Journal of Experimental Botany, Vol. 52, No. 364, pp. 2115-2126,
November 1, 2001
© 2001 Oxford University Press
Original Papers |
Cadmium-induced changes in the growth and oxidative metabolism of pea plants
1 Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Apartado 419, E-18080 Granada, Spain
2 Departamento de Agroecología y Protección Vegetal, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Apartado 419, E-18080 Granada, Spain
Received 22 January 2001; Accepted 1 July 2001
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The effect of growing pea (Pisum sativum L.) plants with CdCl2 (0–50 µM) on different plant physiological parameters and antioxidative enzymes of leaves was studied in order to know the possible involvement of this metal in the generation of oxidative stress. In roots and leaves of pea plants Cd produced a significant inhibition of growth as well as a reduction in the transpiration and photosynthesis rate, chlorophyll content of leaves, and an alteration in the nutrient status in both roots and leaves. The ultrastructural analysis of leaves from plants grown with 50 µM CdCl2, showed cell disturbances characterized by an increase of mesophyll cell size, and a reduction of intercellular spaces, as well as severe disturbances in chloroplast structure. Alterations in the activated oxygen metabolism of pea plants were also detected, as evidenced by an increase in lipid peroxidation and carbonyl-groups content, as well as a decrease in catalase, SOD and, to a lesser extent, guaiacol peroxidase activities. Glutathione reductase activity did not show significant changes as a result of Cd treatment. A strong reduction of chloroplastic and cytosolic Cu,Zn-SODs by Cd was found, and to a lesser extent of Fe-SOD, while Mn-SOD was only affected by the highest Cd concentrations. Catalase isoenzymes responded differentially, the most acidic isoforms being the most sensitive to Cd treatment. Results obtained suggest that growth of pea plants with CdCl2 can induce a concentration-dependent oxidative stress situation in leaves, characterized by an accumulation of lipid peroxides and oxidized proteins as a result of the inhibition of the antioxidant systems. These results, together with the ultrastructural data, point to a possible induction of leaf senescence by cadmium.
Key words: Cadmium, catalase, oxidative stress, pea, SOD, peroxidase.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cadmium is a toxic trace pollutant for humans, animals and plants which enters the environment mainly from industrial processes and phosphate fertilizers and then is transferred to the food chain (Wagner, 1993
). Studies carried out in different plant species have revealed that Cd is strongly phytotoxic and causes growth inhibition and even plant death, although the mechanisms involved in its toxicity are still not completely understood. Cadmium produces alterations in the functionality of membranes by inducing changes in lipid composition (Ouariti et al., 1997) and by affecting the enzymatic activities associated with membranes, such as the H+-ATPase (Fodor et al., 1995). Photosynthesis is also sensitive to Cd, chlorophyll being one of the targets (Somashekaraiah et al., 1992) as well as the enzymes involved in CO2 fixation (Greger and Ögren, 1991; De Filippis and Ziegler, 1993). Cadmium toxicity is also correlated with disturbances in the uptake and distribution of macro and micronutrients in plants (Gussarson et al., 1996).
Certain heavy metals like copper and iron can be toxic by their participation in redox cycles producing hydroxyl radicals (·OH) which are extremely toxic to living cells (Stochs and Bagchi, 1995). By contrast with those metals, Cd is a non-redox metal unable to participate in Fenton-type reactions. The enzymes superoxide dismutase (SOD), and catalase (CAT) and peroxidases are involved in the detoxification of O2 ·-, and H2O2 respectively, thereby preventing the formation of ·OH radicals. Ascorbate peroxidase (APX) and glutathione reductase (GR), as well as glutathione, are important components of the ascorbate–glutathione cycle responsible for the removal of H2O2 in different cellular compartments (Jiménez et al., 1997). Glutathione is also the substrate for the biosynthesis of phytochelatins, which are involved in heavy metal detoxification (Zenk, 1996).
In animal tissues it has been demonstrated that cadmium induces changes of the antioxidant status either by increasing superoxide radical production and lipid peroxidation, or by decreasing the enzymatic and non-enzymatic antioxidants (Stochs and Bagchi, 1995). However, less information is available in plants. In Phaseolus vulgaris, Phaseolus aureus, and Helianthus annuus the toxicity of Cd has been related with the increase of lipid peroxidation and alterations in antioxidant systems (Somashekaraiah et al., 1992; Shaw, 1995; Gallego et al., 1996).
In this work the effect of growing pea plants with CdCl2 on different physiological parameters and enzymatic antioxidants was studied in order to know the possible involvement of cadmium in the generation of oxidative stress.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant material and growth conditions
Pea (Pisum sativum L., cv. Lincoln) plants were obtained from Royal Sluis (Enkhuizen, Holland). Plants were grown in the greenhouse in aerated full nutrient media under optimum conditions (del Río et al., 1985
) for 14 d, and then media were supplemented with different CdCl2 concentrations (0, 10, 20, 30, 40, and 50 µM) and plants were grown for 28 d. Four replicates for each treatment were prepared to give a total of 24 pots. Leaves (type medium and old) were washed and homogenized in 50 mM Tris-HCl buffer (pH 7.5), containing 0.1 mM EDTA, 2 mM DTT, 0.2% (v/v) Triton X-100, and 1 mM PMSF (1/4, w/v). Homogenates were centrifuged at 27 000 g for 20 min and the supernatants were used for protein and enzymic determinations.
