Cd toxicity effects on growth, mineral and chlorophyll contents and activities of stress related enzymes

Độc tính của Cd đến sinh trưởng và phát triển thông qua ảnh hưởng của enzyme

Plant and Soil 200: 241–250, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands. 241 Cadmium toxicity effects on growth, mineral and chlorophyll contents, and activities of stress related enzymes in young maize plants (Zea mays L.) A. Lagriffoul 1,3 , B. Mocquot 1 , M. Mench 1 and J. Vangronsveld 2 1 Agronomy Unit, INRA Bordeaux Aquitaine Research Center, BP 81, F-33883 Villenave d’Ornon cedex, France and 2 Limburgs Universitair Centrum, Department SBG, Universitaire Campus, B-3590 Diepenbeek, Belgium. 3 Corresponding author ∗ Received 3 October 1997. Accepted in revised form 4 February 1998 Key words: biomarker, cadmium, maize (Zea mays L.), peroxidase Abstract Plants were cultivated in a nutrient solution containing increasing cadmium concentrations (i.e. 0.001–25 µM), under strictly controlled growth conditions. Changes in both growth parameters and enzyme activities, directly or indirectly related to the cellular free radical scavenging systems, were studied in roots and leaves of 14-day-old maize plants (Zea mays L., cv. Volga) as a result of Cd uptake. A decrease in both shoot lengthand leaf dry biomass was found to be significant only when growing on 25 µM Cd, whereas concentrations of chlorophyll pigments in the 4th leaf decreased from 1.7 µM Cd on. Changes in enzyme activities occurred at lower Cd concentrations in solution leading to lower threshold values for Cd contents in plants than those observed for growth parameters. Peroxidase (POD; E.C. 1.11.1.7) activity increased in the 3rd and 4th leaf, but not in roots. In contrast, glucose- 6-phosphate dehydrogenase (G6PDH; E.C. 1.1.1.49), isocitrate dehydrogenase (ICDH; E.C. 1.1.1.42) and malic enzyme (ME; E.C. 1.1.1.40) activities decreased in the 3rd leaf. According to the relationship between the POD activity and the Cd content, a toxic critical value was set at 3 mg Cd per kg dry matter in the 3rd leaf and 5 mg Cd per kg dry matter in the 4th. Anionic POD were determined both in root and leaf protein extracts; however, no changes in the isoperoxidase pattern were detected in case of Cd toxicity. Results show that in contrast with growth parameters, the measurement of enzyme activities may be included as early biomarkers in a plant bioassay to assess the phytotoxicity of Cd-contaminated soils on maize plants. Abbreviations: AAS – atomic absorption spectrometry, Chl a – chlorophyll a, Chl b – chlorophyll b, DM – dry matter, FW – fresh weight, DMF – N,N-dimethylformamide, GDH – glutamate dehydrogenase, G6PDH – glucose-6-phosphate dehydrogenase, GPOD – guaiacol-peroxidase, GSH – reduced glutathione, HNS – Hoagland nutrient solution, ICDH – isocitrate dehydrogenase, L3 – third leaf, L4 – fourth leaf, ME – malic enzyme, POD – peroxidases, SOD – superoxide dismutases Introduction Cadmium (Cd) is a trace element ubiquitous in the soil. However, anthropogenic activities such as the non-ferrous metal industry, mining, production, use and disposal of batteries, metal-contaminated wastes ∗ FAX No: (33) 556 84 30 54. E-mail: Lagriffo@bordeaux.inra.fr and sludge disposal, application of pesticides and phosphate fertilizers lead to dispersion of Cd (Al- loway, 1995). This non-essential element is taken up through the roots of many species and accumulated in all plant parts including root, shoot, fruit and grain (Page et al., 1981). Taken up in excess, Cd becomes poisonous and can cause serious health hazards to most living organisms (Jackson and Alloway, 1992). Cadmium accumulation through the trophic levels of 242 the food chain constitutes a risk for humans (Wagner, 1993). The potential phytotoxicity of Cd-contaminated soils is usually monitored by chemical analysis and germination tests. Data from chemical analyses pro- vide an estimate of bothtotal Cd contentin the soil and its extractability, but yielding no information on either the fraction of Cd available to plants or the amount taken up by roots and translocated to the aerial parts. Phytotoxicity is due to interference with metabolic processes in plants (Van Assche et al., 1988). Mecha- nisms and effects of phytotoxicity must thus be consid- ered. The dose-response relationships established up to now by agronomists in plant bioassays for the eval- uation of metal contaminated soils are mainly based on visual symptoms, such as chlorosis, necrosis, leaf epinasty