- Email: [email protected]
Antioxidative responses of wheat treated with realistic concentration of cadmium
Environmental and Experimental Botany 50 (2003) 265 /276 www.elsevier.com/locate/envexpbot
Antioxidative responses of wheat treated with realistic concentration of cadmium M. Teresa Milone a, Cristina Sgherri a, Herman Clijsters b, Flavia NavariIzzo a,* a
Dipartimento di Chimica e Biotecnologie Agrarie, Via del Borghetto, 80-56124 Pisa, Italy b Limburgs Universitaire Centrum, Department SBG, 3590 Diepenbeek, Belgium
Received 3 December 2002; received in revised form 26 March 2003; accepted 26 March 2003
Abstract Two cvs. of wheat differently sensitive to many stress factors (cv. Ofanto less sensitive than cv. Adamello) were grown in a controlled environment with cadmium near threshold concentrations supplying the metal at equal-effect concentrations. Cd excess determined in both cvs. a reduction in water and turgor potential but a maintenance of relative water content. Cv Ofanto showed a higher capacity of Cd exclusion from roots but a higher translocation to shoots in comparison with cv. Adamello. Notwithstanding the higher metal concentration in leaves of cv. Ofanto, K leakage was more pronounced in Adamello suggesting that mechanisms of Cd detoxification and tolerance such as vacuolar compartmentalisation were activated in the first one. In Adamello plants, ethylene rose at the lowest metal concentration and the activation in roots of the antioxidative enzymes catalase, ascorbate peroxidase and guaiacol peroxidase came into play whereas in Ofanto ethylene and catalase did not change. Following cadmium treatment, superoxide dismutase activity was reduced or remained at the control value in roots and in leaves. For both cultivars ascorbate peroxidase, syringaldazine peroxidase and guaiacol peroxidase activities were always higher in roots than in leaves. These activities were induced by Cd in Ofanto leaves, whereas in Adamello leaves they remained at control levels or increased somewhat at the highest metal concentration. Cadmium changed the peroxidase isozyme pattern in both cultivars. Cv. Ofanto showed, as for other stress such as drought, salinity, nickel and copper, a co-tolerance towards Cd. Analogies in the response to other metals such as copper could be found in activation of catalase at the lower metal concentration in cv. Adamello and in the induction of ascorbate peroxidase in leaves of cv. Ofanto. # 2003 Elsevier B.V. All rights reserved. Keywords: Catalase; Ethylene; Peroxidases; Superoxide dismutase; Triticum durum
1. Introduction
* Corresponding author. Tel.: /39-050-97-1921; fax: /39050-59-8614. E-mail address: [email protected] (F. Navari-Izzo).
Cadmium is a widely spread pollutant; it can reach high levels in agricultural soils and is easily assimilated by plants. Cadmium has no known biological function and is extremely toxic, even at
S0098-8472/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0098-8472(03)00037-6
266
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
low concentration. It induces various visible symptoms of phytotoxicity, e.g. leaf roll, chlorosis and growth reduction of root and shoot. Usually inhibition of root elongation is the most sensitive parameter of Cd toxicity (Guo and Marschner, 1995). Redox metals such as Cu, Fe, Hg, etc. may directly produce free radicals (Van Assche and Clijsters, 1990), which can also be formed indirectly by non-redox metals such as Cd by mediation of lipoxygenase (LOX, EC 1.13.11.12) (Van Assche and Clijsters, 1990; Stohs and Bagchi, 1995; Navari-Izzo and Quartacci, 2001). Indeed Cd was found to induce oxidative stress in cells (Hendry et al., 1992; Somashekaraiah et al., 1992). Depending on concentration cadmium can either inhibit or stimulate the activity of several antioxidative enzymes before any visible symptom of toxicity appears. In sunflower leaves Cd decreased the activity of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1. 11. 1. 7), glutathione reductase (GR, EC 1. 6. 4. 2) and dehydroascobate reductase (DHAR, EC 1. 8. 5. 1), while LOX, which mediates lipid peroxidation, increased (Gallego et al., 1996). Upon Cd exposure lipid peroxidation raised also in pea plants (LozanoRodriguez et al., 1997), in Phaseolus aureus (Shaw, 1995) and Phaseolus vulgaris (Chaoui et al., 1997), whereas in Daucus carota no lipid peroxidation was observed (Sanita` di Toppi and Gabbrielli, 1999). Both P. vulgaris and P. aureus showed enhanced guaiacol peroxidase (GPOD, EC 1.11.1.7) activity (Shaw, 1995; Chaoui et al., 1997), but in P. aureus (Shaw, 1995) the activity of CAT decreased, as was also observed in pea plants, where a reduction in SOD and GPOD activities was also demonstrated (Sandalio et al., 2001). Although, peroxidases have been extensively studied, their physiological function remains unclear. In plants several functions are ascribed to these enzymes: auxin metabolism, lignification, wound healing and defence against pathogens. Their cellular distribution (cell walls, endoplasmic reticulum, Golgi apparatus and vacuoles), is presumibly connected with different physiological functions (Gaspar et al., 1991). An involvement
in the protection against oxidative stress, induced by metals, has been postulated. Ethylene is a natural plant growth regulator involved in the control of a wide range of developmental responses. Gora and Clijsters (1989) reported that toxic concentrations of metals stimulate ethylene production in plants. Cd has been shown to interfere specifically with ethylene biosynthesis in bean leaf tissue (Rauser, 1978). In P. vulgaris , low concentrations of Cd stimulated ethylene biosynthesis (Adams and Yang, 1979), but Cd-induced ethylene stimulation declined with increasing Cd concentration (Bhattacharjee, 1997). Cd concentrations above 1 mM stimulated ACC synthase activity and increased the activity of GPODs within 5/10 h after application, but ethylene production gradually declined to control levels. Stimulation of ACC synthase has also been found in response to mechanical wounding, IAA and Cu treatments (Fuhrer, 1982; Sgherri et al., 2001). It is not clear whether these different stimuli regulate ACC synthase through common mechanisms. Oxygen free radicals and hydroperoxides, generated by LOX, might stimulate the conversion of ACC into ethylene (Weckx et al., 1993). In the literature, the Cd concentrations studied are frequently not realistic when compared with the levels of contamination in the natural environment (Sanita` di Toppi and Gabbrielli, 1999); in addition, the metal was generally supplied to plants previously grown without Cd, which makes the experimental conditions different from those of natural crops. Two cvs. of wheat differently sensitive to drought (cv. Ofanto less sensitive than cv. Adamello) were studied for the response to other stress factors i.e. nickel, copper and NaCl. In all cases, cv. Ofanto appeared more tolerant than cv. Adamello (Quartacci et al., 1995; Pandolfini et al., 1996; Ciscato et al., 1997; Loggini et al., 1999; Meneguzzo et al., 2000; Sgherri et al., 2001). In particular, when the two cvs. were subjected to realistic concentrations of copper, the uptake of the metal was higher in the sensitive cv. Since different intracellular Cu concentrations could cause different activation or inhibition of antioxidative systems, the two cvs. were subjected to equal-effect concentrations of copper on the basis of the same root growth inhibition, which is the
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
expression of metal toxicity (Sgherri et al., 2001). The antioxidative response revealed that the leaves of the more tolerant cv. (Ofanto) responded to Cu with an induction of peroxidases, whereas in the more sensitive cv. (Adamello) there were increases in CAT (Sgherri et al., 2001). In the present study cv. Ofanto and cv. Adamello were analysed in order to establish if there is also a co-tolerance against Cd supplied at realistic concentrations. As for Cu (Sgherri et al., 2001), the two cvs. were subjected to equal-effect concentrations of Cd and also in this last case, we expect that differences in the biochemical responses occur in the two cvs. at equal-effect exposure levels. The antioxidative responses were focused studying the changes in the activities of guaiacol and syringaldazine dependent peroxidases, APOD, SOD and CAT. Ethylene emission was also monitored. Metal toxicity impairs membrane function and ion balance (Sandalio et al., 2001), and could affect plant water relations; for this reason leaf water status was also monitored.
2. Materials and methods 2.1. Plant material Two cvs of wheat (Triticum durum Desf.) differently sensitive to drought and heavy metals (Quartacci et al., 1995; Ciscato et al., 1997; Loggini et al., 1999; Sgherri et al., 2001) with cv. Ofanto less sensitive than cv. Adamello, were grown for 10 days under controlled conditions at 200 mE m 2 s 1 PAR with a 16 h photoperiod, 75% R.H. and day/night temperatures of 20/16 8C. Light was supplied by fluorescent tubes (Osram L. 140 W/20) and incandescent lamps (Philips 25 W). Seeds were surface sterilised with 2% sodium hypochlorite for 10 min, rinsed in distilled water and imbibed for 4 h with running tap water. They were sown in a beaker containing 1 l of a half strength Hoagland’s solution containing micronutrients (Hoagland and Arnon, 1950) and 400 ml sterilised perlite as inert support. Increasing concentrations of Cd, selected on the basis of a rooting test (Fig. 1), were added as Cd(SO)4 to the nutrient solution from sowing on. Metal
267
Fig. 1. Root length in two wheat cvs. treated with increasing concentrations of Cd. Each value is the mean of six replicates9/ S.E. S.E. is smaller than the size of symbols.
concentrations, giving 5 and 20% of root growth inhibition, were 0.25 and 2.2 mM for Adamello and 0.5 and 4.7 mM for Ofanto. The nutrient solution was bubbled with sterile air and changed on alternate days to avoid depletion of nutrients as well as Cd. 2.2. Cadmium analysis After harvest fresh and dry weights of the roots and leaves were measured and plant samples were thoroughly washed with sterile distilled water. Cd content of roots and leaves was determined by atomic absorption spectroscopy after wet digestion of dried material in HNO3. Metal content was also determined on roots following desorption and on desorbing solution. After a fast washing with water to remove perlite, roots were immersed in 5 mM Pb(NO3)2 for 30 min at 4 8C. 2.3. Leakage of K ions Potassium leakage was measured according to Quartacci et al. (2001). Roots and leaves of seedlings from each treatment were excised and washed twice to remove the contents of the cut cells. Samples were incubated in 25 ml of double
268
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
distilled water and shaken at 21 8C for 24 h, and aliquots for K determination were taken. Samples were then immersed for 5 min in liquid nitrogen and placed in the same vial containing the leachate and shaken for an additional hour prior to the measurements of total K leached from the killed cells. Potassium released was measured by atomic absorption spectrophotometry with a hollow-cathode lamp.
