Identification et caractérisation fonctionnelle d'un gène codant un facteur de transcription de type WRKY cherzla vigne, VvWRKY1. Implicitation dans les mécanismes de défense.

Afin de comprendre le rôle des facteurs de transcription WRKY dans les réactions de défense de la Vigne (Vitis vinifera) contre des agents pathogènes, nous avons identifié un ADNc, VvWRKY1, codant une protéine présentant de fortes homologies avec les membres de cette famille multigénique. Après avoir vérifié la fonctionnalité de la protéine, traduite par sa capacité à se lier à l'ADN spécifiquement au niveau des boîtes W, l'étude de l'expression de ce gène chez la Vigne a été réalisée. VvWRKY1 est ainsi fortement régulé au cours du développement des feuilles et des baies de raisin. La blessure et des molécules de stress comme l'acide salicylique, l'éthylène, le peroxyde d'hydrogène induisent son expression dans les feuilles de Vigne alors que l'acide jasmonique la réprime. L'analyse fonctionnelle de VvWRKY1 a été entreprise par sur-expression chez le Tabac, Arabidopsis thaliana et la Vigne. Les résultats suggèrent une réelle implication de cette protéine dans la défense contre des champignons pathogènes. Effectivement, une sensibilité différente est observée dans les plantes transgéniques par rapport aux plantes sauvages vis-à -vis de différents champignons. Toutefois, les mécanismes sous-jacents n'ont pas encore été identifiés mais des expériences d'hybridation sur puces à ADN comparant les Vignes transgéniques et les Vignes sauvages ont permis d'identifier des gènes potentiellement régulés par VvWRKY1 dont deux gènes codant des lipoxygénases.In order to understand the function of WRKY transcription factor in defense mechanisms in Grapevine towards pathogens, we identified a full-length cDNA encoding a protein which shares high homology with members of this multigenic family. After checking the ability of the protein to bind DNA on W boxes, gene expression was studied on grape. VvWRKY1 gene is regulated during grapeberry and leaves development. Wounding and stress molecules such as salicylic acid, ethylene and hydrogen peroxyde induce its expression in leaves whereas jasmonic acid repress it. Functional analysis of VvWRKY1 protein was performed by overexpression in tobacco, Arabidopsis thaliana and grapevine. Results suggested its real involvement in defense mechanisms against fungal pathogens. Indeed, different susceptibility towards several fungi was observed in transgenic plants compared to wild type plants. Mechanisms underlying its response stay unclear but microarrays experiments comparing transformed grapevines to wild type plants revealed potential target genes of VvWRKY1 transcription factor and particularly two genes encoding lipoxygenases

Identification et caractérisation fonctionnelle d'un gène codant un facteur de transcription de type WRKY chez la Vigne, VvVRKY1 (implication dans les mécanismes de défense)

Afin de comprendre le rôle des facteurs de transcription WRKY dans les réactions de féfense de la Vigne (Vitis vinifera) contre des agents pathogènes, nous avons identifié un ADNc, VvWRKY1, codant une protéine présentant de fortes homologies avec les membres de cette famille multigénique. Après avoir vérifié la fonctionnalité de la protéine, traduite par sa capacité à se lier à l'ADN spécifiquement au niveau des boîtes W, l'étude de l'expression de ce géne chez la Vigne a été réalisée. VvWRKY1 est ainsi fortement régulé au cours du développement des feuilles et des baies de raisin. Des molécules de stress comme l'acide salicylique, l'éthylène, le peroxyde d'hydrogène induisent son expression dans les feuilles de Vigne alors que l'acide jasmonique la réprime. L'analyse fonctionnelle de VvWRKY1 a été entreprise par sur-expression chez le Tabac, Arabidopsis thaliana et la Vigne. Les résultats suggèrent une réelle implication de cette proteine dans la défense contre des champignons pathogènes. Effectivement, une sensibilité différente est observée dans les plantes transgéniques par rapport aux plantes sauvages vis-à-vis de différents champignons. Toutefois, les mécanismes sous-jacents n'ont pas encore été identifiés mais des expériences d'hybridation sur puces à ADN comparant les Vignes transgéniques et les Vignes sauvages ont permis d'identifier des gènes potentiellement régulés par VvWRKY1 dont deux gènes codant des lipoxygénasesBORDEAUX1-BU Sciences-Talence (335222101) / SudocSudocFranceF

