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First published online December 29, 2005; 10.1104/pp.105.073734 Plant Physiology 140:613-623 (2006) © 2006 American Society of Plant Biologists Transgenic Tobacco Overexpressing Glyoxalase Pathway Enzymes Grow and Set Viable Seeds in Zinc-Spiked Soils1Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India (S.L.S.-P., S.K.Y., M.K.R., S.K.S.); and Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India (A.P.)
We reported earlier that engineering of the glyoxalase pathway (a two-step reaction mediated through glyoxalase I and II enzymes) enhances salinity tolerance. Here we report the extended suitability of this engineering strategy for improved heavy-metal tolerance in transgenic tobacco (Nicotiana tabacum). The glyoxalase transgenics were able to grow, flower, and set normal viable seeds in the presence of 5 mM ZnCl2 without any yield penalty. The endogenous ion content measurements revealed roots to be the major sink for excess zinc accumulation, with negligible amounts in seeds in transgenic plants. Preliminary observations suggest that glyoxalase overexpression could confer tolerance to other heavy metals, such as cadmium or lead. Comparison of relative tolerance capacities of transgenic plants, overexpressing either glyoxalase I or II individually or together in double transgenics, evaluated in terms of various critical parameters such as survival, growth, and yield, reflected double transgenics to perform better than either of the single-gene transformants. Biochemical investigations indicated restricted methylglyoxal accumulation and less lipid peroxidation under high zinc conditions in transgenic plants. Studies employing the glutathione biosynthetic inhibitor, buthionine sulfoximine, suggested an increase in the level of phytochelatins and maintenance of glutathione homeostasis in transgenic plants during exposure to excess zinc as the possible mechanism behind this tolerance. Together, these findings presents a novel strategy to develop multiple stress tolerance via glyoxalase pathway engineering, thus implicating its potential use in engineering agriculturally important crop plants to grow on rapidly deteriorating lands with multiple unfavorable edaphic factors.
Environmental factors such as heat, cold, drought, salinity, and heavy metal result in a massive loss of crop yield all over the world. Although metals are required as structural and catalytic components of enzymatic proteins involved in various physiological processes, they can still be toxic to a plant if present at supraoptimal concentrations (Clemens, 2001  ). Because without intervention heavy metals stay in soil for centuries, efficient remediation strategies are needed. Large parts of agricultural soil are contaminated with zinc by natural and anthropogenic activities, including mining and industrial processes and agricultural practices such as the use of fertilizers containing various heavy metals (Ross, 1994). The pollution of soil by zinc has been a major environmental concern (Dudka et al., 1996; Zarcinas et al., 2004), and the capacity of certain plants to accumulate heavy metals has long been used for phytoremediation. Modulation in the ion transporters for influx or efflux of heavy-metal ions, or chelation and sequestration of excess metal ions by binding with metal-chelating molecules such as metallothioneins, ferritin, or phytochelatins (PCs), are some of the possible strategies for counteracting heavy-metal stress (Clemens, 2001; Clemens et al., 2002; Hall, 2002; Pilon-Smits and Pilon, 2002; Eapen and D'Souza, 2005). This would help to maintain the concentration of essential metal ions in different cellular compartments within the narrow physiological range and to minimize the damage caused by the entry of nonessential metal ions into the cytosol. Zinc plays a critical role in the defense system of cells against reactive oxygen species (ROS) and thus represents an excellent protective agent against the oxidation of several vital cell components, such as membrane lipids and proteins, chlorophyll, sulfhydryl (SH)-containing enzymes, and DNA (Cakmak, 2000).
Plants use a variety of methods to prevent heavy metals from affecting their growth. Ligands such as PCs and metallothioneins bind heavy metals within the cell, thereby reducing the damage these metals would otherwise cause. PCs, small metal-binding peptides derived from reduced glutathione (GSH), represent one of the main metal-chelation and detoxification mechanisms and play an essential role in heavy-metal detoxification in plants (Grill et al., 1985
Glyoxalases I and II, zinc-binding enzymes of the glyoxalase pathway, carry out catabolism of methylglyoxal (MG), which is a cytotoxic compound formed primarily as a by-product of carbohydrate and lipid metabolism. We have reported recently that salinity stress leads to accumulation of MG in plants (Yadav et al., 2005a
In this study, three independent lines of single-gene transformants (overexpressing either glyoxalase I or II alone) and double transgenics (overexpressing glyoxalase I and glyoxalase II together), which showed significant accumulation of glyoxalase proteins and were used previously for the analysis of salinity tolerance (Singla-Pareek et al., 2003 ), were employed. All the experiments described below were carried out on all three independent transgenic lines. Statistically similar results were obtained for each of the three different lines; however, data for only one of the lines of each type are presented (line NtBIS-11 for glyoxalase I, line 72 for glyoxalase II, and line K for the double transgenic).
