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P - Posters P10-026: EFFECTS OF CPPU TREATMENT ON CYTO- KININ HOMEOSTASIS AND GRAIN YIELD IN WHEAT Yao, Z.*1 - Song. J2 - Jameson, P.E.2 1College of Agronomy, Agricultural University of Hebei 2 School of Biological Sciences, University of Canterbury *Corresponding author, e-mail: yzhj201@hebau.edu.cn Artificial disturbance of the endogenous cytokinin level dramatically affects economically important traits including plant architecture, organ size and life span, tolerance to biotic and abiotic stress, and particularly seed yield. Forchlorfenuron (CPPU), which is known to significantly increase the size of fruit, was applied to wheat at anthesis and/or two days after anthesis. The effect of CPPU on endogenous mRNA levels of cytokinin regulatory genes from four multi-gene families (isopentenyl transferase (IPT), cytokinin oxidase/dehydrogenase (CKX), zeatin glucosyltransferases (ZOG) and β-glucosidase (GLU)) was quantified using real-time RT-PCR.Substantial changes in the expression profiles of different family members were observed within four hours of spraying, and the disturbed expression of some gene family members was apparent up to seven days after spraying. Contrasting expression changes between leaf and grain were also detected. The effect of CPPU treatment on final seed yield and on yield components will also be reported. P10-027: STUDYING OF THE «CHITIN-SPECIFIC» DO- MAIN OF PLANT PEROXIDASES Kuzmina, O.* Institute of Biochemistry and Genetics, Ufa Scientific Centre, Russian Academy of Sciences *Corresponding author, e-mail: phyto@anrb.ru Studying of wheat origin as the important agricultural crop has the essential scientific and practical value caused by problems of selection. A variety of wild species of wheat and aegilops together with intraspecific polymorphism allow to assume the presence of the significant amount of stress genes induced in response to many disease agents in them. Among variety of protective genes the specific place is occupied with the genes coding pathogen-induced peroxidase isoforms among which concern and «сhitin-specific» forms. Probably they are important in the lignification of pathogen - damaged plant tissues. The purpose of research is the molecular and genetic organisation analysis of the chitin-specific site of the peroxidase gene in wheat and aegilops. Comparison of sequenced peroxidase gene fragments with known a soft wheat nucleotide sequence Triticum aestivum TC151917 has revealed the 90 % homology with Tr. fungicidum and T. petropavlovskyi and 84 % homology with T. araraticum. The obtained data can be considered from an evolutionary position. So, T. fungicidum and T. petropavlovskyi, also as well as T. aestivum relate to subgenus Urartu. The three species are Au and B genomes carriers. Whereas, T. araraticum, T. militinae and T. boeoticum relate to Boeoticum subgenus and are Ab and G genomes carriers. It is shown a «chitin-specific» peroxidase domain possessed ability to bind a chitin. We have created a gene-engineering design composed of the dahlia mosaic virus 35S promoter and the wheat anionic peroxidase cDNA. It will allow us to find out a plant peroxidase role in protective reactions mechanisms against pathogenes. P10-028: A NAC TRANSCRIPTION FACTOR WITH A ROLE IN ABA SIGNALING MODULATES NITRIC OXI- DE LEVELS IN ARABIDOPSIS Osuna, D. 1 * - Fernández-Arbaizar, A. 1 - Albertos, P. 1 - Godoy, M. 2 - Franco, J. M. 2 - Solano, R. 2 - Carrasco, J.L. 3 - Vera, P. 3 - Lorenzo, O. 1 1 Departamento de Fisiología Vegetal (CIALE). Universidad de Salamanca. 2 Laboratorio de Genómica., Centro Nacional de Biotecnología, CSIC. 3 Instituto de Biología Molecular y Celular de Plantas. Universidad Politécnica de Valencia *Corresponding author, e-mail: daniel.osuna@usal.es The molecular basis of the abscisic acid (ABA) and nitric oxide (NO) crosstalk promoting seed germination and early development are currently unknown. The identification of the elements that participate in this response is, thus, essential to understand the NO perception and signalling by the plant. By means of a genetic screening in 3μM (+)-S-ABA that simulates the effect of NO scavenging by cPTIO, we have isolated the gap1 mutant (Nambara et al., 2002), showing an ABA- and cPTIO-insensitive phenotypes in the transition from dormancy to germination. GAP1 encodes a NAC TF nuclear localized, able to form homodimeric complexes, and it is also a potent transcriptional activator (Osuna et al., 2010). Whole genome transcriptional profiling of gap1 mutant versus Col-0 stratified Arabidopsis seeds revealed several hierarchical clusters with different function in germination and stress responses, highlighting the ABA and NO crosstalk. In addition, the DNA binding specificity of the NAC TF was provided by overexpression in both, Escherichia coli and Nicotiana benthamiana, followed by hybridization to oligonucleotide arrays, and complemented with gel shift assays, which has allowed us the identification of the consensus cis-regulatory sequences responsible of gene expression contained in NAC-regulated genes. Taken together, this data showed this NAC TF as a relevant ABA signaling pathway modulating NO levels during seed germination and stress responses. Finally, the identification of the cis-regulatory element recognized by NAC shed light on new molecular components downstream of this signaling network. Acknowledgements: To Dr. Eiji Nambara group (Universidad de Toronto, Canada) for his collaboration in the mutant isolation. P10-029: INTERACTION STUDIES OF PROTEINS IN- VOLVED IN OSMOSENSING AND CYTOKININ SIGNA- LING PATHWAYS IN POPULUS Héricourt, F.