Enzymic assays
Total SOD activity (EC 1.15.1.1) was assayed according to the ferricytochrome c method (McCord and Fridovich, 1969). SOD isoenzymes were individualized by native-PAGE on 10% acrylamide gels and were localized by a photochemical method (Beauchamp and Fridovich, 1971). Catalase activity (EC 1.11.1.6) was determined as described previously (Aebi, 1984) and native-PAGE was carried out on 6% acrylamide gels. Catalase activity was localized in the gels as described earlier (Woodbury et al., 1971). Catalase isozymes were also analysed by isoelectric focusing (IEF) in 5% polyacrylamide gels in the pH range 5–7 (Pharmacia Biotech ampholites) using a Mini Protean apparatus (Bio-Rad) as described previously (Zelitch et al., 1991). Catalase activity in the gels was localized as described previously (Clare et al., 1984). Guaiacol peroxidase activity was determined according to Quessada and Macheix (Quessada and Macheix, 1984), and glutathione reductase activity was assayed as described by Jiménez et al. (Jiménez et al., 1997).
Western blotting
Proteins were separated by SDS-PAGE in a Mini-Protean II slab cell (Bio-Rad) in 15% (w/v) separating gels and 4% stacking (w/v) gels and transferred onto PVDF membranes. Electrophoretic transfer was carried out in a Bio-Rad Semi-dry Transfer Cell according to Corpas et al. (Corpas et al., 1998). Proteins were detected with antibodies against catalase from cucumber (Yamaguchi and Nishimura, 1984), glutathione reductase from pea (Edwards et al., 1990) and CuZn-SOD from watermelon (Bueno et al., 1995). Goat anti-rabbit IgG with alkaline phosphatase was used as the second antibody. Reactivity was detected with BCIP/NBT (Corpas et al., 1998).
Macronutrient and micronutrient determination
Samples of leaves (type medium and old) and roots were washed and then mineralized with perchloric acid (AOAC, 1984). Metal concentrations (µg g-1 DW) were estimated by atomic absorption spectrometry (CIIAF, 1973). For macronutrient determination (g g-1 DW), samples were digested with sulphuric acid and H2O2 using an open-focused microwave system. N and P were determined by colorimetry, Ca and Mg by atomic absorption spectrometry, and Na and K by flame photometry (CIIAF, 1973). Results were expressed as total content or uptake of the element calculated as the concentration of each element per dry weight of root or leaf, and for each treatment.
Light and electron microscopy
For microscopy studies, leaves of control and 50 µM Cd-treated plants were cut in 1 mm2 segments and fixed in 2.5% glutaraldehyde solution in 50 mM potassium phosphate (pH 6.8) for 2.5 h at room temperature. Samples were post-fixed for 30 min in 1% OsO4 in 50 mM sodium cacodylate (pH 7.2), dehydrated in a graded ethanol series (30–100%; v/v), and embedded in Spurr resin. For light microscopy, semithin sections were stained with methylene blue, and for electron microscopy ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Zeiss EM 10C transmission electron microscope. For scanning electron microscopy, samples were fixed in 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2), dehydrated in a graded ethanol series (30–100%, v/v), and were critical-point-dried through carbon dioxide, mounted on stubs, and coated with gold. The material was observed at 20 KV in a DM950 Carl Zeiss scanning electron microscope.
Other assays
The photosynthesis rate was assayed according to Coombs et al. (Coombs et al., 1985). Transpiration rate, and water use efficiency were determined as described earlier (Ludlow and Muchow, 1990). A portable integrated infrared carbon dioxide analyser (ADC-LCA3) was used for these determinations. Lipid peroxidation was determined by measuring the concentration of thiobarbituric acid-reacting substances (TBARS) as described previously (Buege and Aust, 1972). Carbonyl-groups were assayed using the dinitrophenyl hydrazine method (Levine et al., 1991). Proteins were determined according to Bradford (Bradford, 1976) using BSA as standard, and chlorophyll was measured as described by Arnon (Arnon, 1949). Data were subjected to one-way analysis of variance for each parameter. When the effect was significant (P<0.05), differences between means were evaluated for significance by using Duncan's multiple-range test (P<0.05).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Effect of Cd treatment on plant growth
Increasing concentrations of Cd in the nutrient solution produced a significant growth inhibition of pea plants, measured as dry weight (Table 1
), the greatest adverse effect being on leaves while root growth was only significantly affected by 50 µM CdCl2 (Table 1). The decrease in dry weight of leaves was parallel to a reduction in the leaf area (Table 1) but no visible symptoms of toxicity, except growth reduction, were observed. The decrease of root growth with 50 µM Cd was characterized by a reduction of lateral roots. The effect of higher Cd concentrations ranged between 75 to 150 µM was also studied, but plants were considerably damaged with 75 µM (data not shown), for this reason the study was focused on 50 µM as the highest Cd concentration.
|
The growth inhibition of pea plants was accompanied by a significant decrease in the photosynthesis rate, which was about six times reduced at the highest Cd concentration in comparison with control plants (Table 2
). The chlorophyll content was also affected by Cd, showing a reduction which was proportional to the Cd concentration in the nutrient solution (Fig. 1). The transpiration rate and water use efficiency were also affected by Cd treatment, undergoing a significant and progressive decrease with increasing Cd concentrations in the nutrient solution (Table 2).
|
|
Nutrient distribution
The nutrient composition of some elements in roots and leaves was modified by the Cd treatment (Table 3). Cadmium was mainly accumulated in roots and there was a linear relationship between metal concentration in nutrient solutions and metal content in leaves and roots (Table 3). The content of Zn in leaves was decreased very significantly as a result of Cd treatment, while in roots an increase was observed, although differences were not statistically significant. The content of Fe did not change significantly in roots while in leaves a significant decrease was detected only with 50 µM Cd. The content of Mn in leaves decreased very significantly, but in roots a significant decrease was only detected with 50 µM Cd. In its turn, Cu content did not show significant changes in roots, but in leaves a significant reduction was determined with 50 µM Cd. The contents of macronutrients were also altered by Cd in both leaves and roots (Table 4). The content of Ca were significantly reduced in leaves but did not show significant changes in roots, while the content of Mg was significantly reduced in roots and leaves. The content of N was diminished by the Cd treatment in leaves but in roots a slight increase was detected. The contents of P and K showed a significant reduction by the Cd treatment in leaves while in roots decreases in both macronutrients were only significant at 40 and 50 µM Cd (Table 4).
