• Cadmium (Cd) stress increases cell metabolic demand for sulfur, reducing equivalents, and carbon skeletons, to sustain phytochelatin biosynthesis for Cd detoxification. In this condition the induction of potentially acidifying anaplerotic metabolism in root tissues may be expected. For these reasons the effects of Cd accumulation on anaplerotic metabolism, glycolysis, and cell pH control mechanisms were investigated in maize (Zea mays) roots.
• The study compared root apical segments, excised from plants grown for 24 h in a nutrient solution supplemented, or not, with 10 µm CdCl2, using physiological, biochemical and 31P-nuclear magnetic resonance (NMR) approaches.
• Cadmium exposure resulted in a significant decrease in both cytosolic and vacuolar pH of root cells and in a concomitant increase in the carbon fluxes through anaplerotic metabolism leading to malate biosynthesis, as suggested by changes in dark CO2 fixation, metabolite levels and enzyme activities along glycolysis, and mitochondrial alternative respiration capacity. This scenario was accompanied by a decrease in the net H+ efflux from the roots, probably related to changes in plasma membrane permeability.
• It is concluded that anaplerotic metabolism triggered by Cd detoxification processes might lead to an imbalance in H+ production and consumption, and then to cell acidosis.
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.
Materials and Methods
Plant material and growth conditions
Maize (Z. mays L. cv. Dekalb DK 300) caryopses were sown on filter paper saturated with distilled water and incubated in the dark at 26°C. Three days later, seedlings were transplanted into 5 l plastic tanks (18 seedlings per tank) containing a complete nutrient solution (Nocito et al., 2002). Seedlings were kept for 3 d in a growth chamber maintained at 26°C and 80% relative humidity during a 16-h light period and at 22°C and 70% relative humidity during the 8-h dark period. Photosynthetic photon flux density was 200 µmol m−2 s−1. At the end of this period the nutrient solution was renewed and supplemented with 10 µm CdCl2. After 24 h of Cd exposure, roots were washed for 10 min in ice-cold 5 mm CaCl2 solution to displace extracellular Cd and then rinsed in distilled water. Apical root segments 20 mm long were obtained from the whole-root system of each plant. Before the in vivo experiments, segments were randomized and further washed for 30 min in an aerated 0.5 mm CaCl2 solution maintained at 26°C.
Determinations of Cd, nonprotein thiols, malate, phosphoenolpyruvate, thiobarbituric acid-reactive metabolites and inorganic phosphate
For Cd determination, samples of c. 400 mg FW were mineralized at 120°C in 5 ml 14.4 m HNO3, clarified with 1.5 ml 33% H2O2 and finally dried at 80°C. The mineralized material was solubilized in 5 ml 1 m HNO3 and filtered on a 0.45 µm nylon membrane. Cadmium content was measured by inductively-coupled plasma techniques (Liberty AX sequential ICP-OES; Varian, Palo Alto, CA, USA) using calibration solutions in the range 0.01–1 µg ml−1.
Total nonprotein thiols (NPTs) were determined according to Nagalakshmi & Prasad (2001). Results were expressed as nanomoles of GSH equivalents.
Malate and phosphoenolpyruvate (PEP) were extracted from root apical segments according to Hohorst (1963). Malate concentration was determined by measuring NAD+ reduction at 340 nm, as described by Morgutti et al. (1984). The PEP concentration was determined by measuring NADH oxidation, at 25°C, in a reaction mixture containing 25 mm Tris-HCl (pH 7.20), 20 mm KCl, 10 mm MgCl2, 2 mm dithiothreitol (DTT), 0.15 mm NADH, 2 mm ADP, 2 U ml−1 lactate dehydrogenase and 2 U ml−1 pyruvate kinase.
The levels of 2-thiobarbituric acid (TBA)-reactive metabolites were determined according to Hodges et al. (1999).
