to occur as a consequence of pathogenicRNA infection (1).
To explore the molecular basis of this phenomenon, we use immunoprecipitation and
bisulfite sequencing of rDNA to show that, in infected plants, HSVd recruits and functionally
subverts the histone deacetylase 6 (HDA6) (a regulator involved in the maintenance and
de novo CG and CHG methylation of TEs, rDNA and transgenes via its interaction with
DNA METHYLTRANSFERASE 1) to promote host-epigenetic alterations that trigger the
transcriptional alterations observed during viroid-pathogenesis (2). This notion is
supported by the demonstration that, during infection, the HSVd-HDA6 complex occurs in
vivo and that endogenous HDA6 expression is increased in HSVd-infected cells.
Moreover, transient overexpression of recombinant HDA6 reverts the hypomethylation
status of rDNA observed in HSVd-infected plants and reduces viroid accumulation. We
hypothesize that the host-transcriptional alterations induced as a consequence of viroidmediated HDA6 recruitment favor spurious recognition of HSVd-RNA as an RNA Pol II
template, thereby improving viroid replication. These results constitute the first description
of a physical and functional interaction between a pathogenic-RNA and a component of
the host RNA silencing mechanism, providing novel evidence of the potential of these
pathogenic lncRNAs to physically redesign the host-cell environment and reprogram their
regulatory mechanisms.
References:
1- Martinez, G, et al. (2014). Nucleic Acids Res. 42: 1553-1562
2- Castellano, M, et al. (2016) New Phytologist doi:NPH14001
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Poster 01 / SVI P01
Spider mite-plant interplay: characterization of putative defence genes
Ana Arnaiz, Mª Estrella Santamaria, Manuel Martinez, Isabel Diaz
Centro de Biología y Genómica de Plantas (CBGP-UPM-INIA), Campus de Montegancedo,
Autovía M-40 (km 38), 28223 Pozuelo de Alarcón, Madrid, Spain
The polyphagous two-spotted spider mite Tetranychus urticae is one of the most important
mite pests in agriculture, feeding on a broad range of hosts including greenhouse crops,
ornamentals and annual and perennial plants worldwide. Spider mite short generation time
and high fecundity rates produce an exceptional ability to develop pesticide resistance.
Our group is a cofounder of the international consortium, GAP-M (Genomics in Agricultural
Pest Management, http://www.spidermite.org/gapm/), composed by researchers from
different scientific institutions, involved in the plant-spider mite interplay. The consortium
has demonstrated a proliferation of detoxifying genes and digestive enzymes in the
genome of the spider mite that allow its feed on many hosts (Gribc et al. 2011; Santamaria
et al. 2012a). In this scenario, it is crucial to identify and characterize alternative control
systems to confer plant resistance against mite attack. T. urticae is able to rear on
Arabidopsis plants which offer important advantages for basic research in genetics and
molecular biology. After T. urticae infestation, plants activate a complex signalling network
to generate defence at different levels combining basal constitutive with inducible
defences.
Based on the information previously generated by the consortium about differential gene
expression in resistant and susceptible Arabidopsis accessions after T. urticae infestation,
candidate genes putatively involved in plant defence have been selected to be
characterized (Zhurov et al. 2014). Molecular and biochemical assays have been
performed to demonstrate their participation in plant defence pathways and genetic
approaches have been developed to corroborate the in vivo function. Bioassays with T.
urticae population have complemented a good set of data about their defence role
(Santamaria et al. 2012b, 2015).
References:
Grbic M, et al. (2011). Nature vol, 479: 487-492.
Santamaria ME, et al. (2012a). BMC Genomics vol, 13:307.
Santamaria ME, et al. (2012b). PLoS ONE vol, 7(8): e43011.
Santamaria ME, et al. (2015). PLoS ONE vol, 10(6): e0128323
Zhurov V, et al. (2014). Plant Phys vol, 164: 384-399
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Poster 02 / SVI P02
GCN2: A protein kinase necessary for effective defence response
against necrotrophic phytopathogen Botrytis cinerea
Marta Berrocal-Lobo , Mar Castellano Moreno
CBGP (UPM-INIA), Pozuelo de Alarcón, Madrid, Spain
Several stress conditions such as drought, cold, heat shock, amino acid starvation,
wounding or ultraviolet light produce a global rapid inhibition on protein translation in
plants. This inhibition allows the selective translation of selected mRNAs necessary for
survival (1). This process is dependent on the phosphorylation of translation initiation factor
EIF2α, which is specifically phosphorylated by GCN2, a protein kinase stimulated by
uncharged tRNAs, present in all eukaryotes. The implication of GCN2 in human innate
immunity and diseases such as cancer and Alzheimer´s is well established (2). In plants
GCN2 is involved in the response to stress, producing EIF2α phosphorylation after salicylic
acid, Methyl jasmonate or ACC treatments or after wounding (3).
In this work we demonstrate that the presence of the necrotrophic fungus Botrytis cinerea
(B.c) as the presence of chitin, a well known elicitor of plant defence forming part of fungal
spores, induce a fast EIF2α phosphorylation and protein translation inhibition, showing
that the process is dependent on GCN2 activity. Additionally, GCN2 insertion mutants
show an impaired capacity to defence against B.c.
Our results suggest that in parallel to the well-characterized signalling pathways operating
during plant defence to B.c, a selective protein translation inhibition, mediated by GCN2,
allows the plant to activate an effective immunity response to this fungus.
References:
(1) Echevarría-Zomeño et al., (2013). Int. J. Mol. Sci, 14(3), 4670-4683
(2) Tsalikis et al., (2013). Cell Microbiol. 15(10):1632-41.
(3) Lageix et al., (2008). BMC Plant Biol. Vol. 24.(8):134.
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Poster 03 / SVI P03
Identification and functional validation of novel miRNAs involved in
rice immunity
Rosany Camargo, Lidia Campos-Soriano, Mauricio Soto-Suárez, Blanca San Segundo.
Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Edifici CRAG Campus UAB, 08193, Barcelona, Spain.
Plants have developed efficient mechanisms to protect themselves from pathogen attack.
Emerging evidence support the notion that microRNAs (miRNAs) have a regulatory role in
plant defense responses to pathogen infection. miRNAs are small non-coding RNAs that
play important functions in the regulation of developmental processes, and responses to
biotic and abiotic stresses in different plant species. miRNAs are produced from precursors
with stem-loop structures which are sequentially processed by DICER-like (DCL) activities.
MiRNAs regulate gene expression by triggering sequence-specific cleavage or translation
repression of the target transcripts. In rice, the fungus Magnaporthe oryzae causes rice
blast, one of the most devastating diseases of cultivated rice worldwide. In this work, we
addressed the lack of knowledge on miRNAs controlling the rice responses to infection by
M. oryzae. For this, small RNA libraries were constructed from rice (Oryza sativa cv.
Nipponbare) tissues that had been treated, or not, with M. oryzae elicitors which were then
subjected to high-throughput sequencing. Elicitor treatment was found to be accompanied
by dynamic alterations in the expression of a significant number of known miRNAs. This
study also allowed us to identify small RNA sequences representing novel miRNA
candidates. Criteria used for selection and validation of the most promising novel miRNA
candidates included: i) predicted precursor structure; ii) predicted target gene(s) and mode
of action (miRNA cleavage vs translational inhibition), and iii) availability of rice mutants
for candidate miRNAs and/or target genes. To confirm that miRNA candidates indeed
represent novel miRNAs, we examined their level of accumulation in rice mutants affected
in small RNA biogenesis (e.g. dcl1 mutants). The biological significance of miRNA
candidates was investigated in transgenic rice plants overexpressing the corresponding
precursor sequence. For this, the miRNA precursor sequence for each miRNA candidate
was cloned, and its functionality confirmed through transient expression assays in N.
benthamiana leaves. The transgenic rice lines were assayed for their properties of
resistance/susceptibility to infection by the rice blast fungus M. oryzae. MiRNAs
functioning as positive or negative regulators in the rice response to M. oryzae infection
have been identified. The knowledge gained in this study will help in understanding
miRNA-mediated regulatory mechanisms in rice immunity.
References:
• Campo S, et al. 2013. New Phytol. 199(1):212-27.
• Baldrich P, et al. 2015. RNA Biology 12(8):847-63.
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Poster 04 / SVI P04
Steryl glycoside metabolism as a novel target for improving stress
tolerance in tomato
Nidia Castillo1, Karla Ramírez-Estrada1, Victor Flors2, Montserrat Arró1,3 Albert
Boronat1,4, Albert Ferrer1,3 and Teresa Altabella1,5
1
Center for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Campus UAB, Barcelona,
España; 2Dpto de Ciencias Agrarias y del Medio Natural, UJI, Castellón, España; 3Dpto de
Bioquímica y Fisiología, UB, Barcelona, España; 4Dpto de Bioquímica y Biomedicina Molecular,
UB, Barcelona; España, 5Dpto de Biología, Sanidad y Medio Ambiente, UB, Barcelona, España.
Sterols are found in nature as free sterols (FE) and conjugated as steryl esters (SE), steryl
glycosides (SG) and acylated steryl glycosides (ASG). Among these, FE, SG and ASG are
vital to cell membrane structure and function. Recent studies have shown that free and
conjugated sterols play an essential role not only in plant growth and development but also
in their responses to different types of stress. Tomato (Solanum lycopersicum) is one of
the most widely grown crops that provide key nutrients to the human diet. Tomato, along
with other species of the Solanaceae family, shows an atypical profile of conjugated
sterols, accumulating particularly high amounts of SG and ASG (Duperon et al., 1984;
Whitaker, 1988). However, the biological significance of this peculiar sterol composition is
currently unknown, and the knowledge about the enzymes involved in the synthesis of
conjugated sterols is still very limited. These include the UDP-glucose:sterol
glucosyltransferases (SGTs) that catalyze the transfer of sugar molecules, most commonly
glucose, to the C3-hydroxyl of the sterol backbone to form SG (Ury et al. 1989). We have
identified four tomato (Solanum lycopersicum cv. Micro-Tom) genes coding for putative
SGTs, referred to as SlSGT1-4. The corresponding cDNAs have been cloned and the
functional identity of the encoded enzymes demonstrated by SGT activity assays of the
proteins expressed in E. coli. The expression of the four genes is differentially modulated
in different tomato organs and fruit ripening stages, as well as in response to different
exogenous stimuli (abscisic acid, salycilic acid, methyl jasmonate and flagellin). Moreover,
expression of the SlSGTs fused to YFP in agroinfiltrated N. benthamiana leaves revealed
that at least three tomato SGTs (SGT1-3) are cytosolic proteins. Finally, data will be
presented about the involvement of SGTs and SGs in plant response to different types of
stress (biotic and abiotic). All these data will set the basis for further studies aimed at
understanding the role of glycosylated sterols 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 AGL201313522-R) and the Generalitat de Catalunya (grant number 2014SGR 1434). N.C. is recipient of predoctoral
fellowships from the CONACYT (México). K.R-E. is recipient of a postdoctoral fellowship from the CONACYT
(México).
References
Duperon et al. (1984). Phytochemistry, 23: 743-746
Whitaker BD. (1988). Phytochemistry, 27: 3411-3416.
Ury et al. (1989). Plant Physiol. 91: 567–573.
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Poster 05 / SVI P05
The β-subunit of the Heterotrimeric G protein Modulates Arabidopsis
Immune Responses to Different PAMPs
Chandra M. Singh1,2, Marta García1,2, Antonio Molina1,2, Miguel-Ángel Torres1,2
1
Centro de Biotecnología y Genómica de Plantas (UPM-INIA), Universidad Politécnica Madrid
(UPM) – Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus
Montegancedo, 28223-Pozuelo de Alarcón, Madrid, SPAIN. 2Departamento de BiotecnologíaBiología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de
Biosistemas, 28040-Madrid, SPAIN
Arabidopsis heterotrimeric G protein mediates the activation of PAMP (pathogenassociated molecular pattern)-triggered immunity (PTI) and the establishment of disease
resistance responses against different types of pathogens. We have investigated the role
of the β subunit of the Arabidopsis heterotrimeric G protein, AGB1, in the establishment of
PTI. We generated double mutants agb1 fls2 (impaired in the receptor of bacterial flagellin
PAMP, flg22) and agb1 cerk1 (defective in the receptor of chitin, a fungal PAMP). These
double mutants were used to monitor the activation of PTI in response to individual PAMPs
(flg22 or chitin) or to combinations of PAMPs present in extracts of whole bacteria
(Pseudomonas syringae pv. tomato DC3000) or fungi (Plectosphaerella cucumerina
BMM). PTI markers included the production of reactive oxygen species (ROS), MAPK
activation or defense gene expression. Interestingly, the double agb1 fls2 shows complete
depletion of ROS production upon treatment with the bacterial extracts, suggesting that
additional PAMPs other than flg22 are present in the bacteria extract used and than PTI
activated by this mixture of PAMPs was dependent on AGB1. In line with these data, we
found that agb1 fls2 displays enhanced bacterial growth and additive effects in P. syringae
driven stomatal closure compared to individual mutants. Our experiments suggest that the
β subunit of the Arabidopsis heterotrimeric G protein acts as a signaling hub in the
activation of PTI in response to different PAMPs. Our recent progress in the
characterization of the molecular mechanism determining AGB1 function in PTI will be
presented.
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Poster 06 / SVI P06
Design and validation of an infectious clone of ToLCNDV, an emergent
virus in South-East Spain
Dina Cifuentes1, Josefina Contreras1, Miguel Aranda2, César Petri Serrano1
1
Departamento of Producción Vegetal, Universidad Politécnica de Cartagena, 30203-Cartagena,
Murcia, Spain. 2 Departamento de Biología del Estrés y Patología Vegetal, CEBAS-CSIC, 30100,
Murcia, Spain
Tomato leaf curl New Delhi virus (ToLCNDV) (Geminiviridae, genus Begomovirus) is an
important pathogen that severely affects tomato and cucurbits production. Original from
the Indian Subcontinent, it has been recently detected in Spain. ToLCNDV genome
consists of two single-stranded circular DNA molecules, denominated as DNA-A and DNAB, of approximately 2.7 kb each. Partial dimeric DNA-A and DNA-B clones were
constructed in a binary vector and used to agroinoculate zucchini (cv. ‘Brillante’) and
pumpkins (cv. ‘Avalon’) plants. Symptoms were observed in plants of both species at 100%
and 50% frequency for zucchini and pumpkin plants, respectively. First symptoms
appeared 7 days post-inoculation, and they were clearly observed at 14 days postinoculation. Visual observation was confirmed by molecular techniques, such as molecular
hybridization and qPCR.