2.4. Leaf water status Leaf water status (relative water content, RWC; water potential, Cw; turgor potential, Cp; osmotic potential at full turgor, C100 p ; osmotic potential, Cp) was determined by pressure-volume curves using a pressure chamber. Curve analysis was performed as previously described (Navari-Izzo et al., 1993). Measurements of water status were carried out on ten plants.
2.5. Ethylene analysis Ethylene release was measured with a continuous air flow technique as previously described (Sgherri et al., 2001). Ethylene determination was carried out on a Intermat IGC 120 FB gas chromatograph (Courtry, France) equipped with a Porapak-R column (50 /80 mesh) and a flame ionisation detector. The beaker, containing plants with roots submerged in water, was placed in a cylindrical gas cuvette and aereated (10 l h 1) with a synthetic air mixture N2/O2 (80/20). The gas inlet ended in the water of the beaker; the outlet was located at the top of the cuvette. The cuvettes were put in a small growth chamber where a temperature of 25 8C and a light intensity of 105 mmol m 2 s 1 were maintained. To trap the ethylene produced, the gas outlet of the cuvette was connected with a cooled ( /95 8C) precolumn filled with Porapak-S (50 /80 mesh; Waters, Milford, USA). After 10 min, this column was inserted into the carrier gas stream of a gas chromatograph. The ethylene was totally released and swept into the analytical column with the carrier gas by heating the precolumn to 100 8C in boiling water.
2.6. Enzyme extraction and assays The enzyme activities were determined as described in Sgherri et al. (2001). Plant material was ground in an ice-cold mortar and for each enzyme an appropriate ice-cold extraction buffer at 4 8C was used. The homogenate was filtered through a Miracloth (Calbiochem) and centrifuged at 20 000 /g for 15 min at 4 8C. The protein contents (Lowry et al., 1951) and the enzyme assays were measured in the supernatant at 25 8C. SOD activity was assayed as previously described in Sgherri et al. (2001) in terms of the ability to inhibit the photochemical reduction of nitroblue tetrazolium. One unit of SOD is the amount of extract that gives half maximum inhibition. CAT activity was assayed polarographically with a Clark-type electrode (Hansatech, Kings Lynn, Norfolk, UK). The oxygen produced by the extract was calculated after correction for the autoproduction of oxygen from H2O2. One unit of CAT is the amount of enzyme producing 1 mM O2 min 1. POD activities were measured spectrophotometrically using as substrates ascorbate (APOD), guaiacol (GPOD) and syringaldazine (SPOD). APOD was measured at 298 nm according to Sgherri et al. (2001). One unit of APOD is the amount of enzyme oxidising 1 mmol of ascorbate min 1. GPOD was determined following Sgherri et al. (2001). One unit of GPOD is the amount of enzyme oxidising 1 mmol of guaiacol min 1. SPOD activity was determined according to Imberty et al. (1985). One unit of SPOD is the amount of enzyme oxidising 1 mmol min 1. 2.7. Electrophoretic POD separation Before loading on the gels, the protein extracts were dialysed for 24 h in 20 mM Tris /HCl, pH 7.8, concentrated with PEG and centrifuged at 12 000 /g for 15 min. The electrophoretic separation of the peroxidase bands was performed on 10% PAGE using Tris /HCl buffer 1.5 M, pH 8.8. The gels were run at 200 V for 45 min with a constant current of 35 mA per gel. Samples of leaves and roots contained 0.07 enzyme units. To visualise the band patterns the gels were incubated
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
in the dark for 90 min at 37 8C in 1 M Na-acetate buffer pH 4.6 containing 0.04% benzidine and 20 ml of hydrogen peroxide. Gels were densitometrically scanned and analysed with the SIGMA GEL software. 2.8. Statistical analysis Unless specified otherwise, results are the means of three independent replicates and repeated in at least two independent experiments. For comparison among means, performed using the Newman /Keuls test, one-way ANOVA was used. Means followed by different letters are significantly different at the P 5/0.01 level.
3. Results In the cv. Adamello 5 and 20% of root growth inhibition was observed with, respectively, 0.25 and 2.2 mM Cd in the nutrient solution, while for Ofanto the same percentages of root growth inhibition were observed with 0.5 and 4.7 mM Cd (Fig. 1). Cadmium-treated plants of cv. Adamello demonstrated a marked reduction in leaf and root fresh and dry weight whereas K leakage increased. In contrast, the dry matter of Ofanto roots and K leakage of Ofanto leaves remained unchanged with the treatment (Table 1). In comparison to the control plants, a decreasing trend in water potential was found in both
269
cultivars as the Cd concentration rose (Table 2). Osmotic potential did not change in Ofanto, while in Adamello it increased by 9% at the highest Cd concentration. In Adamello Cd did not affect the osmotic potential at full turgor, while 0.5 mM Cd induced a significant increase in Ofanto plants. Turgor potential maintained always positive values in both cultivars, but it appeared to decrease with the rise of the metal concentration. In both cultivars, the RWC remained at the control value for every Cd concentration tested (Table 2). Root length decreased proportionally with increasing Cd concentration in both cvs. but the slope was less steep for cv. Ofanto (Fig. 1). No Cd was detected in the control roots and leaves of each cultivar, grown in Cd-free conditions. When the metal level rose in the nutrient solution, however, the internal leaf and root Cd concentration also increased, as well as the amount of Cd desorbed from the roots (Fig. 2). At the lowest Cd concentration applied, the root metal uptake was similar for the two cultivars, but with increase of the external metal concentration the roots of Adamello absorbed a higher amount of Cd (Fig. 2); anyway the highest equal-effect concentration determined in roots of cv. Ofanto a higher metal concentration since the same entity of metal was desorbed (about 10%) by both cvs. In the leaves the Cd taken up was always higher in Ofanto than in Adamello (Fig. 2). The lowest Cd concentration (0.25 mM) induced an increase by 62% of the ethylene emission in
Table 1 Fresh weight, dry weight (mg) and K leakage (percent of total K ) in plants of two cultivars of wheat treated with cadmium Adamello
Ofanto
Control
0.25 mM
2.2 mM
Control
0.5 mM
4.7 mM
Leaves Fresh weight Dry weight K leakage
150.79/0.07c 11.89/0.33b 18.29/0.50c
138.99/0.41b 10.79/0.06b 25.39/0.31b
93.19/1.98a 7.69/0.15a 36.59/0.78a
118.29/0.33c 8.89/0.02c 19.39/0.61a
90.69/0.14b 7.89/0.11b 21.59/0.93a
63.09/0.01a 6.89/0.19a 25.39/0.72a
Roots Fresh weight Dry weight K leakage
100.79/0.54c 4.59/0.09c 31.59/0.80c
81.49/0.06b 3.69/0.14b 45.89/0.75b
62.19/0.67a 2.79/0.14a 60.39/1.35a
74.89/0.42c 3.39/0.12a 30.29/0.93b
57.49/0.61b 3.29/0.01a 34.39/1.45b
40.09/0.82a 2.99/0.03a 41.69/0.88a
Each value is the mean of three replicates9/S.E. Means in rows followed by different letters are significantly different at P 5/0.01.
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
270
Table 2 Water potential (Cw), osmotic potential (Cp), osmotic potential at full turgor (C100 p ) and relative water content (RWC) in plants of two cultivars of wheat treated with cadmium Adamello
Cw (MPa) Cp (MPa) C100 (MPa) p Cp (MPa) RWC (%)
Ofanto
Control
0.25 mM
2.2 mM
Control
0.5 mM
4.7 mM
/0.439/0.15b /0.999/0.01a /0.949/0.03a 0.569/0.01c 979/0.23a
/0.489/0.15ab /0.969/0.02a /0.929/0.02a 0.489/0.01b 979/0.19a
/0.529/0.10a /0.909/0.01b /0.869/0.02a 0.389/0.01a 979/0.21a
/0.379/0.10b /0.989/0.02a /0.989/0.01a 0.609/0.01c 989/0.20a
/0.499/0.14a /1.009/0.01a /0.929/0.01b 0.509/0.01b 989/0.15a
/0.499/0.14a /0.969/0.01a /0.939/0.01ab 0.469/0.01a 979/0.21a
Each value is the mean of six replicates9/S.E. Means followed by different letters are significantly different at P 5/0.01.
Adamello; at the highest metal concentration, however, ethylene emission dropped to a value comparable with that found in the control (Fig. 3). In Ofanto, Cd did not show any effect on ethylene emission as compared with the control (Fig. 3). In control as well as in Cd treated roots of both cultivars, the activities of SOD, APOD, SPOD, and GPOD were higher than in leaves (Figs. 4, 6 / 8). On the contrary, leaves exhibited higher CAT activity than roots (Fig. 5). The constitutive activities of SOD and APOD were higher in leaves of Adamello than in those of Ofanto. In control roots, cv. Ofanto showed a higher activity of
Fig. 2. Cd contents in leaves and roots of two wheat cvs. treated with increasing concentrations of Cd. Each value is the mean of six replicates9/S.E. S.E. is as for Fig. 1.
Fig. 3. Ethylene production in plants of two wheat cvs. treated with Cd. Each value is the mean of six replicates9/S.E. S.E. is as for Fig. 1. Means followed by different letters are different at P 5/0.01 level.
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
271
Fig. 4. Superoxide dismutase activity in leaves and roots of two wheat cvs. treated with Cd. Legends as in Fig. 3.
Fig. 5. Catalase activity in leaves and roots of two wheat cvs. treated with Cd. Legends as in Fig. 3.
