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Cadmium is thought to be taken up by plants via iron (Fe) and zinc (Zn) transporters (Clemens, 2001; Hall & Williams, 2003) or calcium (Ca) channels (Perfus-Barbeoch et al., 2002). Accumulation of Cd in plant tissues may cause a variety of toxicity symptoms ranging from chlorosis, wilting and growth reduction, to cell death. Cadmium cellular toxicity may result from interactions with the carboxyl or thiol groups of proteins (Sanità di Toppi & Gabbrielli, 1999), genesis of free radicals inducing oxidative stress (Schützendübel & Polle, 2002) or interference with the regulation and functionality of calcium-dependent processes (Rivetta et al., 1997; Perfus-Barbeoch et al., 2002).
The intracellular chelation of Cd by glutathione (GSH) and phytochelatins (PCs) represent an ubiquitous detoxification strategy adopted by a wide number of plant species (Zenk, 1996; Cobbett & Goldsbrough, 2002). Phytochelatins are synthesized nontranslationally from GSH in a transpeptidation reaction catalysed by the enzyme PC synthase (PCS; γ-Glu-Cys dipeptidyl transpeptidase; Rea et al., 2004). Synthesis of PC is induced within minutes following exposure to different metals or metalloids; among these, Cd is the strongest inducer (Grill et al., 1987; Maitani et al., 1996).
It has been convincingly shown that massive PC production is accompanied by a coordinated transcriptional induction of activities involved in sulfate uptake (Nocito et al., 2002) and assimilation (Lee & Leustek, 1999), and in GSH biosynthesis (Schäfer et al., 1998; Xiang & Oliver, 1998; Saito, 2004). In these conditions, the need to maintain a balance between GSH biosynthesis and PCs production is suggested by the finding that transgenic plants of Brassica juncea overexpressing GSH synthetase or γ-glutamylcysteine synthetase were found to be more tolerant to Cd stress (Zhu et al., 1999a,b; Wawrzyński et al., 2006), whereas transgenic Arabidopsis lines overexpressing PCS were hypersensitive to Cd since these were probably depleted in cell GSH pools and thus more susceptible to Cd-related oxidative stress (Lee et al., 2003; Li et al., 2004).
Recently, transcriptomic, proteomic and metabolomic studies have given more insight into the overall metabolic consequences of Cd exposure in plants (Bailey et al., 2003; Roth et al., 2006; Sarry et al., 2006). In addition to sulfur metabolism, nitrogen and carbon metabolic pathways were also found to be affected by the presence of Cd within the cells (Sarry et al., 2006). In particular, the expression of enzymes involved in glycolysis, the pentose phosphate pathway and in the tricarboxylic acid (TCA) cycle were amplified. Such a stimulation has been interpreted as necessary to sustain the increase in the reducing power demand required for sulfate assimilation and, above all, to provide the carbon skeletons required for the synthesis of the γ-Glu-Cys moiety of GSH and glycine which in turn accumulate in the Cd-PC complexes (Sarry et al., 2006).
Since both glycolysis and pentose phosphate pathways are H+-producing processes (Sakano, 2001) the physiological responses to Cd could potentially induce cell acidosis. Cell pH regulation in plants is ensured by a control not only based on the activity of both vacuolar and plasma-membrane H+-ATPases (PM-H+-ATPase) which actively extrude H+ from the cytosol, but also on fine-tuning of the biochemical mechanisms based on malate synthesis and degradation (Davies, 1986; Sakano, 1998, 2001). When the H+-extruding activities are inhibited, the resulting cytosolic acidification should impair the anaplerotic malate production with a consequent reduction of the carbon flow along the glycolytic pathway (Sakano, 2001). Since Cd has been described as inhibiting PM-H+-ATPase activity (Fodor et al., 1995; Astolfi et al., 2005), a question arises: how do cells deal with the opposing effects of Cd on the metabolism sustaining PC synthesis and the mechanisms regulating pH? With the aim of answering this point, in the present investigation we have evaluated physiological processes and enzyme activities involved in carbon metabolism and cell pH control, together with both cytosolic and vacuolar pH changes by means of 31P-nuclear magnetic resonance (31P-NMR) techniques, in the roots of maize (Zea mays) plants grown for 24 h in the presence of 10 µm Cd.