and red-brownish discoloration, on biomass reduction, yield decrease and changes in mineral com- position (Van Assche and Clijsters, 1990a). However, these types of symptoms mostly characterize high lev- els of phytotoxicity. These approaches are insufficient to evaluate the soil quality. A better understanding of the Cd effects on plants needs to concern much more sensitive parameters, such as cellular metabolic com- pounds that may reflect, specifically if possible, the physiological and biochemical state of the plant. Cd directly or indirectly inhibits physiological processes such as respiration, photosynthesis, water relations and gas exchange (Van Assche and Cli- jsters, 1990a). Cd may be preferentially accumulated in chloroplasts. Photosynthesis is inhibited at several levels: CO 2 -fixation, stomatal conductance, chloro- phyll synthesis, electron transport and enzymes of the Calvin cycle (Ernst, 1980). Changes in cellular metabolism can be observed even at low levels of Cd, before visual symptoms become evident. Enzyme activities have been used as early diagnostic criteria to evaluate the phytotoxicity of metal-contaminated soils (Mench et al., 1994; Vangronsveld and Clijsters, 1994). One of the main toxic effects of trace met- als, such as copper (Cu) and Cd is oxidative stress, linked with a lipid peroxidation of cellular membranes (De Vos et al., 1991; Ernst et al., 1992). Oxidative stress is defined as all of the effects such as cellu- lar damage, caused by the active forms of oxygen such as superoxide (O ·− 2 ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH ·− ) and singlet oxygen ( 1 O 2 ) (Kappus, 1985). Lipophilic (carotenoids, tocopherols) or water-soluble antioxidants (ascorbate, glutathione), and many scavenging enzymes such as POD, SOD and catalases enable the cell to quench these reac- tive oxygen species (Polle and Rennenberg, 1994). Peroxidases induction is a general response in higher plants to the uptake of toxic amounts of metals, and is likely to be related to oxidative reactions at the plasma membrane. Peroxidases induction has been correlated with the level of Zn and Cd in bean (Van Assche et al., 1988) and to the level of Cu in maize (Mocquot et al., 1996). Enzymes involved in the intermediary metabolism are also altered by toxic amounts of Zn and Cd in bean (Van Assche et al., 1988), e.g. ME, ICDH, GDH and G6PDH. Ernst (1980)suggested that the activity of these enzymes directly or indirectly involved in the respiratory Krebs cycle and pentose phosphate pathway is possibly stimulated to compen- sate for the decrease of ATP and NADPH normally provided by the metal-sensitive photosynthetic reac- tions. According to Slaski et al. (1996), the pentose phosphate pathway could play a role in mediating metal resistance, since several of its intermediates, such as pentoses, erythrose 4-phosphate and NADPH, are precursors in the synthesis of substances poten- tially involved in the alleviation of metal stress (amino acids, nucleic acids, coenzymes and lignin). Studies on Cd toxicity in maize seedlings have largely been concerned with photosynthesis (Feretti et al., 1993), but data on Cd-induced changes in en- zyme activities related to either energetic or tolerance metabolism are scarce. In this paper, the effects of Cd uptakeand partitioning in maizerootsand leaves, from plant grown in hydroponic culture, were investigated using enzymes involved in the antioxydant defence, i.e. GPOD, SOD, and in the intermediary metabolism, i.e. ME, ICDH, G6PDH and GDH. Materials and methods Plant growth and plant samples analysis Plant growth conditions, measurements and analysis, were performed as described previously (Mocquot et al., 1996). Zea mays L. cv. Volga seeds were surface- sterilized with hydrogen peroxide 3% (vol/vol), and rinsed with distilled water. Following germination on filter paper soaked with 2 mM CaSO 4 , seedlings were transferred to a modified HNS that was continuously aerated and renewed every two days. Cd(NO 3 ) 2 was added to the HNS after a two-day acclimation phase, at concentrations derived from a preliminary study: 1, 5, 10, 25, 50, 100, 250, 500, 700 nM and 1, 1.7, 3, 5, 10 and 25 µM. Plants were cultivated in 243 a growth chamber at the following conditions: 25/20 ◦ C day/night temperature, 75% relative humidity, 16/8 h photoperiod and 400 µmol m −2 s −1 photosynthetic photon flux density at leaf level. Plants were harvested 11 days after germination, when they reached the stage of full expansion of the L4. Growth