Caractérisation et expression d'homologues de transporteurs de glutathion chez Arabidopsis thaliana

POITIERS-BU Sciences (861942102) / SudocSudocFranceF

Biologie des agents de l'esca et impacts sur la vigne (Vitis vinefera L. cv Ugni blanc)

L esca est une maladie de dépérissement de la vigne provoquée par des champignons intraxylémiens, dont Phaeomoniella chlamydospora et Phaeoacremonium aleophilum. Nous avons tout d abord précisé l influence de facteurs environnementaux tels que la température et le pH sur la croissance des deux champignons et défini des critères cyto-physiologiques permettant leur distinction in vitro. A l issue de leur étude in planta, dans des boutures infectées sélectivement, nous avons observé que ces agents colonisent des tissus distincts : P. chlamydospora se développe essentiellement dans les vaisseaux et les fibres xylémiennes alors que P. aleophilum est détecté préférentiellement dans le protoxylème et la moelle. Ce dernier présente une capacité de dégradation des différentes couches pariétales lignifiées alors que P. chlamydospora ne désorganise que les parois pectocellulosiques. De plus, des expériences de co-inoculation montrent l effet synergique des deux champignons dans leur capacité d envahissement du xylème et d altération des parois. Sur les sarments et les feuilles de Vitis vinifera cv Ugni blanc nous avons défini des caractéristiques phénotypiques et structurales propres à l esca, comparativement à celles du black dead arm et de l eutypiose. Puis, dans les feuilles nous avons mis en évidence l impact de l esca sur le glutathion et sur l expression des gènes d enzymes participant à son métabolisme, dont des glutathion S-transférases. Au delà des altérations précoces du métabolisme primaire, la teneur et l état rédox du glutathion et l expression des glutathion S-transférases représentent des marqueurs de niveau de stress dans la vigne : leur suivi pourrait être un indicateur de l apparition de la maladie au vignoble.Esca disease is a grapevine damage induced by pathogenic fungi localized in the xylem especially Phaeomoniella chlamydospora and Phaeoacremonium aleophilum. We evaluated the effect of various environmental factors, such as temperature and pH on the development of these two fungal pathogens and determined the corresponding cyto-physiological criteria leading to their in vitro characterization. In selectively infected cuttings we observed that these agents invaded different areas: P. chlamydospora developed mainly in vessels and fibres whereas P. aleophilum was detected predominantly in protoxylem and pith areas. Our data also highlighted P. aleophilum capacity to degrade lignified wall layers whereas P. chlamydospora only disorganized pectocellulose walls. In addition, co-inoculations of the two fungi showed the synergistic capacity of these two fungi to invade wood and cause cell wall damage. In canes and leaves of Vitis vinifera cv Ugni blanc we determined specific phenotypic and structural features of the esca disease and compared this to those of black dead arm and eutypa dieback. We observed the impact of esca on glutathione in leaves, focusing on gene expression corresponding to enzymes involved in its metabolism, as well as glutathione S-transferases. Apart from an initial perturbation in primary metabolism, levels and redox state of glutathione and expression of glutathione S-transferases can be considered as stress markers in vine: their detection might constitute an early indicator for infection of grapevine by esca disease in the field.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

AtOPT6 transports glutathione derivatives and is induced by primisulfuron

The oligopeptide transporter (OPT) family contains nine members in Arabidopsis. While there is some evidence that AtOPTs mediate the uptake of tetra- and pentapeptides, OPT homologs in rice (Oryza sativa; OsGT1) and Indian mustard (Brassica juncea; BjGT1) have been described as transporters of glutathione derivatives. This study investigates the possibility that two members of the AtOPT family, AtOPT6 and AtOPT7, may also transport glutathione and its conjugates. Complementation of the hgt1met1 yeast double mutant by plant homologs of the yeast glutathione transporter HGT1 (AtOPT6, AtOPT7, OsGT1, BjGT1) did not restore the growth phenotype, unlike complementation by HGT1. By contrast, complementation by AtOPT6 restored growth of the hgt1 yeast mutant on a medium containing reduced (GSH) or oxidized glutathione as the sole sulfur source and induced uptake of [3H]GSH, whereas complementation by AtOPT7 did not. In these conditions, AtOPT6-dependent GSH uptake in yeast was mediated by a high affinity (Km 5 400 mM) and a low affinity (Km 5 5 mM) phase. It was strongly competed for by an excess oxidized glutathione and glutathione-N-ethylmaleimide conjugate. Growth assays of yeasts in the presence of cadmium (Cd) suggested that AtOPT6 may transport Cd and Cd/GSH conjugate. Reporter gene experiments showed that AtOPT6 is mainly expressed in dividing areas of the plant (cambium, areas of lateral root initiation). RNA blots on cell suspensions and real-time reverse transcription-PCR on Arabidopsis plants indicated that AtOPT6 expression is strongly induced by primisulfuron and, to a lesser extent, by abscisic acid but not by Cd. Altogether, the data show that the substrate specificity and the physiological functions of AtOPT members may be diverse. In addition to peptide transport, AtOPT6 is able to transport glutathione derivatives and metal complexes, and may be involved in stress resistance

