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.     

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.     

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.     

Synthesis of low molecular weight thiols in response to Cd exposure in Thlaspi caerulescens

In this study, we investigated the accumulation of phytochelatins (PCs) and other low molecular weight (LMW) thiols in response to Cd exposure in two contrasting ecotypes differing in Cd accumulation. Using a root elongation test, we found that the highly accumulating ecotype Ganges was more tolerant to Cd than the low Cd-accumulation ecotype Prayon. l -buthionine-(S,R)-sulphoximine (BSO), a potent inhibitor of the γ -glutamylcysteine synthetase ( γ -ECS) (an enzyme involved in the PC biosynthetic pathway), increased the Cd sensitivity of Prayon, but had no effect on Ganges. Although PC accumulation increased in response to Cd exposure, no significant differences were observed between the two ecotypes. Cd exposure induced a dose-dependent accumulation of both Cys and a still unidentified LMW thiol in roots of both ecotypes. Root accumulation of Cys and this thiol was higher in Ganges than in Prayon; the ecotypic differences were more pronounced when the plants were treated with BSO. These findings suggest that PCs do not contribute to the Cd hypertolerance displayed by the Ganges ecotype of Thlaspi caerulescens , whereas Cys and other LMW thiols might be involved.     

Overexpression of enzymes involved in glutathione synthesis enhances tolerance to organic pollutants in Brassica juncea

Transgenic Indian mustard (Brassica juncea) overexpressing y-glutamylcysteine synthetase (ECS) or glutathione synthetase (GS) were shown previously to have two-fold higher levels of glutathione and total nonprotein thiols, as well as enhanced cadmium tolerance and accumulation. Here, the hypothesis was tested that these transgenics have enhanced tolerance to organic pollutants, based on the reasoning that many organic xenobiotics are detoxified via conjugation to glutathione. Both the ECS and GS transgenics showed enhanced tolerance to atrazine: while root growth of wildtype seedlings was inhibited 50% by 100 mg L(-1) atrazine, ECS and GS root growth was inhibited 20-30% (P < 0.05). The tolerance of the transgenics to CDNB (1-chloro-2,4-dinitrobenzene). metolachlor, and phenanthrene was also somewhat higher than wild type, but these differences were not as pronounced. Each of the organics treatments significantly enhanced total nonprotein thiol levels in all plant types (2 to 12-fold). Overall, these results suggest that GSH biosynthesis is limiting for atrazine detoxification in Indian mustard and that overexpression of enzymes involved in GSH biosynthesis offers a promising approach to create plants with the enhanced capacity to tolerate not only heavy metals, but also certain organics.     

Enhanced arsenic tolerance of transgenic eastern cottonwood plants expressing gamma-glutamylcysteine synthetase

Arsenic is a metalloid that occurs naturally at parts per million (ppm) levels in the earth's crust. Natural and human activities have contributed to arsenic mobilization and increased concentration in the environment, such that World Health Organization guidelines for arsenic levels in drinking water are exceeded at many locations, worldwide. This translates into an increased risk of arsenic-related illnesses for millions of people. Recent studies demonstrate that increasing thiol-sinks in transgenic plants by overexpressing the bacterial gamma-glutamylcysteine synthetase (ECS) gene results in a higher tolerance and accumulation of metals and metalloids such as cadmium, mercury, and arsenic. We used Agrobacterium-mediated transformation to genetically engineer eastern cottonwood with a bacterial ECS gene. Eastern cottonwood plants expressing ECS had elevated thiol group levels, consistent with increased ECS activity. In addition, these ECS-expressing plants had enhanced growth on levels of arsenate toxic to control plants in vitro. Furthermore, roots of ECS-expressing plants accumulated significantly more arsenic than control roots (approximately twice as much), while shoots accumulated significantly less arsenic than control shoots (approximately two-thirds as much). We discuss potential mechanisms for shifting the balance of plant arsenic distribution from root accumulation to shoot accumulation, as it pertains to arsenic phytoremediation.     

