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.     

Enhanced tolerance of transgenic poplar plants overexpressing gamma-glutamylcysteine synthetase towards chloroacetanilide herbicides.

A wild-type poplar hybrid and two transgenic clones overexpressing a bacterial gamma-glutamylcysteine synthetase in the cytosol or in the chloroplasts were exposed to the chloroacetanilide herbicides acetochlor and metolachlor dispersed in the soil. The transformed poplars contained higher gamma-glutamylcysteine and glutathione (GSH) levels than wild-type plants and therefore it was supposed that they would have an elevated tolerance towards these herbicides, which are detoxified in GSH-dependent reactions. Phenotypically, the transgenic and wild-type plants did not differ. The growth and the biomass of all poplar lines were markedly reduced by the two chloroacetanilide herbicides. However, the decrease of shoot and root fresh weights caused by the herbicides was significantly smaller in the transgenic than in wild-type plants. In addition, the growth rate of poplars transformed in the cytosol was reduced to a significantly lesser extent than that of wild-type plants following herbicide treatments. The effects of the two herbicides were similar. Herbicide exposures markedly increased the levels of gamma-glutamylcysteine and GSH in leaves of each poplar line. The increase in the foliar amounts of these thiols was stronger in the transgenic lines than in the wild type, particularly in the upper leaves. Considerable GST activities were detected in leaves of all poplar plants. Exposure of poplars to chloroacetanilide herbicides resulted in a marked induction of GST activity in upper leaf positions but not in middle and lower leaves. The extent of enzyme induction did not differ significantly between transgenic and wild-type poplars. Although the results show that the transgenic poplar lines are good candidates for phytoremediation purposes, the further improvement of their detoxification capacity, preferably by transformation using genes encoding herbicide-specific GST isoenzymes, seems to be the most promising way to obtain plants suitable for practical application.     

Light-dependent modulation of foliar glutathione synthesis and associated amino acid metabolism in poplar overexpressing γ-glutamylcysteine synthetase

Glutathione (GSH), γ-glutamylcysteine (γ-EC) and major free amino acids were measured in darkened and illuminated leaves from untransformed poplars (Populus tremula × P. alba) and poplars expressing Escherichia coli genes for γ-glutamylcysteine synthetase (γ-ECS; EC 3.2.3.3) and glutathione reductase (GR; EC 1.6.4.2). In poplars overexpressing γ-ECS, foliar γ-EC contents and GSH contents were markedly enhanced compared to poplars lacking the bacterial gene for the enzyme. However, the quantitative relationship between the foliar pools of γ-EC and GSH in these transformants was markedly dependent on light. In the dark, GSH content was relatively low and γ-EC content high, the latter being higher than the foliar GSH contents of untransformed poplars in all conditions. Hence, this transformation appears to elevate γ-EC from the ranks of a trace metabolite to one of major quantitative importance. On illumination, however, γ-EC content decreased fourfold whereas GSH content doubled. Glutathione was also higher in the light in untransformed poplars and in those overexpressing GR. In these plants, γ-EC was negligible in the light but increased in the dark. Cysteine content was little affected by light in any of the poplar types. No light-dependent changes in the extractable activities of γ-ECS, glutathione synthetase (EC 3.2.3.2) or GR were observed. In contrast, both the activation state and the maximum extractable activity of nitrate reductase (EC 1.6.6.1) were increased by illumination. In all poplar types, glutamate and aspartate were the major amino acids. The most marked light-induced increases in individual amino acids were observed in the glutamine, asparagine, serine and glycine pools. Illumination of leaves from poplars overexpressing γ-ECS at elevated CO₂ or low O₂ largely abolished the inverse light-dependent changes in γ-EC and GSH. Low O₂ did not affect foliar contents of cysteine or glutamate but prevented the light-induced increase in the glycine pool. It is concluded that light-dependent glycine formation through the photorespiratory pathway is required to support maximal rates of GSH synthesis, particularly under conditions where the capacity for γ-EC synthesis is augmented. Received: 17 December 1996 / Accepted: 28 January 1997     

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.     