For inorganic phosphate (Pi) determination, samples of whole-root systems or apical root segments were homogenized in four volumes of 10% (v : v) ice-cold trichloracetic acid and centrifuged at 13 000 g for 15 min. Inorganic phosphate was determined in the supernatant according to Fiske & Subbarow (1925).
Twenty root apical segments were transferred into a thermoregulated (26°C) airtight cuvette containing 4 ml of the nutrient solution without Cd and their oxygen consumption rate was measured using a Clark-type electrode (YSI Incorporated, Yellow Spring, OH, USA). The cytochrome and the alternative pathways of the mitochondrial respiration were inhibited with 1 mm KCN or 4 mm salicylhydroxamic acid (SHAM), respectively. The oxygen consumption rates of the segments were measured for 5 min after a 5-min period of preincubation in the absence or in the presence of each inhibitor. Without inhibitors the O2 consumption rate of root segments from either control or Cd-exposed plants remained constant for more than 45 min. With alternative oxidase (AOX) capacity we refer to the KCN-resistant O2 consumption after correction for the residual component of O2 consumption measured in the presence of both 1 mm KCN and 4 mm SHAM.
Nuclear magnetic resonance spectroscopy
The 31P-NMR spectra were recorded as described by Espen et al. (2000). Experiments were carried out by packing c. 40 root apical segments in a 10-mm diameter NMR tube equipped with a perfusion system connected to a peristaltic pump in which the aerated, thermoregulated (26°C) basal medium (0.5 mm CaSO4, 1 mm 3-(N-morpholino) ethanesulfonic acid-Bis-tris propane (MES-BTP) pH 6.50) flowed (10 ml min−1). Before starting the experiments, the root apical segments were washed for 1 h in the basal medium.
Root apical segments (c. 2 g FW) were homogenized at 4°C in 4 ml of a buffer containing 50 mm 3-(N-morpholino) propanesulfonic acid-Bis-tris propane (MOPS-BTP) (pH 7.50), 330 mm sucrose, 3 mm ethylene glycol-tetraacetic acid (EGTA), 5 mm DTT, 1 mm phenylmethylsulphonyl fluoride (PMSF) and 10 mg ml−1 leupeptin. The homogenate was filtered on Miracloth and then centrifuged at 13 000 g for 15 min; the supernatant was further centrifuged at 100 000 g for 30 min and the new supernatant chromatographed through a Sephadex G-25 Fine column (2.5 cm diameter, 25 cm length; Amersham Bioscience, GE Healthcare Europe GmbH, München, Germany) equilibrated and eluted with the same buffer; the soluble extracted proteins were used for measuring the enzyme activities. To obtain the root mitochondria-enriched fraction, the 13 000 g pellet was resuspended in a large volume of the homogenization buffer, centrifuged and finally resuspended in 250 µl of the same buffer.
Phosphoenolpyruvate carboxylase (PEPC, EC 126.96.36.199) activity was determined according to De Nisi & Zocchi (2000). ATP-dependent phosphofructokinase (ATP-PFK, EC 188.8.131.52), NAD-dependent glyceraldehydes 3-phosphate dehydrogenase (NAD-GA3PDH, EC 184.108.40.206) and pyruvate kinase (PK, EC 220.127.116.11) activities were assayed as described by Espen et al. (2000). Fructose-bisphosphate aldolase (ALDO, EC 18.104.22.168) was assayed according to Hodgson & Plaxton (1998). Malate dehydrogenase (MDH, EC 22.214.171.124) was determined in a buffer containing 50 mm Tris-HCl (pH 9.50), 0.1 mm NADH, and 0.4 mm oxaloacetate. For all the assays NADH oxidation was determined at 340 nm in a Cary-50 spectrophotometer (Varian) at 26°C.