|
|
Structural and ultrastructural studies
As a consequence of Cd treatment a reduction in the intercellular spaces of pea leaves was observed, as well as a statistically significant increase in the size of cells, specially of the pallisade cells, and a reduction in the number of chloroplasts (Fig. 2A, C; Table 5). The analysis by EM showed disturbances in the cell structure of Cd-treated plants, characterized by a disruption of chloroplast ultrastructure (Fig. 2B, D). Chloroplasts from Cd-treated plants showed disorganized thylakoid systems, and an increase in the size and number of plastoglobuli as well as in the size of starch grains (Fig. 2B, D). Invaginations of the tonoplast into the vacuole and nucleus condensations were also observed in cells from Cd-treated plants, as well as the appearance of mielin bands (Fig. 2E, F, G).
|
|
The analysis by SEM of the abaxial side of pea leaves showed stomatal closure in plants treated with 50 µM Cd, while in control plants most stomata were open (Fig. 3
).
|
Effect of Cd on activated oxygen metabolism
As an indicator of lipid peroxidation the content in thiobarbituric acid-reacting substances (TBARS) was measured. In general, increasing concentrations of cadmium caused a linear enhancement of TBARS (Fig. 4). The increase in the content of carbonyl-groups can be considered as evidence of the oxidative modification of proteins and as an oxidative stress index. Cadmium concentrations of 10–30 µM slightly increased the content of carbonyl-groups in crude extracts of leaves compared with control plants, but statistically significant increases were observed with 40 and 50 µM Cd (Fig. 4). In leaves, the activity of the antioxidative enzymes catalase and guaiacol peroxidase was depressed with increasing concentrations of Cd, although differences from controls were only statistically significant for catalase (Fig. 5). Glutathione reductase activity did not show statistically significant changes with Cd, either in activity (Fig. 5) or in protein level measured by immunoblot (data not shown), although an increase could be observed with 10 µM Cd. The analysis of catalase activity by native-PAGE showed only one widespread band of activity which was slightly decreased with increasing concentrations of cadmium (Fig. 6A). However, IEF analysis of catalase showed three bands of activity (CAT-1 to CAT-3) and the most acidic isoforms (CAT-2 and CAT-3) were enhanced by low Cd concentrations (10–30 µM Cd), but depressed with the highest concentrations of Cd (Fig. 6B). Analysis by immunoblot with the antibody against catalase from cucumber demonstrated a Cd concentration-dependent reduction in the protein content measured by densitometric scan (Fig. 6C).
|
|
|
When the total superoxide dismutase activity was measured in leaf homogenates by the cytochrome c method, interferences in the assay were found. Thus, samples had the ability to reduce cytochrome c in the absence of the O2·- generator, xanthine oxidase, and the reduction rate increased with the Cd concentration in the nutrient solution up to 2.4 times at 50 µM Cd (data not shown). For this reason, the results of total SOD activity were not considered reliable. When the pattern of SOD isoenzymes was analysed by native-PAGE a considerable decrease in the activity of all isoenzymes was detected even at a 10 µM Cd concentration (Fig. 7A
). CuZn-SODs were the isozymes more sensitive to the Cd treatment, specially the cytosolic CuZn-SOD I, whose activity decreased by about 80% at 10 µM Cd and almost disappeared at 40 µM Cd (Fig. 7A). The chloroplastic isoform (CuZn-SOD II) lost 60% of its activity at 10 µM Cd and more than 95% at 50 µM Cd. Fe-SOD was more resistant than CuZn-SODs although its activity also showed a progressive reduction with the Cd concentration in the nutrient solution. Mn-SOD was the most resistant isoform to the Cd treatment and was only reduced by 50% at 40 µM Cd. To determine if the observed reduction in the SOD activities was due to a direct interaction of Cd with the enzymes, incubation of commercial CuZn-SOD and pea leaf extracts with 100 µM Cd for 24 h and 72 h was carried out. In these conditions, no effect by Cd on the CuZn-SOD activity was detected even after 72 h incubation (data not shown). The analysis by immunoblot with the antibody against CuZn-SOD from watermelon showed a Cd concentration-dependent reduction in the protein content of both Cu,Zn-SOD isoforms measured by densitometric scanning (Fig. 7B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The aim of this work was to evaluate the effect of Cd on the growth and activated oxygen metabolism of pea plants. Cadmium produced a significant reduction in the growth of pea plants expressed as dry weight. This effect was mainly observed in leaves while roots were apparently only damaged by the highest Cd concentrations (50 µM Cd) the most affected part being the lateral roots. Higher Cd concentrations (75–150 µM) were also assayed but the plant material was severely damaged (data not shown). For this reason 0–50 µM Cd was chosen to carry out the study. The lower sensitivity of roots to Cd could be explained by a capacity to accumulate the metal in a non-active form and to the mobility of Cd within the plant, which facilitates its transport to the aerial part of the plant (Barceló et al., 1988
). On the other hand, no visual symptoms of Cd toxicity were observed in pea leaves, except for the reduction of growth, while in other plant species symptoms similar to those of ferric chlorosis have been described (Ouzounidou et al., 1997).