New insights on the organization and regulation of the fatty acid biosynthetic network in the model higher plant Arabidopsis thaliana

In the plastids of plant cells, fatty acid (FA) production is a central biosynthetic process. It provides acyl chains for the formation of a variety of acyl lipids fulfilling different biological functions ranging from membrane synthesis to signaling or carbon and energy storage. The biochemical pathway leading to the synthesis of FA has been described for a long time. Over the last 15 years, and after the genome of the model higher plant Arabidopsis thaliana has been sequenced, the scientific community has deployed approaches of functional genomics to identify the actors comprising this pathway. One of the puzzling aspects of the emerging molecular biology of FA synthesis resided in the occurrence of multigene families encoding most enzymes of the pathway. Studies carried out to investigate these families led to the conclusion that most members have acquired non-redundant roles in planta. This is usually the consequence of divergent expression patterns of these isogenes and/or of different substrate specificities of the isoforms they encode. Nevertheless, much remains to be elucidated regarding the molecular bases underpinning these specificities. Protein biochemistry together with emerging quantitative proteomic technologies have then led to a better understanding of the structure of the network, which is composed of multiprotein complexes organized within the stromal compartment of plastids: whereas growing evidence suggests that the early steps of the pathway might be associated to the inner envelope membrane, several late enzymes might be localized next to the thylakoids. The question of the existence of a large integrated protein assembly channeling substrates through the whole pathway that would span the stroma remains uncertain. Finally, recent discoveries regarding the post-translational regulation of the pathway open new research horizons and may guide the development of relevant biotechnological strategies aimed at monitoring FA production in plant systems

Characterization, functional validation and gene expression patterns of two 14-3-3 isoforms from Vitis vinifera

In eukaryotic cells, 14-3-3 proteins constitute a family of conserved regulatory proteins which bind to a large number of phosphorylated partners. Although a great body of evidences accumulated in herbaceous species indicates that 14-3-3s play key roles in regulation of plant growth, information about function in tree plants is still very scarce. In this paper, we report the characterization of two novel 14-3-3s from grapevine (Vitis vinifera 'Cabernet Sauvignon') named VV1_CS and VV2_CS. The VV1_CS and VV2_CS cDNA were expressed in Escherichia coli and the recombinant proteins were shown to be functional proteins using plasma membrane H+-ATPase as target. Transcript analysis during grape development or under elicitor-induced stress revealed differential expression of the two 14-3-3 isoforms

Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants

Nitrate is both an important nutrient and a signalling molecule for plants. Although several components of the nitrate signalling pathway have been identified, their hierarchical organization remains unclear. Here we show that the localization of NLP7, a member of the RWP-RK transcription factor family, is regulated by nitrate via a nuclear retention mechanism. Genome-wide analyses revealed that NLP7 binds and modulates a majority of known nitrate signalling and assimilation genes. Our findings indicate that plants, like fungi and mammals, rely on similar nuclear retention mechanisms to instantaneously respond to the availability of key nutrients

Sugar metabolism and the plant target of rapamycin kinase: a sweet operaTOR?

In eukaryotes, the ubiquitous TOR (target of rapamycin) kinase complexes have emerged as central regulators of cell growth and metabolism. The plant TOR complex 1 (TORC1), that contains evolutionary conserved protein partners, has been shown to be implicated in various aspects of C metabolism. Indeed Arabidopsis lines affected in the expression of TORC1 components show profound perturbations in the metabolism of several sugars, including sucrose, starch, and raffinose. Metabolite profiling experiments coupled to transcriptomic analyses of lines affected in TORC1 expression also reveal a wider deregulation of primary metabolism. Moreover recent data suggest that the kinase activity of TORC1, which controls biological outputs like mRNA translation or autophagy, is directly regulated by soluble sugars

Over-expression of VvWRKY1 in grapevines induces expression of jasmonic acid pathway-related genes and confers higher tolerance to the downy mildew