For investigating whether the levels of glyoxalase I and II proteins are regulated by zinc, western-blot analysis of the total proteins isolated from the cotyledonary leaves of Brassica juncea seedlings (source of the glyoxalase I cDNA used in this study), as well as from shoots of rice (Oryza sativa; source of the glyoxalase II cDNA) exposed to different concentrations of ZnCl2, was carried out using anti-glyoxalase I and anti-glyoxalase II antibodies. Significant accumulation of both glyoxalase I and II proteins was noted in response to various concentrations of ZnCl2 (Fig. 1, A and B). To analyze whether the transgenic tobacco lines used in this study retain glyoxalase enzyme activity when exposed to zinc, glyoxalase I and II enzyme activities were measured in seedlings maintained under water or 5 mM ZnCl2 for 24 h. For both enzymes, a higher activity was noted in the transgenic plants grown in water, which showed further enhancement in their activity in response to ZnCl2, indicating the regulation of endogenous glyoxalase enzymes by Zn2+ (Fig. 1, C and D). These transgenic plants showed an overall 15% to 50% enhancement in glyoxalase I activity, whereas glyoxalase II activity increased significantly by 300% to 400%.
Glyoxalase Transgenic Plants Can Tolerate High Levels of Zinc
The potential of glyoxalase transgenic plants for relative tolerance toward ZnCl2 was assessed employing a rapid bioassay based on detached leaf senescence. Isolated leaf discs of wild-type, glyoxalase I and II, and double-transgenic plants were incubated in various concentrations of ZnCl2. A clear contrast in the tolerance limits of the single- and double-gene transformants was observed at a higher concentration of ZnCl2 ( Incubation of the leaf discs in 5, 10, 15, and 20 mM ZnCl2 solution for 5 d showed an early bleaching of wild-type leaf discs compared to those from transgenic plants (Fig. 2A). The double-transgenic plants exhibited significantly more enhanced zinc tolerance than any of the single-gene transformants (Fig. 2, A and B). The wild-type plants exhibited almost a total loss of chlorophyll within 5 d in the presence of zinc (90% loss as compared to its water controls), whereas the double transformants experienced only a 22% decline in chlorophyll at 20 mM ZnCl2 concentration (Fig. 2B). The ability of the transgenic plants to maintain chlorophyll under zinc stress was taken as an index for measuring stress-induced injury. These observations establish a positive relationship between the overexpression of glyoxalase pathway enzymes and zinc stress tolerance in leaf tissues.
Zinc tolerance of T1 generation transgenic seedlings was further checked by transferring them onto growth media supplemented with various concentrations of ZnCl2 (5, 10, and 20 mM) and compared with growth over a period of 25 d on normal media. All four types of seedlings (i.e. wild type, glyoxalases I and II, and double transgenics) showed similar growth in the absence of ZnCl2 (Fig. 2C, a). On media supplemented with 5 mM ZnCl2, the single-gene transgenics of glyoxalase I or II grew well, but again, the double-transgenic lines showed fewer symptoms of stress. However, at this concentration of zinc, severe chlorosis and stunted phenotype of the wild-type seedlings was observed (Fig. 2C, b). On 10 mM ZnCl2-supplemented media, glyoxalase I and II transgenic seedlings also showed a reduction in growth, whereas the double-transgenic seedlings showed minimal visual symptoms of stress-induced damage (Fig. 2C, c). Although there was a slight reduction in the overall growth of all the transgenic seedlings, growth of double transgenics remained least affected under high levels of ZnCl2 (Fig. 2C, d), thus indicating that overexpression of the entire glyoxalase pathway is better at facilitating enhanced zinc tolerance. Previously, transgenics overexpressing specific heavy-metal transporter proteins have been shown to tolerate zinc in the range of 100 µM to 4 mM (Van der Zaal et al., 1999 ; Blaudez et al., 2003; Lee et al., 2003; Verret et al., 2004). Engineering of the glyoxalase pathway shows that a previously unexplored metabolic pathway has been implicated to play a role in zinc tolerance.
