Welcome addresses. Committees

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3 Welcome addresses As President of the International Society for Seed Science (ISSS) it is my pleasure to invite you to our latest International Workshop on Molecular Aspects of Seed Dormancy and Germination in Paris, France in July This is the 4th in this very popular triennial series of workshops that has attracted experts in the field from around the globe. Once again there will be a combination of leading science and social interactions, which will be exciting and instructive for all participants. The workshop is a key component of the Society's aim to foster and promote research, education and communication in the scientific understanding of seed. Bill Finch-Savage, President of the International Society for Seed Science Welcome to the 4 th International Workshop on Molecular Aspects of Seed Dormancy and Germination in Paris! This three day Workshop was previously held in York (2010), Salamanca (2007) and Wageningen (2003). On behalf of the International Society for Seed Science (ISSS) we are very happy to welcome you in Paris to discuss recent progress and developments in this exciting field. Keynotes lectures will cover major aspects of research on germination and dormancy and we have also given the opportunity to many researchers to present highly interesting case-studies and work in progress, which should lead to a stimulating meeting. We do hope that you will enjoy the workshop and the Parisian life and are convinced that your contribution will help to make this workshop exciting and memorable. The Organizing Committee Committees Scientific committee C. Bailly, UPMC Paris, France R. Finkelstein, UCSB Santa Barbara, USA F. Gubler, CSIRO Canberra, Australia H. Hilhorst, Wageningen University, The Netherlands A. Marion-Poll, IJPB INRA Versailles, France E. Nambara, Toronto University, Canada S. Penfield, Exeter University, UK W. Soppe, Max Planck Institute Köln, Germany Organizing Committee C. Bailly, UPMC Paris, France H. Bouteau, UPMC Paris, France F. Corbineau, UPMC Paris, France E. Gendreau, UPMC Paris, France J. Leymarie, UPMC Paris, France P. Meimoun, UPMC Paris, France Sponsors We are grateful to Mairie de Paris, ISSS, UPMC, Faculté de Biologie de l'upmc, Gautier Semences, Limagrain, GNIS, Clinisciences for their financial help to organize this congress. 1

4 PROGRAMME TUESDAY 9 JULY Registration WEDNESDAY 10 JULY Welcome Christophe Bailly, UPMC, France Bill Finch-Savage (ISSS president), University of Warwick, UK Opening talk François Burgaud, GNIS, Paris, France Research into plant breeding is a sine qua non condition for achieving a sustainable and competitive agriculture Session 1: Hormones and Cell Signaling Chair: F. Corbineau Invited speaker: Dave Nelson, University of Georgia, Athens, USA Smoke and hormone mirrors: Karrikins and strigolactones activate seed germination through a shared genetic pathway Break Eiji Nambara, University of Toronto, Canada NLP8 regulates nitrate-induced ABA catabolism during Arabidopsis seed germination Dolores Rodriguez, University of Salamanca, Spain Deciphering the interaction between PP2Cs and CHR24 in Arabidopsis Hiro Nonogaki, Oregon State University of, USA Spontaneous amplification of the ABA biosynthesis and signaling pathways through a positive feedback mechanism imposes deep seed dormancy Julio Maia De Oliveira, University of Wageningen, The Netherlands Abscisic Acid (ABA) sensitivity regulates desiccation tolerance in germinated Arabidopsis seeds Kent Bradford, University of Davis, USA A DOG1-like gene in lettuce may regulate thermoinhibition of germination LUNCH 2

5 Session 2: Omics and Genetics Chair: E. Nambara Invited speaker: George Bassel, University of Birmingham, UK Integrating genome-wide network models with 3D cellular morphodynamics Manuela Nagel, IPK, Germany Genetics of seed longevity comparisons between dry storage, controlled seed deterioration and elevated partial pressure of oxygen (EPPO) using Oregon Wolfe Barley mapping population Erwann Arc, University of Innsbruck, Austria; IJPB, France A combined proteomic and metabolomic profiling of contrasted states of dormancy in imbibed Arabidopsis seeds Raquel Iglesias Ferna ndez, University of Madrid, Spain The bzip transcription factor AtbZIP44 during seed germination: Regulation of the mannanase encoding gene AtMAN Pablo Albertos, University of Salamanca, Spain Insights into Nitric Oxide and ABA crosstalk during seed dormancy and germination Break Session 3: Role of environmental factors in seed germination and dormancy Chair: F. Gubler Invited speaker: Kathleen Donohue, Duke University, USA Dormancy and the rest of life: Germination phenology, pleiotropy, and plant life cycles Antje Voegele, Royal Holloway University of London, UK The molecular biomechanics of the micropylar endosperm as a major mediator of Lepidium sativum seed germination responses to sub- and supra-optimal temperatures POSTERS THURSDAY 11 JULY Session 3: Role of environmental factors in seed germination and dormancy Chair: KJ Bradford Dana-MacGregor, University of Exeter, UK Temperature controlled seed dormancy in Arabidopsis requires an impermeable seed 3

