gamma-Glutamyl transpeptidase GGT4 initiates vacuolar degradation of glutathione S-conjugates in Arabidopsis.

The xenobiotic monochlorobimane is conjugated to glutathione in the cytosol of Arabidopsis thaliana, transported to the vacuole, and hydrolyzed to cysteine S-bimane [Grzam, A., Tennstedt, P., Clemens, S., Hell, R. and Meyer, A.J. (2006) Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase. FEBS Lett. 580, 6384-6390]. The work here identifies gamma-glutamyl transpeptidase 4 (At4g29210, GGT4) as the first step of vacuolar degradation of glutathione conjugates. Hydrolysis of glutathione S-bimane is blocked in ggt4 null mutants of A. thaliana. Accumulation of glutathione S-bimane in mutants and in wild-type plants treated with the high affinity GGT inhibitor acivicin shows that GGT4 is required to initiate the two step hydrolysis sequence. GGT4:green fluorescent protein fusions were used to demonstrate that GGT4 is localized in the lumen of the vacuole.

Localization of members of the gamma-glutamyl transpeptidase family identifies sites of glutathione and glutathione S-conjugate hydrolysis.

gamma-Glutamyl transpeptidases (GGTs) are essential for hydrolysis of the tripeptide glutathione (gamma-glutamate-cysteine-glycine) and glutathione S-conjugates since they are the only enzymes known to cleave the amide bond linking the gamma-carboxylate of glutamate to cysteine. In Arabidopsis thaliana, four GGT genes have been identified based on homology with animal GGTs. They are designated GGT1 (At4g39640), GGT2 (At4g39650), GGT3 (At1g69820), and GGT4 (At4g29210). By analyzing the expression of each GGT in plants containing GGT:beta-glucuronidase fusions, the temporal and spatial pattern of degradation of glutathione and its metabolites was established, revealing appreciable overlap among GGTs. GGT2 exhibited narrow temporal and spatial expression primarily in immature trichomes, developing seeds, and pollen. GGT1 and GGT3 were coexpressed in most organs/tissues. Their expression was highest at sites of rapid growth including the rosette apex, floral stem apex, and seeds and might pinpoint locations where glutathione is delivered to sink tissues to supplement high demand for cysteine. In mature tissues, they were expressed only in vascular tissue. Knockout mutants of GGT2 and GGT4 showed no phenotype. The rosettes of GGT1 knockouts showed premature senescence after flowering. Knockouts of GGT3 showed reduced number of siliques and reduced seed yield. Knockouts were used to localize and assign catalytic activity to each GGT. In the standard GGT assay with gamma-glutamyl p-nitroanilide as substrate, GGT1 accounted for 80% to 99% of the activity in all tissues except seeds where GGT2 was 50% of the activity. Protoplasting experiments indicated that both GGT1 and GGT2 are localized extracellularly but have different physical or chemical associations.

The role of 5'-adenylylsulfate reductase in controlling sulfate reduction in plants.

Cysteine is the first organic product of sulfate assimilation and as such is the precursor of all molecules containing reduced sulfur including methionine, glutathione, and their many metabolites. In plants, 5'-adenylylsulfate (APS) reductase is hypothesized to be a key regulatory point in sulfate assimilation and reduction. APS reductase catalyzes the two-electron reduction of APS to sulfite using glutathione as an electron donor. This paper reviews the experimental basis for this hypothesis. In addition, the results of an experiment designed to test the hypothesis by bypassing the endogenous APS reductase and its regulatory mechanisms are described. Two different bacterial assimilatory reductases were expressed in transgenic Zea mays, the thioredoxin-dependent APS reductase from Pseudomonas aeruginosa and the thioredoxin-dependent 3'-phosphoadenylylsulfate reductase from Escherichia coli. Each of them was placed under transcriptional control of the ubiquitin promoter and the protein products were targeted to chloroplasts. The leaves of transgenic Z. mays lines showed significant accumulation of reduced organic thiol compounds including cysteine, gamma-glutamylcysteine, and glutathione; and reduced inorganic forms of sulfur including sulfite and thiosulfate. Both bacterial enzymes appeared to be equally capable of deregulating the assimilative sulfate reduction pathway. The reduced sulfur compounds accumulated to such high levels that the transgenic plants showed evidence of toxicity. The results provide additional evidence that APS reductase is a major control point for sulfate reduction in Z. mays.