To assess whether the enhanced expression of the glyoxalase enzymes would allow plants to grow, mature, and set seeds in the presence of high zinc, all four types of plants (wild type, glyoxalases I and II, and double transgenics) were grown in the continued presence of 5 mM ZnCl2-spiked soils (Fig. 3A, representative plants are shown). The growth of wild-type plants was severely affected under these conditions as evidenced by their stalled growth and ultimate death. On the other hand, the transgenic plants grew, flowered, and produced normal viable seeds. The growth and survival of double transgenics was much better as compared to the individual transgenic plants. It was found that glyoxalase transgenics that grew well in zinc-spiked soils sequestered the highest amount of zinc in roots (0.34% of dry weight in double transgenics), as compared to leaves, and the lowest amount in seeds (Fig. 3B). Previously, overexpression of a zinc transporter gene in Arabidopsis (Arabidopsis thaliana) led to accumulation of as much as 0.6% of dry weight zinc in roots (Van der Zaal et al., 1999
Further, it was of interest to evaluate how these transgenic plants perform when grown in the continued presence of zinc. Various growth parameters were scored for T1 generation transgenic plants grown in soils spiked with 5 mM ZnCl2 vis-à-vis wild-type plants grown in normal soil with water (Fig. 3C). It should be noted here that similar data for wild-type plants grown in the presence of zinc could not be obtained because these plants failed to sustain growth in the presence of 5 mM ZnCl2 after 40 d. The overall performance and total seed yield of the double-transgenic plants grown in the presence of 5 mM ZnCl2 were found to be comparable to that of the wild type grown in water, strongly indicating the ameliorating effect of glyoxalase transgenes on seed productivity and yield of transgenic plants. The double transgenics grown in the presence of high zinc were able to produce 95% of the total seeds when compared with wild-type plants grown in water, whereas the glyoxalase I and II transgenic lines yielded 84% and 86%, respectively (Fig. 3C). These data document that seed production during high levels of zinc metal exposure is not severely affected in single-gene transgenics (either glyoxalase I or II overexpressing tobacco) as they yielded relatively fewer seeds per pod and also showed reduced plant height under high zinc conditions as compared to the double-transgenic plants. We observed a similar response in a previous study, where, under 200 mM NaCl, the same lines of double transgenic, glyoxalase I, and glyoxalase II yielded 95%, 80%, and 83%, respectively, of the total seeds when compared with that of wild-type plants grown in water (Singla-Pareek et al., 2003 ).
MG is a cytotoxic compound and its level has been reported to increase during various stresses in animal, yeast (Saccharomyces cerevisiae), and bacterial systems (Cooper, 1984
Several biotic and abiotic factors ultimately impose oxidative stress onto the system. Induction of oxidative stress by zinc toxicity has been observed previously (Weckx and Clijster, 1997 ; Prasad et al., 1999; Rao and Sresty, 2000).To carry out similar investigations in our study, endogenous H2O2 concentration was measured. The H2O2 level was found to be similar in both wild-type and transgenic plants under nonstress conditions that increased by almost a similar degree upon exposure to 5 mM ZnCl2 (Fig. 4B), thereby documenting that plants experience oxidative stress when exposed to high levels of heavy metal. Further, to analyze the damage caused by enhanced levels of H2O2, lipid peroxidation in terms of production of malondialdehyde (MDA) was measured. MDA levels were found to increase drastically (by 300%) in wild-type plants when exposed to zinc, whereas its level in the transgenics increased by 68%, 60%, and 17% in glyoxalases I and II and double transgenics, respectively, indicating that wild-type plants undergo a higher degree of lipid peroxidation, leading to a loss of membrane integrity, than the transgenics (Fig. 4C).
