Mechanism of heavy metal ion activation of phytochelatin (PC) synthase: blocked thiols are sufficient for PC synthase-catalyzed transpeptidation of glutathione and related thiol peptides

The dependence of phytochelatin synthase (gamma-glutamylcysteine dipeptidyltranspeptidase (PCS), EC ) on heavy metals for activity has invariably been interpreted in terms of direct metal binding to the enzyme. Here we show, through analyses of immunopurified, recombinant PCS1 from Arabidopsis thaliana (AtPCS1), that free metal ions are not essential for catalysis. Although AtPCS1 appears to be primarily activated posttranslationally in the intact plant and purified AtPCS1 is able to bind heavy metals directly, metal binding per se is not responsible for catalytic activation. As exemplified by Cd(2+)- and Zn(2+)-dependent AtPCS1-mediated catalysis, the kinetics of PC synthesis approximate a substituted enzyme mechanism in which micromolar heavy metal glutathione thiolate (e.g. Cd.GS(2) or Zn.GS(2)) and free glutathione act as gamma-Glu-Cys acceptor and donor. Further, as demonstrated by the facility of AtPCS1 for the net synthesis of S-alkyl-PCs from S-alkylglutathiones with biphasic kinetics, consistent with the sufficiency of S-alkylglutathiones as both gamma-Glu-Cys donors and acceptors in media devoid of metals, even heavy metal thiolates are dispensable. It is concluded that the dependence of AtPCS1 on the provision of heavy metal ions for activity in media containing glutathione and other thiol peptides is a reflection of this enzyme's requirement for glutathione-like peptides containing blocked thiol groups for activity.     

Isolation and characterization of <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> and <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis> phytochelatin synthases

The synthesis of phytochelatins (PC) represents a major metal and metalloid detoxification mechanism in various species. PC most likely play a role in the distribution and accumulation of Cd and possibly other metals. However, to date, no studies have investigated the phytochelatin synthase (PCS) genes and their expression in the Cd-hyperaccumulating species. We used functional screens in two yeast species to identify genes expressed by two Cd hyperaccumulators (Arabidopsis halleri and Thlaspi caerulescens) and involved in cellular Cd tolerance. As a result of these screens, PCS genes were identified for both species. PCS1 was in each case the dominating cDNA isolated. The deduced sequences of AhPCS1 and TcPCS1 are very similar to AtPCS1 and their identity is particularly high in the proposed catalytic N-terminal domain. We also identified in A. halleri and T. caerulescens orthologues of AtPCS2 that encode functional PCS. As compared to A. halleri and A. thaliana, T. caerulescens showed the lowest PCS expression. Furthermore, concentrations of PC in Cd-treated roots were the highest in A. thaliana, intermediate in A. halleri and the lowest in T. caerulescens. This mirrors the known capacity of these species to translocate Cd to the shoot, with T. caerulescens being the best translocator. Very low or undetectable concentrations of PC were measured in A. halleri and T. caerulescens shoots, contrary to A. thaliana. These results suggest that extremely efficient alternative Cd sequestration pathways in leaves of Cd hyperaccumulators prevent activation of PC synthase by Cd²⁺ ions.     

The isochorismate pathway is negatively regulated by salicylic acid signaling in O<Subscript>3</Subscript>-exposed <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phytochelatins (PCs) are heavy metal binding peptides that play an important role in sequestration and detoxification of heavy metals in plants. In this study, our goal was to develop transgenic plants with increased tolerance for and accumulation of heavy metals from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A 35S promoter fused to a FLAG–tagged AtPCS1 cDNA was expressed in Indian mustard, and transgenic lines, designated pc lines, were evaluated for tolerance to and accumulation of Cd and Zn. Transgenic plants with moderate AtPCS1 expression levels showed significantly higher tolerance to Cd and Zn stress, but accumulated significantly less Cd and Zn than wild type plants in both shoot and root tissues. However, transgenic plants with highest expression of the transgene did not exhibit enhanced Cd and Zn tolerance. Shoots of Cd-treated pc plants had significantly higher levels of phytochelatins and thiols than wild-type plants. Significantly lower concentrations of gluthatione in Cd-treated shoot and root tissues of transgenic plants were observed. Moderate expression levels of phytochelatin synthase improved the ability of Indian mustard to tolerate certain levels of heavy metals, but at the same time did not increase the accumulation potential for Cd and Zn.     

Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) plants expressing an <Emphasis Type="Italic">Arabidopsis</Emphasis> phytochelatin synthase (<Emphasis Type="Italic">AtPCS1</Emphasis>) exhibit enhanced As and Cd tolerance

Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants.     

Expression of Caenorhabditis elegans PCS in the AtPCS1‐deficient Arabidopsis thaliana cad1‐3 mutant separates the metal tolerance and non‐host resistance functions of phytochelatin synthases

Phytochelatin synthases (PCS) play key roles in plant metal tolerance. They synthesize small metal‐binding peptides, phytochelatins, under conditions of metal excess. Respective mutants are strongly cadmium and arsenic hypersensitive. However, their ubiquitous presence and constitutive expression had long suggested a more general function of PCS besides metal detoxification. Indeed, phytochelatin synthase1 from Arabidopsis thaliana (AtPCS1) was later implicated in non‐host resistance. The two different physiological functions may be attributable to the two distinct catalytic activities demonstrated for AtPCS1, that is the dipeptidyl transfer onto an acceptor molecule in phytochelatin synthesis, and the proteolytic deglycylation of glutathione conjugates. In order to test this hypothesis and to possibly separate the two biological roles, we expressed a phylogenetically distant PCS from Caenorhabditis elegans in an AtPCS1 mutant. We confirmed the involvement of AtPCS1 in non‐host resistance by showing that plants lacking the functional gene develop a strong cell death phenotype when inoculated with the potato pathogen Phytophthora infestans. Furthermore, we found that the C. elegans gene rescues phytochelatin synthesis and cadmium tolerance, but not the defect in non‐host resistance. This strongly suggests that the second enzymatic function of AtPCS1, which remains to be defined in detail, is underlying the plant immunity function.     

Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd2+ tolerance and accumulation but not translocation to the shoot

Phytochelatins (PCs) are metal binding peptides involved in heavy metal detoxification. To assess whether enhanced phytochelatin synthesis would increase heavy metal tolerance and accumulation in plants, we overexpressed the Arabidopsis phytochelatin synthase gene (AtPCS1) in the non-accumulator plant Nicotiana tabacum. Wild-type plants and plants harbouring the Agrobacterium rhizogenes rolB oncogene were transformed with a 35S AtPCS1 construct. Root cultures from rolB plants could be easily established and we demonstrated here that they represent a reliable system to study heavy metal tolerance. Cd²⁺ tolerance in cultured rolB roots was increased as a result of overexpression of AtPCS1, and further enhanced when reduced glutathione (GSH, the substrate of PCS1) was added to the culture medium. Accordingly, HPLC analysis showed that total PC production in PCS1-overexpressing rolB roots was higher than in rolB roots in the presence of GSH. Overexpression of AtPCS1 in whole seedlings led to a twofold increase in Cd²⁺ accumulation in the roots and shoots of both rolB and wild-type seedlings. Similarly, a significant increase in Cd²⁺ accumulation linked to a higher production of PCs in both roots and shoots was observed in adult plants. However, the percentage of Cd²⁺ translocated to the shoots of seedlings and adult overexpressing plants was unaffected. We conclude that the increase in Cd²⁺ tolerance and accumulation of PCS1 overexpressing plants is directly related to the availability of GSH, while overexpression of phytochelatin synthase does not enhance long distance root-to-shoot Cd²⁺ transport.     