The role of glycine in determining the rate of glutathione synthesis in poplar. Possible implications for glutathione production during stress

The terminal step of glutathione (GSH) synthesis is the condensation of γ-glutamyl-cysteine (γ-EC) with glycine. Relatively little information exists concerning the importance of photorespiratory glycine in determining the rate of conversion of γ-EC to GSH. Consequently, the effect of exogenous glycine and of illumination on foliar contents of γ-EC and GSH was studied in excised leaves and leaf discs from untransformed poplar ( Populus tremula × P. alba ) and poplar overexpressing γ-glutamylcysteine synthetase (γ-ECS; EC 6.3.2.2). Poplars strongly overexpressing γ-ECS (ggs28) had enhanced levels of γ-EC and GSH compared to untransformed poplars. The relationship between γ-EC and GSH contents in ggs28 was light dependent. In illuminated leaves, GSH contents were up to 50-fold higher than γ-EC. On darkening, γ-EC accumulated markedly and GSH declined, so that the GSH:γ-EC ratio was close to 1. These dark-induced changes were prevented by supplying glycine through the petiole or by incubation of leaf discs on glycine. Dark accumulation of γ-EC in leaf discs from untransformed poplar was also prevented by supplying glycine. Supplying cysteine in the dark to discs from untransformed poplar and ggs28 increased γ-EC levels markedly but GSH levels only slightly. Subsequent illumination caused γ-EC to decrease and GSH to increase. Supplying glycine in concert with cysteine had similar effects to illumination. The data suggest that photorespiratory glycine is essential for GSH synthesis, especially under stress conditions, where increased amounts of GSH are required.     

Phytoremediation with transgenic trees

In the present paper actual trends in the use of transgenic trees for phytoremediation of contaminated soils are reviewed. In this context a current field trial in which transgenic poplars with enhanced GSH synthesis and hence elevated capacity for phytochelatin production are compared with wildtype plants for the removal of heavy metals at different levels of contamination and under different climatic conditions. The studies are carried out with grey poplar (Populus tremula x P. alba), wildtype plants and plants overexpressing the gene for gamma-glutamylcysteine synthetase (gshI) from E. coli in the cytosol. The expression of this gene in poplar leads to two- to four-fold enhanced GSH concentrations in the leaves. In greenhouse experiments under controlled conditions these transgenic poplars showed a high potential for uptake and detoxification of heavy metals and pesticides. This capacity is evaluated in field experiments. Further aims of the project are to elucidate (a) the stability of the transgene under field conditions and (b) the possibility of horizontal gene transfer to microorganisms in the rhizosphere. The results will help to assess the biosafety risk of the use of transgenic poplar for phytoremediation of soils.     

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.     

Over-expression of gsh1 in the cytosol affects the photosynthetic apparatus and improves the performance of transgenic poplars on heavy metal-contaminated soil

Recent studies of transgenic poplars over‐expressing the genes gsh1 and gsh2 encoding γ‐glutamylcysteine synthetase (γ‐ECS) and glutathione synthetase, respectively, provided detailed information on regulation of GSH synthesis, enzymes activities and mRNA expression. In this experiment, we studied quantitative parameters of leaves, assimilating tissues, cells and chloroplasts, mesophyll resistance for CO₂ diffusion, chlorophyll and carbohydrate content in wild‐type poplar and transgenic plants over‐expressing gsh1 in the cytosol after 3 years of growth in relatively clean (control) or heavy metal‐contaminated soil in the field. Over‐expression of gsh1 in the cytosol led to a twofold increase of intrafoliar GSH concentration and influenced the photosynthetic apparatus at different levels of organisation, i.e., leaves, photosynthetic cells and chloroplasts. At the control site, transgenic poplars had a twofold smaller total leaf area per plant and a 1.6‐fold leaf area per leaf compared to wild‐type controls. Annual aboveground biomass gain was reduced by 50% in the transgenic plants. The reduction of leaf area of the transformants was accompanied by a significant decline in total cell number per leaf, indicating suppression of cell division. Over‐expression of γ‐ECS in the cytosol also caused changes in mesophyll structure, i.e., a 20% decrease in cell and chloroplast number per leaf area, but also an enhanced volume share of chloroplasts and intercellular airspaces in the leaves. Transgenic and wild poplars did not exhibit differences in chlorophyll and carotenoid content of leaves, but transformants had 1.3‐fold fewer soluble carbohydrates. Cultivation on contaminated soil caused a reduction of palisade cell volume and chloroplast number, both per cell and leaf area, in wild‐type plants but not in transformants. Biomass accumulation of wild‐type poplars decreased in contaminated soil by more than 30‐fold, whereas transformants showed a twofold decrease compared to the control site. Thus, poplars over‐expressing γ‐ECS in the cytosol were more tolerant to heavy metal stress under field conditions than wild‐type plants according to the parameters analysed. Correlation analysis revealed strong dependence of cell number per leaf area unit, chloroplast parameters and mesophyll resistance with the GSH level in poplar leaves.     