parameters were measured immediately: i.e. shoot length, length and width of each leaf. The leaf area was calculated according to Mocquot et al. (1996). After extraction with DMF, Chl a, Chl b and total carotenoids were measured spectrophotometri- cally (Varian Cary 1E) in the extracts from the L4 at 470, 647 and 664.5 nm respectively, as described by Lichtenhaler and Wellburn (1983), and Blanke (1990). Each plant was divided into roots, L3, L4 and the remaining shoots. The fresh weight of all plant parts was determined. Each organ (1 g FW) was sampled twice, immediatly frozen in liquid nitrogen and stored at −80 ◦ C. The remaining tissues were oven-dried at 80 ◦ C for DM determination and then wet digested in nitric acid and hydrogen peroxide. Cadmium con- centrations were determined by AAS (Varian A 20) or by graphite furnace AAS with Zeeman and deu- terium corrections(Varian A 400). Phosphorus, K, Ca, Mg, Fe, Mn and Zn concentrations were measured by inductively coupled plasma emission spectrometry (Varian Liberty 200). Controls were performed using blanks and a reference material (ryegrass BCR 281, Community Bureau of Reference, Commission of the European Communities) treated in the same way. Only one determination of the concentrationof Cd andother elements was realised per sample. All the used chemi- cals were purchased from Prolabo (Normapur),except Cd(NO 3 ) 2 and HNO 3 purchased from Merck (Pro Analysi). Enzyme assays and isoenzymes determination Frozen material (1 g FW) was homogenized with an ice-cooled mortar and ground in 5 mL of 0.1 M Tris-HCl buffer (pH 7.8) containing 1 mM DTT and1mMEDTA, and centrifuged at 12,000 g (4 ◦ C for 10 min). The supernatants were collected and the activities of the following enzymes were mea- sured spectrophotometrically (Mocquot et al., 1996): POD (E.C.: 1.11.1.7), ME (E.C.: 1.1.1.40), G6PDH (E.C.: 1.1.1.49),GDH (E.C.: 1.4.1.2) and ICDH (E.C.: 1.1.1.42). Guaiacol was used for the determination of total POD activity, which is expressed as mU per g FW. One unit (U) equals the amount of substrate (µmol) transformed by the enzyme in one minute at 30 ◦ C. Total soluble proteins were determined using the Bio-Rad protein assay and expressed as mg protein per g FW. Anionic (iso)peroxidases were separated by poly- acrylamide gel electrophoresis on a 7.5/20% gradient slab gel, and stained enzymatically with 0.04% ben- zidine and 0.006% H 2 O 2 for1.5hat37 ◦ C(Van Assche and Clijsters, 1990b). The gels were scanned densitometrically at 632 nm. Data processing The x,y data sets were curve-fitted by the Fig.P pro- gramme (Biosoft, Ferguson, USA). The threshold values for Cd phytotoxicity were defined as the con- centration in the plant tissue above which growth was reduced or metabolism changed (±10%). It was cal- culated from the intersection of the two straight lines, obtained after plotting the logarithm of the studied parameter against the logarithm or the reciprocal of Cd content in plant tissue (Mocquot et al., 1996). Correlations between variables were calculated with STATITCF 4.0 (ITCF, Boigneville, F.). Data for the morphological parameters were analysed using a con- ventionalanalysisof variance,and the Newman–Keuls test at the 5% level. Results Growth and Cd concentration in plant tissues Increasing the Cd concentration of the HNS led to an increase in Cd content of all maize organs (Fig- ure 1). The Cd concentration ranged from 0.7 to 304 mg kg −1 DM in roots, from 0.3 to 123 mg kg −1 DM in L3, and from 0.13 to 73 mg kg −1 DM in L4. The Cd content in plant tissues increased curvilinearly with the Cd concentration of the nutrient solution. The curve plots were best fitted by the classical Michaelis– Menten function Y = Cm ∗ X / (Km + X), where Cm = maximum tissue concentration (mg kg −1 DM), Km = affinity coefficient (µM), Y = Cd concentration in plant and X = Cd concentration in the HNS. Values for these kinetic parameters were estimated from the curvilinear relationships (Figure 1) for roots (Km = 1.7, Cm = 336, r 2 = 0.99), for L3 (Km = 6.5, cm = 147, r 2 = 0.98) and for L4 (Km = 4.9, Cm = 82, r 2 = 0.98). No visual symptoms of Cd toxicity were observed in any organ of the seedlings grown on Cd concen- trations ranging from 1 nM to 10 µM. No significant 244 Figure 1. Cd concentrations (mg kg −1 DM) in L3, L4 and roots of Zea mays L. cv. Volga after two weeks of growth in a modified Hoagland nutrient solution with increasing Cd concentrations. changes in any of the morphological parameters stud- ied occured, i.e. root