During normal functioning of the electron transport chain in chloroplasts and mitochondria as well as in various enzyme-catalyzed redox reactions, ROS are produced (Dat et al., 2000
It has been well established that a complex antioxidant system, the ascorbate-GSH cycle (Zhang and Kirkham, 1996
After growth for 25 d on media containing 5 mM ZnCl2, control plants showed zinc toxicity symptoms, including major loss of chlorophyll and significant reduction in shoot growth. In contrast, under similar conditions, the double transgenics showed plant survival of 70% to 80% (see Fig. 2C). Because glyoxalases utilize GSH and recycle it back into the system, it became imperative to analyze whether GSH biosynthesis plays any role in glyoxalase-mediated zinc tolerance. For this, de novo synthesis of GSH was inhibited by transferring wild-type and transgenic seedlings to the medium supplemented with buthionine sulfoximine (BSO), an inhibitor of
Further, endogenous levels of GSH were measured in wild-type and transgenic seedlings, and a positive correlation could be established between the levels of GSH and zinc tolerance. The basal levels of GSH were higher by about 64% in double-transgenic plants under nonstress conditions, which further increased by 100% during 5 mM ZnCl2 treatment (Fig. 5B), whereas wild-type plants showed only a marginal increase in the GSH levels under 5 mM ZnCl2. In the presence of BSO alone (under nonstress conditions), there was a significant reduction in the level of GSH, while this decline was minimized in transgenic seedlings. In the presence of 5 mM ZnCl2 and BSO together, even the double transgenics were not able to accumulate very high levels of GSH, although they were still higher than the wild-type levels, indicating that de novo synthesis of GSH under zinc stress also contributes toward total GSH levels in the double-transgenic plants. Further evidence for the correlation between GSH levels and zinc tolerance was provided by the experiment where exogenous application of GSH to growth media could confer zinc tolerance in wild-type plants (data not shown), mimicking the effect of GSH biosynthesis and homeostasis. It has been reported previously that exposure to zinc initially resulted in a severe depletion of GSH (Rao and Sresty, 2000 ) caused by an increased consumption of GSH for PC production, whereas other studies have shown that heavy metals such as cadmium stimulated production of GSH (Maier et al., 2003; Sun et al., 2005b). Molecular studies have also shown that heavy metals stimulate expression of genes encoding enzymes involved in GSH synthesis (Schafer et al., 1998). Correlative studies have also shown an increased tolerance of plants toward cadmium with elevated levels of GSH, as well as decreased tolerance in plants with diminished levels of GSH (Zhu et al., 1999a, 1999b; Howden et al., 1995). Therefore, GSH synthesis appears to be crucial for the protection of plants from heavy metals, and it has been thought that GSH possibly could serve as a biomarker of metal toxicity in plants.
In plants, heavy metals induce the formation of acid-soluble nonprotein thiol-rich peptides made up of (
It was further found in this study that PC production is dependent on the presence of heavy metals as exposure to zinc induced about a 280% increase in its levels in double transgenics and about a 155% increase in either of the single-gene transformants (Fig. 5C). However, under similar conditions, this increase was only 32% in wild-type plants. The presence of BSO alone did not affect PC levels, whereas when zinc stress and BSO were applied together, the accumulation of PCs in wild-type plants was significantly reduced, thus leading to their poor survival. However, the double transgenics were able to accumulate some amount of PCs, which possibly imparted a certain degree of tolerance to these plants toward 5 mM ZnCl2. It has been reported that chelation of metal ions with ligands such as PCs or metallothioneins might route metals predominantly toward root sequestration (Evans et al., 1992
With a better understanding of the biochemical mechanisms for heavy-metal tolerance in glyoxalase transgenic plants, we speculate on the involvement of some of the potential target sites, which ultimately helps in tolerance. Because GSH is recycled by the glyoxalase system, it was assumed that an increased level of MG is detoxified efficiently in transgenic plants overexpressing glyoxalase enzymes constitutively, thus creating the possibility of up-regulation of GSH levels at least during stress. The role of glyoxalase overexpression in maintaining GSH homeostasis has already been shown to provide enhanced salinity tolerance (Kumar et al., 2003