6 Hanzi He, University of Wageningen, The Netherlands How do seed maturation environments affect performance? Jose Barrero, CSIRO, Australia Functional analysis of the light inhibition of germination in dormant barley grains Juliette Leymarie, UPMC, France Differential effects of temperature and hypoxia on induction of secondary dormancy in relation with ABA/GAs balance in barley grains Liana Burghardt, Duke University, USA Predicting plant life cycles in seasonal environments: Understanding the role of dormancy Steven Footitt, University of Warwick, UK The response of winter and summer annual Arabidopsis ecotypes to seasonal environmental signals in the soil seed bank Break Chair: W. Soppe Steven Penfield, University of Exeter, UK Control of seed germination by maternal environmental signalling pathways Magdalena Simlat, Krakow University, Poland Expression profiles of VP1 and CPS3 genes during wheat and triticale caryopsis development Session 4: Molecular regulation of seed germination and dormancy Chair: D. Rodriguez Invited speaker: Leonie Bentsink, Wageningen University, The Netherlands How does nature control seed dormancy and longevity? LUNCH Ewa Kalemba, UPMC, France Proteasome involvement in the regulation of sunflower seed dormancy and germination Kai Graeber, Royal Holloway University of London, UK DELAY OF GERMINATION 1 mediates a conserved seed dormancy mechanism regulating endosperm weakening in a temperature- and GA-dependent manner in the Brassicaceae Kazumi Nakabayashi, MPI, Germany Natural variation of DOG1 in self-binding and expression level determines seed dormancy in Arabidopsis 4

7 Yong Xiang, MPI, Germany The protein phosphatase rdo5 is required for seed dormancy in Arabidopsis Break Chair: S. Penfield Hayat El Maarouf-Bouteau, UPMC, France Seed dormancy release does not involve gene expression during dry storage Oscar Lorenzo, University of Salamanca, Spain Seed-specific MADS-box transcription factor AGL67 acts as a repressor of seed germination Kerstin Mueller, University of Nottingham, UK; Simon Fraser University, Canada Epigenetic mechanisms are involved in regulating ABI3 and DOG1 transcription during seed dormancy cycling Fabian Vaistij, University of York, UK Differential control of seed primary dormancy in Arabidopsis ecotypes by the transcription factor SPATULA POSTERS ISSS general assembly CONFERENCE DINNER FRIDAY 12 JULY Session 5: Seed quality, vigour and longevity Chair: H. Hilhorst Invited speaker: Loic Rajjou, IJPB INRA, Versailles, France Decipher the underpinnings of seed vigor to improve agricultural success Andreas Boerner, IPK, Germany Genetic tools for the dissection of quantitative traits seed longevity and dormancy segregation via genome-wide association mapping Franc oise Montrichard, University of Angers, France Evidence for the participation of the methionine sulfoxide reductase repair system in plant seed longevity Barbara Dantas, Brazilian Agricultural Research Corporation, Brazil Germinative metabolism of Caatinga savannah forest species in biosaline agriculture Break 5

8 Chair: B. Finch Savage Sheila Adimargono, MPI, Germany Seed vigour and life cycle events on barley Karima Righetti, University of Angers, France Construction of a stable co-expression network of Medicago truncatula seed development for the prediction of genes modulating longevity Tatiana Veselova, University of Moscow, Russia Non-enzymatic carbohydrate hydrolysis is the cause of air-dry seed deterioration at early aging Wanda Waterworth, University of Leeds, UK DNA damage checkpoints control seed viability, seed vigour and seedling quality Farewell LUNCH 6

9 ORAL PRESENTATIONS 7

10 Opening Talk Research into plant breeding is a sine qua non condition for achieving a sustainable and competitive agriculture François Burgaud Groupement National Interprofessionnel des Semences et plants (GNIS), 44 rue du Louvre, Paris The seed industry is the foundation of agricultural production and as such plays a key role in the competitiveness of the French agriculture. Nevertheless, media and the general public know very little about this sector. And this despite the fact that the French seed industry is one of the major forces of our economy. Indeed, France is the largest European producer and the world s largest exporter of seeds. Production activity in France started a long time ago and the French can look back on a long history of expertise in seed production and processing. Thus, companies from neighbouring countries multiply their seeds in France in order to benefit from the know-how of our seed companies. Today s success is based on a specific balance between historical expertise and innovation. With 72 seedbreeding companies, of which 44 are small- and 21 medium-sized, France benefits from an extremely dynamic network of research companies, which allows expectations of national and international markets to be met. In the field of seed breeding, it is absolutely necessary to be able to leverage a group of cutting-edge researchers to ensure future competitiveness of the seed industry and consequently the entire agriculture sector. Indeed, the obligation to produce more and better, climatic variations, changing consumer tastes and trends are all reasons to make new varieties available to farmers, which ensure the harvest and allow the farmers to continue to meet demands from all different markets (vegetable producers, the processing industry, the end consumer). The challenges which the French seed growers have to meet are very ambitious. They benefit from a number of assets to maintain their competitiveness at a global level and to be capable to bring adequate solutions to the issue of producing more and better. But they need the financial means to pursue research. However, the question of financing varietal research is not straightforward. One way of ensuring future research is to protect newly created varieties by a plant variety certificate ensuring the owner s right of intellectual property, payable as a fee by the farmer when buying certified seeds. Two figures allow the importance of plant breeding for the seed industry to be measured: it takes ten years to develop a new plant variety, and the companies invest approximately 13% of their annual turnover in research. It is essential to continue to support the research effort If we can do this, the French seed industry will have all necessary assets to play the role it deserves which is to actively contribute to the achievement of the sustainable and competitive agriculture Europe aspires to. 8