and leaf weight, plant height and leaf area. The mean values for plants grown on 1.7 µM Cd in the HNS were lower than those for plants cultivated at lower Cd concentrations. How- ever, the decrease was only statistically significant (p ≤ 0.05) above 25 µM Cd in the HNS. Plants exposed to 25 µM Cd showed a significant decrease in shoot length and in fresh weight, when the tissue Cd content reached 123 mg kg −1 DM in L3 and 73 mg kg −1 DM in L4 (Figures 2a, b). Leaf area was reduced at 25 µM Cd in L3 only (Figure 2a). In contrast, a linear ratio between fresh and dry weights with increasing Cd contents in plant tissues shows that water content and dry matter in leaves and roots were not signifi- cantly changed at any of the Cd concentrationsstudied (data not shown). Chlorophyllpigments extracted with DMF from L4 showed a decrease. This effect was ob- served prior to effects on morphological parameters (Figure 3). Sharp decreases in Chl a, Chl b and in total carotenoid contents of L4 were observed when Cd concentrations exceeded 17 mg Cd kg −1 DM. Protein contents The total soluble protein content was examined in leaf and root samples. Roots and L4 showed no significant changes in response to the Cd accumulation. Mean values were 1.4 ± 0.2mgg −1 FW in roots and 10.2 ± 1.3mgg −1 FW in L4. Maize roots contain less soluble protein than leaves. Significant changes in L3 were found. The total soluble protein content of L3 gradually increased from about 4 to 10 mg g −1 FW, up to a Cd concentration of 13 mg kg −1 DM (Figure Figure 2. Changes in morphological parameters, i.e. (a) shoot length (cm) and L3 area (cm 2 ) and (b) L3 and L4 yield (g) of Zea mays L. cv. Volga in relation to Cd concentrations in a modified Hoagland nutrient solution or logarithm of Cd concentrations in plant parts. 4). A constant level of protein about 9 mg g −1 FW was then maintained. Enzyme activities and isoenzyme analysis The activities of the several enzymes were correlated with the leaf Cd content either positively (induction), POD in L3 and L4 (Figures 5a, b), or negatively (inhibition), ICDH, ME and G6PDH in L3 only (Fig- ures 5c–e). In maize roots and leaves, the activity of GDH was not modified by Cd accumulation (data not shown). Foliar POD activity was higher with in- creasing Cd concentrations (Figures 5a, b), whereas no changes were observed in roots (data not shown). 245 Figure 3. Changes in the contents (mg m −2 ) of (a) chlorophyll A, chlorophyll B and (b) carotenoid in L4 of Zea mays L. cv. Volga in relation to the logarithm of Cd concentrations in the tissue, after two weeks of growth in a modified Hoagland nutrient solution. Figure 4. Changes in total protein contents (mg g −1 FW) in L3 of Zea mays L. cv. Volga in relation to the logarithm of Cd concen- trations, after two weeks of growth in a modified Hoagland nutrient solution. In contrast, the activities of the NAD(P)H-reducing enzymes such as G6PDH, ICDH and ME were de- creased, but only in L3 (Figures 5c–e). In L4, no significant effects on enzyme activities related to inter- mediary metabolism were observed (data not shown). In roots, the activities of all the enzymes studied were constant despite Cd increase in this tissue (data not shown). No significant changes in SOD activities were observed in the samples (data not shown). For all enzymes showing a correlation with Cd content in plant tissue, a quadratic function fitted best with the experimental data. Threshold values for leaf Cd con- tents, above which enzyme activity either increased or decreased are shown in Table 1. According to Van Assche et al. (1988), and in con- trast to Figures 5a, b, plotting the logarithm of enzyme activity against the reciprocal of Cd content in the tis- sue allows the calculation of the Cd threshold value of enzyme induction. Cadmium threshold value for POD induction in maize L3 is shown as an example (Figure 6). Apparently, POD activity was stimulated when Cd exceeded 3 mg kg −1 DM in L3 and 5 mg kg −1 DM in L4 (data not shown). As a result of Cd toxicity, the activities of several enzymes involved in or closely related to the Krebs cycle were significantly decreased in L3. Losses in activity were observed from about 15 mg Cd kg −1 DM for G6PDH (Figure 5e) and 22 mg Cd kg −1 DM for ICDH (Figure 5c). In L4, the activity of these enzymes tends to decrease at the highest tissue Cd concentration, but these data were not significantly different from each others (data not shown). Cd-induced changes in total POD activity in