Extracts for measuring carbonic anhydrase (CA, EC 4.2.11) activity were made by grinding at 4°C c. 1 g FW of root apical segments in 7.5 ml of 100 mm Tris-HCl (pH 8.30), 10 mmβ-mercaptoethanol and 1 mm ethylenediaminetetraacetic acid (EDTA). The extract was stirred for 15 min and then centrifuged at 1000 g for 5 min. The supernatant was eluted with 10 ml of the extraction buffer through a Sephadex G-25 column, equilibrated with the same buffer, and the soluble protein fraction collected. All operations were carried out at 4°C. The CA activity was evaluated according to Wilbur & Anderson (1948).
Plasma membrane vesicle isolation and H+-ATPase assay
The plasma membrane vesicles were prepared as described by Palmgren et al. (1990). H+-ATPase phosphohydrolytic activity was evaluated using 40–80 µg of membrane proteins according to Forbusch (1983). The in vitro effect of Cd on PM-H+-ATPase activity was analysed by incubating the plasma membrane vesicles with 1–200 µm CdCl2 for 5 min at 25°C before starting the reaction.
Soluble fraction protein contents were determined by the Bradford procedure using γ-globulin as the standard (Bradford, 1976). Plasma membrane and mitochondrial protein fraction contents were determined by the Quant kit (Amersham Bioscience, GE Healthcare Europe GmbH, München, Germany) according to the manufacturer's protocol, using bovine serum albumin as a standard.
Proteins of soluble, plasma membrane or mitochondrial fractions were separated by tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to a polyvinylidene difluoride filter as described by Espen et al. (2004). Filters were incubated overnight at 4°C with polyclonal antisera raised against a sorghum C4 PEPC isoenzyme (a kind gift from J. Vidal), maize PM-H+-ATPase (a kind gift from R. Serrano) or monoclonal antibodies against Sauromatum guttatum AOX (Elthon et al., 1989), respectively, for proteins in soluble, plasma membrane or mitochondrial fractions. The filters were incubated for 2 h at room temperature with a secondary antibody (alkaline-phosphatase-conjugated antirabbit or antimouse immunoglobulin G). The blot was developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (FAST BCIP/NBT; Sigma-Aldrich, Milan, Italy). Images of blots were scanned and the quantification of the signal was performed by imagequant software (Amersham Bioscience, GE Healthcare Europe GmbH).
In vivo dark 14CO2 fixation
Batches of 20 washed root apical segments were transferred into 50 ml rubber cap sealed-flasks containing 10 ml of the nutrient solution without Cd. The experiments were started by injecting 50 µl of 80 µm NaH14CO3 (54.4 kBq mmol−1) into each flask. The samples were incubated under agitation at 26°C. After 30 min the radioactive medium was quickly drained and the root apical segments blotted with a paper towel and homogenized with 1 ml of 0.1 m HNO3. After bubbling with N2, the homogenates were transferred into scintillation vials containing 10 ml of scintillation cocktail and counted in a LS 7500 (Beckman, Fullerton, CA, USA) scintillation counter.
Net H+ efflux into the medium
Batches of 15 washed root apical segments were transferred into 50 ml flasks containing 10 ml of 0.5 mm CaSO4, 0.1 mm 2-(N-morpholino)-ethanesulfonic acid (MES)-Ca (pH 6.00) and 0.5 mm K2SO4. The samples were incubated under agitation at 26°C. After 30 and 60 min the pH values of the medium were measured and the net H+ efflux was calculated according to Lado et al. (1981).
Cadmium and NPT accumulation
Six-day-old maize plants grown for 24 h in the presence of 10 µm Cd did not show symptoms of toxicity, except for a slight reduction in the elongation of the lateral root branches. About 80% of the total Cd2+ absorbed (0.27 ± 0.01 µmol per plant) was retained in the roots. The metal concentration was higher in the 2-cm long distal portions of the roots than in the whole-root system (Table 1). The presence of Cd induced an increase in the NPT levels of the root, evaluated as GSH equivalents (Table 1). The effect was slightly higher in the root apical segments (4.6-fold) than in the whole-root system (3.9-fold). Under the same conditions, the level of TBA-reactive metabolites of the root was higher in Cd-treated than in control plants; in this case the effect of Cd was also more pronounced in the root apical segments (+59%) than in the whole-root system (+41%).