GPOD, SOD, and APOD than Adamello, whereas SPOD activity was higher in roots of Adamello (Figs. 4, 6 /8). In Cd-treated Adamello leaves, SOD activity decreased to about 50% of the control value at both Cd concentrations (Fig. 4). The metal treatment did not change this activity in Ofanto leaves (Fig. 4). Cd decreased the SOD activity in the roots of both Adamello and Ofanto plants (Fig. 4). Both Cd concentrations induced an increase in the activity of CAT in roots and in leaves of Adamello. In the leaves of Ofanto, the metal did not affect this enzyme; in the roots CAT increased only when 4.7 mM Cd were applied. In leaves of Ofanto APOD activity rose with increasing Cd concentration, whereas in those of Adamello a decrease was observed at the lowest Cd concentration. With 2.2 mM Cd APOD activity did not significantly differ either from the control
values or from those at the lowest Cd concentration. In roots of Adamello, the enzyme activity increased at the lowest Cd concentration, but it dropped when the Cd concentration further increased. In Ofanto roots Cd treatment reduced the APOD activity by 26 and 22% for the two concentrations applied, respectively (Fig. 6). The SPOD activity increased with the Cd treatment level in leaves of Ofanto, whereas in the roots the activity of this enzyme was only increased when the Cd concentration was 0.5 mM. In Adamello, the SPOD activity, which increased in the leaves at 2.2 mM Cd, decreased in the roots of plants treated with 0.25 mM Cd (Fig. 7). The higher Cd concentration in the nutrient solution did not significantly affect GPOD activity in Adamello leaves, but in those of Ofanto this activity rose as a function of the metal concentration. In Adamello roots GPOD activity increased
272
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
Fig. 6. Ascorbate peroxidase activity in leaves and roots of two wheat cvs. treated with Cd. Legends as in Fig. 3.
Fig. 7. Syringaldazine peroxidase activity in leaves and roots of two wheat cvs. treated with Cd. Legends as in Fig. 3.
by 28% at 0.25 mM Cd and dropped to control values at 2.2 mM Cd, while in the roots of Ofanto, the activity decreased by 35% at 0.5 mM Cd without any further change at higher metal concentration (Fig. 8). The isozyme pattern of POD was also affected by Cd. In the control leaves from cv. Adamello (first left line, Fig. 9) and Ofanto (first right line, Fig. 9) the densitometric analysis revealed two POX bands. In the presence of Cd, a new band of higher mobility appeared in both cvs. In Adamello leaves at 0.25 and 2.2 mM Cd, the third band was present for 43 and 49%, respectively, whereas in the leaves of cv. Ofanto at 0.5 and 4.7 mM Cd, the new isoenzyme was present for 23 and 21%, respectively. In the roots of cv. Adamello (left lines, Fig. 9) and Ofanto (right lines, Fig. 9) treated with Cd there was no induction of new bands, in comparison with control, but only modification in
the percentage of the five bands detected there. In Adamello 0.25 mM Cd increased band V by 13% and 2.2 mM increased band II by 15% and decreased band IV by 67%. In the cv. Ofanto band I decreased by 73% at both concentrations, whereas band II, which was unaffected at the lower Cd concentration, rose by 12% at the higher concentration and band IV and V increased at 0.5 mM Cd and decreased at 4.7 mM.
4. Discussion Contrary to the results frequently reported, the data presented here were obtained on plants sown in a medium contaminated with Cd at low realistic concentrations (Sanita` di Toppi and Gabbrielli, 1999; Sgherri et al., 2001). Indeed the metal concentrations applied were near to the threshold
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
Fig. 8. Guaiacol peroxidase activity in leaves and roots of two wheat cvs. treated with Cd. Legends as in Fig. 3.
value, evaluated by inhibition of root elongation, so that the plants were subjected to mild Cd excess
273
from the stage of seed germination onwards. To have the same degree of root growth inhibition, which is an index of Cd toxicity, Ofanto seedlings were subjected to higher Cd doses (Fig. 1), thus indicating its higher tolerance towards Cd treatment involving as first line of defence a more efficient Cd exclusion by roots (Fig. 2). K leakage, as symptom of membrane damage, and the inhibitory effect of Cd on plant growth, which was likely due at least in part to suppression of the cellular elongation growth (Guo and Marschner 1995), could be considered as indices of stress intensity. At the highest equal-effect concentration roots of Ofanto showed a higher Cd concentration in comparison with Adamello and Ofanto was more efficient than Adamello in the transfer of Cd from roots to leaves (Fig. 2), as it was also observed for Cu (Sgherri et al., 2001). This finding is in contrast with literature where it is reported that leaves of sensitive plants have higher metal concentrations than those of a tolerant ecotype (Chardonnens et al., 1999). Since, notwithstanding the same inhibition of root elongation in the two cvs., the reduction in biomass and the increase in K leakage (Table 1) were more pronounced in Adamello, we can suppose in Ofanto an efficient Cd detoxification played by vacuolar compartmentalisation, which prevents the free circulation of Cd ions in the cytosol and forces them into a limited area (Sanita` di Toppi
Fig. 9. PAGE of peroxidases from leaves and roots of two wheat cvs. treated with increasing Cd concentration. Samples of leaves and roots contained 0.07 units of enzyme. Left lines, cv. Adamello subjected to 0, 0.25 and 2.2 mM Cd; right lines, cv. Ofanto subjected to 0, 0.5 and 4.7 mM Cd.
274
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
and Gabbrielli, 1999) thus determining a lower stress intensity. This might explain why K leakage in Ofanto leaves did not increase with the treatment (Table 1). Metal toxicity can affect the plasma membrane permeability (Table 1) causing a reduction in water content; in particular Cd has been reported to interact with the water balance (Barcelo´ et al., 1986; Poschenrieder et al., 1989; Costa and Morel, 1994). In our experiments, the reduction in water potential (Table 2) as a result of Cd uptake could be due to the observed reduction in root growth. The maintenance of RWC, and of a positive leaf turgor in both cvs (Table 2) might represent an adaptive mechanism to avoid leaf water deficit associated with metal toxicity, so that the changes observed in Cd-treated plants should not be considered as a result of water imbalance. Since Cd is a non-redox metal, unable to perform single electron transfer reactions, it does not produce directly reactive oxygen species via Fenton and/or Haber Weiss reactions. The induction of LOX in the presence of a non-redox metal might explain its effect on the cellular redox status since this enzyme produces O2+ by oxidising NADPH (Quartacci et al., 2001). As it was previously observed for other stresses (Sgherri and Navari-Izzo, 1995; Sgherri et al., 2001), activation or inhibition of antioxidative enzymes depends not only on stress intensity but also on the tissue type (Table 1). Likely control roots, being in direct contact with metals in the nutrient solution could be subjected to a higher oxidative pressure than leaves (Sgherri et al., 2002). As a consequence, in roots of both Adamello and Ofanto constitutive antioxidative enzymes were activated compared with leaves with the exception of CAT (Figs. 4 /8), which might have a major role in the antioxidative defence of leaves as it was previously observed for copper (Sgherri et al., 2001). When stress intensity was more severe (Table 1 Sgherri and Navari-Izzo, 1995), as it could be in Adamello roots subjected to the highest Cd concentration, antioxidative enzyme activities, i.e. SOD, APOD, GPOD, were inhibited (Figs. 4, 6 and 8). Data from literature, clarifying the relationship between ethylene production and Cd stress, are
scanty. Stimulated ethylene emission by this metal might represent a signal regulating the expression of genes involved in antioxidative enzyme activation. The stimulation of ethylene emission upon the lowest Cd treatment and its decrease when the Cd concentration rose, as observed in cv. Adamello (Fig. 3), agrees with data obtained on Amaranthus lividus seedlings (Bhattacharjee, 1997). The increase in CAT (Fig. 5), APOD (Fig. 6) and GPOD (Fig. 8) in Adamello roots at the lowest Cd concentration might be indicative for a signalling response in which ethylene emission could be involved. The decreasing trend in ethylene emission in Ofanto (Fig. 3) could be the result of an efficient Cd-sequestering in vacuole, which diminished the Cd stress in comparison with Adamello. The lack in activation and/or the decrease in SOD activity observed in both roots and leaves of Adamello and Ofanto (Fig. 4) was also found in P. vulgaris (Somashekaraiah et al., 1992), Helianthus annuus (Gallego et al., 1996), Pisum sativum (Sandalio et al., 2001) and in several yeast strains (Romandini et al., 1992). It can not be ruled out that Cd affects the activity of SOD either by inhibition of its synthesis or by inactivation by H2O2, produced in several cellular compartments by various non-enzymatic and enzymatic processes which may be responsible for an increase in oxidative stress. This could be true for Cu/ZnSOD and Fe-SOD which are irreversible inhibited by H2O2 (Scandalios, 1993) but not for Mn-SOD, which is unaffected by this activated oxygen species. In fact Sandalio et al. (2001) found that Mn-SOD was the isoform more resistant to Cd exposure, whereas CuZn-SOD and Fe-SOD showed a linear reduction in response to increasing Cd concentration in the nutrient solution. Since more than one isoform contributes to the total SOD activity in a crude plant extract, the decrease in one isoform might be masked by a compensatory increase in another type of isoform giving the overall values reported in Fig. 4. A significant stimulation of the Mn- and Fe-SOD and an almost complete inhibition of the CuZn-SOD without any change in total SOD activity has been described in Cd treated bean plants (Cardinaels et al., 1984).
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
The different responses to Cd of CAT (Fig. 5) and APOD (Fig. 6) activities in the leaves of the two cvs might reveal different targets of oxidative damage. In Ofanto leaves, the stimulation of APOD activity with increasing in Cd concentration may suggest that H2O2 is predominantly eliminated in the chloroplasts. The increase in CAT activity in Adamello leaves could be necessary to scavenge H2O2 in peroxisomes and cytosol, where it might have diffused from chloroplasts (Scandalios, 1994). After penetration through the cell membranes, Cd might have supplementary toxic effects in the latter cv. such as higher peroxide production in the cytosol itself. Under water stress the membrane complex configuration of Adamello is damaged more than that of Ofanto (Quartacci et al., 1995). The superoxide production was also increased in Adamello (Quartacci et al., 1994); this might be due to a release of hydroperoxides from the membrane lipids into the cytosol in this cv, which is also less drought tolerant. Peroxidases are able to prevent oxidative damage to the plasma membrane by quenching peroxide radicals (Van Assche and Clijsters, 1990). Although the roots did not show a clear pattern in the difference of SPOD and GPOD activities between the two cvs, the activity of these enzymes was enhanced by Cd in the leaves of Ofanto, contrary to those of Adamello (Figs. 7 and 8). This increase in activity could represent an appropriate protection against overproduction of peroxides when Cd accumulates in Ofanto. Sgherri et al. (2001) already reported that Cu toxicity also enhances peroxidases in leaves of Ofanto. Finally, the changes in peroxidase isozyme pattern in leaves and roots of both cvs (Fig. 9) confirm that the plants exposed to stress induced by mild Cd toxicity initiate a series of acclimative responses that may provide increased protection against more severe stress. In conclusion, cv. Ofanto showed, as for other stress such as drought, salinity, nickel and copper, a co-tolerance towards Cd and the different changes in the activities of antioxidative enzymes represent, at equal-effect concentrations of Cd, the different biological response of the two cvs. to Cd excess. Analogies in the response to other metals such as copper (Sgherri et al., 2001) could be found
275
in the fact that at the lower metal concentration CAT showed activation in cv. Adamello whereas APOD was induced in leaves of cv. Ofanto.