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Poster 07 / SVI P07
The tomato HAIRPLUS gene is a regulator of trichome density
Rocío Fonseca1, Jorge Luis Quispe1, Ricardo Lebrón2, Cristina Gómez-Martín3, Michael
Hackenberg2, José L. Oliver3, Rafael Lozano1, Juan Capel1
1
Área de Genética. Centro de Investigación en Biotecnología Agroalimentaria (BITAL).
Universidad de Almería. 04120 Almería, España. 2 Dpto. de Genética, Facultad de Ciencias,
Universidad de Granada, Campus de Fuentenueva s/n, 18071-Granada, España. 3 Laboratorio
de Bioinformática, Centro de Investigación Biomédica, PTS, Avda. del Conocimiento s/n, 18100Granada, España
The Solanaceae family comprises about 3,000 species, many of them of agricultural
interest, from which tomato (Solanum lycopersicum L) is the most important among fleshy
fruit crops. Due to the process of domestication, the genetic variability of tomato traits
associated with the response to biotic and abiotic stresses is scarce. For tomato breeding,
resistance genes have been introgressed from wild related species due to compatibility
among species of the Lycopersicon section of Solanum. However, interspecific hybrids
show numerous undesirable agronomic traits and too many backcross generations are
required to recover the favourable traits of cultivated tomato.
In order to increase genetic and phenotypic variability in tomato, our research group has
initiated a program of chemical mutagenesis with ethyl methyl sulfonate (EMS). As part of
this program, we have identified the hairplus mutant (hap), whose main phenotypic trait is
the high density of trichomes in all the aerial organs of the plants. Trichomes are structures
differentiated from epidermal cells whose nature can be glandular, when they have
chemical-secreting glands, or non-glandular. Both these types are observed in tomato,
where non-glandular trichomes act as a physical barrier to the movement and spread of
pests while glandular trichomes secrete sticky or toxic substances immobilizing or repelling
insects. The hairplus mutation increases trichome density, but does not alter the identity
of the two types of trichomes present in tomato. However, the mutation seems to be
pleiotropic and to dramatically reduce the rate of fruit set due to the formation of a small
amount of functional pollen. Genetic analysis in segregating populations indicates that the
hairplus phenotype seems to be inherited as a single recessive Mendelian trait. Molecular
characterization from transcriptomic analysis data performed in the mutant plants allows
us to conclude that the HAIRPLUS gene is a new regulator of tomato trichome density.
Trabajo financiado por el Ministerio de Economía y Competitividad (proyectos AGL2013-49090-C02-1P y AGL2013-49090-C02-2-P). Agradecemos al Campus de Excelencia Internacional Agroalimentario
(CeiA3) su apoyo en actividades científicas.
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Poster 08 / SVI P08
Mediated Plastid RNA Editing in Plant Immunity
Javier García-Andrade, Vicente Ramírez, Ana López, Pablo Vera
Instituto de Biologıía Molecular y Celular de Plantas, Universidad Politécnica de ValenciaC.S.I.C, Ciudad Politécnica de la Innovación, Ingeniero Fausto Elio, Valencia, Spain
Plant regulatory circuits coordinating nuclear and plastid gene expression have evolved in
response to external stimuli. RNA editing is one of such control mechanisms. We
determined the Arabidopsis nuclear-encoded homeodomain-containing protein OCP3 is
incorporated into the chloroplast, and contributes to control over the extent of ndhB
transcript editing. ndhB encodes the B subunit of the chloroplast NADH dehydrogenaselike complex (NDH) involved in cyclic electron flow (CEF) around photosystem I. In ocp3
mutant strains, ndhB editing efficiency decays, CEF is impaired and disease resistance to
fungal pathogens substantially enhanced, a process recapitulated in plants defective in
editing plastid RNAs encoding NDH complex subunits due to mutations in previously
described nuclear-encoded pentatricopeptide-related proteins (i.e. CRR21, CRR2).
Furthermore, we observed that following a pathogenic challenge, wild type plants respond
with editing inhibition of ndhB transcript. In parallel, rapid destabilization of the plastidial
NDH complex is also observed in the plant following perception of a pathogenic cue.
Therefore, NDH complex activity and plant immunity appear as interlinked processes.
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Poster 09 / SVI P09
Signwalling: plant immunity regulated by cell wall integrity
Hugo Mélida1, Laura Bacete1, Eva Miedes1,2, Antonio Molina1,2
1
(UPM) – Instituto Nacional de Investigación y Tecnología Agraría y Alimentaria (INIA) Campus
Montegancedo UPM, 28223-Pozuelo de Alarcón, Madrid, SPAIN. 2 Departamento de
Biotecnología y Biología Vegetal. Escuela Técnica Superior de Ingeniería Agronómica,
Alimentaria y de Biosistemas, UPM. Avda. Complutense 3, 28040, Madrid, SPAIN.
Plant innate immunity can be triggered by pathogen-associated molecular patterns
(PAMPs) and by plant “self” damage-associated molecular patterns (DAMPs). DAMPs
comprise plant cell wall-derived molecules or peptides that are released or synthesized,
respectively, upon pathogen infection or wounding. Despite the relevant role of wall
derived DAMPs in plant-pathogen interactions, a very limited number of them, such as
oligogalacturonides (OGs), has been well characterized. A biased resistance screening of
Arabidopsis thaliana cell wall mutants allowed us to identify a high number of mutants with
altered susceptibility/resistance to four different pathogens with distinct colonization styles,
further corroborating the relevance of cell wall in resistance to pathogens. We
characterized the molecular bases of the wall-mediated resistance found in these mutants,
and several wall fractions from these plants were tested for their capacity to activate
immunity. Interestingly, we found that mutant wall fractions enriched in different wall
components were more active than the corresponding wild-type ones in activating immune
responses. These included intracellular calcium accumulation, phosphorylation by
mitogen-activated protein kinases and transcriptional regulation of immunity genes. The
immune active components in these fractions were shown to be of a carbohydrate nature.
Monosaccharide and glycosidic linkage analyses combined with further fractionation
procedures (size exclusion and ion exchange chromatography) are being performed in
order to elucidate the structures of such immune active DAMPs. Our data indicate that
these novel wall DAMPs activate specific immune signaling pathways that seems to differ
from those triggered by OGs. Our recent progress on the characterization of these novel
DAMPs and their role in the regulation of plant immunity will be presented.
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Poster 10 / SVI P10
The expression and function of root meristem genes during nematode
induced gall development
Rocío Olmo1, Javier Cabrera1, Alejandra Garcia1, María Fe Andrés2, Miguel Ángel
Moreno-Risueño3, Carmen Fenoll1, Carolina Escobar1
1
Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Toledo,
2
Spain, Departamento Protección Vegetal, Instituto Ciencias Agrarias CSIC, Madrid,
3
Spain. Departmento de Biotecnología, Centro de Biotecnología y Genómica de Plantas,
Universidad Politécnica de Madrid INIA, Madrid, Spain.
Plant endoparasitic nematodes constitute a major threat for agriculture due to their impact
on plant productivity and to the gradual banishment of effective, but contaminant chemical
nematicides used for their control. Among the most harmful are root-knot nematodes
(RKNs; Meloidogyne spp.). RKNs induce a group of characteristic cells, called Giant Cells
(GCs) from vascular cell precursors that serve as a feeding site within the root. GCs are
abnormally large cells induced by nematode effectors in the vascular cylinder that undergo
repeated mitosis with aborted cytokinesis, and become transfer-like cells that the
nematode uses as sinks for plant nutrients. Additionally, vascular cells around the GCs
divide, and cortex cells become hypertrophic forming the typical galls or knots in the roots
infected by these nematodes (Cabrera et al., 2015).
Using laser capture microdissection, we obtained the transcriptomes of early-developing
GCs (at 3 days post inoculation) induced by M. javanica in Arabidopsis. We studied the
GC molecular signatures by analysing their transcriptional profiles as compared to control
uninfected tissues (Barcala et al., 2010). Two genes encoding heat-shock proteins and a
heat-shock transcription factor (HSF) were up-regulated. Interestingly, the HSFB4, namely
SCHIZORIZA, is not related to the heat-stress responses, but to developmental processes
within the root meristem.
In addition, we compared the specific transcriptomes of GCs to those of different root cell
types (Cabrera et al., 2014). Results indicated that the transcriptomes of undifferentiated
root cell types, as the quiescent center and genes characteristic of lateral root initial cells,
shared some gene expression changes with GC and gall transcriptomes. The protein
products encoded by these genes have varied functions, such as cell cycle regulation and
cytoskeleton or cell wall remodelling. Among them is also SCHIZORIZA (SCZ),
characteristic of the quiescent center cells transcriptome. We thence studied the
expression of other genes with essential roles in the root QC establishment and stem cell
maintenance like SCARECROW (SCR), SHORT ROOT (SHR) and WUSCHEL-RELATED
HOMEBOX 5 (WOX5). All were induced in galls formed by M. javanica in Arabidopsis
showing different patterns. We will discuss their roles during gall/GC development.
References
Barcala M, et al. (2010) Plant J. 61(4): 698-712.
Cabrera J, et al. (2014) New Phytol. 203(2): 632-645.
Cabrera J, et al. (2015) Plant Signal Behav. 10(3): e990825.
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Poster 11 / SVI P11
Jasmonate-Auxin crosstalk in plant stress responses
Marta-Marina Pérez-Alonso, Mathias Hentrich2, Beatriz Sánchez-Parra3,Stephan
Pollmann4
1
Centre for Plant Biotechnology and Genomics (CBGP) U.P.M., Madrid, Spain, 2 Department of
Plant Physiology, Ruhr-University Bochum, Bochum, Germany, 3 CBGP, U.P.M., Madrid, Spain. 4
CBGP, U.P.M., Madrid, Spain.
Auxin is a plant hormone associated with the regulation of different plant process, ranging
from cell division and elongation, differentiation, tropisms, apical dominance,
senescence, leaf and fruit abscission, to flowering (Hentrich et al., 2013a). Actually,
among the multiple routes proposed for auxin biosynthesis, i.e several tryptophandependent routes and a tryptophan-independent pathways, the two-step indole-3-pyruvic
acid (IPA) pathway is considered the main source of indode-3-acetic acid (IAA) in plants.
This pathway involves the action of two classes of enzymes, tryptophan-pyruvate
aminotransferases (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1
(TAA1)/TRYPTOPHAN AMINOTRANSFERASE RELATED (TAR)) and flavin
monooxygenases (YUCCA) (Zhao, 2010). Among these gene families, the presence of
the YUCCA genes in multiple plant genomes: Petunia, Rice, Tobacco, Tomato, Maize,
and Popular, point out an essential role in auxin biosynthesis and plant development.
Nevertheless the molecular mechanisms that control how YUCCA gene expression is
regulated and its physiological role are still under scrutiny. We previously showed the
transcriptional regulation of two Arabidopsis thaliana YUCCA genes, YUCCA8 and
YUCCA9, by methyl-jasmonate, indicating a crosstalk between these two seemingly
antagonistic phytohormones (Hentrich et al., 2013b). Our recent data, obtained by qPCR
analysis and transient gene expression analysis of YUCCA8 and YUCCA9 promoterreporter lines, reveal that both YUCCA promoters are direct targets of the basic helixloop-helix MYC2 transcription factor, as well as its closely related MYC3 and MYC4
transcription factors. On the other hand, similar to other auxin overproduction mutants,
YUC8 and YUC9 gain-of-function lines, YUCCA8ox and YUCCA9ox, have been shown
to contain elevated free IAA levels and display typical high-auxin related phenotypes
(Hentrich et al., 2013a). Here, we report that 35S:YUC8 and 35S:YUC9 transgenic plants
display substantially increased secondary growth. Microscopic examination and
histochemical analyses demonstrate that the increased IAA content provoked a
significant expansion of both the xylem and interfascicular fibers. In-depth cell wall
analyses revealed that the induced auxin overproduction in the transgenic lines led to an
abnormally strong lignification, which is seemingly mediated by the induction of ethylene
biosynthesis. The considerably pronounced lignification was accompanied by the
significantly increased expression of several XTH genes, which are known to be involved
in cell wall remodelling. In addition, our results show that YUC8 and YUC9 mediate a
positive defence response against bacterial pathogens (Pseudomonas syringae DC300)
and herbivore insects (Tetranychus urticae). Also, we observed an improved drought
tolerance for YUC9ox, thereby underlining a role of auxin in plant responses towards
biotic and abiotic stresses.
References:
Hentrich,M, et al. (2013b). Plant J., 74(4): 626-637.
Hentrich, M, et al. (2013a). Plant Signal. Behav., 8(11): e26363.
Zhao, Y, (2010) Annu. Rev. Plant Biol., 2(61): 49–64.
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Poster 12 / SVI P12
Identification and characterization of a Receptor-Like protein Kinase
involved in the immune response mediated by Cbl/Cipk module.
José María Personat1*, Yolanda Pareja-Jaime1*, Emilio Gutiérrez-Beltrán1, Olga del
Pozo1.
1
Instituto de Bioquímica Vegetal y Fotosíntesis (IBVF), Consejo Superior de Investigaciones
Científicas (CSIC), Sevilla, Spain.
* These authors contributed equally to this work.
Plants respond to pathogen infection by activating a two layered immune response
consisting of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Both
PTI and ETI share early signaling events upon pathogen perception that result in the
activation of defense responses. Recently, our group reported the identification and
characterization of two tomato (Solanum lycopersicum) proteins involved in plant
immunity: Cbl10 (Calcineurin B-Like Protein 10) and Cipk6 (Calcineurin B-Like Interacting
Protein Kinase 6). We demonstrated for the first time the participation of a Cbl/Cipk module
in biotic stress signaling in plants.
In order to investigate SlCipk6 downstream signaling molecular mechanisms, a yeast two
hybrid approach (Y2H) was used to identify SlCipk6-interacting proteins. We carried out
two separate screenings using a tomato cDNA prey library previously developed and
SlCipk6 and a mutant derivative, SlCipk6 (T172D), as baits. Among SlCipk6 interactors, a
Receptor-Like Protein Kinase (SlRlk) cDNA partial clone was identified. Because SlRlk
might be involved in immediate-early signaling events, we decided to follow up its
characterization. We obtained full length SlRlk ORF and confirmed its interaction with
SlCipk6 by Y2H. Here, we present SlCipk6 and SlRlk in planta interaction using coimmunoprecipitation and bimolecular fluorescence complementation assays and the
phosphorylation relationship between both kinases using an in vitro kinase assay in the
presence of [γ-32P]-ATP. We also present results that indicate that SlRlk is a novel
participant in PTI after bacterial challenge. Altogether, we present for the first time the
interaction between a CIPK and a Receptor-Like protein Kinase indicating that CIPKs
might regulate very early molecular eventgs in plant immunity.