Methionine and threonine synthesis are limited by homoserine availability and not the activity of homoserine kinase in Arabidopsis thaliana.

Homoserine kinase (HSK) produces O-phospho-l-homoserine (HserP) used by cystathionine gamma-synthase (CGS) for Met synthesis and threonine synthase (TS) for Thr synthesis. The effects of overexpressing Arabidopsis thaliana HSK, CGS, and Escherichia coli TS (eTS), each controlled by the 35S promoter, were compared. The results indicate that in Arabidopsis Hser supply is the major factor limiting the synthesis of HserP, Met and Thr. HSK is not limiting and CGS or TS control the partitioning of HserP. HSK overexpression had no effect on the level of soluble HserP, Met or Thr, however, when treated with Hser these plants produced far more HserP than wild type. Met and Thr also accumulated markedly after Hser treatment but the increase was similar in HSK overexpressing and wild-type plants. CGS overexpression was previously shown to increase Met content, but had no effect on Thr. After Hser treatment Met accumulation increased in CGS-overexpressing plants compared with wild type, whereas HserP declined and Thr was unaffected. Arabidopsis responded differentially to eTS expression depending on the level of the enzyme. At the highest eTS level the Thr content was not increased, but the phenotype was negatively affected and the T1 plants died before reproducing. Comparatively low eTS did not affect phenotype or Thr/Met level, however after Hser treatment HserP and Met accumulation were reduced compared with wild type and Thr was increased slightly. At intermediate eTS activity seedling growth was retarded unless Met was supplied and CGS expression was induced, indicating that eTS limited HserP availability for Met synthesis.

Enhancement of the primary flavor compound methional in potato by increasing the level of soluble methionine.

The primary flavor compound in potato, methional, is synthesized from methionine by the Strecker degradation reaction. A major problem associated with potato processing is the loss of methional. Methional or its precursor, methionine, is not added back during potato processing due to high costs of production. A novel approach to enhance the methional level in processed potato would be to increase the production of its precursor, soluble methionine (Met). Cystathionine gamma-synthase (CGS) is a key enzyme regulating methionine biosynthesis in plants. To increase the level of soluble methionine in potato, Arabidopsis thaliana CGS cDNA was introduced under transcriptional control of the cauliflower mosaic virus 35S promoter into Russet Burbank potato by Agrobacterium-mediated transformation. Ten different transgenic potato lines (CGS1-10) were analyzed. Immunoblot analysis demonstrated that Arabidopsis CGS is expressed in the leaves, tubers, and roots of transgenic potato plants. CGS enzymatic activity was higher in the leaves and roots of the transgenic potato lines compared to the wild-type potato. Methionine levels in the leaves, roots and tubers of transgenic potato lines were enhanced as high as 6-fold compared to those in wild type potato plants. The methional level in baked tubers of field-grown transgenic potato lines was increased between 2.4- and 4.4-fold in lines CGS1, CGS2, and CGS4. The increase observed in methional levels correlated with the soluble methionine level in the tubers from the same lines measured before processing. These results provide the first evidence that the methional level can be enhanced in processed potatoes by increasing the production of its precursor, methionine.

PATHWAYS AND REGULATION OF SULFUR METABOLISM REVEALED THROUGH MOLECULAR AND GENETIC STUDIES.

Sulfur is essential for life. Its oxidation state is in constant flux as it circulates through the global sulfur cycle. Plants play a key role in the cycle since they are primary producers of organic sulfur compounds. They are able to couple photosynthesis to the reduction of sulfate, assimilation into cysteine, and further metabolism into methionine, glutathione, and many other compounds. The activity of the sulfur assimilation pathway responds dynamically to changes in sulfur supply and to environmental conditions that alter the need for reduced sulfur. Molecular genetic analysis has allowed many of the enzymes and regulatory mechanisms involved in the process to be defined. This review focuses on recent advances in the field of plant sulfur metabolism. It also emphasizes areas about which little is known, including transport and recycling/degradation of sulfur compounds.