Overexpression of phytochelatin synthase in tobacco: distinctive effects of AtPCS1 and CePCS genes on plant response to cadmium

Phytochelatins, heavy-metal-binding polypeptides, are synthesized by phytochelatin synthase (PCS) (EC 2.3.2.15). Previous studies on plants overexpressing PCS genes yielded contrasting phenotypes, ranging from enhanced cadmium tolerance and accumulation to cadmium hypersensitivity. This paper compares the effects of overexpression of AtPCS1 and CePCS in tobacco (Nicotiana tabacum var. Xanthi), and demonstrates how the introduction of single homologous genes affects to a different extent cellular metabolic pathways leading to the opposite of the desired effect. In contrast to WT and CePCS transformants, plants overexpressing AtPCS1 were Cd-hypersensitive although there was no substantial difference in cadmium accumulation between studied lines. Plants exposed to cadmium (5 and 25 muM CdCl2) differed, however, in the concentration of non-protein thiols (NPT). In addition, PCS activity in AtPCS1 transformants was around 5-fold higher than in CePCS and WT plants. AtPCS1 expressing plants displayed a dramatic accumulation of gamma-glutamylcysteine and concomitant strong depletion of glutathione. By contrast, in CePCS transformants, a smaller reduction of the level of glutathione was noticed, and a less pronounced change in gamma-glutamylcysteine concentration. There was only a moderate and temporary increase in phytochelatin levels due to AtPCS1 and CePCS expression. Marked changes in NPT composition due to AtPCS1 expression led to moderately decreased Cd-detoxification capacity reflected by lower SH:Cd ratios, and to higher oxidative stress (assessed by DAB staining), which possibly explains the increase in Cd-sensitivity. The results indicate that contrasting responses to cadmium of plants overexpressing PCS genes might result from species-dependent differences in the activity of phytochelatin synthase produced by the transgenes.     

Nonspecific binding of monoclonal anti-FLAG M2 antibody in Indian mustard (Brassica juncea)

Chimeric constructs with the hydrophilic octapeptide FLAG epitope (DYKDDDDK) have been widely used as multipurpose tags for identification, detection, and purification of FLAG fusion proteins. Constructs consisting of C-terminal FLAG-tagged genomic and cDNA clones of anArabidopsis phytochelatin synthase gene,AtPCS1, were used in developing transgenic lines of Indian mustard. Presence and expression ofAtPCS1 in transgenic lines were confirmed by using PCR and Northern blot analyses. However, immunoblot analysis revealed strong nonspecific binding of a monoclonal anti-FLAG M2 antibody to an endogenous protein in both shoot and leaf tissues of wild-type Indian mustard (85-kDa) that masked presence of the phytochelatin synthase (PCS) protein of interest (55-kDa). Further analysis revealed absence of a nonspecific protein in root tissues of transgenic plants, thus allowing detection of the FLAG-tagged PCS protein.     

Chloroplast targeting of phytochelatin synthase in Arabidopsis: effects on heavy metal tolerance and accumulation

The enzymatically synthesized thiol peptide phytochelatin (PC) plays a central role in heavy metal tolerance and detoxification in plants. In response to heavy metal exposure, the constitutively expressed phytochelatin synthase enzyme (PCS) is activated leading to synthesis of PCs in the cytosol. Recent attempts to increase plant metal accumulation and tolerance reported that PCS over-expression in transgenic plants paradoxically induced cadmium hypersensitivity. In the present paper, we investigate the possibility of synthesizing PCs in plastids by over-expressing a plastid targeted phytochelatin synthase (PCS). Plastids represent a relatively important cellular volume and offer the advantage of containing glutathione, the precursor of PC synthesis. Using a constitutive CaMV 35S promoter and a RbcS transit peptide, we successfully addressed AtPCS1 to chloroplasts, significant PCS activity being measured in this compartment in two independent transgenic lines. A substantial increase in the PC content and a decrease in the glutathione pool were observed in response to cadmium exposure, when compared to wild-type plants. While over-expressing AtPCS1 in the cytosol importantly decreased cadmium tolerance, both cadmium tolerance and accumulation of plants expressing plastidial AtPCS1 were not significantly affected compared to wild-type. Interestingly, targeting AtPCS1 to chloroplasts induced a marked sensitivity to arsenic while plants over-expressing AtPCS1 in the cytoplasm were more tolerant to this metalloid. These results are discussed in relation to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.     