Table 1. Concentrations of cadmium (Cd), nonprotein thiols (NPTs) and 2-thiobarbituric acid (TBA)-reactive metabolites in the roots of Zea mays
Root apical segments
Six-day-old plants were incubated for 24 h in a nutrient solution supplemented with 10 µm CdCl2. About 2-cm long segments were excised from main and lateral roots (root apical segments). Data are means ± SE of three experiments run in triplicate (n = 9). ND, not detectable. Values indicated by asterisks are different from control at P ≤ 0.05.
Cd (µmol g−1 DW)
8.6 ± 0.3
10.2 ± 0.3
NPTs (µmol glutathione eq. g−1 DW)
4.6 ± 0.2
18.2 ± 0.3*
6.8 ± 0.4
31.6 ± 0.6*
TBA-reactive metabolites (nmol g−1 DW)
42.1 ± 3.1
59.2 ± 4.3*
75 ± 1.2
119 ± 6.2*
These preliminary results indicate that maize root apical segments are particularly suitable for studying cell metabolic responses to Cd exposure and accumulation. For this reason, the remaining part of this study focused only on the root apical segments excised from the root systems of control and Cd-treated plants.
In vivo 31P-NMR analysis of root apical segments
Figure 1 shows two representative 31P-NMR spectra of root apical segments from Cd-treated and control plants. The 31P-NMR spectrum of Cd-treated root apical segments showed (Figs 1, 2a) an increase in the peak area assigned to glucose 6-phosphate (G6P; +25%) and a decrease in that assigned to uridine diphosphoglucose (UDPG; –20%); no significant differences were observed in the peak areas assigned to fructose 6-phosphate (F6P). Cadmium treatment strongly increased the vacuolar Pi content of the root cells (+127%) without inducing any significant change in the cytosolic Pi concentration (Fig. 2b). The effect of Cd on total Pi concentration was confirmed by means of a colorimetric assay performed on extracts of the tissues: the concentration of the Pi moved from 0.68 ± 0.01 µmol g−1 FW (control) to 1.21 ± 0.03 µmol g−1 FW (Cd).
The pH values of cytosolic and vacuolar compartments (pHc and pHv) evaluated from the chemical shifts of the corresponding Pi peaks are reported in Fig. 1. The growth for 24 h in the presence of 10 µm Cd2+ induced significant decreases in both pHc and pHv values: –0.27 and –0.52 units, respectively, with respect to the control.
Anaplerotic metabolism and glycolytic enzyme activities following Cd exposure
The capacity to fix CO2 in the dark was higher in Cd-treated (+42%) than in control root apical segments (Table 2). Such a behavior was associated with a higher level of malate (+110%), whereas the PEP level was not affected by Cd (Table 2).
Table 2. Anaplerotic metabolism of root apical segments of Zea mays
CA, carbonic anhydrase; MDH, malate dehydrogenase; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase. Six-day-old plants were incubated for 24 h in a nutrient solution supplemented with 10 µm CdCl2. Data are means ± SE of three experiments run in triplicate (n = 9). Values indicated by asterisks are different from control at P ≤ 0.05.
In vivo dark CO2 fixation (nmol h−1 g−1 FW)
29.1 ± 0.2
41.3 ± 0.2*
CA activity (µmol H+ released min−1 mg−1 protein)
8.9 ± 0.3
11.9 ± 0.4*
PEPC activity (nmol min−1 mg−1 protein)
50.8 ± 0.8
86.4 ± 1.4*
MDH activity (nmol min−1 mg−1 protein)
25.2 ± 0.6
28.1 ± 0.3*
PEP level (nmol g−1 FW)
17.8 ± 1.2
20.0 ± 1.7
Malate level (µmol g−1 FW)
0.48 ± 0.01
1.01 ± 0.02*
Cadmium exposure resulted in a generalized change in the enzyme activities involved in the dark CO2 fixation pathway. As reported in Table 2, the effect was particularly evident for PEPC, the activity of which increased by c. 70% with respect to the control; although to a lesser extent, the treatment with Cd also produced significant increases in the activities of CA (+34%) and MDH (+11%).