11 Session 1: Hormones and Cell Signaling Invited speaker Smoke and hormone mirrors: Karrikins and strigolactones activate seed germination through a shared genetic pathway Caitlin E. Conn, Drexel Neumann, Kelly A. Dyer, David C. Nelson Department of Genetics, University of Georgia, Athens, GA USA dcnelson@uga.edu Plants have evolved seed germination responses to karrikins and strigolactones, two classes of lactone molecules that signal opposite states of local plant community establishment. Karrikins are chemicals found in smoke that activate seed germination of many species following a fire, when competition for resources is highly reduced. Strigolactones are endogenous plant hormones that are exuded into soil to recruit beneficial associations with arbuscular mycorrhizal fungi. Root parasites in the family Orobanchaceae (e.g. Striga, Orobanche spp.) exploit this recruitment strategy as a means for hostdetection, through a highly sensitive germination response to strigolactones. Specialized germination responses to one of these classes of signals represent important ecological adaptations for fire followers and parasites. Genetic studies in Arabidopsis thaliana have shown that both karrikins and strigolactones signal through a genetic pathway that requires the F-box protein MAX2. Specific responses to karrikins and strigolactones are mediated by two homologous a/b hydrolase proteins: KAI2 is required for promotion of seed germination by karrikins and strigolactones, while D14 is necessary for strigolactone-specific regulation of later stages of development. We have performed molecular evolutionary analyses and cross-species complementation tests to investigate the hypothesis that KAI2 enables host recognition by seed of parasitic plants. Our data add to the growing body of evidence that KAI2 and D14 may function as receptors for specific lactone-type signals. As max2 and kai2 mutants have increased seed dormancy phenotypes that are not observed in strigolactone-deficient mutants, we propose that another endogenous lactone may promote seed germination through this pathway. 9

12 NLP8 regulates nitrate-induced ABA catabolism during Arabidopsis seed germination Dawei Yan and Eiji Nambara Department of Cell & Systems Biology, University of Toronto, 25 Willcocks St, Toronto, Ontario, Canada M5S3B2 Nitrate plays bifunctional roles in both a macronutrient and a signal. As a signal, it induces a global change in a pattern of transcriptome of genes responsible for carbon and nitrogen related metabolisms in a short period of time. NIN-like transcription factors (NLP) are shown to be involved in the nitrate signal transduction, however, it remains unclear how this nitrate signaling is integrated into the physiological context to coordinate growth and development. We used seed germination is a model system to understand plant s responses to nutritional signals. Arabidopsis germination is known to be promoted by nitrate. We previously reported that this involves the nitrate enhanced ABA reduction. We found that nlp8 mutants failed to show the nitrate-induced germination. The nlp8 aba2 double mutants germinated in a nitrate independent manner, suggesting that maintaining high ABA levels is required for the NLP8 function during germination. The nlp8 mutants failed to show the nitrate-induced CYP707A2 expression and concomitant ABA reduction. We conclude that NLP8 plays a key role in the nitrate regulated seed germination through regulating ABA catabolism. 10

13 Deciphering the interaction between PP2Cs and CHR24 in Arabidopsis Natanael Viñegra, Abelardo Modrego, Patricia Fresnillo, Óscar Lorenzo, Mª Dolores Rodríguez Departamento de Fisiología Vegetal. Centro Hispano-Luso de Investigaciones Agrarias (CIALE). University of Salamanca. C/ del Duero, 12. Campus de Villamayor Salamanca, Spain ABA mediated responses are key regulators of seed development and germination and its recently discovered receptor complex is formed by PYR/PYL proteins and cluster A PP2Cs. Our group has focused its research on these PP2Cs for the last years. However, we have recently switched our attention to cluster D PP2Cs. Among the interactors of this group we have found a chromatin remodelling factor highly expressed during a specific stage of seed maturation. Chromatin remodelling factors have been reported to play a key role in regulating many developmental processes, where they act as multicomponent complexes, with its members being variable depending on the regulated process. Although some of them have been already characterized, this is not the case of our object of study, CHR24. By Nicotiana benthamiana transient expression, we could determine both CHR24 location into the cell nucleus, distributed as small speckles, and its interaction with a cluster D PP2C, which also showed a speckled pattern. Now we are trying to determine the specific interaction domain by yeast two hybrid assays. In order to get deeper insight into CHR24 function in seed development and germination, we isolated the chr24 knock-out homozygous mutant SALK line and observed that it exhibits some degree of embryolethality, which will lead our future work to embryogenesis and seed development studies. For the time being, we have determined its insensitivity to low concentrations of ABA and, from now on, we will test this line in different abiotic conditions and hormone responses. We are also developing overexpressing lines in order to elucidate its possible role in the ABA signaling pathway at seed dormancy and germination processes. 11

14 Spontaneous amplification of the ABA biosynthesis and signaling pathways through a positive feedback mechanism imposes deep seed dormancy Mariko Nonogaki, Jessica G. Tran, Khadidiatou Sall, Hiro Nonogaki Oregon State University, Corvallis, OR97331, USA hiro.nonogaki@oregonstate.edu Abscisic acid (ABA) plays an essential role in seed dormancy. ABA biosynthesis is regulated primarily by the rate-limiting enzyme NINE-CIS-EPOXYCAROTENOID DIOXYGENASE (NCED). Expression of NCED increases ABA levels in seeds and induces ABA-regulated transcription factors (TFs), which in turn induce downstream genes by binding to the ABA responsive elements (ABREs) in their promoter regions. It was hypothesized that, if NCED was placed under the control of an ABRE-containing seed-specific promoter, this chimeric gene (pabre:nced) should create a positive feedback loop through the production of ABA and ABA-regulated TFs in seeds. The enhanced expression of TFs by this positive feedback loop is expected to come back through pabre:nced expression, which should trigger spontaneous amplification of the magnitude of the ABA biosynthesis and signaling pathways in a seed-specific manner. To test this idea and examine the potential of this approach for the prevention of preharvest sprouting (PHS) in cereal crops, an NCED gene was isolated from sorghum (Sorghum bicolor) and fused to the wheat (Triticum aestivum) Early Methionine-labeled (EM) promoter, which contains ABREs. The ptaem:sbnced gene caused unusually deep dormancy in Arabidopsis Columbia seeds, which last for more than three months and was similar to deep dormancy observed in Cape Verde Islands (Cvi). These results verified the idea of spontaneous amplification of the ABA pathways for seed dormancy control and also suggested that the mechanisms associated with the regulation of seed dormancy by ABA are highly conserved between monocots and dicots. While the potential of NCED induction for PHS prevention had been demonstrated in the previous study using the Plant Gene Switch System (PGSS), a chemically induced gene expression, the current study advances the robust technology to the next generation, which does not require ligand application and is more practical for agricultural applications. 12