Most WRKY transcription factors activate expression of defence genes in a salicylic acid- and/or jasmonic acid-dependent signalling pathway. We previously identified a WRKY gene, VvWRKY1, which is able to enhance tolerance to fungal pathogens when it is overexpressed in tobacco. The present work analyzes the effects of VvWRKY1 overexpression in grapevine. Microarray analysis showed that genes encoding defence-related proteins were up-regulated in the leaves of transgenic 35S::VvWRKY1 grapevines. Quantitative RT-PCR analysis confirmed that three genes putatively involved in jasmonic acid signalling pathway were overexpressed in the transgenic grapes. The ability of VvWRKY1 to trans-activate the promoters of these genes was demonstrated by transient expression in grape protoplasts. The resistance to the causal agent of downy mildew, Plasmopara viticola, was enhanced in the transgenic plants. These results show that VvWRKY1 can increase resistance of grapevine against the downy mildew through transcriptional reprogramming leading to activation of the jasmonic acid signalling pathway

mTOR Signalling in Health and Disease Regulation of plant growth and metabolism by the TOR kinase

Abstract The TOR (target of rapamycin) kinase is present in nearly all eukaryotic organisms and regulates a wealth of biological processes collectively contributing to cell growth. The genome of the model plant Arabidopsis contains a single TOR gene and two RAPTOR (regulatory associated protein of TOR)/KOG1 (Kontroller of growth 1) and GβL/LST8 (G-protein β-subunit-like/lethal with Sec thirteen 8) genes but, in contrast with other organisms, plants appear to be resistant to rapamycin. Disruption of the RAPTOR1 and TOR genes in Arabidopsis results in an early arrest of embryo development. Plants that overexpress the TOR mRNA accumulate more leaf and root biomass, produce more seeds and are more resistant to stress. Conversely, the down-regulation of TOR by constitutive or inducible RNAi (RNA interference) leads to a reduced organ growth, to an early senescence and to severe transcriptomic and metabolic perturbations, including accumulation of sugars and amino acids. It thus seems that plant growth is correlated to the level of TOR expression. We have also investigated the effect of reduced TOR expression on tissue organization and cell division. We suggest that, like in other eukaryotes, the plant TOR kinase could be one of the main contributors to the link between environmental cues and growth processes

MYB118 Represses Endosperm Maturation in Seeds of Arabidopsis

In the exalbuminous species Arabidopsis thaliana, seed maturation is accompanied by the deposition of oil and storage proteins and the reduction of the endosperm to one cell layer. Here, we consider reserve partitioning between embryo and endosperm compartments. The pattern of deposition, final amount, and composition of these reserves differ between the two compartments, with the embryo representing the principal storage tissue in mature seeds. Complex regulatory mechanisms are known to prevent activation of maturation-related programs during embryo morphogenesis and, later, during vegetative growth. Here, we describe a regulator that represses the expression of maturation-related genes during maturation within the endosperm. MYB118 is transcriptionally induced in the maturing endosperm, and seeds of myb118 mutants exhibit an endosperm-specific derepression of maturation-related genes associated with a partial relocation of storage compounds from the embryo to the endosperm. Moreover, MYB118 activates endosperm-induced genes through the recognition of TAACGG elements. These results demonstrate that the differential partitioning of reserves between the embryo and endosperm in exalbuminous Arabidopsis seeds does not only result from developmental programs that establish the embryo as the preponderant tissue within seeds. This differential partitioning is also regulated by MYB118, which regulates the biosynthesis of reserves at the spatial level during maturation

Regulation of plant growth and metabolism by the TOR kinase

The TOR (target of rapamycin) kinase is present in nearly all eukaryotic organisms and regulates a wealth of biological processes collectively contributing to cell growth. The genome of the model plant Arabidopsis contains a single TOR gene and two RAPTOR (regulatory associated protein of TOR)/KOG1 (Kontroller of growth 1) and G beta L/LST8 (G-protein beta-subunit-like/lethal with Sec thirteen 8) genes but, in contrast with other organisms, plants appear to be resistant to rapamycin. Disruption of the RAPTOR1 and TOR genes in Arabidopsis results in an early arrest of embryo development. Plants that overexpress the TOR mRNA accumulate more leaf and root biomass, produce more seeds and are more resistant to stress. Conversely, the down-regulation of TOR by constitutive or inducible RNAi (RNA interference) leads to a reduced organ growth, to an early senescence and to severe transcriptomic and metabolic perturbations, including accumulation of sugars and amino acids. It thus seems that plant growth is correlated to the level of TOR expression. We have also investigated the effect of reduced TOR expression on tissue organization and cell division. We suggest that, like in other eukaryotes, the plant TOR kinase could be one of the main contributors to the link between environmental cues and growth processes