Sulfur metabolism and HM detoxification are closely related processes  (see Chapter 16) (Figure 15.2). In addition, a possible homeostatic role of PCs towards essential HMs is clearly indicated by the fact that (1) low, but detectable, levels of PCs are present even in the absence of HM exposure; and (2) PC levels increase concomitantly with Cu and Zn depletion upon transfer of cell suspension cultures to a minimal micronutrient medium. In fact, it has been hypothesized that PCs are not simply an HM-detoxification system sensu stricto, especially in the presence of low concentrations of (essential) metals. Instead, under these conditions, PCs may primarily act as key components of metal homeostasis. The constitutive expression of PCS  might also be considered as an indication of a more general role of PCs not exclusively related to HM detoxification.
Further supporting this view is the strong protective effect of PCs against Cd-mediated inacti-vation of metal-sensitive enzymes and the ability of Zn- and Cu-PC complexes, mainly of the PC2/PC3 type, to reactivate metal-depleted or metal-poisoned metalloenzymes (although not more efficiently than the corresponding free salts). In plants, it is thus possible that PCs and MT-like proteins cooperatively act in the homeostasis of essential HMs. PCs have also been proposed as activated sulfate acceptors in the formation of a thiosulfate intermediate leading to sulfite formation upon reduction by thiosulfonate reductase. This hypothesis, however, has been challenged by the fact that no plant thiosulfonate reductase has been identified thus far and by the recent demonstration that the main sulfite-forming pathway in plants relies on an enzyme (adenosine 5'-phosphosulfate reductase) that directly reduces activated sulfate (APS) using an intramolecular glutaredoxin domain .
Although the metal detoxification and homeostatic roles of PCs are not mutually exclusive and may coexist at the whole plant level, the fact that Arabidopsis PC-deficient mutants (cad1) grow well in the presence of Cu and Zn micronutrient concentrations  suggests that the latter role, if real, is dispensable or easily replaceable by other metal-binding components, such as MTs. On the other hand, the idea of an exclusive metal detoxification function of PCs is somewhat weakened by the lack of correlation between the PC content and the HM sensitivity of metal-tolerant and nontolerant ecotypes of Silene vulgaris and Datura innoxia and by similar findings recently reported for the HM hyperaccumulator Thlaspi caerulescens and the closely related, nonaccumulator species Thlaspi arvense [22-24]. Neither PCS activity nor PC turnover upon transfer to a Cd-less medium differed between wild-type and HM-tolerant Silene vulgaris . In addition, although PC2 was the most abundant PC peptide in metal-tolerant plants, the more effective metal chelator, PC3, prevailed in the nontolerant ecotype .
FIGURE 15.2 Sulfur metabolism and HM detoxification mechanisms — a comprehensive view.
Further to this point, a stronger correlation between Cu tolerance and the accumulation of MT mRNAs (r values ranging from 0.89 to 0.998) than with the total amount of intracellular nonprotein thiols, including PCs (r = 0.77), has been reported in Arabidopsis . Therefore, it cannot be excluded that other systems, autonomously or in combination with PCs, may regulate HM homeo-stasis in the plant cell. For instance, Cu-MTs and Cu chaperones (termed Atx1, Lys7, and Cox 17) seem to be involved in Cu ion traffic in yeast cells  and analogous mechanisms might operate in higher plants .
Multiple connections exist between sulfur metabolism and heavy metal detoxification and homeostasis in plants. Metal ions are complexed in the cytosol with GSH and the derived PCs are transported into vacuole. The following functions have been implicated in Cd complexation and transport [30 and the references therein]:
• The ATP-binding cassette-type transport activity at the tonoplast
• The vacuolar-type ATPase generating a proton gradient
Another notable cellular response depicted is that some metals interact with genes that have metal-regulating elements (MRE) at the promoter region, as found for MT genes of animals. For example, in the soybean, similar sequences have been found in the upstream region of heat shock gene coded for 17.5 kDa HSP (heat shock protein ). Some heat shock genes and small HSPs have been induced by Cd ions in soybean [31,32]. These HSCs (heat shock cognates) thus may function as molecular chaperones [33,34].