15 Abscisic Acid (ABA) sensitivity regulates desiccation tolerance in germinated Arabidopsis seeds Julio Maia De Oliviera 1, Bas J.W. Dekkers 1,2, Wilco Ligterink 1 and Henk W.M. Hilhorst 1 1 Wageningen Seed Lab, Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands. 2 Department of Molecular Plant Physiology, Utrecht University, Utrecht, The Netherlands. julio.maiadeoliveira@wur.nl Desiccation tolerance (DT) of seeds is based on a range of complex protection mechanisms that accompany dehydration. The mechanisms involved in DT may be roughly divided in three groups: 1) signalling mechanisms including gene regulation and post transcriptional modifications; 2) metabolic adjustments and antioxidant systems; and 3) macromolecular and mechanical stability. Inspired by the proven complexity of this trait we have developed an experimental system to re-establish DT in germinating Arabidopsis thaliana seeds. We show that the incubation of desiccation sensitive (DS) germinated Arabidopsis seeds in a polyethylene glycol (PEG) solution, in abscisic acid (ABA), or a combination of both, re-activates the mechanisms necessary for expression of DT. By studying this model of loss and reestablishment of DT in ABA-deficient and -insensitive mutants, we prove that ABA is necessary for the reestablishment of DT and hypothesize that the events upstream of ABA signalling are not necessary to rescue DT. Furthermore, we demonstrate that the loss of ABA sensitivity plays a major role in the capacity of re-establishing DT in germinated Arabidopsis seeds. We also show that different components of the signalling pathway might influence the responsiveness of those seeds to ABA and their DT recovery. Finally, it seems that, instead of being synthesized in seeds upon stress stimuli, ABA is constitutively present and changes in its perception will allow the control of signalling cascades. Altogether, these findings challenge simplistic models of hormone level-directed stress responses, highlighting the role of specific signalling components such as receptors, phosphatases, kinases and transcription factors in the response to environmental cues. 13

16 A DOG1-like Gene in Lettuce May Regulate Thermoinhibition of Germination Heqiang Huo and Kent J. Bradford Department of Plant Sciences, University of California, Davis, CA, USA The Delay of Germination 1 (DOG1) gene identified in the Cvi accession of Arabidopsis thaliana is responsive to seed maturation temperature and is involved in regulating seed dormancy and afterripening. We asked whether this gene is also involved in regulating thermoinhibition, or failure of seeds to germinate when they are imbibed at warm temperatures. Loss of function of AtDOG1 in Arabidopsis (Columbia) enables ~80% seed germination at 34ºC, a temperature at which wild-type seeds cannot germinate. As seeds of most lettuce genotypes exhibit thermoinhibition, homologs of AtDOG1 (termed DOG1-like [LsDOG1L]) were isolated from three thermosensitive genotypes, cv. Salinas (Lactuca sativa), PI (L. sativa), and PI (L. saligna), and a thermotolerant accession UC96US23 (L. serriola); these shared ~35% identity to AtDOG1 amino acid sequence. Overexpression of LsDOG1L (PI261653) could restore the thermoinhibited wild-type phenotype in Arabidopsis dog1 mutant seeds and caused strong seed dormancy in PI Conversely, PI seeds in which LsDOG1L expression was silenced by RNAi exhibited a reduction in thermoinhibition at 35ºC. Expression of this LsDOG1L gene is seedspecific and its mrna abundance increased during seed development. Expression of LsDOG1L responds to seed maturation temperature, with a 3-fold higher mrna abundance in seeds matured 25ºC than in seeds matured at 30ºC. This difference in expression due to maturation temperature was not observed in UC96US23 and another thermotolerant accession W48 (SAL-90). In Salinas seeds imbibed at 35ºC, LsDOG1L mrna remained at a high level, while its mrna abundance greatly decreased in a line in which LsNCED4, encoding an ABA biosynthetic enzyme (9-cis-epoxycarotenoid dioxygenase 4), was silenced, and which did not exhibit thermoinhibition. These results indicate that LsDOG1L genes may be involved in both seed dormancy and seed thermoinhibition. Supported by USDA-NIFA Award

17 Session 2: Omics and Genetics Invited speaker Integrating genome-wide network models with 3D cellular morphodynamics George Bassel School of Biosciences, University of Birmingham, Birmingham, United Kingdom Seed germination is an environmentally regulated developmental transition in the life cycle of plants. Using publicly available gene expression data, genome-wide network models have been inferred which describe the co-functional relationships between genes underlying the regulation of this transition. Regulatory interactions are being integrated within the spatial and temporal context of the changes in cell shape that drive the transition from seed to seedling. Using this approach the relationship between the molecular machinery underlying the decision-making process of a plant cell to expand and the gene regulatory networks driving changes in the biophysical properties of plant cells are being linked within space and time. 15