leaves were not reflected in changes in the pattern of isoen- zymes. In constrast to Cu (Mocquot et al., 1996), gels stained for POD activity did not show induction of specific (iso)peroxidases either in leaves or in roots, even at the highest Cd concentration. Mineral composition of plant tissues The contents of essential elements (P, K, Mg, Fe, Zn, Mn, Cu and Ca) were not significantly modified by Cd treatment. Data concerning mean concentrations and standard errorsfor these elements are given in Table 2. The effects of Cd on the concentrations of P and Mn in maize are given in Figures 7a, b, respectively. With increasing Cd accumulation, Mn contents were lower in both leaves and roots, while P contents increased only in leaves. For both elements, significant changes occured above 1.7 µM Cd. The concentration of these 246 Figure 5. Enzyme activities (mU g −1 FW) in leaves of Zea mays L. cv. Volga as a function of the logarithm of Cd concentrations in the leaves: (a–b) (guaiacol)-peroxidase in L3 and L4, (c) isocitrate dehydrogenase in L3, (d) malic enzyme in L3 and (e) glucose-6- phosphate dehydrogenase in L3. elements, except Mg, was higher in L3 than in L4, especially Ca (data not shown). Discussion Morphological toxicity symptoms were only observed at high Cd concentrations in leaf and root tissues (Ta- ble 1). Compared to data obtained with copper on the same maize cultivar and grown under exactly the same conditions (Mocquot et al., 1996), threshold values of tissue metal concentrations at which plant growth was significantly inhibited were 3–7 times higher. Metal threshold values for shoot length reduction were 27 and14mgkg −1 DM in Cu-treated L3 and L4 respec- tively, compared to 123 and 73 mg kg −1 DM in the same organs treated with Cd (Table 1). These results suggest that maize is more tolerant to Cd than to Cu. In another maize cultivar, Galli et al. (1996) reported a strong reduction in root dry weight for a root Cd 247 Table 1. Toxic threshold values (mg Cd kg −1 DM) calculated from the functions fitting the relationship between either growth para- meters, chlorophyllous pigments, total protein contents or enzyme activities and Cd concentrations in the tissues of Zea mays L. cv. Vo lg a Plant organs Roots Third leaf (L3) Fourth leaf (L4) Growth parameters Shoot length 304 123 73 Leaf area ns ns Leaf yield 123 73 Root yield ns Total upper parts 76 48 Total upper parts 123 73 Chlorophyllous pigments Chlorophyll a nd 28 Chlorophyll b nd 28 Total carotenoids nd 28 Enzyme activity POD ns 3 5 ICDH ns 22 ns G-6PDH ns 15 ns ME ns 20 ns GDH ns ns ns ns: not significant. nd: not determined. Figure 6. Logarithm of peroxidase activity in tissue extracts plotted against the reciprocal of Cd concentration in L3 of Zea mays L. cv. Volga, after two weeks of growth in a modified Hoagland nutrient solution. Table 2. Mineral concentrations (mg kg −1 DM) in plant organs of maize (Zea mays L. cv. Volga) cultivated in a modified Hoagland nu- trient solution with Cd concentration ranging from 0.001 to 25 µM: mean and standard deviation values across treatment for element concentrations showing no significant change Plant organs Roots Third leaf (L3) Fourth leaf (L4) Potassium (K) 53024 ± 5952 69364 ± 3005 61850 ± 3882 Magnesium (Mg) 2344 ± 281 953 ± 76 1126 ± 69 Iron (Fe) 5200 ± 810 101 ± 11 79 ± 25 Zinc (Zn) 35 ± 833±832±10 Copper (Cu) 15 ± 214±29±2 Calcium (Ca) 4099 ± 666 3436 ± 353 1684 ± 197 Figure 7. Phosphorus (a) and manganese (b) concentrations (mg kg −1 DM) in L3, L4 and roots of Zea mays L. cv. Volga after two weeks of growth in a modified Hoagland nutrient solution with increasing Cd concentrations. 248 concentration of 562 mg kg −1 DM. This was higher that Cd concentration found in the present study (304 mg kg −1 DM for the highest Cd treatment). Bean seedlings seem to be less tolerant to Cd than maize seedlings. For morphological parameters only, a Cd threshold value of 5.5 mg kg −1 DM in pri- mary leaves of bean was found (Van Assche et al., 1988). This suggests that maize plants seem to possess other and/or more efficient detoxification mechanisms to deal with Cd toxicity than bean, including metal- accumulation, binding and inactivation, and oxidative stress scavenging systems. The increased metal concentrations in the tissues cause increased activities of some enzymes (induc- tion) either as a result of de novo protein synthesis or by the activation of enzymes already present in plant cells (Van Assche and Clijsters, 1990a). Changes in SOD and POD