The growth inhibition produced by Cd could be due mainly to the effect of this heavy metal on the photosynthesis rate, which was reduced 6-fold by 50 µM Cd. This reduction could be due in part to the decrease in chlorophyll content produced by the Cd treatment. In fact, in other plant species the degradation of chlorophyll or the inhibition of its biosynthesis, has been proposed as being responsible for the photosynthesis and growth reduction produced by this metal (Somashekaraiah et al., 1992; Bazzaz et al., 1992). The ultrastructural changes observed in the chloroplasts of Cd-treated plants suggest important disturbances in the metabolic functions of these organelles. Some enzymes of the photosynthetic carbon reduction cycle are sensitive to Cd and could also be responsible for the productivity decrease (De Filippis and Ziegler, 1993). Photosynthesis is also sensitive to disturbances in gas exchange through the stomata and, as shown by the scanning electron microscopy results, Cd stimulates the stomata to close. As result, leaf transpiration rate was significantly decreased by Cd treatment. However, the effects of Cd on transpiration rate are complex and apparently depend on metal concentration, plant species and period of treatment. In this way, Cd produced a decrease in transpiration in Picea abies (Schlegel et al., 1987), while the opposite effect has been observed in Beta vulgaris plants (Greger and Johansson, 1992). Cd also produced disturbances in water balance judging by the observed reduction in the water use eficiency parameter, probably by inhibiting the absorption and translocation of water, as observed in other plant species (Barceló et al., 1988). The water movement in the plant could be affected by a reduction in the size and number of xylem vessels imposed by the metal toxicity, and also by alterations of hormone balance (Poschenrieder and Barceló, 1999).
Pea plants accumulate Cd mainly in roots, although the capacity to accumulate this metal depends on the Cd concentration in the nutrient solution. In the presence of 10 µM Cd, roots accumulate the metal more efficiently than in the presence of higher Cd concentrations in term of concentration (µg Cd g-1 DW), but when the results are expressed as uptake (Cd concentration g-1 DW) Cd accumulation in roots was linear between 0–30 µM Cd, reaching a plateau between 30–50 µM Cd. Roots can accumulate Cd in the apoplast, by ionic interactions with carboxyl and/or sulphhydryl groups from components of the cell wall, and part of the metal can be complexed by phytochelatins and sequestered in the vacuole (Cohen et al., 1998). Although in this work the apoplastic accumulation of Cd has not been determined, Lozano-Rodriguez et al. have reported that pea plants accumulate Cd mainly in the soluble fraction and apparently this distribution is also found in over 50% of leguminous plants (Lozano-Rodriguez et al., 1997). Cohen et al. have also reported in the same plant specie a linear component in the Cd influx associated with the apoplast, and a second one which is saturable and is associated with transporter-mediated Cd influx across the root plasma membrane (Cohen et al., 1998).
The nutritional status of leaves and roots was altered by Cd. The presence of Cd in the nutrient solution produced a decrease in the uptake of Zn and Mn and to a lesser extent of Cu and Fe in the leaf, while in roots the uptake of Mn was only reduced with 50 µM Cd, and no significant changes were observed for the rest of micronutrients. These results suggest that Cd interferes mainly with the translocation of micronutrients to the leaves although Mn uptake was also depressed in roots. The differential uptake of one metal in plant tissue is not well known and different transporters are involved in the excretion of metal into the xylem and finally into the leaves. Cd can inhibit the transporters involved in the translocation into the aerial part of the plant at different levels, such as radial movement in the root, loading into the xylem vessels or absorption to the leaf. Metal translocation is also dependent on the production of phytochelatins which have a variable affinity for specific metals and could determine the metal translocation to the shoot. Morphological changes of the conducting xylem tissue may also contribute to a limited translocation of nutrients from the roots (Barceló et al., 1988). The influx of Mn has also been dramatically affected by Cd in other plant species (Hernández et al., 1998; Yang et al., 1996). The effect of Cd on the Fe content is apparently different depending on the plant species and the experimental conditions. Cd-induced Fe deficiency has been reported in sugar beet (Greger and Lindberg, 1987), maize (Siedlecka and Baszinski, 1993), and birch plants (Gussarson et al., 1996), while the opposite effect was described in bean (Chaoui et al., 1997) and tomato plants (Moral et al., 1994). Hernández et al. have reported that the uptake of Fe was little affected in pea plants grown in 50 µM Cd in long-term experiments, while it was reduced in short-term experiments (Hernández et al., 1998). Birch plants grown in the presence of Cd showed a reduction in the concentration of Fe, Mn and Cu in the shoot, while in roots no significant changes were observed, and the content of Zn did not change either in shoots or in roots (Gussarson et al., 1996). Several factors can determine the transport of metals under Cd treatment: changes in H+-ATPase activity, changes in IRT1 transporter selectivity, or modifications of ferric reductase activity, among others. The activity of H+-ATPase in sunflower and wheat roots was reduced in the presence of Cd and this change could be associated with modifications of the membrane constituents (Fodor et al., 1995), however, in pea plants under Fe deficiency H+-ATPase activity was not associated with an increase in Cd accumulation in the root (Cohen et al., 1998). The IRT1 is a Fe transporter that can also transport Mn, Zn and Cd (Korshunova et al., 1999) and its ion selectivity can change depending on the pH in the nutrient solution (Korshunova et al., 1999). Rogers et al. have reported the inhibition of Fe and Zn uptake in Arabidopsis plants by an excess of cadmium (Rogers et al., 2000). With reference to ferric reductase, several authors have proposed that it regulates ion channel gating through the control of specific sulphydryl groups from ion transporters (Welch et al., 1993). In this work Fe was supplied as EDTA-Fe(III) which has to be reduced to Fe(II) by the reductase, and any modification of its activity by Cd could limit the availability of Fe to the plant.