18 Genetics of seed longevity comparisons between dry storage, controlled seed deterioration and elevated partial pressure of oxygen (EPPO) using Oregon Wolfe Barley mapping population Manuela Nagel 1, Steven P.C. Groot 2, Jan Kodde 2 and Andreas Börner 1 1 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Genebank Department, Corrensstraße 3, Stadt Seeland, Germany 2 Wageningen University and Research centre, Plant Research International, PO Box 619, Wageningen, The Netherlands Nagel@ipk-gatersleben.de The ability of seeds to survive a certain period of time, termed seed longevity, is strongly dependent on the growth conditions of the mother plant, pre-storage and storage conditions and the genetic background. Under ambient storage conditions, seed survival of orthodox seeds can vary between few years (e.g. onion or lettuce) and several decades (e.g. pea). Scientific experiments on seed longevity are usually not designed to last decades. Therefore, methods which accelerate seed ageing rate and, finally, reduce seed vigour are widely used. The current study investigates seed vigour of 94 lines of the Oregon Wolfe Barley (OWB) mapping population after a)three and b) five years of dry cold storage at -18 C, c) controlled seed deterioration using 45 C and 18% seed moisture and modified storage atmospheres by d) increased nitrogen concentration and e)elevated partial pressure of oxygen, shortly EPPO. Highly significant differences in seed vigour were detected between dry cold storage, controlled seed deterioration and the EPPO method. Interestingly, high-pressure oxygen triggers similar morphological ageing symptoms as dry storage. However, a low correlation coefficient (r = 0.14) between dry cold storage and EPPO indicates different underlying biochemical mechanism. Composite interval mapping of seed vigour after different treatments revealed seven highly significant (LOD > quantitative trait loci (QTL) on chromosomes 2H, 3H, 5H, 6H and 7H whereas QTL for three years dry storage could be only found on 2H and 5H; for five years dry storage on 2H, 6H, 7H and for EPPO on 2H, 3H and 7H. The applied EST (expressed sequence tags) markers and the recently published Restriction Site Associated DNA (RAD) linkage map will provide additional information about the comparability of experimental seed ageing methods. 16

19 A combined proteomic and metabolomic profiling of contrasted states of dormancy in imbibed Arabidopsis seeds Erwann Arc 1,2, Gilles Clément 1, Béatrice Godin 1 and Loïc Rajjou 1 1 INRA, Jean-Pierre Bourgin Institute (IJPB, UMR1318 INRA-AgroParisTech), Laboratory of Excellence "Saclay Plant Sciences" (LabEx SPS) ; RD10, F Versailles, France 2 Present address: Institute of Botany, University of Innsbruck, Sternwartestraße 15, A-6020 Innsbruck, Austria Erwann.Arc@uibk.ac.at Despite having very similar initial pools of stored mrnas and proteins in the dry state, mature Arabidopsis seeds can either proceed toward radicle protrusion or stay in a dormant state upon imbibition. Dormancy breaking, a prerequisite to germination completion, can be induced by different treatments though the underlying mechanisms remain elusive. Thus, we investigated the consequence of such treatments using omic approaches to unravel specific protein and metabolites accumulation patterns associated to the maintenance or release of seed dormancy. First, two unrelated dormancy-releasing treatments were applied to dormant seeds namely cold stratification and exogenous nitrates, in combination with differential proteomic tools to highlight the specificities of the imbibed dormant state. The results reveal that both treatments lead to highly similar proteome adjustments. Then, an analysis of the impact of cold stratification on seed metabolome allowed confirming hypothesis inferred by our proteomic data by pinpointing distinctions in metabolite accumulations. Overall, our results demonstrate the relevance of combining omic approaches to unravel how the regulation of specific enzymes determines the metabolic activity depending on the physiological state. Our data suggest that dormancy maintenance is associated to an abscisic-acid-dependent recapitulation of the late maturation program resulting in a higher potential to cope with environmental stresses. This waiting state is also associated with the repression of reserve mobilization and specific energetic pathways. However, our analysis highlighted the previously undescribed differential regulation of RFO metabolism in dormant and non-dormant seeds. Finally, the comparison of the present results with previously published omic datasets reinforces and extends the assumption that post transcriptional, translational and post-translational regulations are determinant for seed germination. 17

20 The bzip transcription factor AtbZIP44 during seed germination: Regulation of the mannanase encoding gene AtMAN7. Raquel Iglesias-Fernández 1, Cristina Barrero-Sicilia 1, Néstor Carrillo-Barral 2, Luis Oñate- Sánchez 1, Pilar Carbonero 1 1 Centro de Biotecnología y Genómica de Plantas (UPM-INIA), ETSI Agrónomos, Universidad Politécnica de Madrid, Campus de Montegancedo, 28223, Pozuelo de Alarcón, Madrid (Spain) 2 Departamento de Fisiología Vegetal, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782, Santiago de Compostela (Spain) raquel.iglesias@upm.es During Arabidopsis thaliana seed germination, the weakening of the endosperm cell walls (CWs) is a key step needed to reach germination sensu stricto. Dismantling of these CWs is driven by hydrolytic enzymes such are endo-β-mannanases (MAN; EC ) that catalyze the cleavage of the β1 4 bonds in the mannan backbone; this activity increases upon seed imbibition. Transcripts of the most highly expressed AtMAN gene during seed germination (AtMAN7) are restricted to the micropylar endosperm and to the radicle tip just before radicle emergence. To gain insight into the cis-, trans- transcriptional regulation of the AtMAN7 gene, a phylogenetic shadowing process has been pursued to find conserved cis-elements in the promoters of the Brassicaceae orthologous MAN7 genes and these conserved motives have been used as baits in a 1-hybrid screening, using as a prey, an arrayed yeast library of circa 1,200 Transcriptional Factor Open Reading Frames (TF ORFs) from A. thaliana. The basic-leucine zipper AtbZIP44, has been thus identified and its regulatory function upon AtMAN7 during seed germination validated by different molecular and physiological techniques and by the establishment of the germination kinetics of both over-expression (oex) and T-DNA insertion mutant lines of AtbZIP44. The transcriptional combinatorial network through which AtbZIP44 regulates AtMAN7 gene expression during seed germination has been further explored through proteinprotein interactions between AtbZIP44 and other TFs. 18