activities, and in the (iso)SOD and (iso)POD patterns have been reported both in leaves and roots of bean plants as a result of Cd toxicity (Van Assche et al., 1988). The ‘stress point’ is defined as the metabolic state where the regulation of pathways towards positive direction for plant fitness is at its lim- its (Elstner et al., 1988), and is probably reached at the toxic threshold level of the metal in the tissue (Van Assche and Clijsters, 1990a). The high affinity of metals for sulphydryl groups (-SH) is suggested to be one of the main mecha- nisms of enzyme inhibition (Karataglis et al., 1991; Weigel and Jäger, 1980). Measured decreases in en- zymes of intermediary metabolism (G6PDH, ICDH and ME) contrast with previous observations. Induc- tion of ICDH, GDH, G6PDH and ME activity has been reportedin bean leaves (Van Assche et al., 1988). Cadmium threshold values were calculated to be 3.1, 4.6, 5.5 and 7.4 mg kg −1 DM, respectively for GDH, ME, ICDH and G6PDH induction. In soybean treated with toxic concentrations of Cd and Pb, a similar in- crease has been reported (Lee et al., 1976). A rapid increase in G6PDH activity has been shown by Slaski et al. (1996) in resistant cultivars of wheat treated with Al and in silene treated with Zn (Mathys, 1975). In contrast, the same author reported a total inhibition of G6PDH activity in isolated preparations of this en- zyme treated with Zn. A review of existing data on the induction of enzymes of the intermediary metabolism by several metals is given by Vangronsveld and Cli- jsters (1994). The fact that these enzymes seem to be inhibited by Cd in maize suggests that enzyme responses can vary between plant species and metal treatments. In principle, two main mechanisms of en- zyme inhibition can occur: (1) binding of the metal to sulfhydryl groups, involved in the catalytic action or structural integrity of enzymes, and (2) deficiency of an essential metal in metalloprotein complexes, com- bined with the substitution of the toxic metal for the deficient element. ICDH and ME require Mn +2 ions for activity, so Mn deficiency could act as a limiting factor for ICDH and ME activity. Cd-induced Mn- deficiency in leaves and roots (vide infra) (Figure 7b) could be responsible for the inhibition of these two en- zymes in maize leaves. However, the threshold value for Mn +2 decrease in leaves appeared at higher Cd concentrations than the threshold values for enzyme inhibition. Similary, G6PDH requires Mg +2 ions for activity, but no significant change in Mg +2 content were observed in maize leaves or roots in response to the Cd accumulation (data not shown). Cho and Joshi (1989) found that the loss of G6PDH activity in baker yeast was due to conformational changes in protein structure by metal-binding to the carboxyl or hydroxyl groups of the enzyme. Our data do not discriminate between the relative importance of direct effects on enzymes, substrate limitation or feedback regulation. The chlorophyll pigment contents found in Cd- contaminated maize L4 are similar to those reported by Mocquot et al. (1996) in young plants treated with Cu. In comparison with the critical values for leaf yield or shoot length reduction of the L4 (Ta- ble 1), the decrease in chlorophyll and carotenoid contents appears to be one of the first visible biomark- ers of Cd toxicity. Two possible mechanisms of Cd toxicity on photosynthesis have been proposed to ex- plain the decrease in chlorophyll pigments. Cadmium can alter both chlorophyll biosynthesis by inhibiting protochlorophyllide reductase and the photosynthetic electron transport by inhibiting the water-splitting en- zyme located at the oxidising site of photosystem II (Van Assche and Clijsters, 1990a). Interactions with SH groups are involved in the inhibition of pro- tochlorophyllide reductase by Cd. Since Mn is essen- tial for optimal water-splitting activity (Baszinsky et al., 1980) the interaction observed between Cd and Mn in the whole maize plant (see Figure 7b) could possibly explain the inhibition of electron transport at the level of the water-splitting complex and hence the decrease in chlorophyll pigment contents. In constrast to the high threshold values for inhi- bition of morphological parameters, POD activity was induced at the low tissue Cd concentrations of 3 and 5 mg kg −1 DM in L3 and L4 respectively (Table 1). This is comparable to the Cd threshold values previously 249 found for POD in primary bean and soybean leaves which were reported to be 5.5 mg kg −1 DM (Van