In general, the concentration of macronutrients was severely reduced in leaves while in roots changes were only significant at 40 and 50 µM Cd. Similar results have been observed in birch (Gussarson et al., 1996) and sugar beet plants (Greger and Lindberg, 1987). It is known that the contents of polyvalent cations can be affected by the presence of Cd through antagonism processes mediated either by competition for binding sites or by transporters (Gussarson et al., 1996). The accumulation of K can also be affected by Cd-dependent modification of ATPases or ferric reductase activities, as described for birch (Gussarson et al, 1996) and pea plants (Welch et al., 1993), respectively.
Cadmium produced an enhancement of TBA-reacting substances in pea leaves (Fig. 4
) which is an index of lipid peroxidation and, therefore, of oxidative stress. Similar increases of TBARS by Cd exposure have been observed in Phaseolus vulgaris (Somashekaraiah et al., 1992; Shaw, 1995; Chaoui et al., 1997), Helianthus annuus (Gallego et al., 1996), and Pisum sativum plants (Lozano-Rodriguez et al., 1997). The peroxidation of cell membranes severely affects its functionality and integrity and can produce irreversible damage to the cell function (Halliwell and Gutteridge, 1989). Lipid peroxidation can be initiated by activated oxygen species such as O2 ·-, ·OH or 1O2 or by the action of lipoxygenase (Halliwell and Gutteridge, 1989). Cd is not a redox metal, like Cu and Fe, and therefore cannot catalyse Fenton-type reactions yielding activated oxygen species. However, Cd can induced oxidative stress indirectly by producing disturbances in chloroplasts. Thus, Cd produces degradation of chlorophyll and carotenoids as well as an inhibition of their biosynthesis (Somashekaraiah et al., 1992; Bazzaz et al., 1992) which can produce disturbances in the electron transport rates of PSI and PSII leading to the generation of oxygen free radicals. Metal deficiency imposed by Cd could promote oxygen free radical production by affecting the water splitting complex or electron transport. The depletion of GSH, by the formation of a Cd–phytochelatin complex, could also contribute to oxidative stress by depressing the antioxidative response (Gallego et al., 1996).
Another possible target of oxygen radicals are proteins. The functionality of proteins can be affected by reactive oxygen species either by oxidation of amino acid side chains or by secondary reactions with aldehydic products of lipid peroxidation (Reinheckel et al., 1998). Both primary and secondary reactions can introduce carbonyl-groups into proteins, and the appearance of such groups is taken as evidence of oxidative stress. In this work, the growth of pea plants with increasing Cd concentrations produced an enhancement of carbonyl-groups content in leaf extracts although, in contrast with the lipid peroxidation rate, differences were only statistically significant at the highest Cd concentrations (40 and 50 µM).
The level of oxygen free radicals in cells can also be enhanced by a decrease of the enzymatic antioxidants involved in their detoxification, such as SOD, CAT, GR or peroxidases. Catalase activity of pea leaves was reduced by increasing concentrations of Cd in the nutrient solutions. The decline of catalase activity has been associated with Cd toxicity in Phaseolus vulgaris (Somashekaraiah et al., 1992; Chaoui et al., 1997), Phaseolus aureus (Shaw, 1995), Helianthus annuus (Gallego et al., 1996), and Secale cereale (Streb et al., 1993). However, a direct effect of Cd on catalase activity was discarded because the incubation of commercial catalase with 100 µM Cd did not produce any changes in CAT activity measured by native-PAGE (data not shown). The immunoblot analysis with antibodies against catalase demonstrated that the reduction of catalase activity in pea plants was due to a decrease in the protein content. Catalase is associated with peroxisomes and these organelles contain proteases, some of which are induced by senescence, catalase being a target of the peroxisomal protease activity (Distefano et al., 1999). One the other hand, Streb et al. have described the photoinactivation of catalase from rye leaves under different stress conditions, including toxic Cd concentrations (Streb et al., 1993). The possibility of Cd-dependent changes in the transcriptional regulation of CAT can not be ruled out and is under study in the author's laboratory.
Analysis of guaiacol peroxidase showed a decline in this activity by Cd, although differences were only significant at the highest Cd concentrations (40 and 50 µM). Under these conditions, where CAT and peroxidases are diminished, the cell is not fully competent to remove H2O2 which would accumulate to toxic levels. Glutathione reductase a key enzyme of the ascorbate–glutathione cycle did not show statistically significant changes with Cd treatment althought a slight increase was observed with 10 µM Cd. Low levels of Cd produced an increase of GR activity in Alyssum argenteum, considered as a metal-hyperaccumulator plant, while this activity was reduced at higher Cd concentrations (Schickler and Caspi, 1999).