21 Insights into Nitric Oxide and ABA crosstalk during seed dormancy and germination Albertos P. 1, Mateos I. 1, Romero-Puertas M. 2, Tatematsu K. 3, Nakabayashi K. 4, Kamiya Y. 5, Nambara E. 6, Lorenzo O. 1 1 CIALE, University of Salamanca, Spain 2 EEZ-Consejo Superior de Investigaciones Científicas, Spain 3 National Institute for Basic Biology, Japan 4 Max Planck Institute for Plant Breeding Research, Germany 5 RIKEN Plant Science Center, Japan 6 University of Toronto, Canada paa@usal.es; oslo@usal.es Seed dormancy and germination are complex traits regulated by the interaction of a plethora of signaling molecules, including phytohormones (abscisic acid, ABA) and key plant growth regulators (nitric oxide, NO). The molecular basis of the ABA and NO crosstalk are mostly unknown. The identification of the molecular players that participate in this response is essential to understand the NO perception and signalling by the plant. We have isolated and characterized several mutants encoding transcriptional factors (TFs) showing ABA- and NO scavenging by cptio- (2-(4-Carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide) insensitive phenotypes in the transition from dormancy to germination. The expression of these TFs was mainly localized in seeds and in the meristem zone of emerging roots during seed germination, and was highly induced after ABA treatment in all the emerging organs. Microarray expression analysis revealed several hierarchical clusters with different function in the onset of seed germination, abiotic stress responses and redox status during germination. Additionally, DNA binding specificity of the TFs following a microarray-based approach is also fully provided demonstrating that can specifically bind to the corresponding cis consensus elements. Gain-of-function and loss-of-function studies strongly support the relevance of these TFs in the ABA and NO crosstalk. Insights into the posttranslational redox modifications of these TFs will be presented together with their physiological relevance. Acknowledgments: Impacts of Environmental Conditions on Seed Quality. "EcoSeed " ERC.KBBE Molecular and Genetic Insights into Arabidopsis Nitric Oxide (NO) Signalling Pathway. MICINN (BIO ) "TRANSPLANTA: Function and Biotechnological Potential of Transcription Factors in Plants". CONSOLIDER Programa (CSD ). 19

22 Session 3: Role of environmental factors in seed germination and dormancy Invited speaker Dormancy and the rest of life: Environmentally induced pleiotropy of life-history genes Kathleen Donohue Duke University, Department of Biology, Box 90338, Durham, NC USA Genes that regulate plant life-history traits, including dormancy, interact with the ecological environment in which plants grow. Seasonal environmental factors experienced by one life stage can influence phenotypic expression and allelic effects of genes that regulate those and subsequent life stages. In turn, genetically based traits, and especially phenological traits such as flowering and germination time, can determine the seasonal environment experienced by subsequent life stages. This interaction can cause allelic effects of genes that regulate one life stage to ramify across subsequent life stages, a phenomenon termed environmentally induced pleiotropy. First, examples are given whereby seasonal environmental factors experienced during seed maturation influence allelic effects and natural selection on genes that regulate germination in the field. Next, an example is given whereby a gene that regulates dormancy, Delay Of Gemination 1, has effects across the life cycle and alters the basic life history that is expressed by Arabidopsis thaliana. Allelic effects of this gene and the direction of natural selection on this gene, in turn, depend on flowering time. These interactions across life stages therefore influence fundamental processes of pleiotropy, life-history expression, natural selection, and the maintenance of genetic variation in genes that influence germination and dormancy. 20