Western blot analysis of the soluble cytosolic protein extracts showed that the antibodies raised against sorghum C4 PEPC isoenzyme were able to react against two polypeptides with apparent molecular masses of 103 kDa and 109 kDa (Fig. 3). Densitometric analysis of the bands revealed a higher level (c.+100%) of the 103 kDa polypeptides in Cd-treated than in control root segment extracts.
As reported in Table 3, Cd exposure increased the specific activity of the enzymes ALDO (+52%) and NAD-GA3PDH (+55%); under the same conditions no changes in ATP-PFK- and PK-specific activities were observed.
Table 3. Specific activity of glycolytic enzymes in root apical segments of Zea mays
Data are means ± SE of three experiments run in triplicate (n = 9). Values indicated by asterisks are different from control at P ≤ 0.05.
73.1 ± 5.7
75.3 ± 5.4
118.7 ± 1.5
180.7 ± 3.8*
76.1 ± 9.6
118.0 ± 7.9*
57.4 ± 3.6
60.9 ± 2.9
Oxygen consumption following Cd exposure
The total O2 consumption rate of root apical segments did not differ significantly between control and Cd-treated plants (Table 4). Cyanide-resistant O2 consumption was approx. 14% and 36% of the total O2 consumption in the control and Cd-treated root apical segments, respectively. SHAM had practically no effect on the O2 consumption rate of both control and Cd-treated root apical segments; however, when SHAM was added in combination with KCN, the residual O2 consumption rate dropped to 8% and 13% of the initial rate in the control and in the Cd-exposed root apical segments, respectively.
Table 4. Oxygen consumption rates of root apical segments of Zea mays
O2 consumption rate (µmol min−1 g−1 FW)
SHAM, salicylhydroxamic acid. Data are means ± SE of three experiments run in triplicate (n = 9). Values indicated by asterisks are different from control at P ≤ 0.05.
991.1 ± 46
1125 ± 48
1 mm KCN
142.6 ± 23
410.3 ± 32*
4 mm SHAM
994.0 ± 32
1174 ± 72*
1 mm KCN + 4 mm SHAM
83.2 ± 17
137.0 ± 20
The AOX capacity (see the Materials and Methods section) was significantly higher in Cd-treated than in control root apical segments (273.3 ± 52 vs 59.4 ± 40 µmol min−1 g−1 FW). Such a finding was consistent with the higher relative level of the 37 kDa reactive peptide constituting the homodimeric AOX observed in the mitochondria protein fraction isolated from the Cd-treated roots (Fig. 3).
In vivo net H+ efflux and in vitro plasma-membrane H+-ATPase activity following Cd exposure
The growth in the presence of Cd resulted in a substantial decrease (–60%) in the net H+ efflux from root apical segments (Fig. 4a). Conversely, the ATP-phosphohydrolysing activity of purified plasma-membrane vesicles from the root apical segments was higher in Cd-treated (+60%) than in control plants (Fig. 4b). Western blot analysis conducted on proteins extracted from the same vesicle preparations revealed a positive effect of Cd exposure on the level of PM-H+-ATPase, as indicated by an higher amount of the protein in Cd treated (+75%) than in control roots (Fig. 4c).
The PM-H+-ATPase phosphohydrolytic activity was not directly affected by the presence of Cd in the assay solution up to 50 µm (Fig. 5); over this value the activity of the enzyme was drastically and progressively inhibited, up to a value c. 25% of the initial one at 200 µm.
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).
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.
This work was supported by grants from the Italian Ministry of Education, University, and Research (Progetti di Ricerca di Interesse Nazionale 2006).