We have recently shown the involvement of GSH homeostasis in glyoxalase I transgenics during abiotic stresses. Higher GSH levels are maintained in transgenics and hence survive under stress conditions, whereas wild-type plants cannot maintain GSH levels and die (Yadav et al., 2005c
For assessing the potential of glyoxalase transgenic plants for relative tolerance toward other heavy metals, leaf discs from wild-type and various transgenic tobacco plants were floated separately on a range of ZnCl2, CdCl2, or PbNO3 solutions for 5 d. Although various lower concentrations of these heavy metals were also used in this study, data for only relatively higher concentrations (20 mM) have been presented here, where a clear phenotypic difference in the tolerance capacities of the wild-type, single-, and double-transgenic plants was obtained. These experiments revealed that heavy metal-induced loss of chlorophyll was lower in glyoxalase-overexpressing lines compared to wild-type plants (Fig. 7A). The damage caused by heavy-metal stress was reflected visually in the degree of bleaching observed in the leaf discs after 5 d. The leaf discs from the wild type showed an early bleaching as compared to the transgenic plants. Measurement of the chlorophyll content of the leaf discs from different transgenic lines and wild type exposed to various heavy metals (Fig. 7B) provided further support for a positive relationship between overexpression of glyoxalase pathway enzymes and tolerance to heavy-metal stress. Wild-type plants exhibited almost a total loss of chlorophyll within 5 d in the presence of different heavy metals (85%–90% loss as compared to its water controls), whereas the double transformants experienced only a 24% to 38% decline in chlorophyll (Fig. 7B). Together, these observations indicated that glyoxalase transgenic plants could tolerate toxic levels of zinc, cadmium, and lead, thereby suggesting a wider applicability of the glyoxalase pathway for engineering tolerance toward a broader category of edaphic factors.
This study suggests a novel strategy for ameliorating heavy-metal, especially zinc, stress in plants. We have reported here the functional validation of enzymes of the glyoxalase pathway in enhanced accumulation and tolerance of toxic levels of zinc in the transgenic system. The glyoxalase transgenics were able to grow, flower, and set seeds in the presence of 5 mM ZnCl2 and sequestered excess zinc in roots. An increase in the level of PCs and maintenance of GSH homeostasis in transgenics during exposure to high Zn2+ levels seem to be the mechanisms behind this tolerance. The high accumulation of Zn2+ in the roots and the low accumulation of Zn2+ in the seeds of transgenic plants under high zinc conditions suggest the potential use of this engineering strategy in agriculture of crop plants on zinc-contaminated soil. The role of this pathway in ameliorating heavy-metal toxicity has not been shown previously. Together with our earlier observations on salinity tolerance, we suggest that glyoxalase pathway engineering can be an effective strategy for developing multiple stress tolerance in plants.
Generation of Transgenic Tobacco Plants
The single-gene transformants of tobacco (Nicotiana tabacum) carrying the glyoxalase I open reading frame (ORF) from Brassica juncea (Y13239) or the glyoxalase II ORF from rice (Oryza sativa; AY054407) and the double transgenics of tobacco carrying both glyoxalase I or II ORFs were raised in our previous study (Singla-Pareek et al., 2003
For western blotting and enzyme assays, 7-d-old seedlings of B. juncea and rice were exposed to 5 mM ZnCl2 for 24 h. Extraction of soluble proteins was essentially carried out as described previously (Singla-Pareek et al., 2003
To compare relative stress tolerance between wild-type and transgenic tobacco plants, a rapid bioassay based on detached leaf senescence was performed. For this, fully expanded leaves (60 d old) were briefly washed in deionized water. Leaf discs of 1 cm in diameter were punched out and floated on a 6-mL solution of ZnCl2 (5–20 mM, 5 d) or sterile distilled water (which served as the experimental control). For a separate experiment, leaf discs were floated on a 6-mL solution of either ZnCl2, CdCl2, or PbNO3 for 5 d (here data for only a 20 mM concentration of each have been presented that show clear visible phenotypic difference between tolerance of wild-type, single-gene transformants, and double transgenics). The chlorophyll content was measured as described previously (Singla-Pareek et al., 2003