Development of surface‐engineered yeast cells displaying phytochelatin synthase and their application to cadmium biosensors by the combined use of pyrene‐excimer fluorescence

The development of simple, portable, inexpensive, and rapid analytical methods for detecting and monitoring toxic heavy metals are important for the safety and security of humans and their environment. Herein, we describe the application of phytochelatin (PC) synthase, which plays a critical role in heavy metal responses in higher plants and green algae, in a novel fluorescent sensing platform for cadmium (Cd). We first created surface‐engineered yeast cells on which the PC synthase from Arabidopsis (AtPCS1) was displayed with retention of enzymatic activity. The general concept for the sensor is based on the Cd level‐dependent synthesis of PC₂ from glutathiones by AtPCS1‐displaying yeast cells, followed by simple discriminative detection of PC₂ via sensing of excimer fluorescence of thiol‐labeling pyrene probes. The intensity of excimer fluorescence increased in the presence of Cd up to 1.0 μM in an approximately dose‐dependent manner. This novel biosensor achieved a detection limit of as low as 0.2 μM (22.5 μg/L) for Cd. Although its use may be limited by the fact that Cu and Pb can induce cross‐reaction, the proposed simple biosensor holds promise as a method useful for cost‐effective screening of Cd contamination in environmental and food samples. The AtPCS1‐displaying yeast cells also might be attractive tools for dissection of the catalytic mechanisms of PCS. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1197–1202, 2013     

Leaf-targeted phytochelatin synthase in Arabidopsis thaliana

One of the key steps in developing transgenic plants for the phytoremediation of metal containing soils is to develop plants that accumulate metals in the aerial tissues. With the goal of changing the distribution of phytochelatin (PC)-dependent cadmium accumulation from roots to the leaves, the phytochelatin synthase (PCS) deficient cad1-3 mutant and wild type (Col-0) Arabidopsis plants were transformed with an Arabidopsis phytochelatin synthase (AtPCS1) under the control of a leaf-specific promoter. Three independent transformant lines from each genetic background were chosen for further analysis and designated cad-PCS and WT-PCS. PCS activity in the cadPCS lines was restored in the leaves, but not in the roots. Additionally, when whole plants were treated with cadmium, PCs were found only in the leaves of cad-PCS plants. Although the inserted AtPCS1 gene was leaf-specific, cad-PCS lines showed an overall decrease in cadmium toxicity evidenced by a partial amelioration of the "brown-root" phenotype and root growth was restored to wild type levels when treated with cadmium and arsenate. WT-PCS lines showed an increase in leaf PCS activity but had only wild type PC levels. In addition, cadmium uptake studies indicated that there was no difference in cadmium accumulation among all types tested. So, while we were able to protect the plants against cadmium by expressing PC synthase only in the leaves, we were not able to limit cadmium accumulation to aerial tissues.     

Phytochelatin is not a primary factor in determining copper tolerance

Phytochelatin (PC) is involved in the detoxification of harmful, non-essential heavy metals and the homeostasis of essential heavy metals in plants. Its synthesis can be induced by either cadmium (Cd) or copper (Cu), and can form stable complexes with either element. This might suggest that PC has an important role in determining plant tolerance to both. However, this is not clearly apparent, as evidenced by a PC-deficient and Cd-sensitiveArabidopsis mutant (cad1-3) that shows no significant increase in its sensitivity to copper. Therefore, we investigated whether the mechanism for Cu tolerance differed from that for Cd by analyzing copper sensitivity in Cd-tolerant transgenics and Cd-sensitive mutants ofArabidopsis. Cadmium-tolerant transgenic plants that over-expressedA. thaliana phytochelatin synthase 1 (AtPCS1) were not tolerant of copper stress, thereby supporting the hypothesis that PC is not primarily involved in this tolerance mechanism. We also investigated Cu tolerance incad2-1, a Cd-sensitive and glutathione (GSH)-deficientArabidopsis mutant. Paradoxically,cad2-1 was more resistant to copper stress than were wild-type plants. This was likely due to the high level of cysteine present in that mutant. However, when the growth medium was supplemented with cysteine, the wild types also exhibited copper tolerance. Moreover,Saccharomyces cerevisiae that expressedAtPCS1 showed tolerance to Cd but hypersensitivity to Cu. All these results indicate that PC is not a major factor in determining copper tolerance in plants.     