Ass- che et al., 1988)and 3 mg kg −1 DM (Lee et al., 1976), respectively. Since morphologicalparametersin maize are only affected at much higher tissue Cd contents, POD activation is a important event in Cd-induced acclimation. Induction of POD is a general stress re- sponse and is not specific to metals. The hypothesis that Cd induces a more general physiological reaction involving formation of H 2 O 2 and/or organic peroxides (Elstner et al., 1988) is supported by these observa- tions. During stress, oxygen derivatives accumulate and this results in the induction of enzymes such as POD, SOD and catalases (Creissen et al., 1994). These enzymes provide antioxidant protection and preserve membrane integrity. The removal of H 2 O 2 produced in chloroplasts is essential to avoid inhibition of the Calvin cycle enzymes (Tanaka et al., 1982). Peroxi- dases transform peroxide into water using an electron donor (R) which is oxidized during catalysis: RH 2 + H 2 O 2 ⇒ R+2H 2 O. Electron donors such as ascor- bate, glutathione, guaiacol and many other reduced compounds are involved in POD reactions. Several reports have shown that the addition of Cd to soils culture decreases the Mn concentration in tis- sues of radish (Khan and Frankland, 1983) and oat (Bjerre and Scierup, 1985). Jalil et al. (1994) reported that 0.5 µM Cd in a nutrient solution significantly decreases the Mn and Zn concentrations in the roots and theshoots of durumwheat. A significant reduction in biomass as a result of Cd application proposed by these authors to explain the similar decrease in Fe and Cu contents in wheat can not be extrapolated to ex- plain the Mn depletion observed in the present study. However, the distinctive decrease in Mn and Zn con- centrations in wheat observed at 0.5 µM Cd in the solution may have been due to competition between Zn, Mn and Cd for transport across the plasmalemma. Below critical values, no competition occurred, but above 0.5 µM Cd in the HNS, tissue Cd concen- trations became 2.5 higher than that of Mn and Zn, and competition may occur on transport sites on the plasmalemma. Alternative explanations are however possible. According to Khan and Khan (1983), Fe and Mn concentrationsincrease significantlyin tomato and decrease in egg-plant when Cd is added to the soil culture. Mn and Fe seem to complex with the same organic ligands as heavy metals in egg-plant. This in- teraction at the root surface results in decreased uptake by the plant. All these reports therefore indicate that important interactions of Cd with other essential met- als can occur, either at the root surface or at the plasma membrane of root cells. Conclusions The variations on growth and mineral contents of Cd-contaminated maize seedlings are not a sensitive parameter to evaluate the consequences of Cd uptake. Changes on morphological parameters occurred only at the highest metal concentration studied. Moreover, the evaluation of a Cd-induced oxidative stress us- ing as biomarkers the enzyme activities involved in the intermediary metabolism seems to be rather in- direct. The first defensive mechanism from oxidative stress is the scavenging of activated oxygen species at the sites where they are generated, especially chloro- plasts where Cd accumulates. The chlorophyll and carotenoid decreases at relatively low Cd concentra- tion can be indicative of a damage at the chloroplast level. The evaluation of the enzyme activities less or more directly linked to the respiratory Krebs cycle should be useful to evaluate the general and secondary effects of Cd toxicity on metabolic processes, but not to test the real induction of an early oxidative stress. The determination of scavenging enzyme activities, antioxidative compounds (ascorbate and glutathione) and primary toxic compoundssuchas hydrogenperox- ide could give more precise information on the induc- tion of an activated oxygen-induced stress. All these compounds will be investigated in further studies. Acknowledgements The authors would like to thank Prof. C Foyer for critical reading of the manuscript and fruitful cor- rections. We are very grateful to Mrs S Bussière and Mr T Prunet from INRA Agronomy Unit, Bor- deaux, France, for technical assistance. This work was supported by grants from Institut National de la Recherche Agronomique, Secteur Environnement Physique & Agronomie. References Alloway B J 1995 Heavy Metals in Soils, 2nd ed. Blackie Academic & Professional Publishers, London. 