Cadmium Tolerance and Accumulation in Indian Mustard Is Enhanced by Overexpressing γ-Glutamylcysteine Synthetase

To investigate rate-limiting factors for glutathione and phytochelatin (PC) production and the importance of these compounds for heavy metal tolerance, Indian mustard (Brassica juncea) was genetically engineered to overexpress the Escherichia coli gshI gene encoding γ-glutamylcysteine synthetase (γ-ECS), targeted to the plastids. The γ-ECS transgenic seedlings showed increased tolerance to Cd and had higher concentrations of PCs, γ-GluCys, glutathione, and total non-protein thiols compared with wild-type (WT) seedlings. When tested in a hydroponic system, γ-ECS mature plants accumulated more Cd than WT plants: shoot Cd concentrations were 40% to 90% higher. In spite of their higher tissue Cd concentration, the γ-ECS plants grew better in the presence of Cd than WT. We conclude that overexpression of γ-ECS increases biosynthesis of glutathione and PCs, which in turn enhances Cd tolerance and accumulation. Thus, overexpression of γ-ECS appears to be a promising strategy for the production of plants with superior heavy metal phytoremediation capacity.     

Manipulation of Glutathione and Amino Acid Biosynthesis in the Chloroplast

Poplars (Populus tremula × Populus alba) were transformed to overexpress Escherichia coli γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase in the chloroplast. Five independent lines of each transformant strongly expressed the introduced gene and possessed markedly enhanced activity of the gene product. Glutathione (GSH) contents were unaffected by high chloroplastic glutathione synthetase activity. Enhanced chloroplastic γ-ECS activity markedly increased γ-glutamylcysteine and GSH levels. These effects are similar to those previously observed in poplars overexpressing these enzymes in the cytosol. Similar to cytosolic γ-ECS overexpression, chloroplastic overexpression did not deplete foliar cysteine or methionine pools and did not lead to morphological changes. Light was required for maximal accumulation of GSH in poplars overexpressing γ-ECS in the chloroplast. High chloroplastic, but not cytosolic, γ-ECS activities were accompanied by increases in amino acids synthesized in the chloroplast. We conclude that (a) GSH synthesis can occur in the chloroplast and the cytosol and may be up-regulated in both compartments by increased γ-ECS activity, (b) interactions between GSH synthesis and the pathways supplying the necessary substrates are similar in both compartments, and (c) chloroplastic up-regulation of GSH synthesis is associated with an activating effect on the synthesis of specific amino acids formed in the chloroplast.     

Synthesis of Glutathione in Leaves of Transgenic Poplar Overexpressing [gamma]-Glutamylcysteine Synthetase

Internode stem fragments of the poplar hybrid Populus tremula x Populus alba were transformed with a bacterial gene (gshl) for [gamma]-glutamylcysteine synthetase ([gamma]-ECS) targeted to the cytosol. Lines overexpressing [gamma]-ECS were identified by northern analysis, and the transformant with the highest enzyme activity was used to investigate the control of glutathione synthesis. Whereas foliar [gamma]-ECS activity was below the limit of detection in untransformed plants, activities of up to 8.7 nmol mg-1 protein min-1 were found in the transformant, in which the foliar contents of [gamma]-glutamylcysteine ([gamma]-EC) and glutathione were increased approximately 10- and 3-fold, respectively, without affecting either the reduction state of the glutathione pool or the foliar cysteine content. A supply of exogenous cysteine to leaf discs increased the glutathione content from both transformed and untransformed poplars, and caused the [gamma]-EC content of the transformant discs to increase still further. The following conclusions are drawn: (a) the native [gamma]-ECS of untransformed poplars exists in quantities that are limiting for foliar glutathione synthesis; (b) foliar glutathione synthesis in untransformed poplars is limited by cysteine availability; (c) in the transformant interactions between glutathione synthesis and cysteine synthesis operate to sustain the increased formation of [gamma]-EC and glutathione; and (d) the foliar glutathione content of the transformant is restricted by cysteine availability and by the activity of glutathione synthetase.     

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.     

Chloroplast parameters differ in wild type and transgenic poplars overexpressing gsh1 in the cytosol

Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best‐characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild‐type poplar hybrid Populus tremula × P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding γ‐glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild‐type plants; soil contamination significantly decreased biomass accumulation in both wild‐type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two‐dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast surface/volume ratio, both at the control and the contaminated site. Chloroplast number per cell did not differ between wild and transgenic poplars at the control site. Soil contamination led to suppression of chloroplast replication in wild‐type plants. From these results, we assume that overexpressing the bacterial gsh1 gene in the cytosol interacts with processes in the chloroplast and that sequestration of heavy metal phytochelatin complexes into the vacuole may partially counteract this interaction in plants grown at heavy metal‐contaminated field sites. Further experiments are required to test these assumptions.     

