Systemic effects on leaf glutathione metabolism and defence protein expression caused by esca infection in grapevines.

Esca is a devastating disease of Vitis vinifera L., caused by fungal pathogen(s) inhabiting the wood. The pathogens induce symptoms in the foliage, which are associated with structural and biochemical changes in leaves. The present study was undertaken to examine the effects of the disease on leaf glutathione metabolism in field-grown plants. The glutathione pool decreased and defence proteins such as PR-proteins and chitinases were expressed in the leaves before the appearance of visible symptoms in esca-infected canes. Glutathione depletion was increased as the disease developed in the leaves. The ratio of glutathione disulfide (GSSG) to the total glutathione pool was slightly decreased in leaves without visible symptoms, but it was significantly increased as the disease progressed. The abundance of γ-glutamylcysteine synthetase (γ-ECS) transcripts and of γ-ECS protein was greatly decreased in leaves exhibiting esca symptoms. Although glutathione reductase and glutathione peroxidase transcripts were largely unchanged by the spread of the esca disease, leaf glutathione S-transferase (GST) activities, the amounts of mRNAs encoding GSTU1 and GSTF2 and the abundance of the GSTU1 and GSTF2 proteins were highest at the early stages of infection and then decreased as visible symptoms appeared in the leaves. The GSTF2 protein, which was more abundant than GSTU1, was found in the nucleus and in the cytoplasm, whereas the GSTU1 protein was found largely in the plastids. These data demonstrate that the fungi involved in the esca disease induce pronounced systemic effects in the leaves before the appearance of visible damage. We conclude that the expression of GSTs, the extent of glutathione accumulation and the ratio of GSSG to total glutathione are early indicators of the presence of the esca disease in grapevine canes and thus these parameters can be used as stress markers in field-grown vines.

Multiple cis-regulatory elements and the yeast sulphur regulatory network are required for the regulation of the yeast glutathione transporter, Hgt1p.

HGT1 encodes a high-affinity glutathione transporter in the yeast Saccharomyces cerevisiae that is induced under sulphur limitation. The present work demonstrates that repression by organic sulphur sources is under the control of the classic sulphur regulatory network, as seen by the absence of expression in a met4delta background. Cysteine appeared to be the principal regulatory molecule, since elevated levels were seen in str4delta strains (deficient in cysteine biosynthesis) that could be repressed by elevated levels of cysteine, but not by methionine or glutathione. Investigations into cis-regulatory elements revealed that the previously described motif, a 9-bp cis element, CCGCCACAC, located at the -356 to -364 region of the promoter could in fact be refined to a 7-bp CGCCACA motif that is also repeated at -333 to -340. The second copy of this motif was essential for activity, since mutations in the core region of the second copy completely abolished activity and regulation by sulphur sources. Activity, but not regulation, could be restored by reintroducing an additional copy upstream of the first copy. A third region, GCCGTCTGCAAGGCA, conserved in the HGT1 promoters of the different Saccharomyces spp, was observed at -300 to -285 but, while mutations in this region did not lead to any loss in repression, the basal and induced levels were significantly increased. In contrast to a previous report, no evidence was found for regulation by the VDE endonuclease. The strong repression at the transport level by glutathione seen in strains overexpressing HGT1 was due to a glutathione-dependent toxicity in these cells.

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 = 400 microm) and a low affinity (Km = 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.