References Adams, D.O., Yang, S.F., 1979. Ethylene biosynthesis: identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. USA 76, 170 /174. Barcelo´, J., Poschenrieder, C., Andreu, I., Gunse´, B., 1986. Cadmium-induced decrease of water stress resistance in bush bean plants (Phaseolus vulgaris L. cv Contender). I. Effects on water potential, relative water content and cell wall elasticity. J. Plant Physiol. 125, 17 /25. Bhattacharjee, S., 1997. Membrane lipid peroxidation, free radical scavengers and ethylene evolution in Amaranthus as affected by lead and cadmium. Biol. Plant. 40, 131 /135. Cardinaels, C., Put, C., Van Assche, F., Clijsters, H., 1984. The superoxidedismutase as a biochemical indicator, discriminating between zinc and cadmium toxicity. Arch. Intern. Physiol. Biochim. 92, 27 /28. Chaoui, A., Mazhoudi, S., Ghorbal, M.H., El Ferjani, E., 1997. Cadmium and zinc induction of lipid peroxidation and effects on antioxidant enzymes activities in bean (Phaseolus vulgaris L.). Plant Sci. 127, 139 /147. Chardonnens, A.N., ten Bookum, W.A., Vellinga, S., Schat, H., Verkleij, J.A.C., Ernst, W.H.O., 1999. Allocation patterns of zinc and cadmium in heavy metal tolerant and sensitive Silene vulgaris . J. Plant Physiol. 155, 778 /787. Ciscato, M., Valcke, R., Van Loven, K., Clijsters, H., NavariIzzo, F., 1997. Effects of in vivo copper treatment on the photosynthetic apparatus of two Triticum aestivum cultivars with different stress sensitivity. Physiol. Plant. 100, 901 / 908. Costa, G., Morel, J.L., 1994. Water relations, gas exchange and amino acid content in Cd-treated lettuce. Plant Physiol. Biochem. 32, 561 /570. Fuhrer, J., 1982. Early effects of excess cadmium toxicity in leaf tissue of beans (Phaseolus vulgaris L.). Plant Physiol. 70, 162 /167. Gallego, S.M., Benavides, M.P., Tomaro, M.L., 1996. Effect of heavy metal ion excess on sunflower leaves: evidence for involvement of oxidative stress. Plant Sci. 121, 151 /159. Gaspar, T., Penel, C., Hagege, T., Greppin, H., 1991. Peroxidases in plant growth, differentiation and development processes. In: Lobarzewski, J., Greppin, H., Penel, C., Gaspar, T. (Eds.), Biochemical, Molecular and Physiological Aspects of Plant Peroxidases. University M. CurieSklodowska Lublin. Poland and University of Geneva, Switzerland, pp. 249 /280. Gora, L., Clijsters, H., 1989. Effects of copper and zinc on the ethylene metabolism in Phaseolus vulgaris L. In: Clijsters, H., De Proft, M., Marcelle, R., Van Poucke, M. (Eds.), Biochemical and Physiological Aspects of Ethylene Produc-
276
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
tion in Lower and Higher Plants. Kluwer Academic Publishers, Dordrecht, pp. 219 /228. Guo, Y., Marschner, H., 1995. Uptake, distribution and binding of cadmium and nickel in different plant species. J. Plant Nutr. 18, 2691 /2706. Hendry, G.A.F., Baker, A.J.M., Ewart, C.F., 1992. Cadmium tolerance and toxicity oxygen radical processes and molecular damage in cadmium-tolerant and cadmium-sensitive clones of Holcus lanatus . Acta Bot. Neerl. 41, 271 /281. Hoagland, D.R., Arnon, D.I., 1950. The water-culture method for growing plants without soil. Calif. Agric. Exp. Stn. Circ. 347, 1 /39. Imberty, A., Goldberg, R., Catesson, A.M., 1985. Isolation and characterisation of Populus isoperoxidases involved in the last step of lignin formation. Planta 164, 221 /226. Loggini, B., Scartazza, A., Brugnoli, E., Navari-Izzo, F., 1999. Antioxidative defense system, pigment composition, and photosynthetic efficiency in two wheat cultivars subjected to drought. Plant Physiol. 119, 1091 /1099. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265 /275. Lozano-Rodriguez, E., Hernandez, L.E., Bonay, P., CarpenaRuiz, R.O., 1997. Distribution of Cd in shoot and root tissues of maize and pea plants: physiological distribution. J. Exp. Bot. 48, 123 /128. Meneguzzo, S., Navari-Izzo, F., Izzo, R., 2000. NaCl effects on water relations and accumulation of mineral nutrients in leaves, roots and cell sap of wheat seedlings. J. Plant Physiol. 156, 711 /716. Navari-Izzo, F., Quartacci, M.F., 2001. Phytoremediation of metals: mechanisms against oxidative stress. Minerva Biotechnol. 13, 73 /83. Navari-Izzo, F., Quartacci, M.F., Melfi, D., Izzo, R., 1993. Lipid composition of plasma membranes isolated from sunflower seedlings grown under water-stress. Physiol. Plant. 87, 508 /514. Pandolfini, T., Gabbrielli, R., Ciscato, M., 1996. Nickel toxicity in two durum wheat cultivars differing in drought sensitivity. J. Plant Nutr. 19, 1611 /1627. Poschenrieder, C., Gunse´, B., Barcelo´, J., 1989. Influence of cadmium on water relations, stomatal resistance, and abscisic acid content in expanding bean leaves. Plant Physiol. 90, 1365 /1371. Quartacci, M.F., Sgherri, C.L.M., Pinzino, C., Navari-Izzo, F., 1994. Superoxide radical production in wheat plants differently sensitive to drought. Proc. Roy. Soc. Edinb. 102B, 287 /290. Quartacci, M.F., Pinzino, C., Sgherri, C.L.M., Navari-Izzo, F., 1995. Lipid composition and protein dynamics in thylakoids of two wheat cultivars differently sensitive to drought. Plant Physiol. 108, 191 /197.
Quartacci, M.F., Cosi, E., Navari-Izzo, F., 2001. Lipids and NADPH-dependent superoxide production in plasma membrane vescicles from roots of wheat grown under copper deficiency and excess. J. Exp. Bot. 52, 77 /84. Rauser, W.E., 1978. Early effects of phytotoxic burdens of cadmium, cobalt, nickel and zinc in white beans. Can. J. Bot. 546, 1744 /1749. Romandini, P., Tallandini, L., Beltramini, M., Salvato, B., Manzano, M., de Bertoldi, M., Rocoo, G.P., 1992. Effects of copper and cadmium on growth, superoxide dismutase and catalase activities in different yeast strains. Comp. Biochem. Physiol. 103C, 255 /262. Sandalio, L.M., Dalurzo, H.C., Gomez, M., Romero-Puertas, M.C., del Rio, L.A., 2001. Cadmium-induced changes in the growth and oxidative metabolism of pea plants. J. Exp. Bot. 52, 2115 /2212. Sanita` di Toppi, L., Gabbrielli, R., 1999. Response to cadmium in higher plants. Environ. Exp. Bot. 41, 105 /130. Scandalios, J.G., 1993. Oxygen stress and superoxide dismutases. Plant Physiol. 101, 7 /12. Scandalios, J.G., 1994. Regulation and properties of plant catalases. In: Foyer, C., Mullinaux, P. (Eds.), Causes of Peroxidative Stress and Amelioration of Defence System in Plants. CRC Press, Boca Raton, FL, pp. 275 /315. Sgherri, C.L.M., Navari-Izzo, F., 1995. Sunflower seedlings subjected to increasing water deficit stress: oxidative stress and defence mechanisms. Physiol. Plant. 93, 25 /30. Sgherri, C.L.M., Milone, A.M.T., Clijsters, H., Navari-Izzo, F., 2001. Antioxidative enzymes in two wheat cultivars, differently sensitive to drought and subjected to subsymptomatic copper doses. J. Plant Physiol. 158, 1439 /1447. Sgherri, C., Quartacci, M.F., Izzo, R., Navari-Izzo, F., 2002. Relation between lipoic acid and cell redox status in wheat grown in excess copper. Plant Physiol. Biochem. 40, 591 / 597. Shaw, B.P., 1995. Effect of mercury and cadmium on the activities of antioxidative enzymes in the seedlings of Phaseolus aureus . Biol. Plant. 37, 587 /596. Somashekaraiah, B.V., Padmaja, K., Prasad, A.R.K., 1992. Phytotoxicity of cadmium ions on germinating seedlings of mung bean (Phaseolus vulgaris ): involvement of lipid peroxides in chlorophyll degradation. Physiol. Plant. 85, 85 /89. Stohs, S.J., Bagchi, D., 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18, 321 /336. Van Assche, F., Clijsters, H., 1990. Effect of metal on enzyme activity in plants. Plant Cell Environ. 13, 195 /206. Weckx, J., Vangronsveld, J., Clijsters, H., 1993. Heavy metal induction of ethylene production and stress enzymes. I Kinetics of responses. In: Pech, J.C., Latche´, A., Balagne´, C. (Eds.), Cellular and Molecular Aspects of Plant Hormone Ethylene. Kluwer Academic Publishers, Dordrecht, pp. 238 /239.