23 The molecular biomechanics of the micropylar endosperm as a major mediator of Lepidium sativum seed germination responses to sub- and supra-optimal temperatures Antje Voegele 1,2, Tina Steinbrecher 1 Anita Kleiber 2, Danouše Tarkowská 3, Terezie Urbanová 3, Veronika Turečkova 3, Karin Weitbrecht 2, Kai Graeber 1,2, Miroslav Strnad 3,4 and Gerhard Leubner-Metzger 2 1 Royal Holloway University of London, School of Biological Sciences, Plant Molecular Science, Egham, Surrey, TW20 0EX, United Kingdom, Web: 'The Seed Biology Place' University of Freiburg, Faculty of Biology, Institute for Biology II, Botany / Plant Physiology, D Freiburg, Germany 3 Laboratory of Growth Regulators, Faculty of Science, Palacky University and Institute of Experimental Botany ASCR, v.v.i., Šlechtitelů 11, CZ , Olomouc, Czech Republic 4 Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacky University, Šlechtitelů 11, CZ , Olomouc, Czech Republic antje.voegele@rhul.ac.uk Appropriate seed germination timing allows plants to withstand harsh environmental conditions, such as low temperatures and limited water availability. The mature seeds of the closely related Brassicaceae Arabidopsis thaliana and Lepidium sativum both have a testa and a thin endosperm layer surrounding their embryos, which regulate germination timing by acting as a constraint to radicle protrusion. Prior to endosperm rupture and radicle protrusion, the embryo radicle/hypocotyl axis (RAD) elongates and the micropylar endosperm (CAP) weakens. Endosperm weakening is initiated by an early embryo signal and is promoted by gibberellins (GA) and inhibited by abscisic acid (ABA). Molecular mechanisms of endosperm CAP weakening depend on hormonally and environmentally regulated expression of cell wall remodelling proteins. L. sativum seeds are non-dormant, do not require light for germination and are thus perfectly suited to study the germination responses over the entire ambient temperature range. Their larger seed size facilitates spatial investigation of the underlying molecular and biomechanical mechanisms in a close up view on the two decisive seed compartments CAP and RAD. With a comparative approach involving heterologous CAP and RAD microarrays, a detailed population-based threshold modelling, quantification of GA and ABA contents and biomechanics, we show that the mechanisms by which sub-optimal and supra-optimal temperatures inhibit germination differ considerably. Both cold and warm temperatures do have the transcriptomes, hormone contents and biomechanical properties of the CAP as a major target. Interestingly, sub- and supra-optimal temperatures providing the same degree of germination delay compared to the temperature optimum, are associated with expression of distinct groups of cell wall remodelling proteins and distinct biomechanical CAP properties. The CAP therefore has a decisive role in mediating cold and heat stress responses of seeds. 21

24 Temperature controlled seed dormancy in Arabidopsis requires an impermeable seed Dana R MacGregor, Sarah Kendall, Steven Penfield Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK d.macgregor@exeter.ac.uk Summer annuals must spend the duration of the cold period as un-germinated seeds in the soil. Arabidopsis accomplishes this by setting the level of primary seed dormancy by the temperature experienced during seed maturation, where dormancy increases with decreasing maturation temperatures. How this process is accomplished at the molecular level is beginning to be understood, but little is known about how these molecular events are manifested in the seed s physiology. Here we show that the permeability of the seed coat is determined by maturation temperature and that it is inversely correlated with primary dormancy. Seeds known to have highly permeable seed coats, such as ap2 or tt4, are less able to respond to the decreasing seed maturation temperature and have lower dormancy levels at all temperatures tested. Likewise, seeds that are highly dormant even at higher temperatures, such as Ws, exhibit low seed coat permeability. We demonstrate that temperature-induced changes in seed permeability are correlated to changes in expression of genes involved in the flavonoid biosynthesis pathway. These changes in gene expression are manifested as changes in the levels of anthocyanins, soluble and insoluble proanthocyanidins. We are currently working on determining how the temperature signal is being recognized and utilized by the plant in order to correlate maturation temperature with seed permeability and dormancy. 22

25 How do seed maturation environments affect performance? Hanzi He 1, Leónie Bentsink 2,1 and Henk Hilhorst 1 1 Wageningen Seedlab, Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands 2 Molecular Plant Physiology Group, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands hanzi.he@wur.nl Seed quality is highly dependent on environmental cues during seed formation and filling. It is not clear which environmental factors are the most dominant in this respect. We study the influence of light intensity, day length, temperature, and nutritional conditions during seed filling, on a number of seed quality attributes, with a strong emphasis on dormancy and longevity. Several genotypes were analysed to investigate the environmental effects, including a set of near isogenic lines (NILs, NILDOG1, NILDOG2, NILDOG3, NILDOG6 and NILDOG22), representing different dormancy and longevity pathways and some abscisic acid related mutants (cyp707a1, cyp707a2, Atnced6-Atnced9 double mutant), as well as two NILDOG1 mutants. Phenotyping results clearly indicate that the seed maturation environments affect seed performance. Correlation analysis between phenotypes and metabolites shows highly positive correlations of some metabolites with seed longevity, especially in varying light intensity environments. The metabolite profiles suggest that the Carbon-Nitrogen balance correlates with plant phenotypes. This indicates that the effect of the maternal light environment on seed quality is mediated by the C/N balance and may be causal for the plant phenotypes. 23

26 Functional analysis of the light inhibition of germination in dormant barley grains Jose Barrero 1, Bruce Downie 2 Jake Jacobsen 1 and Frank Gubler 1 1 CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia 2 Department of Horticulture, University of Kentucky, Lexington, Kentucky , USA jose.barrero@csiro.au It is well known that abscisic acid (ABA) plays a central role in the regulation of seed dormancy and that transcriptional regulation of genes encoding ABA biosynthetic and degradation enzymes is responsible for control of ABA content. However, little is known about upstream signalling pathways that lead to transcriptional regulation of ABA content and in particular about how environmental signals (e.g. light and cold) direct expression of ABA metabolism genes. We are interested in these processes in cereal grains, particularly in relation to the development of strategies for controlling pre-harvest sprouting in barley and wheat. Our previous studies have indicated that light is a key environmental signal inhibiting germination in dormant grains of barley, wheat and Brachypodium, and that this effect disappears with after-ripening. We found that the blue component of the light spectrum inhibits germination by inducing the expression of the ABA biosynthetic gene NCED1, thus increasing ABA content in the grain. We have now created barley transgenic lines silencing the blue light receptors CRYTOCHROME (CRY) 1 and 2. Our results demonstrate that CRY1 is the key receptor transducing the blue light signalling in dormant grains. 24

