Andrée Bourbouloux

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.

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 μm) 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.

Glutathione depletion leads to delayed growth stasis in Saccharomyces cerevisiae: evidence of a partially overlapping role for thioredoxin.

Abstract Disruption of the first enzyme of glutathione biosynthesis in both Saccharomyces cerevisiae and Schizosaccharomyces pombe leads to a glutathione auxotrophy phenotype on plates. However, growth experiments in liquid medium revealed that the cessation of growth resulting from glutathione depletion in these yeasts is very delayed in S. cerevisiae compared to S. pombe. Glutathione metabolism was investigated to understand this delayed growth stasis in S. cerevisiae. The assimilation of reduced and oxidized glutathione, the intracellular storage pools of glutathione and the turnover of this compound were investigated and found to be similar in both yeasts. A possible overlapping role of intracellular thioredoxin in causing delayed stasis was studied. Yeast thioredoxin was overexpressed in S. cerevisiae and was found to partially relieve the dependence of S. cerevisiae glutathione auxotrophs on extracellular glutathione in glucose-grown cultures, as well as in glycerol-grown cultures where conditions of increased glutathione requirements exists in the cell. By partially, but not completely, compensating for glutathione deficiency in this yeast, thioredoxin thus appeared to be the major factor that was causing the delayed growth stasis following glutathione depletion in this yeast.

Cutting, ageing and expression of plant membrane transporters

The activity and the expression of sucrose, hexose and amino acid transporters were studied with fresh, cut or aged tissues and plasma membrane vesicles (PMV) of mature sugar beet (Beta vulgaris L.) leaves. Cutting and ageing both induced an increase of the transcripts coding for sucrose transporters and hexose transporters. No significant effect could be detected on the amino acid transporter transcripts with the probe used (aap1). A polyclonal serum directed against the Arabidopsis thaliana sucrose transporter (AtSUC1) reacted with a 42 kDa band of the sugar beet PMV, confirming previous biochemical identification of this band as a sucrose transporter. ELISA assays run with microsomal fractions and PMV using the AtSUC1 sucrose transporter probe indicated that ageing, and to a lesser extent cutting, increased the amount of sucrose transporter present in the plasma membrane. However, while cutting strongly stimulated proton-motive force driven uptake of sucrose in PMV, ageing only resulted in a slight stimulation. These data give evidence for transcriptional, post-transcriptional and post-translational controls of the activity of the sucrose transporter by mechanical treatments. Proton-motive force driven uptake of 3-O-methylglucose and valine in PMV was strongly stimulated in PMV from aged tissues, although previous data had shown that cutting did not affect theses processes. Therefore, the plant cells possess various levels of control mechanisms that allow them to regulate fluxes of the main assimilates across the plasma membrane when their natural environment is directly or indirectly altered.

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 met4Δ background. Cysteine appeared to be the principal regulatory molecule, since elevated levels were seen in str4Δ 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.

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.

Affinity purification of sucrose binding proteins from the plant plasma membrane.

Purified plasma membranes from sugar beet leaves were solubilized by 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate and loaded on a sepharose 6 B column substituted with sucrose. Elution with sucrose at pH 5.2 yielded a peak that represented 0.2% of the loaded protein. This peak did not appear when the samples were pretreated with either 0.5 mM N-ethylmaleimide (NEM) or 0.5 mM para-chloromercuribenzenesulfonic acid. It was also absent when palatinose, a sucrose analogue not recognized by the sucrose transporter, was used as the affinity ligand. The peak specifically eluted by sucrose from the sucrose-Sepharose column exhibited sucrose transport activity after reconstitution into proteoliposomes. This peak was further fractionated by ion-exchange chromatography on a Mono-Q column, and the different fractions obtained were differentially labeled by [3H]NEM in the presence of sugars recognized (sucrose, maltose) or not recognized (palatinose) by the sucrose transporter. The data allowed to identify two fractions that were enriched with two polypeptides (56 and 41 kDa) differentially labeled by NEM in the presence of sucrose.

Characterization of leucine-leucine transport in leaf tissues

Uptake of the dipeptide [(3)H]Leu-Leu into leaf discs from mature broad bean (Vicia faba L.) was characterized. Uptake was maximal at pH 6.0 and appeared to be mediated by three systems with apparent K(m) values of 20 µM, 350 µM and 43 mM, respectively. Leu-Leu uptake was sensitive to N-ethylmaleimide, p-chloromercuribenzenesulphonic acid, diethylpyrocarbonate, and carbonyl-cyanide-m-chlorophenylhydrazone. Nitrate did not compete with peptide uptake, although the peptide transporter and the nitrate transporter have been reported to be homologous. The ability of leaf tissues to take up peptides strongly decreased with leaf age, and the phloem export of peptides as measured by exudation experiments was very low. It is concluded that the leaf tissues contain a peptide transporter that may take up some peptides with a high affinity, but that this transporter is not involved in the long-distance transport of nitrogen under the form of di- or tri-peptides.

Differential occurrence of suberized sheaths in canes of grapevines suffering from black dead arm, esca or Eutypa dieback

Compared to healthy canes of Ugni-Blanc grapevines, structural modifications were observed in August in wood of growing canes showing foliar symptoms induced by esca and Eutypa Dieback. The observed changes appeared attenuated in canes of the current season sampled in December. In contrast, Black Dead Arm (BDA) did not induce significant modifications compared to healthy canes. A seasonal pattern in suberization was observed in control canes since a first suberized sheath occurred in mid-July at the interface xylem ring/pith and a second sheath was built up in August till December at the interface primary phloem/cortex. The same pattern was observed in BDA-attacked vines. In contrast, these continuous structural barriers were not formed in July and August in canes of the current season in grapevines attacked by esca or Eutypa dieback, but restored in the canes observed in December. These structural modifications were quantified and these events were discussed in the scope of plant physiology and pathogenicity of the implied fungi.