The shoot-specific expression of gamma-glutamylcysteine synthetase directs the long-distance transport of thiol-peptides to roots conferring tolerance to mercury and arsenic

Thiol-peptides synthesized as intermediates in phytochelatin (PC) biosynthesis confer cellular tolerance to toxic elements like arsenic, mercury, and cadmium, but little is known about their long-distance transport between plant organs. A modified bacterial gamma-glutamylcysteine synthetase (ECS) gene, S1ptECS, was expressed in the shoots of the ECS-deficient, heavy-metal-sensitive cad2-1 mutant of Arabidopsis (Arabidopsis thaliana). S1ptECS directed strong ECS protein expression in the shoots, but no ECS was detected in the roots of transgenic plant lines. The S1ptECS gene restored full mercury tolerance and partial cadmium tolerance to the mutant and enhanced arsenate tolerance significantly beyond wild-type levels. After arsenic treatment, the root concentrations of gamma-glutamylcysteine (EC), PC2, and PC3 peptides in a S1ptECS-complemented cad2-1 line increased 6- to 100-fold over the mutant levels and were equivalent to wild-type concentrations. The shoot and root levels of glutathione were 2- to 5-fold above those in wild-type plants, with or without treatment with toxicants. Thus, EC and perhaps glutathione are efficiently transported from shoots to roots. The possibility that EC or other PC pathway intermediates may act as carriers for the long-distance phloem transport and subsequent redistribution of thiol-reactive toxins and nutrients in plants is discussed.     

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.     

Thiol synthetases of legumes: immunogold localization and differential gene regulation by phytohormones

In plants and other organisms, glutathione (GSH) biosynthesis is catalysed sequentially by γ-glutamylcysteine synthetase (γECS) and glutathione synthetase (GSHS). In legumes, homoglutathione (hGSH) can replace GSH and is synthesized by γECS and a specific homoglutathione synthetase (hGSHS). The subcellular localization of the enzymes was examined by electron microscopy in several legumes and gene expression was analysed in Lotus japonicus plants treated for 1-48 h with 50 μM of hormones. Immunogold localization studies revealed that γECS is confined to chloroplasts and plastids, whereas hGSHS is also in the cytosol. Addition of hormones caused differential expression of thiol synthetases in roots. After 24-48 h, abscisic and salicylic acids downregulated GSHS whereas jasmonic acid upregulated it. Cytokinins and polyamines activated GSHS but not γECS or hGSHS. Jasmonic acid elicited a coordinated response of the three genes and auxin induced both hGSHS expression and activity. Results show that the thiol biosynthetic pathway is compartmentalized in legumes. Moreover, the similar response profiles of the GSH and hGSH contents in roots of non-nodulated and nodulated plants to the various hormonal treatments indicate that thiol homeostasis is independent of the nitrogen source of the plants. The differential regulation of the three mRNA levels, hGSHS activity, and thiol contents by hormones indicates a fine control of thiol biosynthesis at multiple levels and strongly suggests that GSH and hGSH play distinct roles in plant development and stress responses.     

Lateral gene transfer and parallel evolution in the history of glutathione biosynthesis genes

Background  

Glutathione is found primarily in eukaryotes and in Gram-negative bacteria. It has been proposed that eukaryotes acquired the genes for glutathione biosynthesis from the alpha-proteobacterial progenitor of mitochondria. To evaluate this, we have used bioinformatics to analyze sequences of the biosynthetic enzymes γ-glutamylcysteine ligase and glutathione synthetase.     

Overexpression of Glutathione Synthetase in Indian Mustard Enhances Cadmium Accumulation and Tolerance

An important pathway by which plants detoxify heavy metals is through sequestration with heavy-metal-binding peptides called phytochelatins or their precursor, glutathione. To identify limiting factors for heavy-metal accumulation and tolerance, and to develop transgenic plants with an increased capacity to accumulate and/or tolerate heavy metals, the Escherichia coli gshII gene encoding glutathione synthetase (GS) was overexpressed in the cytosol of Indian mustard (Brassica juncea). The transgenic GS plants accumulated significantly more Cd than the wild type: shoot Cd concentrations were up to 25% higher and total Cd accumulation per shoot was up to 3-fold higher. Moreover, the GS plants showed enhanced tolerance to Cd at both the seedling and mature-plant stages. Cd accumulation and tolerance were correlated with the gshII expression level. Cd-treated GS plants had higher concentrations of glutathione, phytochelatin, thiol, S, and Ca than wild-type plants. We conclude that in the presence of Cd, the GS enzyme is rate limiting for the biosynthesis of glutathione and phytochelatins, and that overexpression of GS offers a promising strategy for the production of plants with superior heavy-metal phytoremediation capacity.     