Antioxidative responses of wheat treated with realistic concentration of cadmium M. Teresa Milone a, Cristina Sgherri a, Herman Clijsters b, Flavia NavariIzzo a,* a
Dipartimento di Chimica e Biotecnologie Agrarie, Via del Borghetto, 80-56124 Pisa, Italy b Limburgs Universitaire Centrum, Department SBG, 3590 Diepenbeek, Belgium
Received 3 December 2002; received in revised form 26 March 2003; accepted 26 March 2003
Abstract Two cvs. of wheat differently sensitive to many stress factors (cv. Ofanto less sensitive than cv. Adamello) were grown in a controlled environment with cadmium near threshold concentrations supplying the metal at equal-effect concentrations. Cd excess determined in both cvs. a reduction in water and turgor potential but a maintenance of relative water content. Cv Ofanto showed a higher capacity of Cd exclusion from roots but a higher translocation to shoots in comparison with cv. Adamello. Notwithstanding the higher metal concentration in leaves of cv. Ofanto, K leakage was more pronounced in Adamello suggesting that mechanisms of Cd detoxification and tolerance such as vacuolar compartmentalisation were activated in the first one. In Adamello plants, ethylene rose at the lowest metal concentration and the activation in roots of the antioxidative enzymes catalase, ascorbate peroxidase and guaiacol peroxidase came into play whereas in Ofanto ethylene and catalase did not change. Following cadmium treatment, superoxide dismutase activity was reduced or remained at the control value in roots and in leaves. For both cultivars ascorbate peroxidase, syringaldazine peroxidase and guaiacol peroxidase activities were always higher in roots than in leaves. These activities were induced by Cd in Ofanto leaves, whereas in Adamello leaves they remained at control levels or increased somewhat at the highest metal concentration. Cadmium changed the peroxidase isozyme pattern in both cultivars. Cv. Ofanto showed, as for other stress such as drought, salinity, nickel and copper, a co-tolerance towards Cd. Analogies in the response to other metals such as copper could be found in activation of catalase at the lower metal concentration in cv. Adamello and in the induction of ascorbate peroxidase in leaves of cv. Ofanto. # 2003 Elsevier B.V. All rights reserved. Keywords: Catalase; Ethylene; Peroxidases; Superoxide dismutase; Triticum durum
1. Introduction
* Corresponding author. Tel.: /39-050-97-1921; fax: /39050-59-8614. E-mail address: [email protected] (F. Navari-Izzo).
Cadmium is a widely spread pollutant; it can reach high levels in agricultural soils and is easily assimilated by plants. Cadmium has no known biological function and is extremely toxic, even at
S0098-8472/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0098-8472(03)00037-6
266
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
low concentration. It induces various visible symptoms of phytotoxicity, e.g. leaf roll, chlorosis and growth reduction of root and shoot. Usually inhibition of root elongation is the most sensitive parameter of Cd toxicity (Guo and Marschner, 1995). Redox metals such as Cu, Fe, Hg, etc. may directly produce free radicals (Van Assche and Clijsters, 1990), which can also be formed indirectly by non-redox metals such as Cd by mediation of lipoxygenase (LOX, EC 1.13.11.12) (Van Assche and Clijsters, 1990; Stohs and Bagchi, 1995; Navari-Izzo and Quartacci, 2001). Indeed Cd was found to induce oxidative stress in cells (Hendry et al., 1992; Somashekaraiah et al., 1992). Depending on concentration cadmium can either inhibit or stimulate the activity of several antioxidative enzymes before any visible symptom of toxicity appears. In sunflower leaves Cd decreased the activity of superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1. 11. 1. 7), glutathione reductase (GR, EC 1. 6. 4. 2) and dehydroascobate reductase (DHAR, EC 1. 8. 5. 1), while LOX, which mediates lipid peroxidation, increased (Gallego et al., 1996). Upon Cd exposure lipid peroxidation raised also in pea plants (LozanoRodriguez et al., 1997), in Phaseolus aureus (Shaw, 1995) and Phaseolus vulgaris (Chaoui et al., 1997), whereas in Daucus carota no lipid peroxidation was observed (Sanita` di Toppi and Gabbrielli, 1999). Both P. vulgaris and P. aureus showed enhanced guaiacol peroxidase (GPOD, EC 1.11.1.7) activity (Shaw, 1995; Chaoui et al., 1997), but in P. aureus (Shaw, 1995) the activity of CAT decreased, as was also observed in pea plants, where a reduction in SOD and GPOD activities was also demonstrated (Sandalio et al., 2001). Although, peroxidases have been extensively studied, their physiological function remains unclear. In plants several functions are ascribed to these enzymes: auxin metabolism, lignification, wound healing and defence against pathogens. Their cellular distribution (cell walls, endoplasmic reticulum, Golgi apparatus and vacuoles), is presumibly connected with different physiological functions (Gaspar et al., 1991). An involvement
in the protection against oxidative stress, induced by metals, has been postulated. Ethylene is a natural plant growth regulator involved in the control of a wide range of developmental responses. Gora and Clijsters (1989) reported that toxic concentrations of metals stimulate ethylene production in plants. Cd has been shown to interfere specifically with ethylene biosynthesis in bean leaf tissue (Rauser, 1978). In P. vulgaris , low concentrations of Cd stimulated ethylene biosynthesis (Adams and Yang, 1979), but Cd-induced ethylene stimulation declined with increasing Cd concentration (Bhattacharjee, 1997). Cd concentrations above 1 mM stimulated ACC synthase activity and increased the activity of GPODs within 5/10 h after application, but ethylene production gradually declined to control levels. Stimulation of ACC synthase has also been found in response to mechanical wounding, IAA and Cu treatments (Fuhrer, 1982; Sgherri et al., 2001). It is not clear whether these different stimuli regulate ACC synthase through common mechanisms. Oxygen free radicals and hydroperoxides, generated by LOX, might stimulate the conversion of ACC into ethylene (Weckx et al., 1993). In the literature, the Cd concentrations studied are frequently not realistic when compared with the levels of contamination in the natural environment (Sanita` di Toppi and Gabbrielli, 1999); in addition, the metal was generally supplied to plants previously grown without Cd, which makes the experimental conditions different from those of natural crops. Two cvs. of wheat differently sensitive to drought (cv. Ofanto less sensitive than cv. Adamello) were studied for the response to other stress factors i.e. nickel, copper and NaCl. In all cases, cv. Ofanto appeared more tolerant than cv. Adamello (Quartacci et al., 1995; Pandolfini et al., 1996; Ciscato et al., 1997; Loggini et al., 1999; Meneguzzo et al., 2000; Sgherri et al., 2001). In particular, when the two cvs. were subjected to realistic concentrations of copper, the uptake of the metal was higher in the sensitive cv. Since different intracellular Cu concentrations could cause different activation or inhibition of antioxidative systems, the two cvs. were subjected to equal-effect concentrations of copper on the basis of the same root growth inhibition, which is the
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
expression of metal toxicity (Sgherri et al., 2001). The antioxidative response revealed that the leaves of the more tolerant cv. (Ofanto) responded to Cu with an induction of peroxidases, whereas in the more sensitive cv. (Adamello) there were increases in CAT (Sgherri et al., 2001). In the present study cv. Ofanto and cv. Adamello were analysed in order to establish if there is also a co-tolerance against Cd supplied at realistic concentrations. As for Cu (Sgherri et al., 2001), the two cvs. were subjected to equal-effect concentrations of Cd and also in this last case, we expect that differences in the biochemical responses occur in the two cvs. at equal-effect exposure levels. The antioxidative responses were focused studying the changes in the activities of guaiacol and syringaldazine dependent peroxidases, APOD, SOD and CAT. Ethylene emission was also monitored. Metal toxicity impairs membrane function and ion balance (Sandalio et al., 2001), and could affect plant water relations; for this reason leaf water status was also monitored.
2. Materials and methods 2.1. Plant material Two cvs of wheat (Triticum durum Desf.) differently sensitive to drought and heavy metals (Quartacci et al., 1995; Ciscato et al., 1997; Loggini et al., 1999; Sgherri et al., 2001) with cv. Ofanto less sensitive than cv. Adamello, were grown for 10 days under controlled conditions at 200 mE m 2 s 1 PAR with a 16 h photoperiod, 75% R.H. and day/night temperatures of 20/16 8C. Light was supplied by fluorescent tubes (Osram L. 140 W/20) and incandescent lamps (Philips 25 W). Seeds were surface sterilised with 2% sodium hypochlorite for 10 min, rinsed in distilled water and imbibed for 4 h with running tap water. They were sown in a beaker containing 1 l of a half strength Hoagland’s solution containing micronutrients (Hoagland and Arnon, 1950) and 400 ml sterilised perlite as inert support. Increasing concentrations of Cd, selected on the basis of a rooting test (Fig. 1), were added as Cd(SO)4 to the nutrient solution from sowing on. Metal
267
Fig. 1. Root length in two wheat cvs. treated with increasing concentrations of Cd. Each value is the mean of six replicates9/ S.E. S.E. is smaller than the size of symbols.
concentrations, giving 5 and 20% of root growth inhibition, were 0.25 and 2.2 mM for Adamello and 0.5 and 4.7 mM for Ofanto. The nutrient solution was bubbled with sterile air and changed on alternate days to avoid depletion of nutrients as well as Cd. 2.2. Cadmium analysis After harvest fresh and dry weights of the roots and leaves were measured and plant samples were thoroughly washed with sterile distilled water. Cd content of roots and leaves was determined by atomic absorption spectroscopy after wet digestion of dried material in HNO3. Metal content was also determined on roots following desorption and on desorbing solution. After a fast washing with water to remove perlite, roots were immersed in 5 mM Pb(NO3)2 for 30 min at 4 8C. 2.3. Leakage of K ions Potassium leakage was measured according to Quartacci et al. (2001). Roots and leaves of seedlings from each treatment were excised and washed twice to remove the contents of the cut cells. Samples were incubated in 25 ml of double
268
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
distilled water and shaken at 21 8C for 24 h, and aliquots for K determination were taken. Samples were then immersed for 5 min in liquid nitrogen and placed in the same vial containing the leachate and shaken for an additional hour prior to the measurements of total K leached from the killed cells. Potassium released was measured by atomic absorption spectrophotometry with a hollow-cathode lamp.
2.4. Leaf water status Leaf water status (relative water content, RWC; water potential, Cw; turgor potential, Cp; osmotic potential at full turgor, C100 p ; osmotic potential, Cp) was determined by pressure-volume curves using a pressure chamber. Curve analysis was performed as previously described (Navari-Izzo et al., 1993). Measurements of water status were carried out on ten plants.
2.5. Ethylene analysis Ethylene release was measured with a continuous air flow technique as previously described (Sgherri et al., 2001). Ethylene determination was carried out on a Intermat IGC 120 FB gas chromatograph (Courtry, France) equipped with a Porapak-R column (50 /80 mesh) and a flame ionisation detector. The beaker, containing plants with roots submerged in water, was placed in a cylindrical gas cuvette and aereated (10 l h 1) with a synthetic air mixture N2/O2 (80/20). The gas inlet ended in the water of the beaker; the outlet was located at the top of the cuvette. The cuvettes were put in a small growth chamber where a temperature of 25 8C and a light intensity of 105 mmol m 2 s 1 were maintained. To trap the ethylene produced, the gas outlet of the cuvette was connected with a cooled ( /95 8C) precolumn filled with Porapak-S (50 /80 mesh; Waters, Milford, USA). After 10 min, this column was inserted into the carrier gas stream of a gas chromatograph. The ethylene was totally released and swept into the analytical column with the carrier gas by heating the precolumn to 100 8C in boiling water.
2.6. Enzyme extraction and assays The enzyme activities were determined as described in Sgherri et al. (2001). Plant material was ground in an ice-cold mortar and for each enzyme an appropriate ice-cold extraction buffer at 4 8C was used. The homogenate was filtered through a Miracloth (Calbiochem) and centrifuged at 20 000 /g for 15 min at 4 8C. The protein contents (Lowry et al., 1951) and the enzyme assays were measured in the supernatant at 25 8C. SOD activity was assayed as previously described in Sgherri et al. (2001) in terms of the ability to inhibit the photochemical reduction of nitroblue tetrazolium. One unit of SOD is the amount of extract that gives half maximum inhibition. CAT activity was assayed polarographically with a Clark-type electrode (Hansatech, Kings Lynn, Norfolk, UK). The oxygen produced by the extract was calculated after correction for the autoproduction of oxygen from H2O2. One unit of CAT is the amount of enzyme producing 1 mM O2 min 1. POD activities were measured spectrophotometrically using as substrates ascorbate (APOD), guaiacol (GPOD) and syringaldazine (SPOD). APOD was measured at 298 nm according to Sgherri et al. (2001). One unit of APOD is the amount of enzyme oxidising 1 mmol of ascorbate min 1. GPOD was determined following Sgherri et al. (2001). One unit of GPOD is the amount of enzyme oxidising 1 mmol of guaiacol min 1. SPOD activity was determined according to Imberty et al. (1985). One unit of SPOD is the amount of enzyme oxidising 1 mmol min 1. 2.7. Electrophoretic POD separation Before loading on the gels, the protein extracts were dialysed for 24 h in 20 mM Tris /HCl, pH 7.8, concentrated with PEG and centrifuged at 12 000 /g for 15 min. The electrophoretic separation of the peroxidase bands was performed on 10% PAGE using Tris /HCl buffer 1.5 M, pH 8.8. The gels were run at 200 V for 45 min with a constant current of 35 mA per gel. Samples of leaves and roots contained 0.07 enzyme units. To visualise the band patterns the gels were incubated
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
in the dark for 90 min at 37 8C in 1 M Na-acetate buffer pH 4.6 containing 0.04% benzidine and 20 ml of hydrogen peroxide. Gels were densitometrically scanned and analysed with the SIGMA GEL software. 2.8. Statistical analysis Unless specified otherwise, results are the means of three independent replicates and repeated in at least two independent experiments. For comparison among means, performed using the Newman /Keuls test, one-way ANOVA was used. Means followed by different letters are significantly different at the P 5/0.01 level.
3. Results In the cv. Adamello 5 and 20% of root growth inhibition was observed with, respectively, 0.25 and 2.2 mM Cd in the nutrient solution, while for Ofanto the same percentages of root growth inhibition were observed with 0.5 and 4.7 mM Cd (Fig. 1). Cadmium-treated plants of cv. Adamello demonstrated a marked reduction in leaf and root fresh and dry weight whereas K leakage increased. In contrast, the dry matter of Ofanto roots and K leakage of Ofanto leaves remained unchanged with the treatment (Table 1). In comparison to the control plants, a decreasing trend in water potential was found in both
269
cultivars as the Cd concentration rose (Table 2). Osmotic potential did not change in Ofanto, while in Adamello it increased by 9% at the highest Cd concentration. In Adamello Cd did not affect the osmotic potential at full turgor, while 0.5 mM Cd induced a significant increase in Ofanto plants. Turgor potential maintained always positive values in both cultivars, but it appeared to decrease with the rise of the metal concentration. In both cultivars, the RWC remained at the control value for every Cd concentration tested (Table 2). Root length decreased proportionally with increasing Cd concentration in both cvs. but the slope was less steep for cv. Ofanto (Fig. 1). No Cd was detected in the control roots and leaves of each cultivar, grown in Cd-free conditions. When the metal level rose in the nutrient solution, however, the internal leaf and root Cd concentration also increased, as well as the amount of Cd desorbed from the roots (Fig. 2). At the lowest Cd concentration applied, the root metal uptake was similar for the two cultivars, but with increase of the external metal concentration the roots of Adamello absorbed a higher amount of Cd (Fig. 2); anyway the highest equal-effect concentration determined in roots of cv. Ofanto a higher metal concentration since the same entity of metal was desorbed (about 10%) by both cvs. In the leaves the Cd taken up was always higher in Ofanto than in Adamello (Fig. 2). The lowest Cd concentration (0.25 mM) induced an increase by 62% of the ethylene emission in
Table 1 Fresh weight, dry weight (mg) and K leakage (percent of total K ) in plants of two cultivars of wheat treated with cadmium Adamello
Ofanto
Control
0.25 mM
2.2 mM
Control
0.5 mM
4.7 mM
Leaves Fresh weight Dry weight K leakage
150.79/0.07c 11.89/0.33b 18.29/0.50c
138.99/0.41b 10.79/0.06b 25.39/0.31b
93.19/1.98a 7.69/0.15a 36.59/0.78a
118.29/0.33c 8.89/0.02c 19.39/0.61a
90.69/0.14b 7.89/0.11b 21.59/0.93a
63.09/0.01a 6.89/0.19a 25.39/0.72a
Roots Fresh weight Dry weight K leakage
100.79/0.54c 4.59/0.09c 31.59/0.80c
81.49/0.06b 3.69/0.14b 45.89/0.75b
62.19/0.67a 2.79/0.14a 60.39/1.35a
74.89/0.42c 3.39/0.12a 30.29/0.93b
57.49/0.61b 3.29/0.01a 34.39/1.45b
40.09/0.82a 2.99/0.03a 41.69/0.88a
Each value is the mean of three replicates9/S.E. Means in rows followed by different letters are significantly different at P 5/0.01.
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
270
Table 2 Water potential (Cw), osmotic potential (Cp), osmotic potential at full turgor (C100 p ) and relative water content (RWC) in plants of two cultivars of wheat treated with cadmium Adamello
Cw (MPa) Cp (MPa) C100 (MPa) p Cp (MPa) RWC (%)
Ofanto
Control
0.25 mM
2.2 mM
Control
0.5 mM
4.7 mM
/0.439/0.15b /0.999/0.01a /0.949/0.03a 0.569/0.01c 979/0.23a
/0.489/0.15ab /0.969/0.02a /0.929/0.02a 0.489/0.01b 979/0.19a
/0.529/0.10a /0.909/0.01b /0.869/0.02a 0.389/0.01a 979/0.21a
/0.379/0.10b /0.989/0.02a /0.989/0.01a 0.609/0.01c 989/0.20a
/0.499/0.14a /1.009/0.01a /0.929/0.01b 0.509/0.01b 989/0.15a
/0.499/0.14a /0.969/0.01a /0.939/0.01ab 0.469/0.01a 979/0.21a
Each value is the mean of six replicates9/S.E. Means followed by different letters are significantly different at P 5/0.01.
Adamello; at the highest metal concentration, however, ethylene emission dropped to a value comparable with that found in the control (Fig. 3). In Ofanto, Cd did not show any effect on ethylene emission as compared with the control (Fig. 3). In control as well as in Cd treated roots of both cultivars, the activities of SOD, APOD, SPOD, and GPOD were higher than in leaves (Figs. 4, 6 / 8). On the contrary, leaves exhibited higher CAT activity than roots (Fig. 5). The constitutive activities of SOD and APOD were higher in leaves of Adamello than in those of Ofanto. In control roots, cv. Ofanto showed a higher activity of
Fig. 2. Cd contents in leaves and roots of two wheat cvs. treated with increasing concentrations of Cd. Each value is the mean of six replicates9/S.E. S.E. is as for Fig. 1.
Fig. 3. Ethylene production in plants of two wheat cvs. treated with Cd. Each value is the mean of six replicates9/S.E. S.E. is as for Fig. 1. Means followed by different letters are different at P 5/0.01 level.
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
271
Fig. 4. Superoxide dismutase activity in leaves and roots of two wheat cvs. treated with Cd. Legends as in Fig. 3.
Fig. 5. Catalase activity in leaves and roots of two wheat cvs. treated with Cd. Legends as in Fig. 3.
GPOD, SOD, and APOD than Adamello, whereas SPOD activity was higher in roots of Adamello (Figs. 4, 6 /8). In Cd-treated Adamello leaves, SOD activity decreased to about 50% of the control value at both Cd concentrations (Fig. 4). The metal treatment did not change this activity in Ofanto leaves (Fig. 4). Cd decreased the SOD activity in the roots of both Adamello and Ofanto plants (Fig. 4). Both Cd concentrations induced an increase in the activity of CAT in roots and in leaves of Adamello. In the leaves of Ofanto, the metal did not affect this enzyme; in the roots CAT increased only when 4.7 mM Cd were applied. In leaves of Ofanto APOD activity rose with increasing Cd concentration, whereas in those of Adamello a decrease was observed at the lowest Cd concentration. With 2.2 mM Cd APOD activity did not significantly differ either from the control
values or from those at the lowest Cd concentration. In roots of Adamello, the enzyme activity increased at the lowest Cd concentration, but it dropped when the Cd concentration further increased. In Ofanto roots Cd treatment reduced the APOD activity by 26 and 22% for the two concentrations applied, respectively (Fig. 6). The SPOD activity increased with the Cd treatment level in leaves of Ofanto, whereas in the roots the activity of this enzyme was only increased when the Cd concentration was 0.5 mM. In Adamello, the SPOD activity, which increased in the leaves at 2.2 mM Cd, decreased in the roots of plants treated with 0.25 mM Cd (Fig. 7). The higher Cd concentration in the nutrient solution did not significantly affect GPOD activity in Adamello leaves, but in those of Ofanto this activity rose as a function of the metal concentration. In Adamello roots GPOD activity increased
272
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
Fig. 6. Ascorbate peroxidase activity in leaves and roots of two wheat cvs. treated with Cd. Legends as in Fig. 3.
Fig. 7. Syringaldazine peroxidase activity in leaves and roots of two wheat cvs. treated with Cd. Legends as in Fig. 3.
by 28% at 0.25 mM Cd and dropped to control values at 2.2 mM Cd, while in the roots of Ofanto, the activity decreased by 35% at 0.5 mM Cd without any further change at higher metal concentration (Fig. 8). The isozyme pattern of POD was also affected by Cd. In the control leaves from cv. Adamello (first left line, Fig. 9) and Ofanto (first right line, Fig. 9) the densitometric analysis revealed two POX bands. In the presence of Cd, a new band of higher mobility appeared in both cvs. In Adamello leaves at 0.25 and 2.2 mM Cd, the third band was present for 43 and 49%, respectively, whereas in the leaves of cv. Ofanto at 0.5 and 4.7 mM Cd, the new isoenzyme was present for 23 and 21%, respectively. In the roots of cv. Adamello (left lines, Fig. 9) and Ofanto (right lines, Fig. 9) treated with Cd there was no induction of new bands, in comparison with control, but only modification in
the percentage of the five bands detected there. In Adamello 0.25 mM Cd increased band V by 13% and 2.2 mM increased band II by 15% and decreased band IV by 67%. In the cv. Ofanto band I decreased by 73% at both concentrations, whereas band II, which was unaffected at the lower Cd concentration, rose by 12% at the higher concentration and band IV and V increased at 0.5 mM Cd and decreased at 4.7 mM.
4. Discussion Contrary to the results frequently reported, the data presented here were obtained on plants sown in a medium contaminated with Cd at low realistic concentrations (Sanita` di Toppi and Gabbrielli, 1999; Sgherri et al., 2001). Indeed the metal concentrations applied were near to the threshold
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
Fig. 8. Guaiacol peroxidase activity in leaves and roots of two wheat cvs. treated with Cd. Legends as in Fig. 3.
value, evaluated by inhibition of root elongation, so that the plants were subjected to mild Cd excess
273
from the stage of seed germination onwards. To have the same degree of root growth inhibition, which is an index of Cd toxicity, Ofanto seedlings were subjected to higher Cd doses (Fig. 1), thus indicating its higher tolerance towards Cd treatment involving as first line of defence a more efficient Cd exclusion by roots (Fig. 2). K leakage, as symptom of membrane damage, and the inhibitory effect of Cd on plant growth, which was likely due at least in part to suppression of the cellular elongation growth (Guo and Marschner 1995), could be considered as indices of stress intensity. At the highest equal-effect concentration roots of Ofanto showed a higher Cd concentration in comparison with Adamello and Ofanto was more efficient than Adamello in the transfer of Cd from roots to leaves (Fig. 2), as it was also observed for Cu (Sgherri et al., 2001). This finding is in contrast with literature where it is reported that leaves of sensitive plants have higher metal concentrations than those of a tolerant ecotype (Chardonnens et al., 1999). Since, notwithstanding the same inhibition of root elongation in the two cvs., the reduction in biomass and the increase in K leakage (Table 1) were more pronounced in Adamello, we can suppose in Ofanto an efficient Cd detoxification played by vacuolar compartmentalisation, which prevents the free circulation of Cd ions in the cytosol and forces them into a limited area (Sanita` di Toppi
Fig. 9. PAGE of peroxidases from leaves and roots of two wheat cvs. treated with increasing Cd concentration. Samples of leaves and roots contained 0.07 units of enzyme. Left lines, cv. Adamello subjected to 0, 0.25 and 2.2 mM Cd; right lines, cv. Ofanto subjected to 0, 0.5 and 4.7 mM Cd.
274
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
and Gabbrielli, 1999) thus determining a lower stress intensity. This might explain why K leakage in Ofanto leaves did not increase with the treatment (Table 1). Metal toxicity can affect the plasma membrane permeability (Table 1) causing a reduction in water content; in particular Cd has been reported to interact with the water balance (Barcelo´ et al., 1986; Poschenrieder et al., 1989; Costa and Morel, 1994). In our experiments, the reduction in water potential (Table 2) as a result of Cd uptake could be due to the observed reduction in root growth. The maintenance of RWC, and of a positive leaf turgor in both cvs (Table 2) might represent an adaptive mechanism to avoid leaf water deficit associated with metal toxicity, so that the changes observed in Cd-treated plants should not be considered as a result of water imbalance. Since Cd is a non-redox metal, unable to perform single electron transfer reactions, it does not produce directly reactive oxygen species via Fenton and/or Haber Weiss reactions. The induction of LOX in the presence of a non-redox metal might explain its effect on the cellular redox status since this enzyme produces O2+ by oxidising NADPH (Quartacci et al., 2001). As it was previously observed for other stresses (Sgherri and Navari-Izzo, 1995; Sgherri et al., 2001), activation or inhibition of antioxidative enzymes depends not only on stress intensity but also on the tissue type (Table 1). Likely control roots, being in direct contact with metals in the nutrient solution could be subjected to a higher oxidative pressure than leaves (Sgherri et al., 2002). As a consequence, in roots of both Adamello and Ofanto constitutive antioxidative enzymes were activated compared with leaves with the exception of CAT (Figs. 4 /8), which might have a major role in the antioxidative defence of leaves as it was previously observed for copper (Sgherri et al., 2001). When stress intensity was more severe (Table 1 Sgherri and Navari-Izzo, 1995), as it could be in Adamello roots subjected to the highest Cd concentration, antioxidative enzyme activities, i.e. SOD, APOD, GPOD, were inhibited (Figs. 4, 6 and 8). Data from literature, clarifying the relationship between ethylene production and Cd stress, are
scanty. Stimulated ethylene emission by this metal might represent a signal regulating the expression of genes involved in antioxidative enzyme activation. The stimulation of ethylene emission upon the lowest Cd treatment and its decrease when the Cd concentration rose, as observed in cv. Adamello (Fig. 3), agrees with data obtained on Amaranthus lividus seedlings (Bhattacharjee, 1997). The increase in CAT (Fig. 5), APOD (Fig. 6) and GPOD (Fig. 8) in Adamello roots at the lowest Cd concentration might be indicative for a signalling response in which ethylene emission could be involved. The decreasing trend in ethylene emission in Ofanto (Fig. 3) could be the result of an efficient Cd-sequestering in vacuole, which diminished the Cd stress in comparison with Adamello. The lack in activation and/or the decrease in SOD activity observed in both roots and leaves of Adamello and Ofanto (Fig. 4) was also found in P. vulgaris (Somashekaraiah et al., 1992), Helianthus annuus (Gallego et al., 1996), Pisum sativum (Sandalio et al., 2001) and in several yeast strains (Romandini et al., 1992). It can not be ruled out that Cd affects the activity of SOD either by inhibition of its synthesis or by inactivation by H2O2, produced in several cellular compartments by various non-enzymatic and enzymatic processes which may be responsible for an increase in oxidative stress. This could be true for Cu/ZnSOD and Fe-SOD which are irreversible inhibited by H2O2 (Scandalios, 1993) but not for Mn-SOD, which is unaffected by this activated oxygen species. In fact Sandalio et al. (2001) found that Mn-SOD was the isoform more resistant to Cd exposure, whereas CuZn-SOD and Fe-SOD showed a linear reduction in response to increasing Cd concentration in the nutrient solution. Since more than one isoform contributes to the total SOD activity in a crude plant extract, the decrease in one isoform might be masked by a compensatory increase in another type of isoform giving the overall values reported in Fig. 4. A significant stimulation of the Mn- and Fe-SOD and an almost complete inhibition of the CuZn-SOD without any change in total SOD activity has been described in Cd treated bean plants (Cardinaels et al., 1984).
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
The different responses to Cd of CAT (Fig. 5) and APOD (Fig. 6) activities in the leaves of the two cvs might reveal different targets of oxidative damage. In Ofanto leaves, the stimulation of APOD activity with increasing in Cd concentration may suggest that H2O2 is predominantly eliminated in the chloroplasts. The increase in CAT activity in Adamello leaves could be necessary to scavenge H2O2 in peroxisomes and cytosol, where it might have diffused from chloroplasts (Scandalios, 1994). After penetration through the cell membranes, Cd might have supplementary toxic effects in the latter cv. such as higher peroxide production in the cytosol itself. Under water stress the membrane complex configuration of Adamello is damaged more than that of Ofanto (Quartacci et al., 1995). The superoxide production was also increased in Adamello (Quartacci et al., 1994); this might be due to a release of hydroperoxides from the membrane lipids into the cytosol in this cv, which is also less drought tolerant. Peroxidases are able to prevent oxidative damage to the plasma membrane by quenching peroxide radicals (Van Assche and Clijsters, 1990). Although the roots did not show a clear pattern in the difference of SPOD and GPOD activities between the two cvs, the activity of these enzymes was enhanced by Cd in the leaves of Ofanto, contrary to those of Adamello (Figs. 7 and 8). This increase in activity could represent an appropriate protection against overproduction of peroxides when Cd accumulates in Ofanto. Sgherri et al. (2001) already reported that Cu toxicity also enhances peroxidases in leaves of Ofanto. Finally, the changes in peroxidase isozyme pattern in leaves and roots of both cvs (Fig. 9) confirm that the plants exposed to stress induced by mild Cd toxicity initiate a series of acclimative responses that may provide increased protection against more severe stress. In conclusion, cv. Ofanto showed, as for other stress such as drought, salinity, nickel and copper, a co-tolerance towards Cd and the different changes in the activities of antioxidative enzymes represent, at equal-effect concentrations of Cd, the different biological response of the two cvs. to Cd excess. Analogies in the response to other metals such as copper (Sgherri et al., 2001) could be found
275
in the fact that at the lower metal concentration CAT showed activation in cv. Adamello whereas APOD was induced in leaves of cv. Ofanto.
References Adams, D.O., Yang, S.F., 1979. Ethylene biosynthesis: identification of 1-aminocyclopropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Natl. Acad. Sci. USA 76, 170 /174. Barcelo´, J., Poschenrieder, C., Andreu, I., Gunse´, B., 1986. Cadmium-induced decrease of water stress resistance in bush bean plants (Phaseolus vulgaris L. cv Contender). I. Effects on water potential, relative water content and cell wall elasticity. J. Plant Physiol. 125, 17 /25. Bhattacharjee, S., 1997. Membrane lipid peroxidation, free radical scavengers and ethylene evolution in Amaranthus as affected by lead and cadmium. Biol. Plant. 40, 131 /135. Cardinaels, C., Put, C., Van Assche, F., Clijsters, H., 1984. The superoxidedismutase as a biochemical indicator, discriminating between zinc and cadmium toxicity. Arch. Intern. Physiol. Biochim. 92, 27 /28. Chaoui, A., Mazhoudi, S., Ghorbal, M.H., El Ferjani, E., 1997. Cadmium and zinc induction of lipid peroxidation and effects on antioxidant enzymes activities in bean (Phaseolus vulgaris L.). Plant Sci. 127, 139 /147. Chardonnens, A.N., ten Bookum, W.A., Vellinga, S., Schat, H., Verkleij, J.A.C., Ernst, W.H.O., 1999. Allocation patterns of zinc and cadmium in heavy metal tolerant and sensitive Silene vulgaris . J. Plant Physiol. 155, 778 /787. Ciscato, M., Valcke, R., Van Loven, K., Clijsters, H., NavariIzzo, F., 1997. Effects of in vivo copper treatment on the photosynthetic apparatus of two Triticum aestivum cultivars with different stress sensitivity. Physiol. Plant. 100, 901 / 908. Costa, G., Morel, J.L., 1994. Water relations, gas exchange and amino acid content in Cd-treated lettuce. Plant Physiol. Biochem. 32, 561 /570. Fuhrer, J., 1982. Early effects of excess cadmium toxicity in leaf tissue of beans (Phaseolus vulgaris L.). Plant Physiol. 70, 162 /167. Gallego, S.M., Benavides, M.P., Tomaro, M.L., 1996. Effect of heavy metal ion excess on sunflower leaves: evidence for involvement of oxidative stress. Plant Sci. 121, 151 /159. Gaspar, T., Penel, C., Hagege, T., Greppin, H., 1991. Peroxidases in plant growth, differentiation and development processes. In: Lobarzewski, J., Greppin, H., Penel, C., Gaspar, T. (Eds.), Biochemical, Molecular and Physiological Aspects of Plant Peroxidases. University M. CurieSklodowska Lublin. Poland and University of Geneva, Switzerland, pp. 249 /280. Gora, L., Clijsters, H., 1989. Effects of copper and zinc on the ethylene metabolism in Phaseolus vulgaris L. In: Clijsters, H., De Proft, M., Marcelle, R., Van Poucke, M. (Eds.), Biochemical and Physiological Aspects of Ethylene Produc-
276
M.T. Milone et al. / Environmental and Experimental Botany 50 (2003) 265 /276
tion in Lower and Higher Plants. Kluwer Academic Publishers, Dordrecht, pp. 219 /228. Guo, Y., Marschner, H., 1995. Uptake, distribution and binding of cadmium and nickel in different plant species. J. Plant Nutr. 18, 2691 /2706. Hendry, G.A.F., Baker, A.J.M., Ewart, C.F., 1992. Cadmium tolerance and toxicity oxygen radical processes and molecular damage in cadmium-tolerant and cadmium-sensitive clones of Holcus lanatus . Acta Bot. Neerl. 41, 271 /281. Hoagland, D.R., Arnon, D.I., 1950. The water-culture method for growing plants without soil. Calif. Agric. Exp. Stn. Circ. 347, 1 /39. Imberty, A., Goldberg, R., Catesson, A.M., 1985. Isolation and characterisation of Populus isoperoxidases involved in the last step of lignin formation. Planta 164, 221 /226. Loggini, B., Scartazza, A., Brugnoli, E., Navari-Izzo, F., 1999. Antioxidative defense system, pigment composition, and photosynthetic efficiency in two wheat cultivars subjected to drought. Plant Physiol. 119, 1091 /1099. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265 /275. Lozano-Rodriguez, E., Hernandez, L.E., Bonay, P., CarpenaRuiz, R.O., 1997. Distribution of Cd in shoot and root tissues of maize and pea plants: physiological distribution. J. Exp. Bot. 48, 123 /128. Meneguzzo, S., Navari-Izzo, F., Izzo, R., 2000. NaCl effects on water relations and accumulation of mineral nutrients in leaves, roots and cell sap of wheat seedlings. J. Plant Physiol. 156, 711 /716. Navari-Izzo, F., Quartacci, M.F., 2001. Phytoremediation of metals: mechanisms against oxidative stress. Minerva Biotechnol. 13, 73 /83. Navari-Izzo, F., Quartacci, M.F., Melfi, D., Izzo, R., 1993. Lipid composition of plasma membranes isolated from sunflower seedlings grown under water-stress. Physiol. Plant. 87, 508 /514. Pandolfini, T., Gabbrielli, R., Ciscato, M., 1996. Nickel toxicity in two durum wheat cultivars differing in drought sensitivity. J. Plant Nutr. 19, 1611 /1627. Poschenrieder, C., Gunse´, B., Barcelo´, J., 1989. Influence of cadmium on water relations, stomatal resistance, and abscisic acid content in expanding bean leaves. Plant Physiol. 90, 1365 /1371. Quartacci, M.F., Sgherri, C.L.M., Pinzino, C., Navari-Izzo, F., 1994. Superoxide radical production in wheat plants differently sensitive to drought. Proc. Roy. Soc. Edinb. 102B, 287 /290. Quartacci, M.F., Pinzino, C., Sgherri, C.L.M., Navari-Izzo, F., 1995. Lipid composition and protein dynamics in thylakoids of two wheat cultivars differently sensitive to drought. Plant Physiol. 108, 191 /197.
Quartacci, M.F., Cosi, E., Navari-Izzo, F., 2001. Lipids and NADPH-dependent superoxide production in plasma membrane vescicles from roots of wheat grown under copper deficiency and excess. J. Exp. Bot. 52, 77 /84. Rauser, W.E., 1978. Early effects of phytotoxic burdens of cadmium, cobalt, nickel and zinc in white beans. Can. J. Bot. 546, 1744 /1749. Romandini, P., Tallandini, L., Beltramini, M., Salvato, B., Manzano, M., de Bertoldi, M., Rocoo, G.P., 1992. Effects of copper and cadmium on growth, superoxide dismutase and catalase activities in different yeast strains. Comp. Biochem. Physiol. 103C, 255 /262. Sandalio, L.M., Dalurzo, H.C., Gomez, M., Romero-Puertas, M.C., del Rio, L.A., 2001. Cadmium-induced changes in the growth and oxidative metabolism of pea plants. J. Exp. Bot. 52, 2115 /2212. Sanita` di Toppi, L., Gabbrielli, R., 1999. Response to cadmium in higher plants. Environ. Exp. Bot. 41, 105 /130. Scandalios, J.G., 1993. Oxygen stress and superoxide dismutases. Plant Physiol. 101, 7 /12. Scandalios, J.G., 1994. Regulation and properties of plant catalases. In: Foyer, C., Mullinaux, P. (Eds.), Causes of Peroxidative Stress and Amelioration of Defence System in Plants. CRC Press, Boca Raton, FL, pp. 275 /315. Sgherri, C.L.M., Navari-Izzo, F., 1995. Sunflower seedlings subjected to increasing water deficit stress: oxidative stress and defence mechanisms. Physiol. Plant. 93, 25 /30. Sgherri, C.L.M., Milone, A.M.T., Clijsters, H., Navari-Izzo, F., 2001. Antioxidative enzymes in two wheat cultivars, differently sensitive to drought and subjected to subsymptomatic copper doses. J. Plant Physiol. 158, 1439 /1447. Sgherri, C., Quartacci, M.F., Izzo, R., Navari-Izzo, F., 2002. Relation between lipoic acid and cell redox status in wheat grown in excess copper. Plant Physiol. Biochem. 40, 591 / 597. Shaw, B.P., 1995. Effect of mercury and cadmium on the activities of antioxidative enzymes in the seedlings of Phaseolus aureus . Biol. Plant. 37, 587 /596. Somashekaraiah, B.V., Padmaja, K., Prasad, A.R.K., 1992. Phytotoxicity of cadmium ions on germinating seedlings of mung bean (Phaseolus vulgaris ): involvement of lipid peroxides in chlorophyll degradation. Physiol. Plant. 85, 85 /89. Stohs, S.J., Bagchi, D., 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18, 321 /336. Van Assche, F., Clijsters, H., 1990. Effect of metal on enzyme activity in plants. Plant Cell Environ. 13, 195 /206. Weckx, J., Vangronsveld, J., Clijsters, H., 1993. Heavy metal induction of ethylene production and stress enzymes. I Kinetics of responses. In: Pech, J.C., Latche´, A., Balagne´, C. (Eds.), Cellular and Molecular Aspects of Plant Hormone Ethylene. Kluwer Academic Publishers, Dordrecht, pp. 238 /239.