References:
Boller, T., and Felix, G. (2009. Annu. Rev. Plant Biol. 60: 379–406.
del Pozo, O. et al (2004). EMBO J. 23: 3072–3082.
de la Torre F et al (2013) Plant Cell 25: 2748-64.
Tsuda, K. et al. (2010).Curr. Opin. Plant Biol. 13: 459–465.
Zhou J et al (1995) Cell 83: 925-35.
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Poster 13 / SVI P13
qPCR analysis of differently resistant chestnut clones to P. cinnamomi
infection
Saleta Rico1, Jesús Mª Vielba1, Nieves Vidal1, Conchi Sánchez1, Beatriz Cuenca2
1
Departamento de Fisiología Vegetal. Instituto de Investigaciones Agrobiológicas de Galicia
(IIAG-CSIC). Av. de Vigo s/n 15705 Santiago de Compostela (A Coruña). 2 TRAGSA. Vivero de
Ourense. Ctra Maceda-Valdrey Km 2. 32700 Maceda (Ourense).
Chestnut "ink disease" is caused by Phytophthora cinnamomi, a fungus-like eukaryotic
microorganism that belongs to the oomycota class. This disease has dramatic effects on
the populations of this species in Europe, particularly in zones with water-saturated soils,
where it is associated with a high mortality rate. It can also affect plantlets, therefore
causing severe economic losses to nurseries. Hybrids between European and Asiatic
chestnut species have long been used because of the higher degree of natural resistance
that Chinese and Japanese species show against this pathogen.
We have analyzed the expression patterns of nine defense-related genes in leaves of four
chestnut cultivars with different resistance level to the infection of this pathogen. This in
vitro working system included three Castanea hybrids with different percentage of alleles
of Asiatic origin, and a pure Castanea sativa clone. Plantlets were inoculated with a virulent
strain of P. cinnamomi, and leaf samples were collected 24, 48 and 72 hours after the
inoculation for the qPCR assay. CsGH3-1, a hormone-signaling gene responsive to auxin,
showed its highest expression level in the most resistant clone (PO11), with significant
levels already detected at 24 hours. This was also the case of the related CsGH3-2 gene,
but the latter exhibited a minor variation. Expression levels of transcription factors
belonging to the GRAS and AP2 families were low in the most sensitive clones. Other
genes potentially related to defense mechanisms, like CsTCTP and CsCPE, showed
expression levels that could also be linked to the degree of resistance of the different
chestnut clones.
Results provided relevant insights into the nature of Phytophthora spp. infection and plant
defense mechanisms. Our results suggest that the inoculation triggers a general shift in
the gene expression pattern of the plantlets. The timing of the response is crucial for the
resistance against these pathogens, because the most resistant clone was able to respond
earlier to P. cinnamomi. Besides, a greater transcriptional activation is directly related to
a higher degree of resistance.
To our knowledge, this is the first approach to investigate transcriptomic responses of
chestnut to P. cinnamomi in tissues other than roots. Data obtained from the expression
patterns might be useful for the development of early-detection methods in order to
complement other approaches. Furthermore, these results contribute to a better
understanding of the pathogen-host relation in trees, an under-represented area of study.
This work was funded by the CDTI through the program FEDER-INTERCONECTA 2013/2014
(INTEGRACASTANEA EXP00064828/ITC-20133040).
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Poster 14 / SVI P14
A bacterial acetyltransferase targets the protein kinase ZIP1, a positive
regulator of plant immunity.
Jose S. Rufián, Javier Rueda-Blanco, Diego López-Marquez, Carmen R. Beuzón, Javier
Ruiz-Albert
Instituto de Hortofruticultura Subtropical y Mediterránea, Universidad de Málaga-Consejo
Superior de Investigaciones Científicas (IHSM-UMA-CSIC). Dpto. Biología Celular, Genética y
Fisiología, Campus de Teatinos, Málaga, E-29071, Spain
Pseudomonas syringae is a model bacterial pathogen that penetrates the leaf to reach the
plant apoplast, where it replicates causing disease. In order to do that, the pathogen must
interfere and suppress a two-tiered plant defense response: PTI (PAMP-Triggered
Immunity, or basal resistance) and ETI (Effector-Triggered Immunity). P. syringae uses a
type III secretion system to directly deliver effector proteins inside the plant cell cytosol,
many of which are known to suppress PTI, some of which are known to trigger ETI, and a
handful of which are known to suppress ETI. Bacterial infection can also trigger a systemic
plant defense response that protects the plant against additional pathogen attacks known
as SAR (Systemic Acquired Resistance). We are particularly interested in the molecular
and cellular mechanisms involved in effector-mediated defense evasion by P. syringae, in
particular those involved in the suppression of ETI and SAR, and/or mediation of hormone
signaling.
Here we present data describing effector-mediated interference with plant immunity, by
means of acetylation of a key positive regulator of local and systemic responses. Our
work identifies a novel plant target for effector function, and characterizes its function.
This work illustrates how analyzing the means by which a given effector interferes with its
target can provide novel information regarding eukaryotic molecular mechanisms.
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Poster 15 / SVI P15
Volatile compounds emitted by diverse phytopathogenic
microorganisms promote plant growth and flowering through
cytokinin action: a case of dirty dishes
Ángela María Sánchez-López1, Marouane Baslam1*, Nuria De Diego2*, Francisco José
Muñoz1, Abdellatif Bahaji1, Goizeder Almagro1, Adriana Ricarte-Bermejo1, Pablo GarcíaGómez1, Jun Li1, Jan F. Humplík2, Ondřej Novák3, Lukáš Spíchal2, Karel Doležal2,
Edurne Baroja-Fernández1, and Javier Pozueta-Romero1
1
31192 Mutiloabeti, Nafarroa, Spain. 2Chemical Biology and Genetics Department, Centre of the
Region Haná for Biotechnological and Agricultural Research, Palacký University, Olomouc, CZ78371, Czech Republic. 3Growth Regulators Laboratory, Centre of the Region Haná for
Biotechnological and Agricultural Research, Palacký University and Institute of Experimental
Botany ASCR, Olomouc, CZ-78371, Czech Republic.
It is known that volatile emissions from some beneficial rhizosphere microorganisms
promote plant growth. Here we show that volatile compounds (VOCs) emitted by
phylogenetically diverse rhizosphere and non-rhizhosphere bacteria and fungi (including
plant pathogens) promote growth and flowering of various plant species, including crops.
In Arabidopsis plants exposed to VOCs emitted by the phytopathogen Alternaria alternata,
changes included enhancement of photosynthesis and accumulation of high levels of
cytokinins (CKs) and sugars. Evidence obtained using transgenic Arabidopsis plants with
altered CK status show that CKs play essential roles in this phenomenon, since growth
and flowering responses to the VOCs were reduced in mutants with CK-deficiency
(35S:AtCKX1) or low receptor sensitivity (ahk2/3). Further, we demonstrate that the plant
responses to fungal VOCs are light-dependent. Transcriptomic analyses of Arabidopsis
leaves exposed to A. alternata VOCs revealed changes in the expression of light- and CKresponsive genes involved in photosynthesis, growth and flowering. Notably, many genes
differentially expressed in plants treated with fungal VOCs were also differentially
expressed in plants exposed to VOCs emitted by the plant growth promoting
rhizobacterium Bacillus subtilis GB03, suggesting that plants react to microbial VOCs
through highly conserved regulatory mechanisms.
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Poster 16 / SVI P16
Deciphering the molecular mechanism underlying the broad spectrum
disease resistance of plants expressing YDA MAP3K
Sanjay Swami1,2, Lucía Jordá1,2, Sara Sopeña-Torres1,2, Clara Sánchez-Rodríguez1,2,3,
Viviana Escudero1,2, Yangnan Gu4, and Antonio Molina1,2
1
(UPM) – Instituto Nacional de Investigación y Tecnología Agraría y Alimentaria (INIA), Campus
Montegancedo UPM, 28223-Pozuelo de Alarcón, Madrid, SPAIN. 2Departamento de
Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria
y de Biosistemas, 28040-Madrid, Spain. 3 ETH, Universitätstrasse 2, 8092-Zürich, Switzerland.
4
Department of Biology, P.O. Box 90338, Duke University, Durham NC 27708, USA.
YODA (YDA) is a MAPK kinase kinase (MAP3K) that functions downstream the Receptor
Like Kinase (RLK) ERECTA (ER). YDA-ER pathway control Arabidopsis immune
responses, but also stomatal patterning and other plant developmental-associated
processes. Remarkably, plants expressing a constitutively active form of YDA (CA-YDA)
show broad-spectrum disease resistance to different type of pathogens, including fungi,
oomycetes, and bacteria. CA-YDA-mediated resistance is independent of defensive
pathways regulated by salicylic acid, jasmonic acid or ethylene, and of previously
characterised canonical immune pathways. In deep comparative transcriptomic analyses
of mock and fungal-inoculated wild-type and CA-YDA plants was performed. Interestingly,
we found that CA-YDA plants constitutively express defense-associated genes, such as
those encoding antimicrobial peptides, receptor-like kinases, and uncharacterised
extracellular peptides (ePEPs). We selected Arabidopsis mutants impaired in the
expression of these ePEPs and tested their contribution to resistance against the fungal
pathogen Plectosphaerella cucumerina BMM, the bacterium Pseudomonas syringae pv.
tomato DC3000 and the oomycete Hyaloperonospora arabidopsidis Noco2. Remarkably,
some of these ePEPs were found to contribute to immunity and also have a role in the
regulation of developmental processes. YDA and ER define a novel immune pathway that
orchestrates broad-spectrum resistance by regulating defensive responses that act in
parallel to canonical disease resistance pathways.
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Poster 17 / SVI P17
Copper transport to Medicago truncatula nodules is mediated by
MtCOPT1
Marta Senovilla1, Rosario Castro-Rodríguez1, Juan Imperial1,2 & Manuel GonzálezGuerrero1
1
Universidad Politécnica de Madrid. Centro de Biotecnología y Genómica de Plantas, Pozuelo de
Alarcón, 28223 Madrid. Spain. 2Consejo Superior de Investigaciones Científicas. Madrid. Spain.
Copper is a common redox cofactor for many physiological processes in plants, including
photosynthesis, mitochondrial respiration, and symbiotic nitrogen fixation (SNF). The latter
is the conversion, fixation, of N2 into NH3, a process carried out by endosymbiotic bacteria
(rhizobia) in legume root nodules. SNF requires relatively large amounts of copper as an
essential cofactor of some of the key reactions being carried out by the endosymbiotic
rhizobia (Preisig et al., 1996). This oligonutrient is delivered by the host legume through
the vasculature to the nodules and released in the infection/differentiation zone of them
(where rhizobia differentiate into nitrogen-fixing bacteroids). From there, a plasma
membrane transporter has to introduce copper into the cell for further cuproprotein
assembly (Rodríguez-Haas et al., 2013). COPT family of copper transporters, mediate
high affinity copper transport into the cytosol of eukaryotic cells (Peñarrubia et al., (2015);
Sancenón et al., (2003)). From the eight COPT family genes present in M. truncatula
genome, MtCOPT1 is the only one induced specifically in nodule. MtCOPT1 is able to
restore Saccharomyces cerevisae Δctr1 (defective in copper uptake) capacity to transport
copper. Inmunolocalization and GUS fusion studies localize MtCOPT1 mainly in the
plasma membrane of cells in the infection/differenciation zone of the nodule. In addition, a
Tnt-1-derived knockdown mutant line for MtCOPT1 shows decreased nitrogenase activity
when compared with the wild-type line. This activity is, at least, partially rescued when a
wild-type copy of MtCOPT1 gene is reintroduced. Taken together, these data suggest an
important role of MtCOPT1 copper-mediated transport for SNF.
This work was supported by ERC Starting Grant (ERC-2013-StG-335284) and MINECO Grant (AGL-201232974) to M.G-G.
References:
Peñarrubia L, et al. (2015). Frontiers in Plant Science 6, 255.
Preisig, O., et al. (1996). J. Bacteriol. 178, 1532–1538.
Rodríguez-Haas, B., et al. (2013). Metallomics 5, 1247–1253.
Sancenón V, et al. (2003). Plant Molecular Biology 51, 577–587.
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Poster 18 / SVI P18
Effects of Turnip mosaic virus infections on Arabidopsis thaliana
development
Silvia López-González1, Laura Sinausía1, Flora Sánchez1, Luis F. Pacios1, Fernando
Ponz1
Centro de Biotecnología y Genómica de Plantas. Universidad Politécnica de Madrid (UPM)Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA). Campus
Montegancedo UPM. 28223 Pozuelo de Alarcón (Madrid), Spain
Viral infections in plants frequently induce typical disease symptoms such as
necrosis, mosaics, chlorotic lesions, leaf yellowing, vein clearing and others. Nevertheless,
symptoms related to plant development and growth can also be induced by viruses. Many
of these symptoms are often found jointly in virus-infected plants, but it is also possible to
find differential symptoms between closely related viruses, even between different strains
of the same virus.
Turnip mosaic virus (TuMV) is a potyvirus that infects Arabidopsis. The different
developmental alterations induced by two TuMV strains in this host are the subject of
intense study in our laboratory. Progress in the characterization of these differential
interactions will be presented and discussed, from both plant and viral perspectives.
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Poster 19 / SVI P19
New evidences of molecular responses during the interaction of Ulmus
minor with Ophiostoma novo-ulmi and an endophyte associated to
DED-tolerant elms
Juan Sobrino-Plata1, Iván Fernández2, David Medel1, Sara Ormeño1, Begoña Coira1,
Juan A. Martín1, Carmen Collada1, Corné M. J. Pieterse2, Luis Gil1
1
Forest Genetics, Physiology and History Research Group, Forestry Engineering School,
Technical University of Madrid, Madrid, Spain 2Plant-Microbe Interactions, Department of
Biology, Utrecht University, Utrecht, Netherlands
Dutch elm disease (DED) is possibly the most devastating forest disease worldwide. DED,
caused by the invasive alien pathogen Ophiostoma novo-ulmi, has decimated Ulmus
minor populations in Europe. Many efforts have been dedicated to control this disease
over the last decades and have resulted in important success, such as the recent register
of seven tolerant U. minor clones as reproductive forest materials (Martín et al. 2015).
However, elm breeding programs are still based on traditional methods, and clearly need
to apply recent methodological advances to achieve an effective selection and breeding of
tolerant genotypes.