A reassessment of substrate specificity and activation of phytochelatin synthases from model plants by physiologically relevant metals

Phytochelatin synthases (PCS) catalyze phytochelatin (PC) synthesis from glutathione (GSH) in the presence of certain metals. The resulting PC-metal complexes are transported into the vacuole, avoiding toxic effects on metabolism. Legumes have the unique capacity to partially or completely replace GSH by homoglutathione (hGSH) and PCs by homophytochelatins (hPCs). However, the synthesis of hPCs has received little attention. A search for PCS genes in the model legume Lotus (Lotus japonicus) resulted in the isolation of a cDNA clone encoding a protein (LjPCS1) highly homologous to a previously reported homophytochelatin synthase (hPCS) of Glycine max (GmhPCS1). Recombinant LjPCS1 and Arabidopsis (Arabidopsis thaliana) PCS1 (AtPCS1) were affinity purified and their polyhistidine-tags removed. AtPCS1 catalyzed hPC synthesis from hGSH alone at even higher rates than did LjPCS1, indicating that GmhPCS1 is not a genuine hPCS and that a low ratio of hPC to PC synthesis is an inherent feature of PCS1 enzymes. For both enzymes, hGSH is a good acceptor, but a poor donor, of gamma-glutamylcysteine units. Purified AtPCS1 and LjPCS1 were activated (in decreasing order) by Cd2+, Zn2+, Cu2+, and Fe3+, but not by Co2+ or Ni2+, in the presence of 5 mm GSH and 50 microm metal ions. Activation of both enzymes by Fe3+ was proven by the complete inhibition of PC synthesis by the iron-specific chelator desferrioxamine. Plants of Arabidopsis and Lotus accumulated (h)PCs only in response to a large excess of Cu2+ and Zn2+, but to a much lower extent than did with Cd2+, indicating that (h)PC synthesis does not significantly contribute in vivo to copper, zinc, and iron detoxification.     

Expression of Arabidopsis phytochelatin synthase 2 is too low to complement an AtPCS1-defective Cad1-3 mutant

Phytochelatins play an important role in heavy metal detoxification in plants as well as in other organisms. The Arabidopsis thaliana mutant cad1-3 does not produce detectable levels of phytochelatins in response to cadmium stress. The hypersensitivity of cad1-3 to cadmium stress is attributed to a mutation in the phytochelatin synthase 1 (AtPCS1) gene. However, A. thaliana also contains a functional phytochelatin synthase 2 (AtPCS2). In this study, we investigated why the cad1-3 mutant is hypersensitive to cadmium stress despite the presence of AtPCS2. Northern and Western blot analyses showed that expression of AtPCS2 is weak compared to AtPCS1 in both roots and shoots of transgenic Arabidopsis. The lower level of AtPCS2 expression was confirmed by RT-PCR analysis of wild type Arabidopsis. Moreover, no tissue-specific expression of AtPCS2 was observed. Even when AtPCS2 was under the control of the AtPCS1 promoter or of the cauliflower mosaic virus 35S promoter (CaMV 35S) it was not capable of fully complementing the cad1-3 mutant for cadmium resistance.     