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Among the earliest responses of plants to Cd exposure, the accumulation of cysteine-rich peptides arising from GSH is the most extensively characterized (Cobbett & Goldsbrough, 2002). Following 24 h of exposure to 10 µm Cd2+ the levels of NPTs in maize root apical segments were 4.6-fold higher than that of the control (Table 1). Such a response is mainly caused by the stimulatory effect of Cd on the synthesis of PCs, which rapidly become the most abundant class of thiol compounds in the root cells (Nocito et al., 2002). The Cd-induced burst of PC biosynthesis is usually accompanied by a transient depletion of cell GSH pools that may be perceived by plants as indicating an additional need for sulfur (Nocito et al., 2002, 2007), reducing equivalents and carbon skeletons, mainly phosphoglycerate and 2-oxoglutarate since they are precursors of cysteine, glycine and glutamate, the amino acid monomers of GSH and PCs. In nonphotosynthetic tissues the need for reducing equivalents and carbon skeletons could mainly be supported through a carbohydrate overflow towards glycolysis, the pentose-phosphate pathway and anaplerotic reactions producing TCA cycle intermediates (Fernie et al., 2004).
The 31P-NMR analysis strongly suggests that cell carbohydrate fluxes towards consuming activities are enhanced under Cd stress (Figs 1, 2a). The decrease in UDPG concentration and the concomitant increase in that of G6P (Fig. 2a) should indicate an effect of Cd in enhancing the glycolytic rate of root cells. In fact, the higher level of G6P might be interpreted as a consequence of a preparatory step essential to support increases in both energy and metabolite cell demands. The effect of Cd in promoting the glycolytic rate is further supported by the observed increases in ALDO- and NAD-GA3PDH-specific activities (Table 3). Such behaviors could result from an effect of Cd on the expression levels of these enzymes, as previously reported in studies on both roots and cell suspensions of Arabidopsis (Roth et al., 2006; Sarry et al., 2006).
The metabolic response to Cd does not seem to involve ATP-PFK activity. Moreover, we could exclude a possible allosteric modulation of ATP-PFK, since the cytosolic concentrations of its positive (Pi) and negative (PEP) modulators (Plaxton, 1996) were also unaffected by Cd (Fig. 2b, Table 2). These findings may be expected since long-term responses to stress generally involve coordinated changes in the levels of groups of enzymes along a pathway, rather than allosteric modulations of single regulatory enzymes (Fell, 2005).
The metabolic significance of the glycolytic activation under Cd stress could be related to the need to maintain adequate carbon fluxes through anaplerotic pathways involved in the production of TCA cycle intermediates essential to sustain GSH and PC biosynthesis (Fig. 6). To investigate this hypothesis we measured the in vivo dark CO2 fixation of root apical segments, since this activity is expected to increase in all the metabolic scenarios where TCA cycle is acting in a biosynthetic role (Splittstoesser, 1966). Results show that the dark CO2 fixation was significantly enhanced under Cd stress, as well as the activity of CA, PEPC and MDH, the key enzymes involved in this pathway (Table 2; Ting & Dugger, 1967; Basra & Malik, 1985; Chang & Roberts, 1992). Such behaviors could account for the concomitant accumulation of malate in the root tissues exposed to Cd (Table 2). Interestingly, the effect of Cd on carbon dioxide hydration capacity of the root segments seems to be a typical short-term response involving the CA isoforms of nonphotosynthetic tissues, since photosynthetic isoforms of this enzyme have been reported to be impaired by Cd exposure (Aravind & Prasad, 2004). Furthermore, kinetic and immunological analyses of PEPC provide some evidence to support a direct link between PEPC level and dark CO2 fixation activity in the root