AtOPT6 Transports Glutathione Derivatives and Is Induced by Primisulfuron

The oligopeptide transporter (OPT) family contains nine members in Arabidopsis. While there is some evidence that AtOPTs mediate the uptake of tetra- and pentapeptides, OPT homologs in rice (Oryza sativa; OsGT1) and Indian mustard (Brassica juncea; BjGT1) have been described as transporters of glutathione derivatives. This study investigates the possibility that two members of the AtOPT family, AtOPT6 and AtOPT7, may also transport glutathione and its conjugates. Complementation of the hgt1met1 yeast double mutant by plant homologs of the yeast glutathione transporter HGT1 (AtOPT6, AtOPT7, OsGT1, BjGT1) did not restore the growth phenotype, unlike complementation by HGT1. By contrast, complementation by AtOPT6 restored growth of the hgt1 yeast mutant on a medium containing reduced (GSH) or oxidized glutathione as the sole sulfur source and induced uptake of [(3)H]GSH, whereas complementation by AtOPT7 did not. In these conditions, AtOPT6-dependent GSH uptake in yeast was mediated by a high affinity (K(m) = 400 μm) and a low affinity (K(m) = 5 mm) phase. It was strongly competed for by an excess oxidized glutathione and glutathione-N-ethylmaleimide conjugate. Growth assays of yeasts in the presence of cadmium (Cd) suggested that AtOPT6 may transport Cd and Cd/GSH conjugate. Reporter gene experiments showed that AtOPT6 is mainly expressed in dividing areas of the plant (cambium, areas of lateral root initiation). RNA blots on cell suspensions and real-time reverse transcription-PCR on Arabidopsis plants indicated that AtOPT6 expression is strongly induced by primisulfuron and, to a lesser extent, by abscisic acid but not by Cd. Altogether, the data show that the substrate specificity and the physiological functions of AtOPT members may be diverse. In addition to peptide transport, AtOPT6 is able to transport glutathione derivatives and metal complexes, and may be involved in stress resistance

A novel family of transporters mediating the transport of glutathione derivatives in plants

Uptake and compartmentation of reduced glutathione (GSH), oxidized glutathione (GSSG), and glutathione conjugates are important for many functions including sulfur transport, resistance against biotic and abiotic stresses, and developmental processes. Complementation of a yeast (Saccharomyces cerevisiae) mutant (hgt1) deficient in glutathione transport was used to characterize a glutathione transporter cDNA (OsGT1) from rice (Oryza sativa). The 2.58-kb full-length cDNA (AF393848, gi 27497095), which was obtained by screening of a cDNA library and 5'-rapid amplification of cDNA ends-polymerase chain reaction, contains an open reading frame encoding a 766-amino acid protein. Complementation of the hgt1 yeast mutant strain with the OsGT1 cDNA restored growth on a medium containing GSH as the sole sulfur source. The strain expressing OsGT1 mediated [3H]GSH uptake, and this uptake was significantly competed not only by unlabeled GSSG and GS conjugates but also by some amino acids and peptides, suggesting a wide substrate specificity. OsGT1 may be involved in the retrieval of GSSG, GS conjugates, and nitrogen-containing peptides from the cell wall

A Novel Family of Transporters Mediating the Transport of Glutathione Derivatives in Plants

Uptake and compartmentation of reduced glutathione (GSH), oxidized glutathione (GSSG), and glutathione conjugates are important for many functions including sulfur transport, resistance against biotic and abiotic stresses, and developmental processes. Complementation of a yeast (Saccharomyces cerevisiae) mutant (hgt1) deficient in glutathione transport was used to characterize a glutathione transporter cDNA (OsGT1) from rice (Oryza sativa). The 2.58-kb full-length cDNA (AF393848, gi 27497095), which was obtained by screening of a cDNA library and 5′-rapid amplification of cDNA ends-polymerase chain reaction, contains an open reading frame encoding a 766-amino acid protein. Complementation of the hgt1 yeast mutant strain with the OsGT1 cDNA restored growth on a medium containing GSH as the sole sulfur source. The strain expressing OsGT1 mediated [(3)H]GSH uptake, and this uptake was significantly competed not only by unlabeled GSSG and GS conjugates but also by some amino acids and peptides, suggesting a wide substrate specificity. OsGT1 may be involved in the retrieval of GSSG, GS conjugates, and nitrogen-containing peptides from the cell wall