Superoxide dismutase is a key enzyme in protecting cells against oxidative stress. This enzyme catalyses the dismutation of O2 ·- to H2O2 and O2. When SOD was analysed by native-PAGE a considerable decrease in the activity of all isoenzymes present in leaves was detected. CuZn-SOD was the isoform more sensitive to Cd exposure, specially the cytosolic isoform, followed by the chloroplastic one (Fig. 5A). Fe-SOD was also affected by the treatment while Mn-SOD, located in the mitochondria and peroxisomes was the most resistant isoform. The analysis by Western blot with the anti-CuZn-SOD demonstrated a linear reduction in the protein content of CuZn-SOD in response to increasing of Cd concentrations in the nutrient solution. The incubation of CuZn-SOD from bovine erythrocytes and leaf extracts with 100 µM Cd demonstrated that this metal has no direct effect on the SOD activity, even after 72 h incubation. Fe-SOD and CuZn-SOD are both sensitive to H2O2 whereas Mn-SOD is resistant to this oxidant. This suggests that both isoforms could be inactivated by an excess of H2O2 produced in different cell compartments as a result of Cd toxicity. The oxidized protein could be selectively cleaved by proteases induced by oxidative stress, as discussed previously for catalase activity (Distefano et al., 1999). However, translational or post-translational modifications of SODs can not be discarded. The reduction of Cu, Zn, Fe, and Mn uptake in leaves by Cd treatment could affect the synthesis of the isozymes containing those metals (del Río et al., 1991). A Cd-dependent reduction of SOD activity has also been observed in Phaseolus vulgaris (Somashekaraiah et al., 1992), Helianthus annus (Gallego et al., 1996), and different yeast strains (Romandini et al., 1992), while an enhancement in SOD activity in response to moderate Cd concentrations has been reported in Lemna plants (Srivastave and Tel-Or, 1991).
Results obtained in this work suggest that CdCl2 induces a concentration-dependent oxidative stress situation in pea leaves characterized by an accumulation of lipid peroxides, oxidized proteins and, possibly, H2O2 as a result of the inhibition of the antioxidative enzymes catalase and peroxidase. In a recent work it was demonstrated that Cd produces about twice the amount in the H2O2 level in peroxisomes purified from pea plants exposed to 50 µM Cd (Romero-Puertas et al., 1999), and Piqueras et al. have detected a fast generation of H2O2 in tobacco cell cultures in response to 5 mM Cd, as well as an increased peroxidation of lipids and in the activities of SOD and APX (Piqueras et al., 1999).
The oxidative deterioration is considered an intrinsic feature of the senescence process of leaves (del Río et al., 1998). Senescence is also characterized by a cessation of photosynthesis, disintegration of organelle structures, increases in lipid peroxidation and membrane leakiness, as well as other processes (Buchanan-Wollaston, 1997). In senescent leaves, chloroplasts show an increase in the number and size of plastoglobuli, as well as disturbances in their membrane structure (Smart, 1994). Other events that take place during senescence include a reduction in the size of chloroplasts, invagination of the tonoplast into the vacuole, chromatin condensation in the nucleus, and loss of cytoplasmic components (Inada et al., 1998). All these symptoms have been observed in this work in pea plants growing under 50 µM CdCl2, and suggest that Cd toxicity induces leaf senescence.
In conclusion, the growth of pea plants with CdCl2 can induce a concentration-dependent oxidative stress situation in leaves as well as leaf senescence. The generation of oxidative stress could be a characteristic of the mechanism of Cd toxicity in plants. However, more information is needed at the subcellular and molecular levels in order to get deeper insights into the mechanistic explanations of Cd toxicity.
|
Acknowledgments |
|---|
HC Dalurzo and MC Romero-Puertas acknowledge a fellowship from ICI and Junta de Andalucía (Spain), respectively. The authors acknowledge the Center of Scientific Instrumentation of the University of Granada for technical assistance in the EM analysis. The generous donation of antibodies against catalase from cucumber by Dr Mikio Nishimura (National Institute for Basic Biology, Okazaki, Japan) and glutathione reductase from pea by Dr Philip M Mullineaux (John Innes Centre, Norwich, UK) is appreciated. The skilful technical assistance of Mr Carmelo Ruiz is also acknowledged. This work was supported by grants PB98-0493-01 and IFD97-0889-01 from the DGESIC (Ministry of Education and Culture) and by the Junta de Andalucía (Research Group CVI 192), Spain.
|
Notes |
|---|
3 To whom correspondence should be addressed. Fax: +34 958 129600. E-mail: lmsanda{at}eez.csic.es
|
Abbreviations |
|---|
APX, ascorbate peroxidase; BSA, bovine serum albumin; CAT, catalase; DTT, dithiothreitol; EM, electron microscopy; GR, glutathione reductase; IEF, isoelectric focusing; LSD, least significant diference; MDA, malondialdehyde; PAGE, polyacrylamide gel electrophoresis; PMSF, phenyl methylsulphonyl fluoride; SEM, scanning electron microscopy; SOD, superoxide dismutase; TBARS, thiobarbituric acid reacting substances..
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Aebi H. 1984. Catalase in vitro. Methods in Enzymology 105, 121–126.[ISI][Medline]
AOAC. 1984. Official methods of analysis, 14th edn. Willians S, ed. Arlington, VI, USA: Association of Official Analytical Chemists.
Arnon DI. 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology 24, 1–15.
Barceló J, Vázquez MD, Poschenrieder Ch. 1988. Structural and ultrastructural disorders in cadmium-treated bush bean plants. The New Phytologist 108, 37–49.
Bazzaz FA, Rolfe GL, Carlson RW. 1992. Effect of cadmium on photosynthesis and transpiration of excised leaves of corn and sunflower. Physiologia Plantarum 32, 373–377.
Beauchamp C, Fridovich I. 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry 44, 276–287.[ISI][Medline]
Buege JA, Aust SD. 1972. Microsomal lipid peroxidation. Methods in Enzymology 52, 302–310.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.[ISI][Medline]
Buchanan-Wollaston V. 1997. The molecular biology of leaf senescence. Journal of Experimental Botany 48, 181–199.
Bueno P, Varela J, Giménez-Gallego G, del Río LA. 1995. Peroxisomal copper, zinc superoxide dismutase. Characterization of the isoenzyme from watermelon cotyledons. Plant Physiology 108, 1151–1160.[Abstract]
Chaoui A, Mazhoudi S, Ghorbal MH, El Ferjani E. 1997. Cadmium and zinc induction of lipid peroxidation and effects on antioxidant enzymes activities in bean (Phaseolus vulgaris L.). Plant Science 127, 139–147.