apical segments (Table 2, Fig. 3). Western blot analysis (Fig. 3) shows the presence of two reactive peptides in the root extracts, corresponding to the 103 kDa and 108 kDa PEPC isoforms (Osuna et al., 1996). The amount of the 103 kDa isoform was increased by Cd accumulation, whereas the concentration of the 108 kDa isoform did not seem to be related to enhancement in the dark CO2 fixation capacity of the root segments. The greater amount of 103 kDa PEPC could be related to the need for maintaining a high level of carboxylase activity in a cell's metabolic status where malate concentration is increasing. Since the end product of the dark CO2 fixation pathway acts as an allosteric inhibitor of cytosolic PEPC activity (Izui et al., 2004), a fine regulation of PEPC level could allow root cells to move the sensitivity threshold for malate feedback inhibition, and thus maintain a over-flux along the pathway even in the presence of high malate concentration. Moreover, since PEPC of maize root cells plays primarily an anaplerotic role by replenishing C4-dicarboxylic acids in the TCA cycle (Chollet et al., 1996), it seems reasonable to assume that continuous malate withdrawal from the cytosol by transport into mitochondria may significantly alleviate its inhibitory effect on PEPC activity (Fig. 6). Finally, the effect of Cd on the accumulation of G6P, a positive effector of PEPC, could partly override the negative feedback exerted by malate, promoting oxaloacetate production (Izui et al., 2004).
Figure 6. Effect of cadmium (Cd) on glycolysis, anaplerotic metabolism and cell pH. PC-based Cd detoxification processes increase cell metabolic demand for carbon skeletons, mainly 2-oxoglutarate and 3-phosphoglycerate, to sustain glutathione (GSH) biosynthesis. The genesis of an ‘additional sink’ for carbon induces a carbohydrate overflow towards glycolysis and anaplerotic reactions involving carbonic anhydrase (CA), phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) to produce oxaloacetate and malate essential for replenishing C4-dicarboxylic acids used for both energy and biosynthetic metabolism in the TCA cycle. Metabolic changes induced by Cd also involve alternative oxidase (AOX) protein level and capacity, whose increases may ensure high turnover rates of carbon skeletons in both cytosol and TCA cycle, and adequate NAD+ recycling, bypassing the metabolic adenylate-control. The effect of Cd on plasma membrane passive permeability to H+ along with a H+ glycolytic overproduction may lead to an imbalance in H+ production and consumption, and then to cell acidosis. Asterisks and tinted boxes indicate metabolites and enzyme activities, whose levels are significantly increased by Cd exposure. G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; F1,6BP, fructose 1,6-bisphosphate; G3P, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; 1,3DPGA, 1,3-diphosphoglycerate; 3PGA, 3-phosphoglycerate; 2PGA, 2-phosphoglycerate; PEP, phosphoenolpyruvate; Cyt pathway, cytochrome pathway of the mitochondrial respiration.
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Mitochondrial cyanide-resistant alternative respiration pathway is crucial in anaplerotic metabolism, since it enables high turnover rates of carbon skeletons in both cytosol and TCA cycle, bypassing the adenylate-control (Arnholdt-Schmitt et al., 2006). Although the use of specific inhibitors in discriminating the actual activities of cytochrome and alternative respiration pathways has been widely debated (Day et al., 1996), the fourfold increase in in vivo AOX capacity (see the Results and Table 4), together with the higher amount of AOX protein in Cd-treated root segments (Fig. 3), strongly suggest that the alternative pathway was enhanced following Cd exposure. Such a response may be functional with the withdrawal of glycolysis and TCA cycle intermediates in order to bypass the adenylate control in providing carbon skeletons for GSH and PC biosynthesis (Fig. 6).