One of the main constraints in searching DED-tolerant genotypes is the long period
needed to evaluate the susceptibility of each tree. This screening should be done in
experimental plots with trees of at least 4 years old, since at lower ages trees show juvenile
tolerance. Improving elm breeding and efficacy in restoring elms at a large scale requires
first to shorten and simplify the selection process. On the other hand, the role of endophytic
fungi in tree tolerance to biotic and abiotic stress is receiving increasing attention. In
relation to DED, we have recently observed that the abundance of certain endophytes is
strongly associated to the tolerance level of their host tree to O. novo-ulmi. We hypothesize
that endophytes could play a key role in plant tolerance to the pathogen.
Using the transcriptome from a previous work published by our team (Perdiguero et al.
2015) we studied changes in expression of selected genes related to biotic interaction
responses, as well as concomitant changes in the physiology of the plants. This study was
performed with elm plantlets grown in vitro of well characterized tolerant and susceptible
elm clones. We studied plant responses to inoculation with O. novo-ulmi and an endophytic
fungus associated to DED-tolerant clones. By comparison of two tolerant and two
susceptible clones differences in the expression of genes that codify for two pathogenesisrelated proteins (PRs) were determined. Concretely a higher expression of PR4 was
observed in roots of tolerant clones compared to susceptible ones, while PR1 was
overexpressed in shoots of susceptible clones. Moreover we observed an overexpression
of phenylalanine ammonia lyase (PAL) gene in all the studied genotypes when they were
inoculated with O. novo-ulmi. This effect was attenuated when the plants were preinoculated with the endophyte, suggesting a beneficial effect. Additionally, the inoculation
of this fungus promoted the growth of the plants compared to non-treated plants or those
inoculated with O. novo-ulmi.
These results contribute to understand the molecular basis of U. minor tolerance to DED
opening new prospects for a future molecular-assisted selection of tolerant genotypes.
References:
Martín JA, et al. (2015) Forest 8: 172-180.
Perdiguero P, et al. (2015) Front Plant Sci 6: 541-553
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164
Sesión VII: Temas y Técnicas Emergentes
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Comunicaciones Sesión VII.
Temas y Técnicas Emergentes
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Ponencia Invitada / SVII PI
Sugars plays an important role in cuticle metabolism and cell wall
architecture of tomato and affects shelf-life softening
José G. Vallarino1, Yeats TH2,Rose JK2, Alisdair R. Fernie3, and Sonia Osorio1 *.
1
Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, University of MalagaConsejo Superior de Investigaciones Científicas, Department of Molecular Biology and
Biochemistry, Campus de teatinos , 29071 Málaga, Spain. 2 Department of Plant Biology. Cornell
University. Ithaca, NY, 14853, USA. 3Max-Planck-Institute für Molekulare Planzenphysiologie,
Am Mühlenberg 1, 14476 Golm, Germany. *Correspondence should be addressed to S.O.
([email protected])
Despite the fact that the sugar content of a fruit is regarded as one of its most commercially
important quality traits when assessed by the consumer, relatively little is known
concerning the physiological importance of sugar metabolism for the fruit shelf-life itself.
Cell wall disassembly is one of the main processes occurring at the end of the ripening
period and its rate and extent are crucial for the maintenance of fruit quality and integrity
(Matas et al., 2009). The cuticle, the lipophilic membrane layer that covers the outer
epidermal cell wall of the aerial parts of higher plants (Jeffree, 2006; Nawrath, 2006), has
been largely disregarded with respect to its putative influence in modulating fruit
development, ripening and postharvest performance. Here, we evaluate the effects of
modifying sugar metabolism by reduction of the activity of cell wall invertase (LIN5; Zanor
et al., 2009) via targeted approaches in tomato (Solanum lycopersicum). Detailed
characterization at transcriptomic, biochemical, histological, and biomechanical levels
revealed several unsual features of RNAi-LIN5 cuticles. These results suggest that
perturbation of endogenous fruit sugar levels affects the composition of the tomato cuticle
and cell wall architecture and reveal the importance of sugar metabolism in tomato fruit
metabolism and postharvest.
References
Jeffree CE, (2006). Biology of the plant cuticle. Oxford: Blackwell Publishing, 11–125
Matas AJ, et al (2009) Curr Opin Biotechnol 20: 197–203.
Nawrath C, (2006) Curr Opin Plant Biol 9: 281–287.
Zanor MI, et al (2009) Plant Physiol, 150: 1204–1218
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Comunicación Oral 1 / SVI ICO1
The integration of physiological, proteomic, and metabolomic levels
reveals new adaptive and stress-responsive mechanisms in Pinus
Luis Valledor1, María Jesús Cañal1, Jesús Pascual1, Mónica Escandón1, Wolfram
Weckwerth2,3, Mónica Meijón1
1
Plant Physiology Lab, Organisms and Systems Biology, University of Oviedo, Asturias, Spain. 2
Ecogenomics and Systems Biology, Faculty of Life Sciences, University of Vienna, Vienna,
Austria. 3Vienna Metabolomics Center (VIME), University of Vienna, Vienna, Austria.
Globally expected changes in environmental conditions, mainly increased temperatures,
irradiation, and droughts threatens plant productivity. Despite some advances towards
more tolerant varieties have been achieved in edible crops, the knowledge about the
specific mechanisms mediating stress adaptive responses in Conifers are scarce. Know
these pathways is crucial for designing new strategies focused on maintaining forest
productivity.
We studied the effect temperature and UV irradiation dosages aiming to mimic future
scenarios based on current models in a time course experiment in greenhouse grown
plantlets. Furthermore, the availability of a common garden, ten origins covering NorthSouth clinal variation and Mediterranean and Atlantic basins, allowed us to have a field
system to exploit natural variation towards deciphering how Pine can adapt to different
environments. Current technology for high-throughput phenotyping at the different -omic
levels and its integrative analysis will revolutionize the way we how we understand tree
biology and forest management.
Two complementary techniques (GC-MS and LC-Orbitrap-MS) were used to identify and
quantify the maximum number of ions corresponding to primary and secondary
metabolites. Very accurate mass, comparison to custom libraries, and in some cases
MS/MS were the strategies employed for identification. Proteins were identified and
quantified following a bottom up approach, employing custom databases.
The metabolome analysis of heat and UV stresses datasets allowed both the definition of
novel responsive pathways and the validation of major stress responses previously
described in other plants. The integration of analysed datasets (metabolome, proteome,
physiology, gene expression) provided a comprehensive picture of stress responses
proposing new metabolites and proteins closely related to specific stress adaptive
responses including kinases and proteases related to signalling and metabolic
coordination. The analysis of phenotypic diversity, following a “population metabolomics”
approach exploiting the common garden, showed how evolution to local environment was
linked to metabolome specialization to adapt to different climates.
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Comunicación Oral 2 / SVII CO2
Development of artificial vision systems for automatic phenotyping
Pedro Javier Navarro-Lorente1; Fernándo Pérez-Sanz2; Julia Weiss2 and Marcos EgeaCortines2
1
División de Sistemas e Ingeniería Electrónica. Campus de la Muralla del Mar, Universidad
Politécnica de Cartagena. 30202. Cartagena, 2 Instituto de Biología Vegetal, Genética Molecular.
Edif. I+D+I, Campus de la Muralla del Mar, Universidad Politécnica de Cartagena 30202,
Cartagena - Murcia, España.
The omic technologies have achieved a major change in the way we approach science as
complete “omes” can be analysed and data determines the major players in a given
biological process. We have developed artificial vision systems to achieve automatic plant
phenotyping. This should help us understand kinetics in all aspects of plant development
that are difficult to identify just by single point sampling. We built an initial vision system
based on two separate cameras capturing images in black and white for morning and
infrared for night pictures. It allowed us to identify several problems including the
movement of plant organs such as nutation, and we were able to identify differences in
growth kinetics of wild type and nana, a mutant affected in the circadian clock (Navarro et
al., 2012). A major issue encountered was the shifting of the pictures caused by the
differing spatial position of the sensors. We built a second system based on a single
camera with two sensors, one capturing RED GREEN BLUE (RGB) for day and a second
one at Near Infrarred (NIR) for night images. We were able to improve substantially the
image acquisition. Applying machine learning algorithms we were able to obtain excellent
automatic segmentation results from above images for rosettes but we had to apply two
different algorithms (kNN and SVM) for day and night images (Navarro et al., 2016). The
degree of automatic image classification was 99.3% of success for a total of 1200 images.
Two major issues encountered were the low capacity to segment images of laterally
growing organs such as flowers and leaves as a result of a low chromatic capacity of light
wavelengths emitted by the LED required for plant growth in chambers. The second was
the inherent organ movement that is itself circadian thus causing a two-fold problem as we
are interested in understanding the effects of circadian regulation on growth and both
processes are interrelated. Our current efforts and data on the effect of loss of function by
knockdown of LHY in Antirrhinum and ZTL in Petunia will be presented.
This work was part of the projects ViSelTR (TIN2012-39279) and MICINN BFU-2013-45148-R.
References:
Navarro PJ et al. (2012). Sensors 12: 15356–15375
Navarro PJ, et al (2016). Sensors (Basel). In press:
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Comunicación Oral 3 / SVII CO3
Next-generation forward genetic screens using mapping-bysequencing
David Wilson-Sánchez, Raquel Sarmiento-Mañús, María Rosa Ponce, José Luis Micol
Forward genetic screens have identified many genes and continue to be powerful tools for
the dissection of gene action and interactions in Arabidopsis and other plant species1.
Moreover, next-generation sequencing (NGS) has revitalized the time-consuming genetic
approaches to identify the mutation causing a phenotype of interest. Mapping-bysequencing combines NGS with classical mapping strategies and allows rapid
identification of point mutations2,3. As in conventional linkage analyses, mapping-bysequencing requires a phenotyped mapping population, but requires only a single round
of crosses to define a very narrow candidate region and the position of the causal mutation
itself. In addition, the mapping populations are pooled for NGS; mapping-by-sequencing
does not require individual genotypes. The single-nucleotide polymorphisms (SNPs)
caused by the chemical mutagenesis can be used as markers, enabling the use of a single
backcross to obtain a mapping population and thus making polymorphic strains
dispensable. Mapping-by-sequencing also does not require previous knowledge of the
wild-type sequence, making this approach useful for non-model species.
We performed several simulations to facilitate the design of mapping-by-sequencing
experiments for the identification of chemically induced point mutations. Through these
simulations, we evaluated first which short-read NGS technology is best suited to
Arabidopsis gene-rich genomic regions, and the minimum sequencing depth required to
confidently call variants. Next, we simulated mapping-by-sequencing experiments for the
identification of point mutations and determined how mapping population size and
sequencing depth affect mapping resolution. We also performed virtual outcrosses and
backcrosses in order to compare natural variations versus chemically induced SNPs as
mapping-by-sequencing tools. We also evaluated the viability of crossing two chemically
induced non-allelic mutants to obtain a mapping population to simultaneously map two
recessive mutations. In addition, we compared different ways of identifying dominant
mutations. Finally, using simulations, we tested a custom protocol to map T-DNA or
transposon insertions with paired-end Illumina-like reads; we assessed its reliability using
low sequencing depths and pooling several mutants together. The results of these
simulations proved useful for the design of real experiments.
References:
1.- Micol, J.L. (2009). Current Opinion in Plant Biology 12, 9-16.
2.- Schneeberger, K. et al. (2009). Nature Methods 6, 550-551.
3.- Candela, H et al. (2015). Journal of Integrative Plant Biology 57, 606-612.
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Comunicacion Oral 4 / SVII CO4
Genome-wide identification of ARGONAUTE-bound target RNAs in
Arabidopsis
Alberto Carbonell1, Noah Fahlgren2, James C. Carrington2, José-Antonio Daròs1
1
Instituto de Biología Molecular y Celular de Plantas (CSIC-Universidad Politécnica de Valencia),
Valencia, Spain, 2Donald Danforth Plant Science Centre, Saint Louis, USA
RNA silencing is an evolutionarily conserved, sequence-specific gene-inactivation system
that regulates several key biological processes such as development, stress responses,
chromatin remodeling and antiviral defense. In eukaryotes, ARGONAUTE (AGO) proteins,
a class of RNaseH-like ribonuclease, associate with small RNAs (sRNAs) of 20~30
nucleotides in length to silence target DNA or RNA at the transcriptional or posttranscriptional level, respectively. In plants and animals, ternary complexes form when
AGO-sRNA complexes recognize target RNAs through sequence-specific interactions
conferred by their guide sRNA. As a result, target RNAs are typically repressed either
through direct cleavage (slicing) or through other mechanisms such as target
destabilization or translational repression.
In plants, the Arabidopsis thaliana genome includes 10 AGO genes, of which at least
AGO1, AGO2, AGO5, AGO7 and AGO10 express proteins with post-transcriptional roles.
The sRNAs associating with each of these AGOs were identified by high-throughput
sequencing of sRNAs from AGO immunoprecipitates. However, similar approaches were
not reported for the genome-wide identification of AGO-bound target RNAs until recently.
We showed that AGO ternary complexes including cellular AGO1, AGO2 or AGO7 and
target RNAs are efficiently captured with catalytically null HA-tagged AGOs (cnAGOs)
expressed under their native promoter, but not with their catalytically active counterparts
(Carbonell et al. 2012). We hypothesized that because CnAGOs do not slice target RNAs,
ternary complexes are stabilized and target RNAs can now be efficiently coimmunoprecipitated. In particular, the RNA immunoprecipitation (RIP) followed by highthroughput sequencing (RIP-Seq) of cnAGO1-bound target RNAs confirmed that ternary
complexes including CnAGOs are enriched with known AGO1 target RNAs compared to
samples including catalytically active AGO1 (Carbonell et al. 2012). However, the
sensitivity of this methodology must be improved to be able to identify new AGO target
RNAs and/or novel mechanisms of regulation of target RNAs by post-transcriptional
AGOs.
We will present recent optimizations in the experimental and computational/statistical
methods of our RIP-Seq methodology. The introduction of a micrococcal nuclease
(MNase) digestion step in cellular extracts prior to the immunoprecipitation has allowed
the efficient trimming of AGO-non-protected nucleotides from target RNAs, what resulted
in an enrichment in AGO-protected RNAs. The computational analysis pipeline was also
further developed to identify authentic target sites (or AGO footprints) in enriched coimmunoprecipitated transcripts, by doing peak calling analysis and assigning a maxima
value to each peak included in target site regions, and assessing the statistical significance
of each peak. Our current methodology has already identified several potential new AGO1
target RNAs that are under investigation.
References:
Carbonell A, et al. (2012) Plant Cell, 24(9): 3613-3629-780.