* - Chefdor, F. - Bertheau, L. - Larcher, M. - Depierreux, C. - Brignolas, F. - Carpin, S. Université d’Orléans, UFR-Faculté des Sciences, Laboratoire de Biologie des Ligneux et des Grandes Cultures *Corresponding author, e-mail: francois.hericourt@univ-orleans.fr The osmosensing pathway in Arabidopsis thaliana is constituted by a multi-step phosphorelay similar to the one of Saccharomyces cerevisiae, involving a Histidine-aspartate Kinase (HK) osmosensor, AHK1, and a Histidine-containing Phosphotransfer (HPt) protein, AHP2. In Populus, we have identified a cDNA encoding a HK, named HK1, and five cDNA encoding HPt proteins, HPt1 to HPt5. Analysis of interaction tests performed with the cytoplasmic domain of HK1 and all HPts in a two-hybrid system revealed a strong interaction between HK1 and HPt2 and weak interactions between HK1 and other HPts. In A. thaliana, AHP2 interacts not exclusively with AHK1 but also with AHK2, AHK3 and AHK4, which are involved in the cytokinin signaling pathway. In order to determine the interconnectivity between these two different signaling pathways in Populus, we studied the interaction between HPts and cytokinin receptors. Therefore, the homologous receptors of AHK2, AHK3 and AHK4 have been isolated from Populus and the cytoplasmic domain of these proteins has been tested in a two-hybrid system for their potential interaction with HPt1 to HPt5. The results indicate that HPts are commonly used by the two signaling pathways as it is the case in A. thaliana, but clearly with distinct affinities. These results suggest also that HPt2 could be the HPt protein preferentially involved in the osmosensing pathway. P10-030: GENE AND PROTEIN EXPRESSION OF CU TRANSPORTERS GMHMA8 IN SOYBEAN PLANTS: EFFECTS OF EXCESS CU P
FESPB 2010 - XVII Congress of the Federation of European Societies of Plant Biology Del Castillo Garza, M. 1 * - Bernal, M. 1 - Risueño, M.C. 2 - Picorel, R. 1 - Testillano, P.S. 2 1 Estación Experimental de Aula Dei (CSIC) 2 Centro de Investigaciones Biológicas (CSIC) *Corresponding author, e-mail: delcastillomb@eead.csic.es The Cu transporter HMA8 is a member of the P1B-ATPase subfamily that plays an important role on Cu allocation and detoxification. We have identified two divergent genes in soybean, named GmHMA8-1 and GmHMA8-2, homologous to PAA2/ HMA8 previously described in Arabidopsis thaliana. A previous work revealed their expression and chloroplastic localization in soybean suspension cultures (Bernal et al., 2007). Both genes are subject to alternative splicing by an intron retention mechanism leading to the formation of four transcripts: HMA8-1, HMA8- 2 and the corresponding non spliced forms, NSP-HMA8-1 and NSP-HMA8-2. The intron retention yields a premature stop codon in the non spliced forms. Thus, four different putative GmH- MA8 proteins with high homology between them may exist in soybean. Thegoal of this work was to analyze their expression in planta at a transcript and protein level by RT-PCR, in situ hybridization (FISH) and immunofluorescence techniques, and confocal analysis to investigate their global expression in each organ of the plant and whether their expression was tissue-specific. For this purpose, soybean plants were grown hydroponically in control conditions (0.12 μM CuSO4) and Cu-treated (10 μM CuSO4). Samples of different organs were frozen for RNA extraction, and fixed and cryoprocessed for FISH and IF essays. Results showed specific patterns of expression, with differences in control and Cu-treated plants. Transcripts were presents in the cytoplasm of mesophyll cells of leaves, whereas GmHMA8 proteins were localized in chloroplasts. These results give news insights of the role of GmHMA8 proteins on Cu homeostasis within the chloroplast in planta. P10-031: WRKY III TRANSCRIPTION FACTOR FAMILY: A COMPLEX TRANSCRIPTIONAL REGULATORY NET- WORK FOR DEFENSE AND SENESCENCE IN ARABI- DOPSIS. Besseau, S.* - Li, J. - Palva, E. T. University of Helsinki - Viikki Biocenter *Corresponding author, e-mail: sebastien.besseau@helsinki.fi Leaf senescence is the last stage of leaf development and leads to leaf death. Senescence is a way to remobilize nutrients from old leaves to new growing and reproductive organs. This step is therefore of pivotal importance for whole plant development. Senescence is an ordered sequence of events under genetic control consisting dismantling of cellular organelles, hydrolysis of macromolecules (such as proteins, nucleic acids, lipids, and chlorophyll), remobilization and transport of nutrients out of the senescing tissue. Various internal and external factors participate in triggering and modulating senescence, including age of leaves, nutrient availability, photoperiod, hormones, abiotic and biotic stresses. At the molecular level, senescence is regulated by a complex transcriptional regulatory network, involving massive reprogramming of gene expression. WRKY group III