Regulation of Glutathione Synthesis by Cadmium in Pisum sativum L

In roots and shoots of pea plants (Pisum sativum L.) cultivated with CdCl₂ concentrations up to 50 micromolar, growth, the content of total acid soluble thiols, and the activity of glutathione synthetase (EC 6.3.2.3) and of adenosine 5′-phosphosulfate sulfotransferase were measured. In addition, the occurrence of Cd-binding peptides (phytochelatins) and the contents of glutathione and cysteine were determined in roots of plants exposed to 20 micromolar Cd and/or 1 millimolar buthionine sulfoximine, an inhibitor of glutathione synthesis. An appreciable increase in activity of glutathione synthetase at 20 and 50 micromolar Cd and of adenosine 5′-phosphosulfate sulfotransferase at 5 micromolar and higher Cd concentrations was detected in the roots. Most of the additional thiols formed due to Cd treatment were eluted from a gel filtration HPLC column together with Cd, indicating the presence of phytochelatins. In plants treated with buthionine sulfoximine and Cd, no phytochelatins could be detected but the cysteine content increased 21-fold. Additionally, a larger increase in both enzyme activities occurred than with Cd alone. Taken together, our results are consistent with the hypothesis that glutathione is a precursor for phytochelatin synthesis.     

Mechanism of cadmium tolerance in Salicornia europaea at optimum levels of NaCl

• Many saline-alkali soils around the world are polluted by the heavy metal Cd, restricting the development of agriculture and ecology in those regions. The halophyte Salicornia europaea L. is capable of growing healthily in Cd-contaminated saline-alkali soil, suggesting that the species is tolerant to stress caused by both salt and heavy metals. In this study, the mechanism of Cd tolerance in this species was explored under 200 mM NaCl.
• Flame spectrophotometric assays for ions content and spectrophotometric for organic soluble substances, antioxidant enzyme activity, phytochelatins (PCs) content and phytochelatin synthase (PCS) activity, the photosynthetic parameters by portable photosynthesis measurement system, genes expression by qRT-PCR analysis were carried out.
• Cd treatment significantly decreased the dry weight, photosynthetic rate, K⁺, Zn²⁺, and Fe^2+/3+ content, while significantly increasing Na⁺ and Cd⁺, soluble organic matter, and reactive oxygen species (ROS) levels. Compared with Cd treatment at 0 mM NaCl, Cd treatment at 200 mM NaCl significantly increased dry weight and photosynthetic rate while significantly decreasing ROS content through increased antioxidant enzyme activity. When exposed to Cd stress, treatment with 200 mM NaCl significantly increased PCs content and PCS activity and up-regulated the expression of the phytochelatin synthase genes CDA1 and PCS1 were, thereby increasing resistance to Cd.
• NaCl treatment increases the tolerance of S. europaea to the heavy metal Cd by growing rapidly, reducing the quantity of Cd²⁺ from entering the plant shoots, increasing the levels of PCs that chelate Cd²⁺, thereby reducing its toxicity.

     

Glutamine synthetase and glutamate dehydrogenase in cadmium-stressed triticale seedlings

The studies were performed on young triticale seedlings grown on a mineral medium containing 5 mM NO ₃ ⁻ as the nitrogen source, with the addition of 0.5 mM CdCl₂. It was determined that cadmium ions accumulated mainly in the plant roots. Decreases in nitrate concentrations both in the roots and shoots of seedlings, as well as decreases in soluble protein contents with simultaneous increases in endopeptidase activity were also observed. Both in roots and shoots significant decreases in glutamic acid were noted. Toxic cadmium ion accumulation in seedlings significantly modified activity of primary nitrogen assimilating enzymes, i.e. glutamine synthetase (GS, EC 6.3.1.2) and glutamate dehydrogenase (GDH, EC 1.4.1.2). There was a significant decrease in GS activity both in roots and in shoots of the stressed plants, in comparison to plants grown without cadmium. In shoots of the control plants and plants subjected to stress two GS isoforms were discovered: cytoplasmatic (GS₁) and chloroplastic (GS₂). Substantial decreases in total glutamine synthetase activity in green parts of seedlings, occurring under stress conditions, result from dramatic decrease in GS₂ activity (by 60 % in relation to the control plants); despite simultaneous increases in the cytoplasmatic isoform (GS₁) activity by approx. 96 %. Cadmium ions accumulating in roots and shoots of seedlings not only increased GDH activity, but also modified its coenzymatic specificity.