CIIAF. 1973. Méthodes de reférence pour le determination dés elements mineraux dans les vegetaux. Oleagineux 28, 87–92.
Clare DA, Duong MN, Darr D, Archibald F, Fridovich I. 1984. Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase. Analytical Biochemistry 140, 532–537.[ISI][Medline]
Cohen CK, Fox TC, Garvin DF, Kochian LV. 1998. The role of iron-deficiency stress responses in stimulating heavy-metal transport in plants. Plant Physiology 116, 1063–1072.
Coombs J, Hall DO, Long SP, Scurlock JMO. 1985. In: Techniques in bioproductivity and photosynthesis, 2nd edn. Oxford: Pergamon Press, 62–93.
Corpas FJ, Barroso JB, Sandalio LM, Distefano S, Palma JM, Lupiáñez JA, del Río LA. 1998. A dehydrogenase-mediated recycling system of NADPH in plant peroxisomes. Biochemical Journal 330, 777–784.
De Filippis LF, Ziegler H. 1993. Effect of sublethal concentrations of zinc, cadmium and mercury on the photosynthetic carbon reduction cycle of Euglena. Journal of Plant Physiology 142, 167–172.
Distefano S, Palma JM, McCarthy I, del Río LA. 1999. Proteolytic cleavage of plant proteins by peroxisomal endoproteases from senescent pea leaves. Planta 209, 308–313.[ISI][Medline]
Edwards EA, Rawsthorne S, Mullineaux PM. 1990. Subcellular distribution of multiple forms of glutathione reductase in leaves of pea (Pisum sativum L.). Planta 180, 278–284.
Fodor A, Szabó-Nagy A, Erdei L. 1995. The effects of cadmium on the fluidity and H+-ATPase activity of plasma membrane from sunflower and wheat roots. Journal of Plant Physiology 14, 787–792.
Gallego SM, Benavides MP, Tomaro ML. 1996. Effect of heavy metal ion excess on sunflower leaves: evidence for involvement of oxidative stress. Plant Science 121, 151–159.
Greger M, Johansson M. 1992. Cadmium effects on leaf transpiration of sugar beet (Beta vulgaris). Physiologia Plantarum 86, 465–473.
Greger M, Lindberg S. 1987. Effects of Cd and EDTA on young sugar beet (Beta vulgaris). II. Net uptake and distribution of Mg, Ca and Fe(II)/Fe(III). Physiologia Plantarum 69, 81–86.
Greger M, Ögren E. 1991. Direct and indirect effects of Cd2+ on photosynthesis in sugar beet (Beta vulgaris). Physiologia Plantarum 83, 129–135.
Gussarson M, Asp H, Adalsteinsson S, Jensén P. 1996. Enhancement of cadmium effects on growth and nutrient composition of birch (Betula pendula) by buthionine sulphoximine (BSO). Journal of Experimental Botany 47, 211–215.
Halliwell B, Gutteridge JMC. 1989. Free radicals in biology and medicine, 2nd edn. Oxford: Clarendon Press.
Hernández LE, Lozano-Rodriguez E, Gárate A, Carpena-Ruiz R. 1998. Influence of cadmium on the uptake, tissue accumulation and subcellular distribution of manganese in pea seedlings. Plant Science 132, 139–151.
Inada N, Sakai A, Kuroiwa H, Kuroiwa T. 1998. Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles. Investigations by fluorescence and electron microscopy. Planta 206, 585–597.[ISI]
Jiménez A, Hernández JA, del Río LA, Sevilla F. 1997. Evidence for the presence of the ascorbate–glutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiology 114, 275–284.[Abstract]
Korshunova YO, Eide D, Clark WG, Guerinot ML, Pakrasi HB. 1999. The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range. Plant Molecular Biology 40, 37–44.[ISI][Medline]
Levine RL, Willians JA, Stadtman ER, Shacter E. 1991. Carbonyl assays for determination of oxidatively modified proteins. Methods in Enzymology 233, 346–363.
Lozano-Rodriguez E, Hernández LE, Bonay P, Carpena-Ruiz RO. 1997. Distribution of Cd in shoot and root tissues of maize and pea plants: physiological disturbances. Journal of Experimental Botany 48, 123–128.
Ludlow MM, Muchow RC. 1990. A critical evaluation of trails for improving crops yield in water-limited environments. Advances in Agronomy 43, 107–153.
McCord JM, Fridovich I. 1969. Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). Journal of Biological Chemistry 244, 6049–6055.
Moral R, Gómez I, Navarro-Pedreño J, Mataix J. 1994. Effects of cadmium on nutrient distribution, yield and growth of tomato grown in soil-less culture. Journal of Plant Nutrition 17, 953–962.
Ouariti O, Boussama N, Zarrouk M, Cherif A, Ghorbal MH. 1997. Cadmium- and copper-induced changes in tomato membrane lipids. Phytochemistry 45, 1343–1350.[ISI][Medline]
Ouzounidou G, Moustakas M, Eleftherious EP. 1997. Physiological and ultrastructural effects of cadmium on wheat (Triticum aestivum L.) leaves. Archives of Environmental Contamination and Toxicology 32, 154–160.[ISI][Medline]
Piqueras A, Olmos E, Martínez-Solano JR, Hellín E. 1999. Cd-induced oxidative burst in tobacco BY2 cells: time-course, subcellular location and antioxidant response. Free Radical Research 31, Supplement, S33–S38.
Poschenrieder Ch, Barceló J. 1999. Water relations in heavy metal stressed plants. In: Prasad MNV, Hagemeyer J, eds. Heavy metal stress in plants: from molecules to ecosystems. Heidelberg: Springer-Verlag, 207–230.