Cui et al.  reported that the elemental sulphur and EDTA amendments increased the extractable fractions of soil Pb and Zn. EDTA was more effective than S. Shoot uptake of Pb and
Zn by Indian mustard and wheat was increased with S and EDTA amendments. Indian mustard shoots took up more Pb and Zn than winter wheat with or without S and EDTA amendment.
15.9 OVEREXPRESSION OF LCT1 (LOW-AFFINITY CATION TRANSPORTER) IN TOBACCO ENHANCED: THE PROTECTIVE ACTION OF CALCIUM AGAINST CADMIUM TOXICITY
Metal-metal interactions have been reported to have ameliorating functions [36-40]. For example, calcium involvement in zinc uptake and detoxification was studied in Zn2+-tolerant and nontolerant populations of Silene maritima. Increasing calcium concentrations reduced Zn toxicity and led to a higher level of zinc accumulation by the roots of the tolerant plants; however, they decreased transport to the shoots of both types . A higher calcium concentration in a medium was also reported to abolish the toxic effects of Cd2+ [42,43] and Pb2+  on the activity of photosystem II. In addition, high Ca status and a high level of tolerance to Ca deficit accompanied enhanced Zn, Pb, Cu, and Al tolerance [45-47].
The regulation of heavy metal uptake and internal transport constitutes part of the basis of plant resistance to their toxicity. The mechanism accounting for the transport of heavy metals across membranes in plants and its regulation is far from understood, however. The general view is that nonessential metals usually cross plasmalemma and internal membranes through cation transporters with a broad substrate specificity or that they use pathways reserved for other ions [48,49]. For example, cadmium and lead were shown to be transported through pathways for calcium ions.
In plants, it has been suggested that putative tonoplast Ca2+/H+ antiporters encoded by CAX1 and CAX2 (calcium exchange protein) from Arabidopsis are involved in the transport of cadmium from the cytoplasm to the vacuole . In turn, LCT1 cloned from wheat roots (however expressed in roots and leaves), a nonselective transmembrane transporter for Na+, K+ [51,52], and Ca2+, also appeared to mediate Cd2+ transport to the cell . Toxic metal ions such as Cd2+ or Pb2+ are known as very effective substituents of Ca2+, e.g., in calmodulin, consequently interfering seriously with the role of this cation in a number of metabolic processes [54-56]. In this context, it seems possible that the regulation of heavy metal uptake/transport in a plant by the presence of calcium in various concentrations in the medium could in part contribute to an ameliorative effect of calcium on heavy metal toxicity.
To gain insight into the molecular mechanism of Ca2+-dependent Cd2+ tolerance, tobacco was transformed with wheat cDNA LCT1, the first cloned plant influx system mediating the uptake of Ca2+ and Cd2+ ions into a cell . Transformants were tested for the possible involvement of LCT1 in diminishing Cd toxicity with the enhancement of Ca2+ concentration in the medium. Antosiewicz and Hennig  also demonstrated that LCT1 is involved in calcium acquisition and in the alleviation of toxic effects of Cd2+ by enhanced external Ca2+ concentration.
Antosiewicz and Hennig  were the first to demonstrate the involvement of LCT1 in calcium acquisition and in the regulation of amelioration of Cd toxicity by calcium. Wheat cDNA LCT1 (low-affinity cation transporter gene), a nonspecific transporter for Ca2+, Cd2+, Na+, and K+, was overex-pressed in tobacco. Transformants were tested for their sensitivity to a range of Ca2+ concentrations [0.01 to 10 mM Ca(NO3)2] with or without the presence of 0.05 mM Cd(NO3)2. LCT1-transformed plants expressed a phenotype distinct from controls only under conditions of low calcium (0.01 to 1 mM Ca2+). They grew significantly better and had slightly higher shoot calcium concentration.