Wounding of Arabidopsis leaves causes a powerful but transient protection against Botrytis infection

Physical injury inflicted on living tissue makes it vulnerable to invasion by pathogens. Wounding of Arabidopsis thaliana leaves, however, does not conform to this concept and leads to immunity to Botrytis cinerea , the causal agent of grey mould. In wounded leaves, hyphal growth was strongly inhibited compared to unwounded controls. Wound-induced resistance was not associated with salicylic acid-, jasmonic acid- or ethylene-dependent defence responses. The phytoalexin camalexin was found to be involved in this defence response as camalexin-deficient mutants were not protected after wounding and the B. cinerea strains used here were sensitive to this compound. Wounding alone did not lead to camalexin production but primed its accumulation after inoculation with B. cinerea , further supporting the role of camalexin in wound-induced resistance. In parallel with increased camalexin production, genes involved in the biosynthesis of camalexin were induced faster in wounded and infected plants in comparison with unwounded and infected plants. Glutathione was also found to be required for resistance, as mutants deficient in γ-glutamylcysteine synthetase showed susceptibility to B. cinerea after wounding, indicating that wild-type basal levels of glutathione are required for the wound-induced resistance. Furthermore, expression of the gene encoding glutathione- S -transferase 1 was primed by wounding in leaves inoculated with B. cinerea . In addition, the priming of MAP kinase activity was observed after inoculation of wounded leaves with B . cinerea compared to unwounded inoculated controls. Our results demonstrate how abiotic stress can induce immunity to virulent strains of B. cinerea , a process that involves camalexin and glutathione.     

Engineering and genetic approaches to modulating the glutathione network in plants

Reduced glutathione (GSH) is the most abundant low-molecular weight thiol in plant cells. It accumulates to high concentrations, particularly in stress situations. Because the pathway of GSH synthesis consists of only two enzymes, manipulation of cellular glutathione contents by genetic intervention has proved to be relatively straightforward. The discovery of a new bacterial bifunctional enzyme catalysing GSH synthesis but lacking feedback inhibition characteristics offers new prospects of enhancing GSH production and accumulation by plant cells, while the identification of γ-glutamyl cysteine and glutathione transporters provides additional possibilities for selective compartment-specific targeting. Such manipulations might also be used to affect plant biology in disparate ways, because GSH and glutathione disulphide (GSSG) have crucial roles in processes as diverse as the regulation of the cell cycle, systemic acquired resistance and xenobiotic detoxification. For example, depletion of the total glutathione pool can be used to manipulate the shoot : root ratio, because GSH is required specifically for the growth of the root meristem. Similarly, chloroplast γ-glutamyl cysteine synthetase overexpression could be used to increase the abundance of specific amino acids such as leucine, lysine and tyrosine that are synthesized in the chloroplasts. Here we review the aspects of glutathione biology related to synthesis, compartmentation and transport and related signalling functions that modulate plant growth and development and underpin any assessment of manipulation of GSH homeostasis from the viewpoint of nutritional genomics.     

The role of thiol species in the hypertolerance of Aspergillus sp. P37 to arsenic