transcription factors (TF) family is known to be induced by SA and is related to plant defense. Some members of this WRKY III familywere already shown as crucial regulators of defense (e.g. WRKY70, 62, 38, 53) as well as regulators of senescence (e.g. WRKY70, 53). This dual function is explained by related physiological processes during defense and senescence. We have initiated a global and systematic study of WRKY III TF family members to address their contribution to the regulation of plant defense and senescence signaling in Arabidopsis. To this aim, expression patterns of the WRKY III TF genes were elucidated, the phenotypic effects of silencing of these genes characterized and the WRKY III interaction network identified by yeast 2-hybrid and co-immunoprecipitation. We report here that WRKY70 and 53 control senescence as part of a complex regulatory network with other WRKY III TFs. P10-032: ROLE OF PP2C INTERACTING PROTEINS IN ABA SIGNALING AND STRESS RESPONSES Rodriguez, D.* - Modrego, A. - Saavedra, X. - Sanz, L. - Fresnillo, P. - Lorenzo, O. Universidad de Salamanca *Corresponding author, e-mail: mdr@usal.es Protein phosphatases type 2C (PP2C) are major components of abscisic acid (ABA) signaling pathway. We have identified two functional PP2C from beechnut (Fagus sylvatica L.): FsPP2C1 and FsPP2C2, a negative regulator of the ABA pathway, and a positive regulator of ABA signaling, respectively. To further analyse the function of these PP2Cs, and by means of yeast two hybrid (Y2H) assay using an Arabidopsis thaliana cDNA library, two members of the recently described RCAR/PYR family, PYL8/RCAR3 and PYL7/RCAR2, were identified as putative interactors of FsPP2C1. Bimolecular fluorescence complementation (BiFC) assays in tobacco (Nicotiana benthamiana L.) plants confirmed the in planta interaction of both proteins and interestingly, resulted in a specific nuclear colocalization of this interaction. Gain-of-function assays by overexpressing PYL8/RCAR3 under a 35S promoter revealed increased ABA hypersensitivity of Arabidopsis transgenic seeds and consequently unability to germinate under osmotic or salt stress conditions. Furthermore, 35S:PYL8/RCAR3 plants showed increased tolerance to abiotic stress and higher expression levels of ABA responsive genes. Taken together, these results suggest that PYL8/RCAR3 positively regulates ABA signaling during seed germination and abiotic stress responses.Additionaly, three putative interactors of FsPP2C2 have been identified by Y2H assays, belonging to the small auxin up-RNA (SAUR) family, the large PPR family and an E3 ubiquitin ligase, respectively. Currenty, these interactions are being confirmed by BiFC assays and the corresponding functional tests are being performed. Interaction of FsPP2C2 orthologs in Arabidopsis (AtPP2Cs) with these new PP2C partners is also under study. P10-033: TGA TRANSCRIPTION FACTORS MEDIATE GLUTAREDOXIN GRXC9 GENE ACTIVATION BY SALI- CYLIC ACID IN ARABIDOPSIS THALIANA Herrera, A. - Carvallo, L. - Blanco, F. - Villarroel, E. - Holuigue, L. P. Universidad Católica de Chile Salicylic acid (SA) is one of the key signals involved in defense responses against biotic and abiotic stresses. GRXC9 gene, coding for a glutaredoxin with antioxidant function, is one of the genes rapidly activated by SA in Arabidopsis. We are interested in elucidate the mechanism of transcriptional activation of this gene by SA. In silico promoter analysis of GRXC9 gene identified two putative SA-responsive as-1-like elements in its proximal region. These elements have been previously described as targets for bZIP factors from the TGA family in promoters of model defense genes. In this work we used a combination of tools to elucidate the function of these elements in the SA-mediated transcriptional activation of GRXC9 gene. Mutants in the TGA 2/5/6 subclass of transcription factors showed impaired GRXC9 activation by SA. In vivo reporter assays, using constructs containing the full length, deletions and mutants of the GRXC9 promoter controlling the expression of GUS reporter gene, indicated requirement of both as-1-like elements for SA-mediated activation of GRXC9 gene. Also, we used yeast two- and one-hybrid assays to study the interaction between TGA transcription factors and their transactivation capacity. Finally, we assessed the association of TGA transcription factors, RNA polymerase II and modified histones to GRXC9 promoter by Chromatin Immunoprecipitation (ChIP) assays. Our results indicate that SA activates the transcription
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POST 04 POSTERS • Environmental S
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Author Index Aarts, M.G. S18-002 Ab
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Cattivelli, L. P01-031 Cawly, J. P1
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Gallego, B. P17-037 Gallego, P.P. P
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Kersten, B. P10-017 Keskin, O. P05-
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Miguel, C. S02-001, P01-122 Milhinh
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Ramiro, M. P09-018 Ramon, M P13-002
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Tanimoto, E. P01-045 Tarakanov, I.
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