Transformants subjected to 0.05 mM Cd(NO3)2 in the presence of 1 mM Ca2+ displayed a substantially higher level of tolerance to cadmium and accumulated less Cd in roots. LCT1 increased calcium and decreased cadmium accumulation in transformed plants. LCT1, the putative plasmalemma nonspecific transporter for Ca2+, Cd2+, Na+, and K+ [51-53], was used for tobacco transformation in order to check whether the toxicity of cadmium administered in a range of Ca2+ concen trations to control and LCT1-transformed plants would be different. LCT1 improved plant performance at low external calcium; LCT1 expression in tobacco improved the growth of plants only within a limited range of calcium concentrations. LCT1 contributed to Ca2+-dependent Cd2+ tolerance.
Numerous authors have described the phenomenon of calcium mitigating heavy metal toxicity [36,41-44]. The reported amelioration constitutes a part of the whole-plant defense system that includes uptake/transport and detoxification/sequestration. Based on the known broad spectrum of the role of Ca2+ in the regulation of metabolic processes , calcium might be an important factor in any of these components. It is not known whether the observed reduced toxicity might result from less cadmium uptake or from more efficient detoxification. For example, Choi et al.  demonstrated the contribution of calcium to the protection mechanism by immobilization of the metal as coprecipitates with calcium and phosphorous. Reduction of cadmium uptake and accumulation by calcium were also reported for several plant species [60-62] as was the opposite effect: inhibition of the accumulation of calcium by cadmium, leading to calcium deficiency .
15.10 OVEREXPRESSION OF ALFALFA ALDOSE/ALDEHYDE REDUCTASE CONFERS TOLERANCE TO CADMIUM STRESS
Wild-type (SR1) tobacco line and transgenic lines ALR1/5 and ALR1/9 overexpressing aldose/alde-hyde reductase were less susceptible to cadmium-induced stress. Based on these observations, Hegedüs et al.  suggested that alfalfa aldose/aldehyde reductase overexpression may generally induce higher stress tolerance. These coworkers transformed tobacco (Nicotiana tabacum cv. Petit Havanna line SR1) by alfalfa aldose/aldehyde reductase cDNA attached to the viral constitutive promoter CaMv35S. The WT (SR1), a nonexpressing transgenic line (ALR1/7), and two transgenic lines (ALR1/5 and ALR1/9) previously shown by Western hybridization to overexpress the alfalfa ALR protein  were used in these experiments.
The ectopic synthesis of a novel alfalfa NADPH-dependent aldose/aldehyde reductase enzyme in transgenic tobacco plants provided substantial tolerance against oxidative stress, such as drought, paraquate, and UV-B [21,22]. Cd treatment caused a significant decrease in the chlorophyll content in all the genotypes tested. However, this decrease was less pronounced in the lines overproducing aldose/aldehyde reductase (72 and 74% for lines ALR1/9 and ALR1/5, respectively) than in the SR1 wild-type plants (52%). Similar tendencies were revealed for changes in the total carotenoid content of tobacco leaves.
The novel molecular methodologies, i.e., genomics and proteomics, have not been brought together in the past to characterize molecular mechanisms related to plant metal accumulation. Because the Arabidopsis genome has been completely sequenced, the full benefit of those data is available for identification of genes and proteins found in plants that hyperaccumulate metals. Various ecotypes of plants that hyperaccumulate metals are being currently investigated in various laboratories to identify metal responsive proteins. Proteomics provides a powerful additional tool for the identification of proteins induced or repressed under metal stress and also for comparison of various ecotypes. Thlaspi is the ideal plant because many of the proteins can be identified based on the homology to Arabidopsis or Brassica genes. On the other hand, it was evident that Thlaspi contains many proteins for which homology was not found from databases. These proteins may be of particular interest for further studies of metal tolerance, uptake, and accumulation.
15.11 HAIRY ROOTS OF HORSERADISH ARE AN IDEAL SYSTEM FOR INDUCTION OF PHYTOCHELATIN HOMOLOGS
When exposed to excess heavy metals, plants induce phytochelatins and related peptides (all designated as PCAs). Horseradish (Armoracia rusticana) was exposed for 3 days to cadmium (1
The wizard plant of phytoremediation electroporation, PEG mediated DNA uptake, particle bombardment, microinjection, and agrobacterium mediated genetic transformations have been successful
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