Enhanced Heavy Metal Tolerance and Accumulation by Transgenic Sugar Beets Expressing Streptococcus thermophilus StGCS-GS in the Presence of Cd,Zn and Cu Alone or in Combination

Phytoremediation is a promising means of ameliorating heavy metal pollution through the use of transgenic plants as artificial hyperaccumulators. A novel Streptococcus thermophilus γ-glutamylcysteine synthetase-glutathione synthetase (StGCS-GS) that synthesizes glutathione (GSH) with limited feedback inhibition was overexpressed in sugar beet (Beta vulgaris L.), yielding three transgenic lines (s2, s4 and s5) with enhanced tolerance to different concentrations of cadmium, zinc and copper, as indicated by their increased biomass, root length and relative growth compared with wild-type plants. Transgenic sugar beets accumulated more Cd, Zn and Cu ions in shoots than wild-type, as well as higher GSH and phytochelatin (PC) levels under different heavy metal stresses. This enhanced heavy metal tolerance and increased accumulation were likely due to the increased expression of StGCS-GS and consequent overproduction of both GSH and PC. Furthermore, when multiple heavy metal ions were present at the same time, transgenic sugar beets overexpressing StGCS-GS resisted two or three of the metal combinations (50 μM Cd-Zn, Cd-Cu, Zn-Cu and Cd-Zn-Cu), with greater absorption in shoots. Additionally, there was no obvious competition between metals. Overall, the results demonstrate the explicit role of StGCS-GS in enhancing Cd, Zn and Cu tolerance and accumulation in transgenic sugar beet, which may represent a highly promising new tool for phytoremediation.     

Characterization of phytochelatin synthase from tomato

The enzyme that synthesizes Cd-binding phytochelatins (PCs), PC synthase, has been studied in tomato ( Lycopersicon esculentum ) cell cultures and plants. This enzyme transfers γ-GluCys from GSH or PC to either GSH or an existing polymer of (γ-GluCys)_nGly. PC synthase from tomato requires GSH or PCs as substrates but cannot utilise γ-GluCys or GSSG. PC synthase is activated both in vivo and in vitro by a variety of heavy metal ions, including Cd²⁺, Ag⁺, Cu²⁺, Au⁺, Zn²⁺, Fe²⁺, Hg²⁺ and Pb²⁺. In crude protein extracts from tomato cells the enzyme has an apparent K_m of 7.7 m M for GSH in the presence of 0.5 m M Cd²⁺, and exhibits maximum activity at pH 8.0 and 35°C. PC synthase is present in tomato cells grown in the absence of Cd. The level of enzyme activity is regulated during the cell culture cycle, with the highest activity occurring 3 days after subculture. Cadmium-resistant tomato cells growing in medium containing 6 m M CdCl₂ have a 65% increase in PC synthase activity compared to unselected cells. PC synthase is also present in roots and stems of tomato plants, but not in leaves or fruits. The distribution of the enzyme in tomato plants and regulation of PC synthase activity in tomato cells indicate that PC synthase, and PCs, may have additional functions in plant metabolism that are not directly related to the formation of Cd-PC complexes in response to cadmium.     

Cadmium tolerance and phytochelatin content of Arabidopsis seedlings over-expressing the phytochelatin synthase gene AtPCS1

Previous studies demonstrated that expression of the Arabidopsis phytochelatin (PC) biosynthetic gene AtPCS1 in Nicotiana tabacum plants increases the Cd tolerance in the presence of exogenous glutathione (GSH). In this paper, the Cd tolerance of Arabidopsis plants over-expressing AtPCS1 (AtPCSox lines) has been analysed and the differences between Arabidopsis and tobacco are shown. Based on the analysis of seedling fresh weight, primary root length, and alterations in root anatomy, evidence is provided that, at relatively low Cd concentrations, the Cd tolerance of AtPCSox lines is lower than the wild type, while AtPCS1 over-expressing tobacco is more tolerant to Cd than the wild type. At higher Cd concentrations, Arabidopsis AtPCSox seedlings are more tolerant to Cd than the wild type, while tobacco AtPCS1 seedlings are as sensitive as the wild type. Exogenous GSH, in contrast to what was observed in tobacco, did not increase the Cd tolerance of AtPCSox lines. The PC content in wild-type Arabidopsis at low Cd concentrations is more than three times higher than in tobacco and substantial differences were also found in the PC chain lengths. These data indicate that the differences in Cd tolerance and in its dependence on exogenous GSH between Arabidopsis and tobacco are due to species-specific differences in the endogenous content of PCs and GSH and may be in the relative abundance of PCs of different length.