Aspergillus sp. P37 is an arsenate-hypertolerant fungus isolated from a river in Spain with a long history of contamination with metals. This strain is able to grow in the presence of 0.2 M arsenate, i.e. 20-fold higher than the reference strain, Aspergillus nidulans TS1. Although Aspergillus sp. P37 reduces As(V) to As(III), which is slowly pumped out of the cell, the measured efflux of oxyanions is insufficient to explain the high tolerance levels of this strain. To gain an insight into this paradox, the accumulation of acid-soluble thiol species in Aspergillus sp. P37 when exposed to arsenic was compared with that of the arsenic-sensitive A. nidulans TS1 strain. Increasing levels of arsenic in the medium did not diminish the intracellular pool of reduced glutathione in Aspergillus sp. P37, in sharp contrast with the decline of glutathione in A. nidulans under the same conditions. Furthermore, concentrations of arsenic that were inhibitory for the sensitive A. nidulans strain (e.g. 50 mM and above) provoked a massive formation of vacuoles filled with thiol species. Because the major fraction of the cellular arsenic was present as the glutathione conjugate As(GS)3, it is plausible that the arsenic-hypertolerant phenotype of Aspergillus sp. P37 is in part due to an enhanced capacity to maintain a large intracellular glutathione pool under conditions of arsenic exposure and to sequester As(GS)3 in vacuoles. High pressure liquid chromatography analysis of cell extracts revealed that the contact of Aspergillus sp. P37 (but not A. nidulans) with high arsenic concentrations (> or =150 mM) induced the production of small quantities of a distinct thiol species indistinguishable from plant phytochelatin-2. Yet, we argue that phytochelatins do not explain arsenic resistance in Aspergillus, and we advocate the role of As(GS)3 complexes in arsenic detoxification.     

The glutathione-deficient mutant pad2-1 accumulates lower amounts of glucosinolates and is more susceptible to the insect herbivore Spodoptera littoralis

Plants often respond to pathogen or insect attack by inducing the synthesis of toxic compounds such as phytoalexins and glucosinolates (GS). The Arabidopsis mutant pad2-1 has reduced levels of the phytoalexin camalexin and is known for its increased susceptibility to fungal and bacterial pathogens. We found that pad2-1 is also more susceptible to the generalist insect Spodoptera littoralis but not to the specialist Pieris brassicae . The PAD2 gene encodes a γ-glutamylcysteine synthetase that is involved in glutathione (GSH) synthesis, and consequently the pad2-1 mutant contains about 20% of the GSH found in wild-type plants. Lower GSH levels of pad2-1 were correlated with reduced accumulation of the two major indole and aliphatic GSs of Arabidopsis, indolyl-3-methyl-GS and 4-methylsulfinylbutyl-GS, in response to insect feeding. This effect was specific to GSH, was not complemented by treatment of pad2-1 with the strong reducing agent dithiothreitol, and was not observed with the ascorbate-deficient mutant vtc1-1 . In contrast to the jasmonate-insensitive mutant coi1-1 , expression of insect-regulated and GS biosynthesis genes was not affected in pad2-1 . Our data suggest a crucial role for GSH in GS biosynthesis and insect resistance.     

Glutathione is an important antioxidant molecule in the yeast Saccharomyces cerevisiae

Abstract The tripeptide γ-l-glutamyl-l-cystinylglycine (glutathione) is one of the major antioxidant molecules of cells and is thought to play a vital role in buffering the cell against reactive oxygen species and toxic electrophiles. We wished to determine the role of glutathione in the protection of the yeast Saccharomyces cerevisiae against oxidative stress. This study shows that glutathione is an important antioxidant molecule in yeast, with γ-glutamylcysteine synthetase ( gshI ) mutants, deficient in glutathione synthesis, being hypersensitive to H₂O₂ and Superoxide anions in both exponential- and stationary-phase cultures. Despite this, these mutants are still able to induce adaptive stress responses to oxidants.     

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.     

Expression of bacterial GshF in Pichia pastoris for glutathione production

Conventionally, two consecutive enzymatic reactions catalyzed by γ-glutamylcysteine synthetase and glutathione synthetase are most commonly used for glutathione production. Here we demonstrate that bacterial bifunctional GshF can be used for glutathione production in a eukaryotic system without accumulation of the intermediate γ-glutamylcysteine.     

Subcellular distribution of glutathione precursors in Arabidopsis thaliana

Glutathione is an important antioxidant and has many important functions in plant development, growth and defense. Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors, cysteine, glutamate, glycine and γ-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis. The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense. The aim of this study was to investigate the compartment-specific importance of glutathione precursors in Arabidopsis thaliana. The subcellular distribution was compared between wild type plants (Col-0), plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant, wild type plants treated with buthionine sulfoximine), and one complemented line (OE3) with restored glutathione synthesis. Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine, glutamate and glycine in most cell compartments including plastids and the cytosol. A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments. These experiments demonstrated that the inhibition of γ-glutamylcysteine synthetase (GSH1) - the first enzyme of glutathione synthesis - causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells.