Book of Abstracts - Geyseco

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03 - S - Selected Abstracts for Oral Presentations cence, and the determination of endogenous concentrations of phytohormones is essential to elucidate the role of a particular hormone in any physiological process. Availability of a sensitive and rapid method to quantify multiple classes of plant hormones simultaneously will greatly facilitate the investigation of hormone-induced signalling networks in controlling specific developmental pathways and physiological responses. Due to the presence of plant hormones at very low concentrations in plant tissues (10-9 M to 10-6 M) and their different chemistries, the development of a high-throughput and comprehensive method for the determination of phytohormones is challenging. The present work reports a rapid, specific and sensitive method using UPLC- MS/MS for the quantitative and simultaneous analysis of the major phytohormones found in plant tissues, including auxins, cytokinins, gibberellins, abscisic acid, 1-amino-cyclopropane-1- carboxyic acid (the ethylene precursor), jasmonic acid and salicylic acid. Sample preparation, extraction procedures and UPLC- MS/MS conditions were optimized for the determination of all plant hormones in a single run. This new method is applicable to the analysis of dynamic changes in endogenous concentrations of phytohormones in different plant tissues to study plant developmental processes or plant responses to biotic and abiotic stresses. An example is shown in which simultaneous analyses of phytohormones is performed in leaves of plants exposed to salt stress, both in the model plant, Arabidopsis thaliana and in an aromatic plant, Salvia officinalis. S03-001: OXYLIPIN-INDUCED TYROSINE PHOS- PHORYLATION OF PLANT PROTEINS Yakusheva, O.* - Karimova, F. Kazan Institute of Biochemistry and Biophysics, RAS *Corresponding author: vov1985@mail.ru Oxylipins are products of oxygenated polyunsaturated fatty acids, biologically active signaling molecules. Nowadays molecular mechanisms of oxylipin effects are the object of close attention. In plant cells the signaling pathway of jasmonates is the most studied. Earlier the researchers of out Institute showed that one of the main products of legume lipoxygenase metabolism is (9Z)-12-hydroxy-9-dodecenoic acid (HDA). It was shown that HDA is a growth stimulator causing an increase in soybean callus biomass up to 400%. Previously we showed HDA-induced Ca 2+ - and cAMP-dependent plant protein phosphorylation for 2h of exposure. Protein tyrosine phosphorylation is known to be critical for cell proliferation and differentiation. In this context plant protein tyrosine phosphorylation is of great interest. We investigated dynamics of HDA effect in vivo on the tyrosine phosphorylation level (TPL) of leaf soluble proteins in pea plants. Our results indicate that TPL quickly changes in control and HDAtreated plants during different time periods. To detect factors critical for HDA effect on TPL plants grown on the nutrient solution without Ca 2+ were used. We showed that Ca 2+ -deficiency in the growth medium caused a decrease in TPL of all polypeptides in comparison with control plants grown on the optimal solution. These data suggest that there is Ca 2+ - dependence of protein phosphorylation/dephosphorylation enzymes activity. The HDA effect on TPL was also Ca 2+ -dependent. As known in vertebrate cells, protein tyrosine phosphatase (PTP) activity is 10-100 times higher than protein tyrosine kinase activity. Using PTP inhibitor phenylarsinoxide we showed contribution of PTP activity to Ca 2+ - dependence of HDA-induced TPL in pea plants. S03-002: ANALYSIS OF THE ARABIDOPSIS GLYCO- PROTEOME Van Der Krol, S.¹* - Song, W.¹ - Mentink, R.² - Henquet, M.³ - Cordewener, J.³ - Bosch, D.³ - America, T.³ ¹Lab. of Plant Physiology, Wageningen University ²Hubrecht Inst. Utrecht Netherlands ³Plant Research International, Wageningen, Netherlands *Corresponding author: sander.vanderkrol@wur.nl Arabidopsis contains ~4500 secreted proteins with one or more of the N-glycosylation consensus site N-x-S/T. However, for only few glycoproteins has the presence of an Nglycan actually been confirmed and mapped experimentally. Here we present the characterization of the glycoproteome from Arabidopsis, as extracted from leaves, seedlings and developing seeds. Extracted proteins were first digested with trypsin, after which (activated) glycopeptides were coupled to Hydrazide resin. After extensive washing, bound peptides were released by the enzyme PNGaseF and were measured by LCMS DDA and MS E . Because PNGaseF converts the N to D, it leaves a ‘glycansignature’ in the peptide sequence to be analyzed. Moreover, PNGaseF cleaves mannose glycans (on glycopeptides in ER), but not complex glycans (on glycopeptides in and from Golgi). This allowed distinguishing between ER- and Golgi-derived glycoproteomes, by comparing glycopeptide profiles from WT (only ER-derived glycopeptides) and glycopeptide profiles from mutant plants without complex glycans (cgl) (full glycoproteome). Using this method we confirmed glycan occupancy of over 800 consensus sites on more than 300 proteins. We show that some glycoproteins (e.g. LRR receptors) have heterogeneous glycosylation (mannose and complex glycans within the same protein). N-glycan site occupancy mapping was also used to correct THMM-predicted membrane protein topology of eight membrane proteins. The results show that our method allows for High Through Put proteomics of this important subset of the plant proteome. This is now used to map changes in the glycoproteome in response to protein secretion stress in seeds and in pathogeneffector/plant interactions. S03-003: A SYSTEM BIOLOGY APPROACH TO UN- DERSTAND FUNCTIONS OF RAPTOR1 IN ARABIDOP- SIS THALIANA Li, Y. * - Caldana, C. - Giavalisco, P. - Leisse, A. - Willmitzer, L. Max-Planck-Institute of Molecular Plant Physiology *Corresponding author: yli@mpimp-golm.mpg.de RAPTOR/KOG1 proteins, conserved WD-40 repeat proteins, are binding partners of the target of rapamycin (TOR) kinase that plays a central role in metabolism, such as cellular growth in response to nutrients, mitogens and growth factors in eukaryotes. In Arabidopsis, RAPTOR1 interacts with TOR and S6K1 in vivo, and overexpression of RAPTOR1 rendered the S6K1 osmotic stress insensitive. We developed computational and experimental methods to identify RAPTOR1 by using artificial microRNA lines. It is shown that, by quantitative RT-PCR, RAPTOR1 expression level decreased after estradiol induction. Interestingly, amiRaptor1 plants were much smaller after transfer to MS medium containing estradiol. Further, I will focus on the function of RAPTOR1 in TOR signaling pathway by tanscriptomics, proteomics and metabolomics data analysis at system-level. S03-004: STEADY-STATE 13C METABOLIC FLUX ANALYSIS: FOCUS ON DEVELOPING BARLEY SEEDS Krach, C. * Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung *Corresponding author e-mail:christian.krach@gmx.de Metabolic Flux Analysis has become a well-established tool in microbial metabolic engineering. It has been successfully adopted to rational redirections of carbon metabolism and so increasing the yield of desired fermentation products. In contrast affords to manipulate a plant’s metabolism beyond the scope of secondary metabolites were less successful, resulting in data hard to interpret. Accordingly a system-wide analysis and a more general understanding of metabolic processes are necessary. A cell’s set of metabolic fluxes represents a very comprehensive phenotype of its metabolic activity and therefore includes extremely important information for the targeted improvement of crop metabolism. Intracellular fluxes themselves cannot be measured S
FESPB 2010 - XVII Congress of the Federation of European Societies of Plant Biology directly but have to be deduced from 13 C steady-state labelling experiments. The additional use of labelled substrate turns it possible to generate detailed flux maps including bidirectional or cyclic fluxes, exceeding the possibilities of the well established flux balancing. Furthermore the application of special software makes it possible to calculate the fluxes from GC-MS fractioning patterns of metabolites and positional isotopomer enrichment. To achieve these goals we set up barley spike- and single grain cultures. After feeding labelled substrates metabolites are extracted and subjected to GC-MS analysis. The data are corrected for natural isotope abundance and then can be used to calculate the fluxes using 13CFlux software. S04-001: ODDSOC2, A MADS BOX FLORAL REPRESSOR THAT IS DOWN-REGULATED BY VERNALIZATION IN TEMPERATE CEREALS Greenup, A.¹* - Sasani, S.² - Oliver, S.¹ - Talbot, M.¹ - Dennis, E.¹ - Hemming, M.¹ - Trevaskis, B.¹ ¹CSIRO ²University of Tehran *Corresponding author e-mail: u4378396@anu.edu.au In temperate cereals the transition from vegetative to reproductive development can be accelerated by exposure to extended periods of cold (vernalization). We investigated the function of a grass-specific MADS box gene ODDSOC2 in the vernalization response of barley (Hordeum vulgare). In barley HvOS2 is expressed in the shoot apex and leaves but is repressed by vernalization. Repression of OS2 can occur independently of the central regulator of vernalization, VERNALIZATION1 (VRN1). In addition to regulating OS2 as part of the vernalization pathway, barleys that carry active alleles of HvVRN1 have reduced expression of HvOS2, suggesting that HvVRN1 down-regulates HvOS2 during development. Ectopic expression of HvOS2 in a ‘spring’ barley delayed flowering and reduced spike, stem and leaf length. Microarray analysis of plants overexpressing HvOS2 revealed that expression of barley homologues of the Arabidopsis thaliana gene Floral Promoting Factor 1 were reduced (FPF1) and expression of RNase-S-like genes was increased. FPF1 promotes floral development and enhances cell elongation in Arabidopsis, rice and tobacco, so down-regulation of FPF1-like genes might explain the phenotypes observed in barley plants over-expressing HvOS2. Based on our findings an extended model of the genetic pathways controlling vernalizationinduced flowering in cereals has been developed. The model describes the regulatory relationships between VRN1, OS2 and FPF1-like genes. These findings further highlight the differences between the vernalization responses of temperate cereals and the model plant Arabidopsis. S04-002: AN ANCIENT PHOTOPERIODIC MECHANISM CONTROLLING DEVELOPMENT AND CARBON ME- TABOLISM IN PLANTS AND ALGAE Valverde, F.