Regulation of glutathione synthesis in leaves of transgenic poplar (Populus tremula X P. alba) overexpressing glutathione synthetase

The poplar hybrid Populus tremula X P. alba was transformed with the Escherichia coli gene for glutathione synthetase ( gsh II ) targetted to the cytosol. Leaves of five lines of transgenic plants exhibited glutathione synthetase activities 15- to 60-fold higher than those of wild-type plants. Total glutathione levels and GSH/GSSG ratios were similar in transgenic and wild-type plants. Precursor feeding experiments with cysteine and γ-glutamylcysteine suggest that glutathione synthesis in the cytoplasm is controlled by a multistep procedure that includes (i) the availability of cysteine, (ii) the availability of γ-glutamylcysteine, and (iii) regulation of the activities of both γ-glutamylcysteine synthetase and glutathione synthetase. However step (ii) may set an upper limit for the cellular glutathione content.     

Cloning of the GSH1 and GSH2 Genes Complementing the Defective Biosynthesis of Glutathione in the Methylotrophic Yeast Hansenula polymorpha

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh⁺ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the -glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh^– phenotype of the gsh2 mutant. The Gsh⁺ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24, having the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme -glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).     

Restricting glutathione biosynthesis to the cytosol is sufficient for normal plant development

Glutathione (GSH) homeostasis in plants is essential for cellular redox control and efficient responses to abiotic and biotic stress. Compartmentation of the GSH biosynthetic pathway is a unique feature of plants. The first enzyme, γ-glutamate cysteine ligase (GSH1), responsible for synthesis of γ-glutamylcysteine (γ-EC), is, in Arabidopsis, exclusively located in the plastids, whereas the second enzyme, glutathione synthetase (GSH2), is located in both plastids and cytosol. In Arabidopsis, gsh2 insertion mutants have a seedling lethal phenotype in contrast to the embryo lethal phenotype of gsh1 null mutants. This difference in phenotype may be due to partial replacement of GSH functions by γ-EC, which in gsh2 mutants hyperaccumulates to levels 5000-fold that in the wild type and 200-fold wild-type levels of GSH. In situ labelling of thiols with bimane and confocal imaging in combination with HPLC analysis showed high concentrations of γ-EC in the cytosol. Feedback inhibition of Brassica juncea plastidic GSH1 by γ-EC in vitro strongly suggests export of γ-EC as functional explanation for hyperaccumulation. Complementation of gsh2 mutants with the cytosol-specific GSH2 gave rise to phenotypically wild-type transgenic plants. These results support the conclusion that cytosolic synthesis of GSH is sufficient for plant growth. The transgenic lines further show that, consistent with the exclusive plastidic localization of GSH1, γ-EC is exported from the plastids to supply the cytosol with the immediate precursor for GSH biosynthesis, and that there can be efficient re-import of GSH into the plastids to allow effective control of GSH biosynthesis through feedback inhibition of GSH1.     

Increased Activity of [gamma]-Glutamylcysteine Synthetase in Tomato Cells Selected for Cadmium Tolerance

Two cell lines of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) were systematically compared for their capacity to tolerate cadmium. Unselected CdS cells died in the presence of 0.3 mM CdCl2. CdR6-0 cells, which were selected from CdS, survived and grew in medium supplemented with 0.3 mM CdCl2. Growth of CdR6-0 cells under this condition was accompanied by synthesis of cadmium-binding phytochelatins and maintenance of cellular glutathione (GSH) levels. CdR6-0 cells also exhibited increased tolerance to buthionine sulfoximine, in both the presence and absence of 0.1 mM CdCl2. The specific activity of [gamma]-glutamylcysteine synthetase (EC 6.3.2.2) was approximately 2-fold higher in CdR6-0 cells than in CdS cells, whereas there was no difference between cell lines in specific activity of GSH synthetase (EC 6.3.2.3). Increased activity of the first enzyme of GSH biosynthesis in CdR6-0 cells, presumably a result of selection for increased cadmium tolerance, provides an enhanced capacity to synthesize GSH and to maintain the production of phytochelatins in response to cadmium. This adaptation may contribute to the enhanced cadmium tolerance of CdR6-0 cells.     

Resistance to Cadmium Ions and Formation of a Cadmium-Binding Complex in Various Wild-Type Yeasts

The resistance to cadmium ions (Cd-resistance) and possibleformation of cadmium-binding complexes were examined in eightdifferent wild-type yeasts. Saccharomyces exiguus, Pichia farinosa,Torulaspora delbrueckii and Schizosaccharomyces octosporus exhibitedpartial Cd-resistance, as compared to the Cd-resistant strain301N and the Cu-resistant but Cd-sensitive strain X2180-1B ofSaccharomyces cerevisiae. Saccharomyces carlsbergensis, Pichiamogii, Zygosaccharomyces rouxii and Kluyveromyces lactis wereall Cd-sensitive. The partially Cd-sensitive species, with theexception of S. exiguus, accumulated Cd²⁺ ions in the cytoplasmicfraction to varying extents. This fraction from S. octosporusincluded a Cd-binding complex that contained (     

Cloning of the GSH1 and GSH2 genes complementing the defective biosynthesis of glutathione in the methylotrophic yeast Hansenula polymorpha

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh+ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the gamma-glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh- phenotype of the gsh2 mutant. The Gsh+ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24 with the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme gamma-glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).     

Recombinant industrial brewing yeast strains with ADH2 interruption using self-cloning GSH1+CUP1 cassette

A self-cloning module for gene knock-out and knock-in in industrial brewing yeast strain was constructed that contains copper resistance and γ-glutamylcysteine synthetase gene cassette, flanked by alcohol dehydrogenase II gene ( ADH2 ) of Saccharomyces cerevisiae . The module was used to obtain recombined strains RY1 and RY2 by targeting the ADH2 locus of host Y1. RY1 and RY2 were genetically stable. PCR and enzyme activity analysis of RY1 and RY2 cells showed that one copy of ADH2 was deleted by GSH1 + CUP1 insertion, and an additional copy of wild type was still present. The fermentation ability of the recombinants was not changed after genetic modification, and a high level of glutathione (GSH) was secreted, resulting from GSH1 overexpression, which codes for γ-glutamylcysteine synthetase. A pilot-scale brewing test for RY1 and RY2 indicated that acetaldehyde content in fermenting liquor decreased by 21–22%, GSH content increased by 20–22% compared with the host, the antioxidizability of the recombinants was improved, and the sensorial evaluation was also better than that of the host. No heterologous DNA was harbored in the recombinants; therefore, they could be applied in the beer industry in terms of their biosafety.     

Heavy Metal Tolerance and Accumulation in Indian Mustard (Brassica Juncea L.) Expressing Bacterial γ-Glutamylcysteine Synthetase or Glutathione Synthetase

The overexpression of either γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase (GS) in Brassica juncea transgenics was shown previously to result in higher accumulation of glutathione (GSH) and phytochelatins (PCs), as well as enhanced Cd tolerance and accumulation. The present study was aimed at analyzing the effects of γ-ECS or GS overexpression on tolerance to and accumulation of other metal/loids supplied individually in agar medium (seedlings) or in hydroponics (mature plants). Also, as pollution in nature generally consists of mixtures of metals, glutamylcysteine synthetase (ECS) and GS seedlings were tested on combinations of metals. Compared to wild-type plants, ECS and GS transgenics exhibited a significantly higher capacity to tolerate and accumulate a variety of metal/loids (particularly As, Cd, and Cr) as well as mixed-metal combinations (As, Cd, Zn/As, Pb, and Zn). This enhanced metal tolerance and accumulation of the ECS and GS transgenics may be attributable to enhanced production of PCs, sustained by a greater availability of GSH as substrate, as suggested by their higher concentrations of GSH, PC2, PC3, and PC4 as compared to wild-type plants. Overexpression of GS and γ-ECS may represent a promising strategy for the development of plants with an enhanced phytoremediation capacity for mixtures of metals.     

Modulation of Exogenous Glutathione in Phytochelatins and Photosynthetic Performance Against Cd Stress in the Two Rice Genotypes Differing in Cd Tolerance

Greenhouse hydroponic experiments were conducted using Cd-sensitive (Xiushui63) and tolerant (Bing97252) rice genotypes to evaluate genotypic differences in response of photosynthesis and phytochelatins to Cd toxicity in the presence of exogenous glutathione (GSH). Plant height, chlorophyll content, net photosynthetic rate (Pn), and biomass decreased in 5 and 50 μM Cd treatments, and Cd-sensitive genotype showed more severe reduction than the tolerant one. Cadmium stress caused decrease in maximal photochemical efficiency of PSII (Fv/Fm) and effective PSII quantum yield [Y(II)] and increase in quantum yield of regulated energy dissipation [Y(NPQ)], with changes in Cd-sensitive genotype being more evident. Cadmium-induced phytochelatins (PCs), GSH, and cysteine accumulation was observed in roots of both genotypes, with markedly higher level in PCs and GSH on day 5 in Bing97252 compared with that measured in Xiushui63. Exogenous GSH significantly alleviated growth inhibition in Xiushui63 under 5 μM Cd and in both genotypes in 50 μM Cd. External GSH significantly increased chlorophyll content, Pn, Fv/Fm, and Y(II) of plants exposed to Cd, but decreased Y(NPQ) and the coefficient of non-photochemical quenching (qN). GSH addition significantly increased root GSH content in plants under Cd exposure (except day 5 of 50 μM Cd) and induced up-regulation in PCs of 5 μM-Cd-treated Bing97252 throughout the 15-day and Xiushui63 of 5-day exposure. The results suggest that genotypic difference in the tolerance to Cd stress was positively linked to the capacity in elevation of GSH and PCs, and that alleviation of Cd toxicity by GSH is related to significant improvement in chlorophyll content, photosynthetic performance, and root GSH levels.     

Protection from Paraquat-Mediated Photo-Oxidative Stress by Glutathione in Poplar (Populus tremula×P. alba) Plants

Abstract: The sensitivity of hybrid poplar (Populus tremula × P. alba) to oxidative stress mediated by paraquat exposure was analysed with leaf discs from wild-type plants and plants expressing the bacterial cDNA of the enzymes of glutathione synthesis, namely gshI, encoding γ-glutamylcysteine synthetase (ECS), or gshII, encoding glutathione synthetase (GS), both in the cytosol. It was expected that leaf discs containing more than 2-fold elevated glutathione concentrations due to over-expression of ECS are less susceptible to paraquat exposure than wild-type plants and transformants over-expressing GS. However, neither over-expression of GS nor of ECS improved paraquat tolerance of the leaves. This result was surprising, because in wild-type plants reduced paraquat sensitivity of young compared with mature leaves coincided with ca. 30 % higher glutathione contents of the young leaves. Apparently, developmental changes in paraquat sensitivity of poplar leaves are controlled by factors different from glutathione contents. Feeding experiments with glutathione and its metabolic precursor γ-glutamylcysteine (EC) plus gly showed that glutathione can provide protection from paraquat-mediated photo-oxidative stress; but at least ca. 5-fold elevated glutathione levels are required for this effect in poplar leaves. Currently, such high glutathione levels have not been achieved by the application of plant molecular biology techniques. The significance of glutathione for the compensation of photo-oxidative stress is discussed.     

Enhanced tolerance to and accumulation of mercury, but not arsenic, in plants overexpressing two enzymes required for thiol peptide synthesis

Arsenic and mercury are among the most toxic elemental pollutants in the environment, endangering human health and ecological integrity. Both elements are found in highly thiol-reactive forms, arsenite and Hg(II), respectively, in plant tissues. Overexpression of Escherichia coli γ-glutamylcysteine synthetase (ECS) or glutathione synthetase (GS) in Arabidopsis thaliana plants provided significant increases in the thiol peptides glutathione (GSH) and γ-glutamylcysteine (γ-EC), and/or phytochelatins (PCs), and some resistance to arsenic and mercury, but no substantial increases in the levels of these elements in above-ground tissues. In contrast, the co-expression of ECS and GS in ECS × GS lines produced significant increases in tolerance to toxic levels of mercury. The ECS × GS co-expression line accumulated 35-fold more biomass and three-fold more mercury aboveground than the wild type (WT) when grown on Hg(II). No increases in arsenic accumulation were detected in the ECS × GS line. Increased resistance to and accumulation of mercury apparently resulted from enhanced root concentrations of PCs in ECS × GS co-expression lines not seen in the wild type or lines expressing ECS or GS alone. Correlations between the levels of arsenic and mercury resistance and accumulation and increases in the accumulation of the various thiol peptides in the ECS, GS and ECS × GS transgenic plant lines are discussed.     

Responses to cadmium in leaves of transformed poplars overexpressing Γ-glutamylcysteine synthetase

Poplars overexpressing a bacterial Γ -glutamylcysteine synthetase ( Γ -ECS) in the cytosol (lines ggs11 and ggs28) had a 30-fold increase in foliar Γ -ECS activity relative to untransformed controls. Foliar Γ -glutamylcysteine ( Γ -EC) was increased by 10-fold while foliar glutathione accumulation increased by up to 3.5-fold in the leaves of the transformants. Untransformed and transformed poplars were grown with different soil concentrations of cadmium (0–1100 μg g⁻¹ soil) for 2 weeks. Cadmium accumulated in the leaves of both transformed and untransformed poplars and growth was inhibited. Growth inhibition and foliar cadmium accumulation were greatest at the highest soil cadmium concentrations in all lines. Exposure to cadmium enhanced the foliar cysteine, Γ -EC and glutathione pools in all lines but less glutathione was present in the leaves of the untransformed controls than the transformants under all growth conditions. Cadmium-induced changes in the activities of malic enzyme, isocitrate dehydrogenase and guaiacol peroxidase were less pronounced in the leaves of the transformed poplars overexpressing Γ -ECS than in the untransformed controls. Glutamate dehydrogenase and glutathione reductase activities were unchanged by exposure to cadmium. We conclude that overexpression of Γ -ECS activity and foliar glutathione accumulation in transformed poplar allows greater tissue cadmium accumulation but has only a marginal effect on cadmium tolerance in poplar.     

Glutathione Deficiency Leads to Riboflavin Oversynthesis in the Yeast Pichia guilliermondii

The Pichia guilliermondii GSH1 and GSH2 genes encoding Saccharomyces cerevisiae homologues of glutathione (GSH) biosynthesis enzymes, γ-glutamylcysteine synthetase and glutathione synthetase, respectively, were cloned and deleted. Constructed P. guilliermondii Δgsh1 and Δgsh2 mutants were GSH auxotrophs, displayed significantly decreased cellular GSH+GSSG levels and sensitivity to tert-butyl hydroperoxide, hydrogen peroxide, and cadmium ions. In GSH-deficient synthetic medium, growths of Δgsh1 and Δgsh2 mutants were limited to 3–4 and 5–6 cell divisions, respectively. Under these conditions Δgsh1 and Δgsh2 mutants possessed 365 and 148 times elevated riboflavin production, 10.7 and 2.3 times increased cellular iron content, as well as 6.8 and 1.4 fold increased ferrireductase activity, respectively, compared to the wild-type strain. Glutathione addition to the growth medium completely restored the growth of both mutants and decreased riboflavin production, cellular iron content, and ferrireductase activity to the level of the parental strain. Cysteine also partially restored the growth of the Δgsh2 mutants, while methionine or dithiothreitol could not restore the growth neither of the Δgsh1, nor of the Δgsh2 mutants. Besides, it was shown that in GSH presence riboflavin production by both Δgsh1 and Δgsh2 mutants, similarly to that of the wild-type strain, depended on iron concentration in the growth medium. Furthermore, in GSH-deficient synthetic medium P. guilliermondii Δgsh2 mutant cells, despite iron overload, behaved like iron-deprived wild-type cells. Thus, in P. guilliermondii yeast, glutathione is required for proper regulation of both riboflavin and iron metabolism.     

λ-Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization

λ-Glutamylcysteine synthetase activity (EC 6.3.2.2) was analysed in Sephacryl S-200 eluents of extracts from cell suspension cultures ofNicotiana tabacum L. cv. Samsun by determination of λ-glutamylcysteine as its monobromobimane derivative. The enzyme has a relative molecular mass (M_r) of 60000 and exhibits maximal activity at pH 8 (50% at pH 7.0 and pH9.0) and an absolute requirement for Mg²⁺. With 0.2mM Cd²⁺ or Zn²⁺, enzyme activity was reduced by 35% and 19%, respectively. Treatment with 5 mM dithioerythritol led to a heavy loss of activity and to dissociation into subunits (M_r 34000). Buthionine sulfoximine andl-methionine-sulfoximine, known as potent inhibitors of λ-glutamylcysteine synthetase from mammalian cells, were found to be effective inhibitors of the plant enzyme too. The apparent K_m values forl-glutamate,l-cysteine, and α-aminobutyrate were, respectively, 10.4mM, 0.19 mM, and 6.36 mM. The enzyme was completely inhibited by glutathione (K_i=0.42 mM). The data indicate that the rate of glutathione synthesis in vivo may be influenced substantially by the concentration of cysteine and glutamate and may be further regulated by feedback inhibition of λ-glutamylcysteine synthetase by glutathione itself. λ-Glutamylcysteine synthetase is, like glutathione synthetase, localized in chloroplasts as well as in the cytoplasm. Chloroplasts fromPisum sativum L. isolated on a Percoll gradient contained about 72% of the λ-glutamylcysteine synthetase activity in leaf cells and 48% of the total glutathione synthetase activity. In chloroplasts ofSpinacia oleracea L. about 61% of the total λ-glutamylcysteine synthetase activity of the cells were found and 58% of the total glutathione synthetase activity. These results indicate that glutathione synthesis can take place in at least two compartments of the plant cell. Dedicated to Professor A. Prison on the occasion of his 80th birthday     

Isolation and initial characterization of glutathione-deficient mutants of Escherichia coli K 12

The thiol-oxidizing agent “diamide” (CH₃)₂NCON=NCON(CH₃)₂ was used to isolate mutants of Escherichia coli K 12 deficient in the biosynthesis of glutathione. A colony-colour technique has been developed for identification of colonies of these mutants. Four glutathione-deficient mutants were isolated. They show normal growth rates in minimal medium without GSH supplementation, indicating that glutathione is not involved in essential metabolic processes. In one mutant, glutathione synthetase was entirely inactive. Three mutants were deficient in γ-glutamylcysteine synthetase; in two of them, this resulted in a complete lack of GSH. These mutants were found to be more susceptible than their parent strains to a wide range of chemical agents, but did not show a greater sensitivity to X-rays. It must be concluded that the protective role of glutathione is only significant when a chemical challenge is present.     

Altered metabolism of glutathione in cells transformed by oncogenes which cause resistance to ionizing radiations

We measured glutathione (GSH) metabolism in normal NIH/3T3 fibroblasts, and in cells transformed by the oncogenes sis, erbB, src, ras, dbl, and raf.erbB,src,ras and raf, but not sis and dbltransformants, showed increased level of total and reduced GSH as compared with normal NIH/3T3 fibroblasts; oxidized GSH was elevated only in src- and ras-transformed cells. Increased total GSH content was associated with decreased activity of the synthetic enzyme γ-glutamylcysteine synthetase, and oxidized GSH level with increased activity of GSH reductase. These data suggest that GSH synthesis was selectively enhanced in cells transformed by specific oncogenes, with resulting down-regulation of its synthetic enzyme; alterations of GSH metabolism appeared to be peculiar of transformation by specific oncogenes, and not trivial epiphenomena of neoplastic transformation. Oncogenic transformants that presented elevated level of GSH were also those reported to be resistant to antineoplastic drugs and ionizing radiations, thus confirming a possible link between altered GSH metabolism and resistance to antineoplastic treatment.     

Evaluation of Transgenic Poplars Over-Expressing Enzymes of Glutathione Synthesis for Phytoremediation of Cadmium

Abstract: Recently, phytoremediation of soils polluted with heavy metals has received a lot of attention. Since glutathione (GSH) and its derivatives (e.g., phytochelatins) play a major role in plant defence against environmental pollutants, we tested the effects of over-expression of bacterial genes for GSH synthesis in poplar on cadmium accumulation. A pilot experiment with CdCl₂ in hydroponics revealed that poplars over-expressing γ-glutamylcysteine synthetase (γ-ECS) accumulated significantly more Cd in root tissue than wild type or glutathione synthetase over-expressing poplars. To test the partitioning of Cd in different organs, poplar lines over-expressing γ-ECS in the cytosol and in chloroplasts were treated with 0.2 mM CdCl₂ in hydroponics. Significant amounts of Cd were translocated to leaves, but significant differences in Cd accumulation were not observed between transgenic and wild type plants. To evaluate these lines for large-scale phytoremediation of cadmium, plants were treated with 2 mM Cd in soil. Over a four-week period, the poplar plants were able to accumulate up to 5.3 mg Cd. Most remarkably, in young leaves of both transgenic lines, Cd was accumulated to concentrations 2.5 - 3 times higher than in the wild type. The increased allocation of cadmium to the young leaves represents a potentional advantage for the phytoremediation process using the same plants over several vegetation periods. The use of transgenic poplar lines with enhanced glutathione production capacity seems to be of particular advantage in highly polluted soils.     

Phytochelatin synthesis in maize seedlings in response to excess zinc

Buthionine sulfoximine (BSO) specifically inhibits γ-glutamylcysteine synthetase and decreases a cellular level of glutathione (GSH) in maize seedling roots. Exogenous GSH restores Zn-phytochelatins synthesis in BSO-treated maize plants.