Quessada MP, Macheix JJ. 1984. Caractérisation d’une peroxidase impliqué specifiquement dans la lignification, en relation avec l'incompatibilité au greffage chez l'abricotier. Physiologie Végetale 22, 533–540.
Reinheckel T, Noack H, Lorenz S, Wiswedel I, Augustin W. 1998. Comparison of protein oxidation and aldehyde formation during oxidative stress in isolated mitochondria. Free Radical Research 29, 297–305.[ISI][Medline]
del Río LA, Pastori GM, Palma JM, Sandalio LM, Sevilla F, Corpas FJ, Jiménez A, López-Huertas E, Hernández JA. 1998. The activated oxygen role of peroxisomes in senescence. Plant Physiology 116, 1195–1200.
del Río LA, Sandalio LM, Yáñez J, Gómez M. 1985. Induction of a manganese-containing superoxide dismutase in leaves of Pisum sativum L. by high nutrient levels of zinc and manganese. Journal of Inorganic Biochemistry 24, 25–34.
del Río LA, Sevilla F, Sandalio LM and Palma JM. 1991. Nutritional effect and expression of SODs: induction and gene expression; diagnostics; prospective protection against oxygen toxicity. Free Radical Research Communications 12–13, 819–827.
Rogers EE, Eide DJ, Guerinot ML. 2000. Altered selectivity in an Arabidopsis metal transporter. Proceedings of the National Academy of Sciences, USA 97, 12356–12360.
Romandini P, Tallandini L, Beltramini M, Salvato B, Manzano M, De Bertoldi M, Rocoo GP. 1992. Effects of copper and cadmium on growth, superoxide dismutase and catalase activities in different yeast strains. Comparative Biochemistry and Physiology 103C, 255–262.
Romero-Puertas MC, McCarthy I, Sandalio LM, Palma JM, Corpas FJ, Gómez M, del Río LA. 1999. Cadmium toxicity and oxidative metabolism of pea leaf peroxisomes. Free Radical Research 31, Supplement, S25–S32.
Schickler H, Caspi H. 1999. Response of antioxidative enzymes to nickel and cadmium stress in hyperaccumulator plants of the genus Alyssum. Physiology Plantarum 105, 39–44.
Schlegel H, Godbold DL, Hüttermann A. 1987. Whole plant aspects of heavy metal induced changes in CO2 uptake and water relations of spruce (Picea abies) seedlings. Physiologia Plantarum 69, 265–270.
Shaw BP. 1995. Effect of mercury and cadmium on the activities of antioxidative enzymes in the seedling of Phaseolus aureus. Biology of Plants 37, 587–596.
Siedlecka A, Baszinsky T. 1993. Inhibition of electron flow around photosystem I in chloroplasts of Cd-treated maize plants is due to Cd-induced iron deficiency. Physiologia Plantarum 87, 199–202.
Smart C. 1994. Gene expression during leaf senescence. New Phytologist 126, 419–448.
Somashekaraiah BV, Padmaja K, Prasad ARK. 1992. Phytotoxicity of cadmium ions on germinating seedlings of mung bean (Phaseolus vulgaris): involvement of lipid peroxides in chlorophyll degradation. Physiologia Plantarum 85, 85–89.
Srivastave A, Tel-Or E. 1991. Effect of some environmental pollutants on the superoxide dismutase activity in Lemna. Free Radical Research Communications 12–13, 601–607.
Stochs SJ, Bagchi D. 1995. Oxidative mechanism in the toxicity of metal ions. Free Radical Biology and Medicine 18, 321–336.[ISI][Medline]
Streb P, Michael-Knauf A, Feierabend J. 1993. Preferential photoinactivation of catalase and photoinhibition of photosystem II are common early symptoms under various osmotic and chemical stress conditions. Physiologia Plantarum 88, 590–598.
Wagner GJ. 1993. Accumulation of cadmium in crop plants and its consequences to human health. Advances in Agronomy 51, 173–212.
Welch RM, Norwell WA, Schaefer SC, Shaff JE, Kochian LV. 1993. Induction of iron(III) and copper(II) reduction in pea (Pisum sativum L.) by Fe and Cu status: does the root-cell plasmalemma Fe(III)-chelate reductase perform a general role in regulating cation uptake? Planta 190, 555–561.
Woodbury W, Spencer AK, Stahmann MA. 1971. An improved procedure using ferricyanide for detecting catalase isozymes. Analytical Biochemistry 443, 301–305.
Yamaguchi J, Nishimura M. 1984. Purification of glyoxysomal catalase and immunochemical comparison of glyoxysomal and leaf peroxisomal catalase in germinating pumpkin cotyledons. Plant Physiology 74, 261–267.
Yang X, Baligar VC, Martens DC, Clark RB. 1996. Cadmium effects on the influx and transport of mineral nutrients in plant species. Journal of Plant Nutrition 19, 643–656.
Zelitch I, Havir EA, McGonigle B, McHale NA, Nelson T. 1991. Leaf catalase mRNA and catalase-protein levels in a high-catalase tobacco mutant with O2-resistant photosynthesis. Plant Physiology 97, 1529–1595.
Zenk MH. 1996. Heavy metal detoxification in higher plants. A review. Gene 179, 21–30.[ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Paradiso, R. Berardino, M. C. de Pinto, L. Sanita di Toppi, M. M. Storelli, F. Tommasi, and L. De Gara Increase in Ascorbate-Glutathione Metabolism as Local and Precocious Systemic Responses Induced by Cadmium in Durum Wheat Plants Plant Cell Physiol., March 1, 2008; 49(3): 362 - 374. |