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Poster 01 / SVII P01
New insights into protein S-Sulfhydration: A large-scale proteomic
study using the Tag Switch Method in Arabidopsis
Ángeles Aroca, Cecilia Gotor, Luis Romero
Sevilla, Spain.
Hydrogen sulfide (H2S) has been referred as the third gasotransmitter in animal and
vegetal cells. In animals, it has been involved in many important physiological and
pathological processes and more recently, its protective effect against oxidative, metal,
heat, osmotic and saline stresses have been reported in plants. Sulfide has also been
involved in the regulation of important physiological processes as the stomatal
closure/aperture, modulation of photosynthesis and autophagy regulation.
Nevertheless, the mechanism of its action is not fully understood yet. The main signaling
mechanism of H2S is through the posttranslational modification S-sulfhydration of reactive
cysteine residues on target proteins by converting the thiol group (-SH) into a persulfide
group (-SSH). A growing number of S-sulfhydrated proteins have been described using
different tagging methods and proteomic approaches (Aroca et al, 2015). However, the
specificity of these methods has been in the spotlight of several authors, arising certain
methodological limitations. Thus, a new approach for the S-sulfhydrated protein detection
has been recently described by Zhang et al. (2014), the Tag Switch Method, which shows
a high specificity and sensibility.
Due to the growing importance of sulfide as a signaling molecule and considering the
knowledge on function and targets of S-sulfhydration is scarce in plants, in this study we
perform a large-scale proteomic study of endogenously S-sulfhydrated proteins in
Arabidopsis wild type and des1 mutant plants using for the first time the Tag Switch Method
in a plant system. Functional evidences of S-sulfhydration in cellular signalling have also
addressed by genetic and chemical methodologies and demonstrated the importance of
this modification in protein subcellular localization.
References
Aroca, Á., et al. (2015). Plant Physiol 168: 334-342.
Zhang, D., et al. (2014). Angewandte Chemie International Edition 53: 575-581.
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Single-cell monitoring of cell cycle progression in whole developing
organs
Bénédicte Desvoyes1, Ainhoa Arana-Echarri1, María Delgado-Barea1, Sofía Otero1,
María Isabel Lopez1, Crisanto Gutierrez1
1
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera 1, 28049 Madrid,
Spain.
Plant organ development is mainly a postembryonic process that occurs in the adult in a
continuous manner. Consequently, a strict balance between cell proliferation and
differentiation is required. In addition, many plant cells undergo one or more
endoreplication cycles as part of their normal differentiation program. Therefore, growing
plant organs consist of populations of dividing, endoreplicating and differentiated cells all
derived from a few stem cells. The transitions between the different cell pools are
integrated and respond to developmental and environmental cues. In fact, cell cycle control
influences greatly developmental programs, e.g. increased or decreased proliferation rates
affect organ growth and shape.
To get insight into the coordination of the different processes that influence organ
development it is important to be able to identify cells progressing in vivo through the cell
cycle in a non-invasive manner. We have used fluorescently-labeled proteins that
unambiguously identify cells in G1, S/G2 and G2 and generated plants that express them
under their own promoters in a single plant. (1) As a G1 marker, we used a CFP-labeled
prereplication complex protein that is loaded in late M/early G1 and rapidly degraded soon
after S-phase initiation in a proteasome-dependent manner. (2) S-phase cells are followed
by the incorporation of a canonical histone H3.1-mRFP, which is maintained through
mitosis in actively dividing cells and excluded late in G2 in cells undergoing their last mitotic
cycle. (3) Finally, G2 cells are labeled with CYCB1;1-GFP, with a maximum in late G2 and
degraded by the APC in anaphase. These markers are also useful to follow cells
undergoing endoreplication cycles. The combination of these three markers in a single
organism allows monitoring all the cell cycle phases and constitutes an important tool to
study cell cycle regulation in a growing organism during normal growth or in response to
internal cues and external challenges.
In order to simplify the use of these marker plant line to monitor cell cycle variation in
mutants we are now using the GoldenBraid system to generate transgenic plants that
express the different markers from a single locus.
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PAMP-triggered immunity against Pseudomonas syringae involves
microRNA-mediated regulation of several uncharacterized R genes
Diego López-Márquez1, Edgar A. Rodríguez-Negrete1, Nieves López-Pagan1, Adela
Zumaquero1, Eduardo R. Bejarano1 and Carmen R. Beuzon1
1
Department of Cellular Biology, Genetics and Physiology. IHSM-UMA-CSIC, Málaga, Spain.
Two main types of noncoding small RNA molecules have been found in plants: microRNAs
(miRNAs) and small interfering RNAs (siRNAs). They differ in their biogenesis and mode
of action, but share similar sizes (20-24 nt). Their precursors are processed by Dicer-Like
RNase III (dcl) proteins present in Arabidopsis thaliana, and in their mature form can act
as negative regulators of gene expression, being involved in a vast array of plant
processes, including plant development, genomic integrity or response to stress. SmallRNA mediated regulation can occurs at transcriptional level (TGS) or at posttranscriptional level (PTGS). In recent years, the role of gene silencing in the regulation of
expression of genes related to plant defence responses against bacterial pathogens is
becoming clearer. Comparisons carried out in our lab between the expression profiles of
different mutants affected in gene silencing, and plants challenged with Pseudomonas
syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes,
belonging to the TIR-NBS-LRR gene family, differentially expressed in these conditions.
Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible
for down-regulating expression of these R genes through the generation of siRNAs. We
have also found that the corresponding pri-miRNA is down-regulated after PAMPperception in a SA-dependent manner. We also demonstrate that plants with altered levels
of miRNA* (knockdown lines or overexpression lines) exhibit altered PTI-associated
phenotypes, suggesting a role for this miRNA* in this defence response against bacteria.
In addition we identify one of the target genes as a negative regulator of defence response
against Pseudomonas syringae.
References:
Li F, et al. (2012). PNAS, 109(5):1790-5.
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Medicago truncatula Ferroportin2 is a nodule-specific transporter that
participates in iron delivery to symbiosomes
Viviana P. Escudero1, Manuel Tejada-Jiménez1, Juan Imperial1,2 & Manuel GonzálezGuerrero1
1
Universidad Politécnica de Madrid. Centro de Biotecnología y Genómica de Plantas, Pozuelo de
Alarcón, 28223 Madrid. Spain. 2Consejo Superior de Investigaciones Científicas. Madrid. Spain.
Symbiotic nitrogen fixation (SNF) is carried out by endosymbiotic rhizobia living within
nodules developed in legume roots. SNF is responsible for an important part of the
atmospheric N2 that is converted to ammonia. As a result of this symbiosis, host plants are
able to colonize soils with low nitrogen content. In addition, potentiating SNF is an
alternative to the overuse of polluting synthetic nitrogen fertilizers in agriculture. Iron is a
critical element for SNF as cofactor of multiple proteins involved in this process
(leghemoglobin, nitrogenase,…). Studies in model legume Medicago truncatula have
shown that this metal is delivered by the vasculature to the infection/maturation zone (zone
II) of the nodule, where it is released to the apoplast (Rodríguez-Haas et al., 2013). From
there, the plasma membrane iron transporter MtNramp1 moves it into rhizobia-containing
cells (Tejada-jiménez et al., 2016). However, for iron to be able to act as an effective
cofactor for SNF, it still has to reach the endosymbiotic rhizobia through the symbiosome
membrane that surrounds them. We propose that Ferroportin2 (FPN2) is carries out this
role in M. truncatula.
Transcriptomic studies show that MtFPN2 is a nodule-specific gene. Yeast
complementation assays in a battery of yeast metal transport mutants indicate that
MtFPN2 lowers cytosolic Fe2+ content, either by efflux out of the cell, or influx into an
organelle. MtFPN2 is located in the nodule vascular bundles and surrounding the
endosymbiotic rhizobia, with a distribution that closely resembles the symbiosome
membrane. All this data point to a role of MtFPN2 in delivering iron to nitrogen fixing
endosymbiotic rhizobia. This is further supported by the phenotypical characterization of a
Tnt1-insertion mutant that shows reduced biomass and nitrogenase activity compared to
wild type plants, the likely result of a deficiency in essential iron.
This work was supported by ERC Starting Grant (ERC-2013-StG-335284) and MINECO Grant (AGL-201232974) to M.G-G.
References:
Rodríguez-Haas, B., et al. (2013). Metallomics 5, 1247–1253.
Tejada-Jiménez, M. et al (2016). Plant Phys 168: 258–272.
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Targeted gene modification in Fragaria vesca mediated by
CRISPR/Cas9 system
Carmen Martín-Pizarro1, David Posé1
1
Instituto de Hortofruticultura Subtropical y Mediterránea, Universidad de Málaga–Consejo
Superior de Investigaciones Científicas, Departamento de Biología Molecular y Bioquímica,
Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain.
Genome editing is becoming an important biotechnological tool for gene function analysis
and crop improvement, being the CRISPR-Cas9 (Clustered Regularly Interspaced Short
Palindromic Repeat-CRISPR associated protein 9) system the most widely used. The
natural CRISPR/Cas9 system has been reduced to two components: a single-guide RNA
(sgRNA) for target recognition via RNA-DNA base pairing, which is commonly expressed
using a promoter for small-RNAs (U6 promoter), and the Cas9 endonuclease for DNA
cleavage (1).
To validate the CRISPR/Cas9 system in strawberry plants, we designed two sgRNAs
directed against the floral homeotic gene APETALA3 (sgRNA-AP3#1 and sgRNA-AP3#2).
This gene was selected because ap3 mutations induce clear developmental phenotypes
in which petals and stamens are missing or partially converted to sepals and carpels
respectively (2).
In this work, we used two different U6 promoters to drive the sgRNA-AP3s expression:
AtU6-26 from Arabidopsis (4), and a U6 promoter from Fragaria vesca (FvU6) (this work).
We also tested two different coding sequences of Cas9: a human- (hSpCas9) (3) and a
plant-codon optimized (pSpCas9) (this work).
Transient expression experiments using both CRISPR/Cas9 systems (AtU6-26:sgRNAAP3#1_35S:hSpCas9_AtU6-26:sgRNA-AP3#2
and
FvU6:sgRNAAP3#1_35S:pSpCas9_FvU6:sgRNA-AP3#2) were performed infiltrating Agrobacterium
tumefaciens into F. vesca fruits. PCR amplification and sequencing analyses across the
target sites showed a deletion of 188-189 bp corresponding to the region comprised
between the two cutting sites of Cas9, confirming that the CRISPR/Cas9 system is
functional in F. vesca. Remarkably, the two systems showed different mutagenic efficiency
that could be related to differences in expression of the U6 promoters as well as
differences in the Cas9 transcripts stability and translation.
Stable transformants for both F. vesca (2n) and Fragaria X ananassa (8n) are currently
being established to test whether is possible to obtain heritable homozygous mutants
derived from CRISPR/Cas9 strategies in strawberry.
Thus, our work offers a promising tool for genome editing and gene functional analysis in
strawberry. This tool might represent a more efficient alternative to the sometimes
inefficient RNAi silencing methods commonly used in this species.
References:
1.
2.
3.
4.
Jinek M, et al (2012). Science. 337(6096): 816-821.
Yang Y, et al (2003) Plant J. 33(1):177-188.
Cong L, et al (2013). Science. 339(6121):819-823.
Feng Z, et al (2013) Cell. Res. 23(19):1229-1232.
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PolyRibo-Seq technique: highlighting the relation between
transcriptomic and proteomic networks in flower development.
Laura Ossorio1, Jose Tomas Matus1, Jose Luis Riechmann1
1
Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB. Campus UAB.
Edifici CRAG, Barcelona
Proteins are key elements of/for cellular function in living organisms. Several efforts have
been focused on understanding the relation between gene transcription and protein
abundance at a genome-wide level. However, recent studies have illustrated a limited
correlation between total mRNA and protein levels, highlighting the importance of posttranscriptional processes that affect protein and peptide abundance. In order to overcome
the limitations of transcriptomic and proteomic studies, all of which report on steady-state
levels of mRNAs and proteins, the recently-developed ribosome profiling technique
enables to globally assess translation rates, obtaining a snapshot of in vivo translation. It
is based on the sequencing of ribosome protected mRNA fragments (known as ribosome
footprints), providing quantitative information on translational efficiency and precise
positioning of ribosomes at the codon level.
The ribosome profiling technique requires the arrest of translation, followed by RNAse I
digestion of mRNA populations in order to isolate ribosome footprints that will be further
used in library construction for deep sequencing (1). An adaptation of this method, termed
Poly-Ribo-Seq (2), allows the purification of polysomes in a sucrose gradient by
ultracentrifugation before nuclease digestion. Under this approach, mRNAs that are truly
being translated are enriched; reducing the presence of ribosome-bound mRNAs that
despite not being translated are still generating ribosome footprints.
Our research team is interested in understanding the initial steps of flower development in
Arabidopsis thaliana. We have previously conducted transcriptomic and genome-wide
binding studies to unravel the role of APETALA 1 (AP1), a transcription factor involved in
floral meristem determination and development (3). We are currently combining the use of
the Poly-Ribo-Seq technique with a floral induction system (pAP1:AP1-GR ap1 cal) (4) to
globally correlate the transcriptomic an proteomic landscape at the network level during
flower development, and to obtain a proteomic chronology of gene expression during that
process. The AP1-GR inducible system enables us to synchronize flower development
and perform time course analyses of the process, coupling shot-gun proteomics and
mRNA-Seq studies to reveal the extent of the regulatory processes determining the levels
of protein expression.
References:
1. Ingolia, N, et al. (2012). Nat Protoc, 7(8), 1534–1550.
2. Juntawong, P, et al. (2015) Method Mol Biol: 139-173.
3. Kaufmann, K, et al. (2010). Science, 328(5974), 85-89.
4. Wellmer, F, et al. (2006). PLoS Genet, 2(7), e117.
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Proteomic analysis of Xanthomonas campestris pv. Camprestris
during an in vitro interaction with Brassica oleracea L.