Alternative glycolytic PEP consumption by PEPC has been described as a component of a biochemical mechanism involved in controlling the cell pH (Davies, 1986). Three of the glycolytic steps leading to PEP synthesis (i.e. the reactions catalysed by esokinase, ATP-PFK and NAD-GA3PDH) produce protons in the cytosol (Fig. 6). According to the revision of the Davies pH-stat model proposed by Sakano (1998), cytosolic acidification results in negative and positive influences on PEPC and malic enzyme activities, respectively. Such a mechanism, since it results in both PEP accumulation and allosteric inhibition of ATP-PFK activity, could reduce glycolytic carbon fluxes and thus H+ production. Conversely, cytosolic alkalinization, stimulating the activity of PEPC and inhibiting that of malic enzyme, could promote carbon fluxes along the glycolytic pathway. Cadmium exposure induces a significant acidification of the cytosol, as indicated by 31P-NMR studies (Fig. 1) and concomitantly increases both glycolytic carbon flux and dark CO2 fixation. From these data we can speculate that the increase in PEPC level is able to partly counterbalance the negative effect exerted by cytosolic acidification on PEPC activity itself. In other words, Cd may affect the pH-stat mechanisms by shifting gene expression and metabolism toward an anaplerotic mode, thus interfering with the efficiency of the Davies pH-stat model.
The PM-H+-ATPase is a ‘master enzyme’ involved in the control of intercellular pH (Palmgren, 2001). The Cd-induced increase in both the amount and the phosphohydrolytic activity of this enzyme (Fig. 4b,c) could be interpreted as a response to counteract the possible direct inhibition exerted by Cd on PM-H+-ATPase activity (Fig. 5). However, taking into account the concentration reached by total Cd in the root tissues (Table 1) and the high affinity of this metal ion for sulfhydryl groups of NPTs, it seems unlikely to suppose an in vivo direct poisoning effect of free Cd ions on the PM-H+-ATPase phosphohydrolytic activity. Nevertheless, the evaluation of the in vivo H+ net efflux from root segments leads to an opposite conclusion, since this activity was significantly inhibited by Cd exposure (Fig. 4a). Such an apparent discrepancy could be caused by an effect of Cd on plasma membrane lipid composition that may result in a reduction of the PM-H+-ATPase passive permeability to H+, as previously described in maize, pea and rice (Ros et al., 1992; Hernández et al., 1997; Astolfi et al., 2005). Such an interpretation is consistent with the high level of TBA-reactive metabolites – diagnostic indicators of lipid peroxidation (Heath & Packer, 1968; Hodges et al., 1999) – detected in Cd-treated root segments (Table 1).
The stimulation of anaplerotic metabolism for sustaining the synthesis of GSH and PCs together with the inhibition of the net H+-extruding capability of cells could be considered the cause of the significant decrease in the intercellular pHs of the root apical segments (Fig. 6).
Finally, 31P-NMR analysis (Figs 1 and 2b) and Pi colorimetric assay (see the Results section) suggests that Cd induced a different allocation of Pi in the root system. In fact, whereas the total amount of Pi evaluated in the whole-root system did not change (data not shown) the concentration of the anion significantly increased in the root apical segments. Such a behavior was mainly the result of an increase in Pi vacuolar concentration of the cells of the root apical segment, as indicated by 31P-NMR spectra analysis (Fig. 1). Considering the vacuole as an active participant in the regulation of pHc (Kurkdjian & Guern, 1989) the higher concentration of the vacuolar Pi in Cd-treated root segments might be interpreted as a physiological response to the cytosolic acidification induced by Cd exposure. As proposed by Zocchi et al. (2007) the shift of the monoprotonated form of Pi () to the diprotonaded one () and its movement into the vacuole should contribute to counteract the increasing amount of H+ in the cytosol consequent on the higher glycolytic flow and the Cd inhibition of PM-H+-ATPase (Fig. 6).
In conclusion, a high-intensity exposure of maize plants to Cd establishes in the roots a metabolic scenario, which, even though it leads to the synthesis of detoxifying molecules, also leads to an imbalance in H+ production and consumption.