* - Ruiz, M.T. - Albi, T. - Cano, B. - Girón, B. - Laureano, A. - Ortiz, M.I. - Ribeiro, M.A. - Said, F.E. - Romero, J.M. Instituto de Bioquímica Vegetal y Fotosíntesis. CSIC-USE *Corresponding author e-mail: federico@ibvf.csic.es The decision to flower is a crucial developmental process in a plant because it determines the reproductive success of the individual. It demands essential energetic resources and failing to flower at the correct time of the year seriously impinges plant vitality. Carbon metabolism in plants is connected to their developmental stage; i.e. Mutants in the photoperiod pathway, that present a longer vegetative growth phase, such as CONSTANS (CO) accumulate more starch than wild type plants. Our group has recently published the role of a CO ortholog (CrCO) in the control of photoperiod in the green alga Chlamydomonas reinhardtii and its influence over several key metabolic and cell cycle processes. To better understand the process, we propose to make a comparative study of the role of photoperiod in the control of carbon metabolism in green algae and plants.Several observations indicate that the effect of CO on leave starch accumulation may be related to its capacity to increase the expression of a starch synthese (GBSSI) and that it could involve a new regulatory mechanism. For this reason we have isolated gbssi mutants, characterized their flowering time phenotype and capacity to synthesize starch. gbssI mutants have been crossed to plants overexpressing CO and their effect on flowering time checked. We will also show GBSSI expression in 35S::CO plants and co mutants in LD and SD conditions to asses the effect of CO on GBSSI expression in a circadian manner. S04-003: IDENTIFICATION AND FUNCTIONAL ANALY- SES OF GENES INVOLVED IN FRUIT SET IN TOMATO Gómez-Mena, C.* - Medina, M. - Cañas, L. - Beltrán, J.P. Instituto de Biología Molecular y Celular de Plantas (IBMCP- CSIC) *Corresponding author e-mail: cgomezm@ibmcp.upv.es Tomato has become a plant model system for fleshy fruit to study fruit set and development. The shift from the static flower ovary to fast-growing young fruit is a phenomenon known as fruit set, and is an important step in the development of all sexually reproducing higher plants. In general, fruit set is induced after pollination and successful fertilization of the egg cells in the ovules. However fruit development can be uncoupled from fertilization and seed development to generate seedless (parthenocarpic) fruits. Male-sterile tomato plants have been obtained by anther ablation at early stages of development (Roué et al 2007). The ovaries of these transgenic plants quickly develop and fruit set is established in the absence of fertilization. Using these lines, we have carried out a genomic approach in order to identify the genes involved in the process. A set of 173 unigenes coding transcription factors are differentially expressed at early stages of ovary development in the male-sterile plants pEND1::barnase. The function of these genes is being investigated using VIGS technology. The identification and characterization of these genes will make possible to develop biotechnological tools to gain control over fruit set in tomato. Roque, E., Gómez, M.D., Ellul, P., Wallbraun, M., Madueño, F., Beltrán, J.P., Cañas, L.A. (2007). Plant Cell Rep. 26: 313-325. S04-004: THE ROLE OF MIRNAS DURING GERM CELL SPECIFICATION IN ARABIDOPSIS POLLEN Borges, F.¹ - Slotkin, R. K.² - Gardner, R.³ - Martienssen, R. A.4 - Becker, J.D.*¹ ¹Plant Genomics, Instituto Gulbenkian de Ciência, Oeiras, Portugal ²Plant Cellular and Molecular Biology, The Ohio State University, Columbus, OH, USA ³Cell Imaging Unit, Instituto Gulbenkian de Ciência, Oeiras, Portugal 4 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA *Corresponding author e-mail: jbecker@igc.gulbenkian.pt Plant meiocytes undergo subsequent mitotic divisions to form the gametes, which must rapidly reprogramme their epigenome before fertilization. In Arabidopsis, the male germline differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell that divides before anthesis to originate two sperm cells. The vegetative nucleus (VN) retains a somatic nature, orchestrates pollen tube growth and does not contribute with genetic material to the next generation. However, recent observations indicated that DNA demethylation and expression of particular transposable element (TE) loci occurs in the VN, producing siRNAs that might reinforce epigenetic silencing of TE activity in the gametes. Transcriptional profiling of FACS-purified mature pollen and sperm cells has shown that transcripts involved in small RNA biogenesis and RNA-directed DNA methylation are enriched in
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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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