27 Differential effects of temperature and hypoxia on induction of secondary dormancy in relation with ABA/GAs balance in barley grains Juliette Leymarie, Hai Ha Hoang, Christophe Bailly and Françoise Corbineau Université Pierre et Marie Curie, Sorbonne Universités, UR5 UPMC- EAC7180 CNRS, 4 place Jussieu, Paris, France juliette.leymarie@upmc.fr Primary dormant barley grains germinate at C, but not at higher temperatures. This inability to germinate at high temperatures (30 C) results from a limitation of oxygen supply to the embryo through the seed envelopes, the oxygen tension in embryos measured with microsensors being 15.8% at 15 C, but only 0.3% at 30 C. Incubation of grains in unfavourable conditions for germination, i.e. 3 days at 30 C in air or at 15 C in hypoxia (5% O2), induces a loss of subsequent germination ability at 15 C, in darkness and in air. This phenomenon is considered as an induction of secondary dormancy. At 30 C, it requires an embryo water content higher than 0.50 g H2O g-1 DM, and is associated with an increase in embryo ABA content after transfer at 15 C, while ABA content is slightly changed by hypoxia. The role of HvNCED1 (ABA synthesis) seems dominant in the induction at 30 C, while HvNCED2 seems more important in the induction by hypoxia. In both cases, the induction of secondary dormancy is associated with a reduction of GA signalling due to increased expression of genes involved in GA catabolism and inhibition of those involved in their synthesis. GA-metabolism genes involved are different according to the type of induction of secondary dormancy, but HvGA2ox3 and HvGA3ox2 appear to play a leading role. Our results clearly show the involvement of the ABA/GA balance, and that the expression pattern of genes involved in ABA/GA metabolism depends on the environmental factors that induce secondary dormancy. Induction by hypoxia at 15 C appears to be more regulated by GA and less by ABA than the induction by high temperature. 25

28 Predicting plant life cycles in seasonal environments: Understanding the role of dormancy Liana T. Burghardt 1, C. Jessica E. Metcalf 2, Amity M. Wilczek 3, Johanna Schmitt 4, and Kathleen Donohue 1 1 Duke University, USA 2 Dept of Zoology, Oxford University, UK 3 Deep Springs College, USA 4 University of California at Davis, USA burgharl@gmail.com As research on the molecular mechanisms of dormancy and seed germination advances, it will become increasingly important to connect genetic variation and environmental responses dissected in the lab to their ecological relevance in natural populations. Similar to flowering and seed dispersal transitions, germination timing, or phenology, is sensitive to external environmental cues. Because of this plasticity, germination timing depends not only on internal seed characteristics, but also on the environmental conditions experienced after dispersal. These environmental conditions are determined by the timing of previous life stage transitions. Because of the connections between life stages, considering dormancy/germination processes outside of context of the whole life cycle may be misleading. We introduce a modeling approach that links eco-physiological models of how separate life stages respond to their environment. We parameterized phenology models for Arabidopsis thaliana a model genetic organism expressing life-cycle variation and used it to predict life cycles in four European locations. We found that simple developmental timing systems can restrict life-cycle expression independent of the effects of fecundity and survival and that environmental variation alone produces a wide breadth of lifecycle phenotypes. This variation was facilitated by small dormancy differences within seed cohorts and by environmental context. When we varied dormancy and flowering parameters in ways inspired by observed natural variation, we found that dormancy may more strongly influence life cycle than floral repression level. In sum, the ILC model allows us to predict for each environment how phenological plasticity, genetic variation, and the sequence of developmental events determines when plants will be in each life stage. We suggest that as knowledge increases this approach can facilitate understanding of how genetic variation in dormancy and germination affects whole life cycles. 26

29 The response of winter and summer annual Arabidopsis ecotypes to seasonal environmental signals in the soil seed bank Steven Footitt and William E. Finch-Savage School of Life Sciences, Wellesbourne Campus, University of Warwick, Warwick CV35 9EF, UK Seeds use environmental cues to sense the seasons and their surroundings to initiate the plants life cycle. Dormancy cycling underlying this process is extensively described, but in the soil seed bank (SSB) the coordination of the molecular mechanisms described in the laboratory is largely unknown. To address this we conducted a detailed eco-physiological characterisation linked to a targeted investigation of gene expression over the annual dormancy cycle of two contrasting Arabidopsis thaliana ecotypes in the SSB; one from the Cape Verdi Islands (Cvi) and one from Ireland (Bur). These ecotypes have different dormancy phenotypes, and emerged at different times in the field reflecting winter (Cvi) and summer annual (Bur) behavior. We show how genes involved in environmental sensing (temporal and spatial) respond to the seasons. The results are consistent with a seed specific seasonal temperature response (temporal sensing) through DELAY OF GERMINATION1 (DOG1) that indicates the correct season; and MOTHER of FLOWERING TIME (MFT) which has an opposite thermal response in seeds of the two ecotypes indicating a role in determining their different dormancy cycling phenotypes. In addition, soil temperature also drives expression of circadian clock genes in the SSB. Temporally driven co-opted mechanisms that sense spatial signals such as nitrate via CBL-INTERACTING PROTEIN KINASE 23 (CIPK23) phosphorylation of the NITRATE TRANSPORTER 1 (NRT1.1) and light via PHYTOCHROME A (PHYA) appear to determine germination potential in the correct temporal window. We also show that chromatin remodeling may be orchestrated in response to temporal and spatial signaling via genes such as HUB1 and KRYPTONITE as dormancy cycles with the seasons. In the SSB, these processes allow seeds to continually respond to a range of environmental signals linking dormancy to the seasonal cycle. These processes determine the time and climate space in which germination and plant establishment occur. 27