To assess the relative zinc tolerance of various plants, wild-type and T1 generation transgenic seeds overexpressing glyoxalase I and glyoxalase II or both were germinated on one-half-strength (0.5x) Murashige-Skoog medium in the presence of appropriate antibiotics. The surviving seedlings (7 d old) were transferred to 0.5x Murashige and Skoog medium supplemented with 5, 10, or 20 mM ZnCl2 for imposing heavy-metal stress or onto plain 0.5x Murashige and Skoog medium that served as the experimental control. The seedlings were maintained under culture room conditions, and their growth was monitored for 25 d under stress. For analyzing the effect of the GSH biosynthesis inhibitor BSO, 7-d-old surviving seedlings were transferred to 0.5x Murashige and Skoog medium supplemented with either 5 mM ZnCl2 or 100 µM BSO or both (5 mM ZnCl2 + 100 µM BSO), and their growth was monitored for 25 d under culture room conditions. In addition to the experiments with seedlings, we carried out the assessment of the transgenic plants for their tolerance toward high levels of zinc throughout their life cycle. For this purpose, wild-type and T1 transgenic seeds were germinated on Murashige and Skoog medium containing appropriate antibiotics. The surviving seedlings were transferred to earthen pots and grown in a greenhouse (16 h light, 8 h dark, 25°C ± 2°C). Starting 2 weeks after transfer, plants were watered biweekly with a 5 mM ZnCl2 solution. Three wild-type and three independent transgenic lines of each type (i.e. glyoxalases I and II and double transformants) with three plants each were distributed in two groups, and each group was watered with either 5 mM ZnCl2 solution or water.
Mature wild-type and transgenic plants grown under water or in 5 mM ZnCl2 in a greenhouse for 150 d were used. Roots, old leaves, young leaves, and seeds were collected from three different plants of each type and thoroughly rinsed in deionized water and the fresh weight of each sample was determined. The samples were then processed for estimation of zinc (Zn2+) content using simultaneous inductively coupled argon-plasma emission spectrometry as described earlier (Singla-Pareek et al., 2003
MG was extracted from leaf tissue (0.3 g) by homogenizing in 3 mL of 0.5 M perchloric acid. One milliliter of total reaction mixture contained 250 µL 7.2 mM 1,2-diaminobenzene, 100 µL 5 M perchloric acid, and 650 µL of the sample, which was added last, and the absorbance of the derivative was read at 336 nm as suggested (Yadav et al., 2005a
TNP-SH compounds were extracted and assayed according to De Vos et al. (1992)
Total and oxidized glutathione (GSSG) analysis was carried out as described previously (Griffith, 1980
The level of PCs was estimated in terms of PC-SH levels calculated by subtracting the amount of GSH from that of TNP-SH compounds. The validity of this method has been investigated (De Knecht et al., 1992 Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers Y13239 and AY054407.
We thank Dr. B. Porter and Dr. F. White, Kansas State University, for the initial glyoxalase II clone, and Dr. V. Rajamani and Dr. J.K. Tripathi, Jawaharlal Nehru University, New Delhi, for helping with the work related to ionic content measurements. We also thank Dr. M.V. Rajam, University of Delhi South Campus, New Delhi, for a critical reading of the manuscript. Received November 2, 2005; returned for revision December 9, 2005; accepted December 9, 2005.
1 This work was supported by internal grants from the International Centre for Genetic Engineering and Biotechnology, New Delhi; the Department of Biotechnology (DBT) Network Project; the International Foundation for Science, Sweden (research grant to S.L.S.-P.); and a DBT postdoctoral fellowship (to S.K.Y.). 
2 Present address: Biotechnology Division, Institute of Himalayan Bioresource Technology, Palampur 176061 (HP), India. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Sneh L. Singla-Pareek (sneh{at}icgeb.res.in). Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.073734. * Corresponding author; e-mail sneh{at}icgeb.res.in; fax 91–011–26162316.
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ABSTRACT
RESULTS AND DISCUSSION
10 mM), at which, however, the leaf discs from wild-type plants could not survive. For the purpose of comparing the contribution of individual glyoxalase genes vis-à-vis the combined effect of overexpression of two genes toward this tolerance, it was important to use much higher concentrations of zinc that essentially do not reflect their tolerance capacities under field conditions. Therefore, for all the experiments presented in this study, relatively higher amounts of zinc have been used to impose heavy-metal stress than those reported in previous studies related to metal tolerance. 
-glutamylcysteine synthetase (Meister, 1988