Cristiane dos Santos1,2,3, Daiane Gonzaga Ribeiro2,4, Osmundo Brilhante Oliveira-Neto2,
André Merlo Murad2, Mariana Rocha Maximiano1,2, Octávio Luís Franco1, Jesús Valentín
Jorrín-Novo3, Angela Mehta2
1
Universidade Federal de Juiz de Fora - UFJF, MG, Brasil; 2EMBRAPARecursos Genéticos e
Biotecnologia, Brasília-DF, Brasil; 3Universidad de Córdoba, España; 4Universidade de Brasilia UnB, Brasilia, Brasil
The phythopathogen Xanthomonas camprestris pv. campestris (Xcc) is the causal agent
of black rot, a disease of great economic impact that affects all cruciferous, including
Brassica oleracea L. This work aimed at studying the Xcc and B. oleracea interaction by
analyzing changes in the pathogen protein profile using an in vivo inoculation system
(Mehta; Rosato, 2010). The objective is to identify proteins involved in the infection process
and those responsible or marker of virulence and aggressiveness. Forty-five days old
plants from the resistant (União) and susceptible (Kenzan) cultivars, were inoculated and
bacterial growth, isolation, and protein profile analysis performed at 48 hours after
inoculation. Bacteria cultured in NYG medium was used as control. Total proteins were
extracted using phenol and precipitated with ammonium acetate/methanol prior to Qubit®
(Invitrogen) quantification and 2D-nanoUPLC/MSE analysis. Protein identification and
quantification the ProteinLynx Global Server (PLGS) platform was used. Sequences
available in UniProt were used for comparison of the data obtained. A total of 1690 proteins
were identified in control, 1138 in susceptible, and 569 in resistant inoculated plants, with
around 350 (susceptible) and 150 (resistant) showing qualitative or quantitative differences
with respect to the control. A model of Xcc-susceptible host interaction is proposed.
Proteomics data for 10 proteins were validated by qRT-PCR. The total RNA was extracted
using hot acid phenol method. The integrity of total RNA treated with Turbo™ DNAse
(Applied Biosystems/Ambion) was confirmed by electrophoresis. cDNA was synthesized
using the kit Go Script ™ Reverse Transcription System (Promega). Both, proteomics and
transcriptomics data showed good correlationship, with nine genes up-regulated and one
down-regulated in the compatible interaction. The first up-group included: argD, xcc_0115,
xcc_0119, xccb100_0502, clp, clpX, ssb, fabH e carB. Out of the ten genes, only
Xccb_1000874 was down regulated.
References
MEHTA, A.; ROSATO, Y. B. (2003) Current Microbiology, v. 47. (400-404).
VORHÖLTER, F. J. et al. (2003) Journal of Biotechnology, v. 106 (193-202).
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Interconnections between 3’-UTR mRNA processing, TOR pathway and
plant pathogenesis in the rice blast fungus
Julio Rodríguez-Romero 1, Marco Marconi 1, Mark Wilkinson1 and Ane Sesma1
1
Centro de Biotecnología y Genómica de Plantas. Universidad Politécnica de Madrid (UPM) Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Campus
Montegancedo UPM28223-Pozuelo de Alarcón (Madrid), Spain.
Rice is the most widely distributed dietary staple in the world. It also represents a
significant percentage of global farm land reaching up to 160 million hectares each year.
One of the most devastating rice pathogen is the blast fungus, Magnaporthe oryzae. Yield
losses caused by blast disease oscillate between 10-30 % per annum, which, even at the
most conservative estimate, are sufficient to feed 60 billion people.
The polyadenylation of mRNAs is a two-step process. Pre-mRNAs are first cleaved at their
3' end and then, the poly (A) tail is added by RNA polymerases during 3’ end formation.
Presence of multiple 3’ end cleavage sites is common in eukaryotic genes, and the
selection of a proper cleavage site represents an important step of regulation of gene
expression. Several proteins of the polyadenylation machinery have been shown to
regulate alternative polyadenylation (APA), including Rbp35/CfI25 complex in
Magnaporthe oryzae and Hrp1 in yeast. The M. oryzae Rbp35/Cfi25 complex regulates
the length of 3’UTRs of transcripts with developmental and virulence-associated functions
(Rodriguez-Romero et al., 2014).
In M. oryzae, Rbp35 regulates APA in ~30% of genes, and nearly 75% of then show a
preference for proximal poly(A) sites. The Δrbp35 mutant lacks precision in the cleavage
and shows an increase of proximal cut sites in pre-mRNAs. In addition, we have observed
that APA is involved in regulating M. oryzae gene expression in response to nutritional
fluctuations.
Significantly, Rbp35 regulates APA predominantly in genes related with signaling. These
include regulatory proteins such as 14-3-3 and several genes related with Target of
Rapamycin (TOR) pathway, which is the most severely affected signaling pathway in
Δrbp35, with at least eighteen genes of the pathway presenting altered 3’UTRs in carbon
depleted cells. TOR is a conserved serine/threonine kinase present in all eukaryotes from
fungi to humans. It is also a key component of the most central nutrient-sensing signal
transduction pathways in eukaryotic cells.
A prominent feature of the 14-3-3 proteins is their ability to bind a multitude of functionally
diverse signaling proteins, including kinases and phosphatases. M. oryzae has two
proteins 14-3-3 (MGG_01588 and MGG_13806) and both mRNAs contain several
polyadenylation sites. Both genes are required for full infection of leaves and roots. The
3’-UTRs of 14-3-3A and 14-3-3B shows a shortening during infection. In addition, delta
mutants of 14-3-3A and 14-3-3B shows altered response under osmotic stress, possibly
connected to MoMsn2 and MoOsm1 (Hog1) pathway. The identification of Rbp35/CfI25
as a component of the alternative polyadenylation machinery and its interconnections with
protein kinase signaling are important step to unravel post-transcriptional networks that
regulate M.oryzae plant colonization.
References:
Rodriguez-Romero, et al (2014). Nucleic Acid Res 43, 179–95.
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Metabolomic profiling of fruit of cultivars and wild species strawberry:
a survey of Fragaria complex
José G. Vallarino1, Zoran Nikoloski2, Carmen Soria3, Patrick Giavalisco2, Alisdair Fernie2,
Sonia Osorio1*
1
Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, University of MalagaConsejo Superior de Investigaciones Científicas, Department of Molecular Biology and
Biochemistry, Campus de teatinos , 29071 Málaga, Spain. 2Max-Planck-Institute für Molekulare
Planzenphysiologie, Am Mühlenberg 1, 14476 Golm, Germany. 3IFAPA, Centro de Churriana,
Cortijo de la Cruz S/N, Churriana, 29140 Malaga, Spain. *Correspondence should be addressed
to S.O. ([email protected])
Strawberry is one of the most valued fruit wordwide. Modern cultivated varieties (Fragaria
x ananassa) exhibit large fruits with intense red color and relative long postharvest. The
increase in productivity on cultivation is, however, somewhat offset by the narrowing of the
crops genetic base which leads to decrease in the intensity and variety of the flavor and
aroma. Since modern breeding programmes are starting to using both, cultivated (Fragaria
x ananassa) and wild species relatives, this exotic germplasm can provide a valuable
source for the improvement of agriculturally important trait. A GC-MS and LC-MS/MS
based survey is presented here of the relative metabolic levels of fruits across
development and ripening of fourteen commercially cultivated strawberry and wild species
that can be crossed with it. Almost 200 semi-polar metabolites were identified.
Here, we present an integrative optimization-based approach by using computational
methods by coupling fruit metabolic network model that successfully revealed the
difference between the covariance structures of the integrated metabolite data sets. This
analysis can predict the metabolic signature of varieties and suggest the existence of
genotype-specific regulatory mechanism underlying the differential metabolite
accumulation. Therefore, the integrated analysis can be used in breeding programs aimed
at improving metabolite-based quality traits such as flavor and health- related metabolites
in strawberry fruits.
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Mapping-by-sequencing accelerates the isolation of causal mutations
from forward genetic screens in tomato
F.J. Yuste-Lisbona1, F. Pérez-Martín1, B. Pineda2, A. Atarés2, A. Fernádez-Lozano1, L.
Castañeda1, E Giménez1, M. García-Alcázar1, R. Fonseca1, C. Capel1, J.L. Quispe1, S.
Bretones1, V. Moreno2, T. Angosto1, J. Capel1, N.A. Müller3, J.M. Jiménez-Gómez3, R.
Lozano1
1
Centro de Investigación en Biotecnología Agroalimentaria (BITAL). Universidad de Almería.
04120 Almería, Spain; 2Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC),
Universidad Politécnica de Valencia. 46022 Valencia, Spain; 3Department of Plant Breeding and
Genetics, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
Forward genetic analysis remains as one of the most powerful tools for assessing gene
functions, although the identification of the causal mutation responsible for a given
phenotype is laborious and time-consuming. Mapping-by-sequencing arises to radically
accelerate the cloning process by combining genetic mapping with whole-genome
sequencing. First described in Arabidopsis (Schneeberger et al., 2009) and later in rice
(Abe et al., 2012), mapping-by-sequencing is based on the examination of genome-wide
allele frequency data in phenotypic bulks of an F2 population developed from a cross
between the mutant and a wild type genotype. We have applied a mapping-by-sequencing
approach to identify the mutations responsible for interesting phenotypes affecting
developmental patterns of tomato (Solanum lycopersicum L.) from the screening of two
collections of artificially induced mutants (T-DNA and EMS). With this aim, several mutants
were firstly selected and crossed with the wild relative species S. pimpinellifolium
(accession LA1589) to generate F2 populations. For each mutant line, a pool was created
with the DNA of the F2 plants showing the mutant traits. These pools were then individually
subjected to deep sequencing and the resulting reads were mapped to the tomato
reference genome. Afterwards, variant calling and filtering were performed with the
different pool samples simultaneously. Bi-allelic variants were used to calculate the allele
frequencies of each mutant pool as follow: non-reference allele counts / total allele counts.
Thereby, the allele frequencies of the F2 mutant pool are expected to be near 0.5, except
for the genomic region where the causal mutation is located (allele frequencies close to
0). Finally, the identified candidate genomic regions were screened for unique mutations.
As example of this approach, we showed the identification of two mutations. The first one
was named as ume1 and corresponds to a SNP mutation in the first exon of a putative
leucine-rich repeat transmembrane-type receptor kinase, which causes a single amino
acid substitution in the protein kinase domain. The second one, spp1 is due to a 1 pb
insertion in the fourth exon of a putative DNA-binding protein resulting in a premature stop
codon. In both cases, mapping-by-sequencing has allowed us to clone a novel regulatory
gene involved in plant development proving the efficiency of this genetic approach for gene
discovery in a model species as tomato.
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:
Abe, A, et al. (2012). Nat Biotechnol, 30: 174-178.
Schneeberger, K, et al. (2009). Nat Methods, 6: 550-551.
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Transcriptional changes during germination and early development of
Quercus ilex seeds
Mª Cristina Romero-Rodrígueza,b,c,d,f, Antonio Archidona-Yustea,b,e,f, Isabel Feitoe,
Carolina de-la-Torree, Rosa Sánchez Lucasa,b, Jesús V. Jorrín-Novoa,b and Nieves Abrila,c
a
Dpt. of Biochemistry and Molecular Biology. Agrifood Campus of International Excelence, ceiA3.
University of Cordoba. Rabanales Campus. Cordoba, Spain. b Agricultural and Plant Proteomics
Research Group, Dpt. of Biochemistry and Molecular Biology, ETSIAM, University of Cordoba,
Spain. c Centro Multidisciplinario de Investigaciones Tecnológicas, Dirección General de
Investigaciones Científicas y Tecnológicas, Universidad Nacional de Asunción, Paraguay d Dpto.
De Fitoquímica, Dirección de Investigación, Facultad de Ciencias Químicas, Universidad
Nacional de Asunción, Paraguay. e Regional Institute for Research and Agro-Food
Developmentin Asturias (SERIDA), “La Mata” Experimental Station, 33820 Grado, Asturias,
Spain. f A. Archidona and C. Romero contributed equally to this work and both should be
considered as “first author”.
We have used a targeted strategy based on reverse transcription of total RNA and real
time-PCR amplification for absolute quantitation of the transcript levels of a group of twelve
genes (Dhn3, Gols, Ocp3, Sdir, Skp1, Pp2c, Nadh6, Gapdh, Fdh, Rbcl, Sod and Gst1) in
mature and germinating acorns of holm oak Holm oak (Quercus ilex subsp. ballota [Desf.]
Samp.). The transcriptional analysis results have been verified and complemented with
the determination of (i) ABA levels and (ii) proteins amounts determined by immunoblotting
(DHN3, GAPDH, RBCL). We found that mature Q. ilex seeds show some of the
characteristics of orthodox seed that included (i) accumulation of insoluble proteins
(DHN3) that contribute to the intracellular vitrified state in seeds; and (ii) accumulation of
transcripts involved in the synthesis of certain osmoregulator raffinose series
oligosaccharides (GolS), the anti-oxidative defence (Sod1, Gst) and the preparation for
the development of an adult plant (RbcL). But the Holm oak mature acorns share with
other recalcitrant seeds the ability to maintain a partially active metabolism, with high level
of glycolytic (Gapdh) and mitochondrial respiratory enzymes (Nadh6) and the absence of
ABA. The results presented here will help to increase the knowledge of the physiological
changes that take place during Q. ilex seed germination, illustrate the importance of
considering the behaviour of seeds for the afforestation projects and restoration
programmes under the impending climate change in Mediterranean regions.
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Evolution and conservation of circadian gene co-expression patterns
in plants
Francisco J. Romero-Campero1, Pedro de los Reyes Rodríguez2, M. Teresa Ruiz2, José M.
Romero2, Federico Valverde2
1
Department of Computer Science and Artificial Intelligence, Universidad de Sevilla, Sevilla,
Spain, 2 Institute for Plant Biochemistry and Photosynthesis, CSIC-Universidad de Sevilla,
Sevilla, Spain
Circadian rhythms play a key role in diurnal transcriptome regulation in higher plants and
unicellular algae. In photosynthetic organisms, regulation of metabolism and development
is determined by light and dark cycles, circadian rhythms being used to anticipate and
adapt to them. Recently, massive amounts of transcriptomic data, including detailed
temporal resolution over 24 h periods, have been generated for several photosynthetic
organisms. These genome-wide studies have shown that in higher plants 30% of genes
exhibit cyclic diurnal patterns, whereas in unicellular algae, cycling genes constitute 80%
of their genome. The availability of massive amounts of transcriptomics data obtained from
different species under equivalent environmental conditions enables the application of
integrative and comparative transcriptomics methodologies such as gene co-expression
networks (1, 2). In this work, we have integrated and analysed RNA-seq and microarray
data generated over 24 h periods in neutral days (12 h light / 12 h dark) and long days (16
h light / 8 h dark) from three different photosynthetic species: Arabidopsis thaliana (3, 4),
Chlamydomonas reinhardtii (5) and Ostreococcus tauri (6). Transcriptomic data integration
and analysis were performed using gene co-expression networks developed in this study.
With the use of different clustering techniques we identified specific gene clusters or
modules consisting of groups of highly co-expressed genes involved in particular biological
processes. This reveals a significant temporal organization of diverse processes such as
carbon/nitrogen metabolism and cell cycle progression into specific gene co-expression
patterns. By comparing the gene modules identified in the different gene co-expression
networks (7) we were able to determine which biological processes exhibit circadian gene
co-expression patterns conserved over the green lineage and which ones have evolved
into different patterns. Additionally, we have developed a web-based tool that will allow
researchers to independently analyse their circadian genes of interest studying the
biological processes they are potentially involved in, the conservation or evolution of the
gene co-expression patterns they follow, as well as the transcription factor binding sites
that are significantly present in their promoters.
This work was funded by project BIO2014-52452-P (MINECO) to FV and JMR and PAI BIO-281 (Junta de
Andalucía).
References:
1- Romero-Campero et al. (2016) BMC Genomics, 17:227.
2.- ChlamyNET web tool, http://viridiplantae.ibvf.csic.es/ChlamyNet/
3- Bläsing, O. et al. (2005) The Plant Cell, 17:3257-3281.
4- Rugnone, ML. Et al. (2013) Proc Natl Acad Sci USA, 110:12120-12125.
5- Zones, J. et al. (2015) The Plant Cell, 27:2743-2769.
6- Monnier, A. et al. (2010) BMC Genomics, 11: 192.
7- Langfelder, P. et al. (2011) PloS Comp Biol, 7:e1001057.
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Índice de Autores
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Índice de Autores
185
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Índice de Autores
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186
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Índice de Autores
A. Atarés
A. Fernádez-Lozano
A. Gómez-Cadenas
A. Hernández
Abdellatif Bahaji
Abel Rosado
Abraham Mas
Adela Zumaquero
Adrián Cabezas-Fuster
Adrian Valli
Adriana Ricarte-Bermejo
Agnieszka Zienkiewicz
Agustín Fernández
Ainhoa Arana-Echarri
Albert Boronat
Albert Ferrer
Alberto Carbonell
Alberto Coego
Alberto de Marcos
Alberto Fuster
Alejandra Garcia
Alejandro Atares
Alejandro Fernández-Arbaizar
Alejandro Lara
Alejandro Rivera
Alejandro Ruiz Bayón
Alejandro Ruiz-Bayón
Alexandra Dubini
Alfonso Mouriz
Alfonso Muñoz
Alfonso Ortega
Alfonso Sánchez
Alicia Moreno-Cortés
Alisdair R. Fernie
Alma Burciaga
Almudena Ferrández Ayela
Álvaro Calderón
Alvaro Sanchez-Corrionero
Amelia Felipo-Benavent
Amr Nassrallah
Ana Arnaiz
Ana B. Castro-Sanz
Ana Belén Sánchez-García
Ana Casañal
Ana Elisa Valdés
Ana López
Ana M. Laureano
Ana Mapelli-Brahm
Ana Pilar Ortega
Ana Rodríguez
André Merlo Murad
Andrea E. Aguilar Jaramillo
Andrei P. Smertenko
Andrés Belver
Ane Sesma
Angel Mª Zamarreño
Ángela María Sánchez-López
Angela Mehta
Ángeles Aroca
Ángeles Gómez-Zambrano
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SVII P10
SVII P10
SV CO4
SV P09
SI CO1
SV CO1
SIV P12
SVII P03
SII P29
SVI CO1
SI CO1
SII P09
SIII CO4
SVII P02
SI P04
SI P04
SVII CO4
SV P02
SII P13
SII P10
SVI P10
SV P01
SIV P08
SI P04
SII P04
SII P34
SII P32
SI PI
SIII P05
SII P26
SII P13
SI P10
SIII CO1
SVII P09
SI P04
SIII P08
SIV P09
SII PI
SIV P05
SIV CO3
SVI CO3
SII P26
SII P01
SI P02
SII P04
SVI P08
SIV P07
SI P05
SV P01
SIV P09
SVII P07
SII CO4
SIV CO2
SV P01
SVI PI
SV P06
SI CO1
SVII P07
SVII P01
SII P07
SI P07
SVI P15
SIII CO3
SI P07 SVI P15
SVI P04
SVI P04
SI P01
SV P13 SV PI
SI P03
SVII PI
SIII P07 SIII P04
SII P20
SVI P01
SVII P08
SI P07
SVI P15
Anna Stepanova
Anne-Laure Le Gac
Antonio Archidona-Yustea
Antonio Cano
Antonio Jesús Castro
Antonio Leyva
Antonio Martínez-Laborda
Antonio Molina
Antonio Vera
Araceli G. Castillo
Araíz Gallo
Arnaldo L. Schapire
Arthur Grossman
Aurora Alaguero
Aurora Galván
B. Pineda
B. Téllez-Robledo
Bárbara Téllez-Robledo
Beatriz Cubero
Beatriz Cuenca
Beatriz Sánchez-Parra
Begoña Coira
Begoña García-Sogo
Begoña Orozco-Navarrete
Begoña Prieto
Belén Mendez-Vigo
Belén Naranjo
Belén Rombolá-Caldentey
Bénédicte Desvoyes
Benito Pineda
Benjamin Hermans
Blanca Craven-Bartle
Blanca San Segundo
Borja Belda-Palazon
Bruno Santos
C. Capel
C. Hafsi
C. Manzano
Caiji Gao
Carl Gunnar Fossdal
Carla Navarro-Quiles
Carla-Dianela Méndez
Carlos Alonso-Blanco
Carlos Hernández-Cortés
Carlos Perea
Carlos Perea-Resa
Carmen Collada
Carmen Fenoll
Carmen Martín-Pizarro
Carmen R. Beuzon
Carmen Soria
Carolina DelaTorre
Carolina Escobar
Catharina Merchante
Cecilia Gotor
Celina Costas
César Petri Serrano
Chandra M. Singh
Chengcai An
Cheol Woong Jeong
187
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SV CO1
SI P01
SII P01
SI PI
SVII P10
SII P24
SIII P03
SII P18
SVI P13
SVI P11
SVI P19
SII P14
SI P02
SIII P02
SIII P01
SI CO2
SV P14
SII P05
SII P14
SI P09
SI CO3
SVI P02
SV P02
SIII CO3
SVII P10
SV P09
SII P24
SIV P03
SIII P09
SII P28
SII P12
SIII P01
SII P02
SIV P08
SV P03
SVI P19
SII P13
SVII P05
SVII P03
SVII P09
SIV P09
SVI P10
SIV CO1
SIV P02
SIII P10
SVI P06
SVI P05
SV P02
SV P07
SVI P09 SVI P16
SIV P12
SV P08
SV P14
SIV P11
SV P01
SIII P05
SV P08
SV P05
SV P01
SVII P02
SV P07
SIV P03
SV CO1
SVI P10
SVI P14
SVII P11
SI P02
SIV P07 SVII P01
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Índice de Autores
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Christopher Dervinis
Christopher Hale
Claire Agius
Clara Echevarría
Clara Sánchez-Rodríguez
Claudio Brandoli
Concepción Almoguera
Concepción Ávila
Concepción Manzano
Concha Gómez-Mena
Conchi Sánchez
Corné M. J. Pieterse
Crisanto Gutierrez
Crisanto Gutierrez
Cristian Carrasco-López
Cristian Mateo
Cristiane dos Santos
Cristina Gómez-Martín
Cristina Navarro
D. M. Pazmiño
D.K. Gupta
Daiane Gonzaga Ribeiro
Daniel Blasco-Espada
Daniel Conde
Daniel Tabas-Madrid
David Alabadí
David Baulcombe
David González-Ballester
David Medel
David Perez-Herguedas
David Posé
David Wilson-Sánchez
David Wilson-Sánchez
Delphine Pott
Detlef Weigel
Diego Gómez-Martínez
Diego López-Marquez
Dina Cifuentes
Doris Wagner
Dorota Komar
E. Giménez
E. Guzmán
Edelín Roque
Edgar A. Rodríguez-Negrete
Eduardo Bueso
Eduardo Mateo-Bonmatí
Eduardo R. Bejarano
Edurne Baroja-Fernández
Eiji Nambara
Elena Carneros
Elena Ramírez-Parra
Emilio Fernández
Emilio Gutierrez-Beltran
Encarnación Rodríguez-Cazorla
Enrique Martínez-Force
Erin Peden
Erundina Ruiz
Erwann Arc
Estela Giménez
Esther Carrera
SIII CO1
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SVI P13
SVI P19
SII CO1
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SV P03
SV P06
SVII P07
SVI P07
SV P06
SV CO4
SV P09
SVII P07
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SIII CO1
SIII P01
SII P10
SIII CO3
SI PI
SVI P19
SVI CO3
SVII P05
SII P33
SVII CO3
SI P03
SV P11
SV P04
SVI P14
SVI P06
SV P07
SII P17
SVII P10
SI P10
SII P19
SVII P03
SV CO3
SII P28
SVII P03
SI CO1
SIV P08
SIII P09
SII P26
SI PI
SIV CO2
SII P22
SI P05
SI P01
SII P06
SIV P08
SII P14
SII P08
SIV P01
SIII P03
SII P05 SIII P10
SV P05 SII P12
SIV P05
SII CO2 SII P31
SVII P03
SIV CO4
SII P11 SII P30
SI P07 SVI P15
SIV P15
SVI P12
SI P06
SI P10
Esther Marín González
Eswarayya Ramireddy
Eugenio G. Minguet
Eva Miedes
Eva Núñez Delegido
Eva Rodríguez-Alcocer
F. Javier Gallego
F. Pérez-Martín
F. Xavier Picó
F.J. Yuste-Lisbona
Fco. René Toribio
Federico Valverde
Felix Ortego
Fernando J. Yuste-Lisbona
Fernando Pérez-Martín
Fernándo Pérez-Sanz
Fernando Ponz
Flora Sánchez
Florian Chevalier
Francisca María Lozano
Francisco J. Colina
Francisco J. Romero-Campero
Francisco J. Romero-Campero
Francisco Javier Cejudo
Francisco José Muñoz
Francisco M. Cánovas
Francisco Merchan
Franziska Turck
Gabriel Castrillo
Gaetano Bissoli
Geert de Jaeger
George Coupland
Gloria Pinto
Goizeder Almagro
Gustavo Gómez
Héctor Candela
Héctor Candela
Helena Fernández
Hugh Cross
Hugo Mélida
Ignacio Rubio Somoza
Igor Yakovlev
Iker Aranjuelo
Ilse Kranner
Inmaculada Moreno
Inmaculada Sánchez-Vicente
Iraida Amaya
Irene Aragüez
Irene García
Irene Martinez
Irma Roig-Villanova
Isabel Allona
Isabel Diaz
Isabel Domínguez
Isabel Feito
Isabel Lopez-Diaz
Isabel Mateos
Iván del Olmo
Iván del Olmo
Iván Fernández
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SIII P01
SVII P10
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SI P08
SVI CO3
SII P14
SII P19
SVII CO2
SVI P18
SVI P18
SIV P04
SII P21
SI P11
SII P25
SVII P12
SI CO2
SI CO1
SI CO3
SIV P14
SII P07
SV P06
SIV CO4
SIV P03
SV P11
SI P11
SI CO1
SVI CO4
SII P02
SII P27
SII P04
SIII P09
SVI P09
SIV PI
SIII P09
SI CO1
SIV P08
SIV P07
SIV P06
SI P03
SIII P10
SIV P02
SIV CO4
SIII CO2
SIII CO1
SVI CO3
SIV P04
SIV P09
SII P08
SIV P06
SII P14
SIII P02
SVI P19
SII P02
SII P21
SIII P06
SVII P12
SII P14
SIV P09
SI P08
SII P07
SI P07
SVI P15
SIV P01
SV CO3
SV CO2
SI P07
SVI P15
SII P06
SII P21
SIV P05
SIV P15
SVI P01
SVII P11
SIV P08 SIV P15
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Índice de Autores
Ivett Bárány
J. Capel
J. León
J.C. del Pozo
J. J. Salas
J. L. Quispe
J. M. Jiménez-Gómez
Jaime F. Martínez-García
James C. Carrington
Jan F. Humplík
Jana Crespo-Trives
Javier Agusti
Javier Barrero-Gil
Javier Brumós
Javier Cabrera
Javier García-Andrade
Javier Paz-Ares
Javier Pérez-Hormaeche
Javier Pozueta-Romero
Javier Rueda-Blanco
Javier Ruiz-Albert
Javier Silva-Navas
Jeonga Yun
Jessica Pérez-Sancho
Jesús Mª Vielba
Jesús Muñoz
Jesús Muñoz-Bertomeu
Jesús Pascual
Jesús Pascual
Jesús Valentín Jorrín-Novo
Joan Villanova
Joana Amaral
Joana Sequeira-Mendes
Joaquín J. Salas
Joëlle K. Mühlemann
Jorge Luis Quispe
Jorge Ruiz-Ramírez
Jorunn E. Olsen
José A. Jarillo
José A. Jarillo
José Alonso
José Antonio Fernández
José Feijó
José G. Vallarino
Jose Julian
José L. Oliver
José León
Jose Luis Jurado-Oller
José Luis Micol
José Luis Micol
José Luis Micol
Jose Luis Riechmann
José M. Álvarez
José M. Franco
José M. Franco-Zorrilla
José M. García-Reyes
José M. Ramos-Sánchez
José M. Romero
Jose Mª García-Mina
José Manuel Jiménez-Gómez
RBMP
SII CO3
SVII P10
SV CO4
SII P24
SI P05
SVII P10
SVII P10
SIII CO2
SVI CO2
SI P07
SII P10
SIII PI
SV P04
SIV CO1
SVI P10
SVI P08
SV P06
SII P18
SI CO1
SVI P14
SVI P14
SII PI
SIV CO1
SV CO1
SVI P13
SIV CO4
SV CO3
SIII CO4
SVI ICO1
SVII P07
SII P01
SI P11
SII CO1
SI P10
SI P09
SVI P07
SII P32
SIII P09
SII P14
SIII P05
SIV CO1
SV CO3
SII P09
SI P03
SV P02
SVI P07
SIV P03
SI PI
SII CO2
SII P28
SII P33
SVII P06
SIV P01
SIV P08
SV P05
SII P26
SIII CO1
SIII P06
SV P06
SV P03
SI P06
SVII CO4
SVI P15
SV P11
SV P08
SI P07
SV P14
SVI P15
SIII P03
SV P10
SIV P09 SV CO2
SIV P10
SVII P11
SII P12
SII P17
SII P03
SIII P10
SIII P02
SVII P09 SVII PI
SII P11 SII P27
SII P30 SII P31
SVII CO3 SII P15
SI P08
SVII P12
José Manuel Pardo
José Manuel Pérez-Pérez
José María Personat
José Pío Beltán
Jose S. Rufián
Jose Tomas Matus
José-Antonio Daròs
Josefina Contreras
José-María Personat
Juan A. Martín
Juan Antonio García
Juan Capel
Juan Carlos del Pozo
Juan Carlos del Pozo
Juan de Dios Alché
Juan F. Campos
Juan Imperial
Juan Jesús Medina
Juan Jordano
Juan Majada
Juan Manuel Pérez-Ruiz
Juan Perianez-Rodriguez
Juan Sobrino-Plata
Juan-José Ripoll
Julia Weiss
Julio Rodríguez-Romero
Julio Salinas
Julio Salinas
Jun Li
Karel Doležal
Karen Chacón
Karla Ramírez-Estrada
Kazumi Nakabayashi
Kerrigan B. Gilbert
Kinia Ameztoy
Kiyoshi Tatematsu
Krzysztof Zienkiewicz
L. B. Pena
L. Castañeda
L. M. Sandalio
L. M. Sandalio
L. Maria Lois
Laura Bacete
Laura de Lorenzo
Laura Ossorio
Laura Sinausía
Lesia Rodriguez
Liam Dolan
Lidia Campos-Soriano
Liwen Jiang
Lothar Willtmizer
Louisa M. Liberman
Lourdes Fernández
Lourdes Fernández-Calvino
Lourdes Rubio
Lucía Arenas-Alfonseca
Lucía Jordá
Lucía Juan-Vicente
Luis C. Romero
Luis Cañas
189
SV P14
SII P01
SVI P12
SII P19
SVI P14
SVII P06
SVII CO4
SVI P06
SIV P14
SVI P19
SVI CO1
SII P14
SII P26
SV P10
SII P09
SII P14
SVI P17
SI P03
SIV P14
SIV P09
SI CO2
SII PI
SVI P19
SII P22
SI P09
SVI PI
SIV P08
SV P04
SI P07
SI P07
SI P08
SI P04
SIV P08
SVI CO2
SI P07
SIV P08
SII P09
SV P09
SVII P10
SV CO4
SV P09
SIV P12
SVI P09
SV P11
SVII P06
SVI P18
SIV P03
SII P35
SVI P02
SIV P03
SV CO1
SII P05
SV PI
SV P13
SV CO3
SIV P02
SVI P16
SII P11
SIV P07
SII P19
SII P18
SII P15
SVI P07
SII P05 SIII P03
SVII P04
SII P16
SII P20
SIV P13 SVII CO2
SVII P08
SV CO1 SV P03
SVI P15
SVI P15
SVI P04
SIV P15
SV P07
SV P12
SII P30
SIV P02
XIII
Índice de Autores
RBMP
Luis E. Hernández
Luis F. Pacios
Luis Gil
Luis Matías Hernández
Luis Romero
Luis Sanz
Luis Valledor
Luis Valledor
Lukáš Spíchal
M. Cristina Romero-Rodrígueza
M. C. Romero-Puertas
M. Romero-Puertas
M. Carmen Bolarin
M. G. Claros
M. García-Alcázar
M. Inouhe
M. Isabel Ortiz-Marchena
M. Mar Castellano
M. Mar Castellano
M. Marconi
M. Rodríguez-Serrano
M. Sanz-Fernández
M. Teresa Ruiz
M. Wilkinson
Mª Belén Pascual
Mª Estrella Santamaria
Mª Fernanda Ruiz
Mª Isabel Puga
Manuel A. Piñeiro
Manuel Acosta
Manuel González-Guerrero
Manuel Martinez
Manuel Piñeiro
Manuel Piñeiro
Manuel Tejada-Jiménez
Mar Castellano Moreno
Mar Martín
Marco Marconi
Marcos Egea-Cortines
Marcos Viejo
Mari C. Risueño
Maria A. Fernandez
María A. Fernández-López
María Carbó
Maria del Carmen Mena
María Delgado-Barea
Maria Dolores Gomez
María Fe Andrés
María Fernández-Marcos
María Garcia
Maria Ghirardi
María G. Fernández-Espinosa
María Isabel Lopez
María Jesús Cañal
María José Asins
María José Jiménez-Quesada
María Luz Annacondia
María M. Martín Trillo
SV P06
SVI P18
SVI P19
SII CO4
SVII P01
SII CO3
SIII CO4
SI P11
SI P07
SVII P11
SV CO4
SIV P15
SII P14
SV CO4
SVII P10
SV P09
SIII P06
SV P12
SV PI
SII P24
SV CO4
SV P09
SI P08
SII P24
SI CO3
SVI P01
SV P04
SV P11
SII P14
SII P01
SVI P17
SVI CO3
SII P17
SIII P05
SVII P04
SVI P02
SII P13
SVI PI
SIV P13
SIII P09
SII CO3
SIV P03
SII P15
SI P11
SI P07
SVII P02
SII P08
SVI P10
SII P05
SI P08
SI P01
SIV P06
SVII P02
SIII CO4
SVI ICO1
SI P11
SV P01
SII P09
SIII CO4
SIII P05
SIV P09 SV CO2
SIV P10 SVI ICO1
SVI P15
SV P09
SI P08
SII P26
SV P13
SVII P12
SVI CO3
SVII P04
SVI P01
SIII P02 SII P03
SII P35
SVII P08
SI P09
SVII CO2
SIV P09 SIV P10
SII P04 SV CO2
María Martín
María Pilar Angarita-Díaz
María Rosa Ponce
María Rosa Ponce
Mariana R. Maximiano
Mariano Perales
Mari-Cruz Castillo
Maricruz Rochina
Marija Savic
Marina A. Ribeiro-Pedro
Mario F Fraga
Mark Wilkinson
Marko Boehm
Marouane Baslam
Marouane Baslam
Marta Berrocal-Lobo
Marta Boter
Marta Carrera-Alesina
Marta García
Marta Godoy
Marta Isabel Terry López
Marta Peirats-Llobet
Marta Senovilla
Marta-Marina Pérez-Alonso
Mathias Hentrich
Matias KirstMauricio Soto-Suárez
Mayte Castellano
Mercedes Diaz-Mendoza
Mercedes Pallero-Baena
Mercedes Ramiro
Michael Hackenberg
Miguel A Blázquez
Miguel A Perez-Amador
Miguel A. Blázquez
Miguel A. Moreno-Risueno
Miguel A. Moreno-Risueno
Miguel Ángel Blázquez
Miguel Ángel Botella
Miguel Aranda
Miguel Gonzalez-Guzman
Miguel-Ángel Torres
Miquel Sendra-Ortolà
Moisés Cabanes Martínez
Mónica Escandón
Mónica Medina
Mónica Meijón
Mónica Meijón
Mónica Pernas
Mónica Venegas-Calerón
Montaña Mena
Montserrat Arró
Moona Rahikainen
Myriam Calonje
N. A. Müller
Natalia Bueno
Natalia Dudareva
Natalia Gómez-Peral
Nathalie Prat-Leonhard
Nidia Castillo
190
SI P04
SII P19
SII P23
SII P32
SVII P07
SIII CO1
SIV P03
SII P19
SIII P01
SIII P06
SIII CO4
SVI PI
SI P01
SI CO1
SI P07
SVI P02
SIII P02
SIV P13
SVI P05
SIV P08
SIV P13
SV P02
SVI P17
SVI P11
SVI P11
SIII CO1
SVI P02
SVI CO4
SVI CO3
SIII P06
SIII P01
SVI P07
SIV P05
SII P08
SII CO3
SII P16
SII CO3
SII P10
SV CO1
SVI P06
SIV P03
SVI P05
SII P28
SIII P08
SIV P10
SII P19
SIII CO4
SV CO2
SIII P05
SI P05
SII P13
SI P04
SIV P10
SII P07
SVII P10
SIV P01
SI P09
SII P02
SV P11
SVI P04
SII P27
SII P34
SII P29
SVII CO3
SVII P08
SVI P15
SII P35
SV P07
SIII P03
SII P20
SII PI
SVI P10
SV P07
SVI ICO1 SV CO2
SIV P09 SIV P10
SVI ICO1
SIII P02 SII P35
SVI P04
SII P25
SII P21
SII P06
XIII
Índice de Autores
Nieves Abril
Nieves López-Pagan
Nieves Vidal
Noah Fahlgren
Noel Blanco-Touriñan
Noelia Arteaga
Noelia Jaime-Pérez
Noemí Ruiz-López
Nuria De Diego
Nuria Fernández
Nuria Fernández-Bautista
Octávio Luís Franco
Olga del Pozo
Ondřej Novák
Oscar Lorenzo
Oscar Lorenzo
Osmundo B. Oliveira-Neto
Pablo Albertos
Pablo García-Gómez
Pablo González-Melendi
Pablo Pujol
Pablo Vera
Panagiotis N. Moschou
Patrick Giavalisco
Paula Ragel
Paula Suárez López
Pedro de los Reyes Rodríguez
Pedro Javier Navarro-Lorente
Pedro L. Rodriguez
Pedro Robles Ramos
Peter V. Bozhkov
Philip N. Benfey
Pilar Cubas
Pilar Prieto-Dapena
Pilar Rojas-Gracia
Pilar S. Testillano
Pilar Teixidor
Qian Wu
R. Bautista
R. Fonseca
R. Lozano
Rafael A. Cañas
Rafael Catalá
Rafael Garcés
Rafael Lozano
Ramón Serrano
Raquel Martins-Noguerol
Raquel Olías
Raquel Sarmiento Mañús
Raquel Sarmiento Mañús
Regina Galarza
Regla Bustos
Riansares Muñoz
Ricardo J. Ordás
Ricardo Lebrón
Rim Hamza
Roberto Solano
Rocío Fonseca
Rocío Olmo
Rodrigo Marí-Ordóñez
RBMP
SVII P11
SVII P03
SVI P13
SVII CO4
SIV P05
SIII P01
SV P01
SV CO1
SI P07
SV PI
SV P12
SVII P07
SVI P12
SI P07
SIV P06
SIV P15
SVII P07
SIV P08
SI P07
SIII CO1
SI CO1
SVI P08
SIV CO2
SVII P09
SII P18
SII CO4
SVII P12
SVII CO2
SV P02
SIII P08
SIV CO2
SII P05
SIV P04
SIV P14
SII P19
SII CO3
SI CO1
SV P02
SV CO4
SVII P10
SVII P10
SI CO3
SV P03
SI P05
SII P14
SV CO3
SI P06
SV P01
SII P34
SII P32
SI CO1
SII P35
SV P06
SIV P01
SVI P07
SII P19
SIV P08
SVI P07
SVI P10
SII P10
SII CO3
SI P10
SVI P15
SV P13
SVI P15
SII CO3 SIV P08
SIV P15
SVI P15
SIV P03 SV P07
SIII P04 SIII P07
SIV P01
SV P04
SI P06
SI P10
SII P19 SVI P07
SIV CO4
SII P23 SII P29
SVII CO3
Rosa Micol-Ponce
Rosa Micol-Ponce
Rosa Sánchez Lucas
Rosana Navajas
Rosany Camargo
Rosario Castro-Rodríguez
Rose JK
S. Bretones
S. Navarro
Saijaliisa Kangasjärvi
Saleta Rico
Samanta Ortuño
Samuel Daniel Lup
Sandra Fonseca
Sanjay Swami
Sara Fontcuberta-Cervera
Sara Jover-Gil
Sara Jover-Gil
Sara Navarro
Sara Navarro-Neila
Sara Ormeño
Sara Sopeña-Torres
Sebastián Martínez-López
Sergio Ciordia
Sergio Ibáñez
Sergio Navarro-Cartagena
Shao Cui
Silvia Hnatova
Silvia López-González
Sofia Madeira
Sofia Otero
Sonia Campo
Sonia Osorio
Sonia Osorio
Sonia Zarco-Fernández
Soon-Ki Han
Soraya Pelaz
Stefan Wyder
Steffen Vanneste
Stephan Pollmann
Stéphane MaurySteven E. Jacobsen
T. Angosto
Tamara González-Costa
Tamara Lechón
Tamara Muñoz-Nortes
Teresa Altabella
Teresa Antón
Thomas Roach
Thomas Schmülling
Thotegowdanapalya C. Mohan
Till Kash
Trinidad Angosto
Ueli Grossniklauss
Ugo Bastolla
V. Moreno
Valeria Gagliardini
Valle Ojeda
Vicente Moreno
Vicente Pallas
191
SII P23
SII P32
SVII P11
SI P07
SVI P02
SVI P17
SVII PI
SVII P10
SII P24
SIV P10
SVI P13
SII P22
SII P15
SIV CO3
SVI P16
SII P32
SII CO2
SII P21
SV P10
SIII P03
SVI P19
SVI P16
SII CO2
SI P07
SII P15
SIII P07
SV P02
SIII CO3
SVI P18
SV P05
SV P05
SVI CO2
SI P03
SV CO1
SV P06
SV P07
SII CO4
SII P04
SV CO1
SIII P03
SIII CO1
SIII P10
SVII P10
SII P27
SII CO3
SII P27
SI P04
SII P14
SIV P08
SV P06
SV P06
SII P22
SII P14
SII P04
SIII P10
SVII P10
SII P04
SI CO2
SII P14
SVI CO4
SII P29
SII P34
SII P02
SII P23
SII P06
SII P31
SII P31
SII P33
SII P33
SVII P02
SVII P09 SVII PI
SIV P11 SVI P11
SVI P04
SII P19
SV P01
XIII
Índice de Autores
RBMP
Vicente Ramírez
Vicente Rubio
Víctor Carrasco
Victor Flors
Víctor Quesada Pérez
Victoria Ruiz-Hernández
Victoriano Valpuesta
Virginia Palomares
Viviana P. Escudero
Vojislava Grbic
Wenqiang Yang
Wiam Merini
Wolfram Weckwerth
Xiangchun Yu
Xiaofeng Wang
Xu Zhang
Yangnan Gu
Yeats TH
YeonKyeon Lee
Yolanda Pareja-Jaime
Yue Zhou
Zaida Andrés
Zaida Vergara
Zoran Nikoloski
SVI P08
SIV CO3
SIII P03
SVI P04
SIII P04 SIII P08 SIII P07 SIII P04
SI P09
SI P02
SII CO3
SVII P04 SVI P16
SVI CO3
SI P01
SII P07 SII P25
SVI ICO1
SV P02
SV P02
SV P02
SVI P16
SVII PI
SIII P09
SVI P12
SII P07
SV P08
SII CO1
SVII P09
192
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Libro de resúmenes - XIII Reunión de Biología Molecular de Plantas

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