368 p. Baszinsky T, Wajda L, Krol M, Wolinska D, Krupa Z and Tukendorf A 1980 Photosynthetic activities of cadmium treated tomato plants. Physiol. Plant. 48, 365-370. 250 Bjerre G K and Schierup H 1985 Uptake of six heavy metals by oat as influenced by soil type and additions of cadmium, lead, zinc and copper. Plant Soil 88, 57–69. Blanke M M 1990 Determination of chlorophyll with DMF. Vitic. Enol. Sci. 45, 76–78. Cho S W and Joshi J G 1989 Inactivation of baker’s yeast glucose- 6-phosphate dehydrogenase by aluminium. Biochemistry 28, 3613–3618. Creissen G P, Edwards E A and Mullineaux P M 1994 Glutathione reductase and ascorbate peroxidase. In Causes of Photooxidative Stress and Amelioration of Defense Systems in Plants. Eds. C H Foyer and P M Mullineaux. pp 344–361. CRC Press, Boca Raton, Florida. De Vos C H R, Schat H, De Waal M A M, Vooijs R and Ernst W H O 1991 Increased resistance to copper induced damage of the root cell plasmalemma in copper-tolerant Silene cucubalus. Physiol. Plant. 82, 523–528. Elstner E F, Wagner G A and Schutz W 1988 Activated oxygen in green plants in relation to stress situations. Curr. Top. Plant Physiol. Biochem. 7, 159–187. Ernst W H O 1980 Biochemical aspects of cadmium in plants. In Cadmium in the Environment. Ed. J O Nriagu. pp 639–653. Wiley and Sons, New York. Ernst W H O, Verkleij J A C and Schat H 1992 Metal tolerance in plants. Acta Bot. Neer. 41, 229–248. Ferretti M, Ghisi R, Merlo L, Dalla Vecchia F and Passera C 1993 Effect of cadmium on photosynthesis and enzymes of photosyn- thetic sulphate and nitrate assimilation pathways in maize (Zea mays L.). Photosynthetica 29, 49–54. Galli U, Schüepp H and Brunold C 1996 Thiols in cadmium and copper-treated maize (Zea mays L.). Planta 198, 139–143. Jackson A P and Alloway B J 1992 The transfer of cadmium from agricultural soils to the human food chain. In Biogeochemistry of Trace Metals. Ed. D C Adriano. pp 109–158. CRC Press, Boca Raton, Florida, USA. Jalil A, Selles F and Clarke J M 1994 Growth and Cd accumulation in two durum wheat cultivars. Commun. Soil Sci. Plant Anal. 25, 2597–2611. Kappus H 1985 Lipid peroxidation: mechanisms, analysis, enzy- mology and biochemical relevance. In Oxidative Stress. Ed. H Sies. pp 273–310. Academic Press I l.c., London. Karataglis S, Moustakas M and Symeonidis L 1991 Effect of heavy metals on isoperoxydases of wheat. Biol. Plant. 33, 3–9. Khan S and Khan N S 1983 Influence of lead and cadmium on the growth and nutrient concentration of tomato (Lycopersicon esculentum) and egg-plant (Solanum melongena). Plant Soil 74, 387–394. Khan D H and Frankland B 1983 Effects of cadmium and lead on radish plants with particular reference to movement of metals through soil profile and plant. Plant Soil 70, 335–345. Lee K C, Cunningham B A, Paulsen G M, Liang G H and Moore R B 1976 Effects of cadmium on respiration rate and activities of several enzymes in soybean seedlings. Physiol. Plant. 36, 4–6. Lichtenhaler H K and Wellburn A R 1983 Determination of total carotenoids and chlorophylls a and b of leaf extracts in differ- ent solvents. Biochem. Soc. Trans. 603rd Meeting, Liverpool 11, 591–592. Mathys W1975 Enzymes of heavy-metal-resistant and non-resistant populations of Silene cucubalus and their interaction with some heavy metals in vitro and in vivo. Physiol. Plant. 33, 161–165. Mench M, Martin E and Solda P 1994 After effects of metals de- rived from a highly metal-polluted sludge on maize (Zea mays L.). Water Air Soil Pollut. 75, 277–291. Mocquot B, Vangronsveld J, Clijsters H and Mench m 1996 Copper toxicity in young maize (Zea mays L.) plants: effects on growth, mineral and chlorophyll contents, and enzyme activities. Plant Soil 182, 287–300. Page A L, Bingham F T and Chang A C 1981 Cadmium. In Effect of Heavy Metal Pollution on Plants. Vol. 1: Effects of Trace Metals on Plant Function. Ed. N W Leep. pp 77–109. Applied Science Publishers, Ripple Road, Barking, Essex, England. Polle A and Rennenberg H 1994 Photooxidative stress in trees. In Causes of Photooxidative Stress and Amelioration of Defense Systems in Plants. Eds. C H Foyer and P M Mullineaux. pp 200– 212. CRC Press, Boca Raton, Florida. Slaski J J, Zhang G, Basu U, Stephens J L and Taylor g 1996 Aluminium resistance in wheat (Triticum aestivum L.) is asso- ciated with rapid Al-induced changes in activities of glucose-6- phosphate dehydrogenase and 6-phosphogluconate dehydroge- nase in root apices. Physiol. Plant. 98, 477–484. Tanaka K, Otsubo T and Kondo N 1982 Participation of hydrogen peroxide in the inactivation of Calvin cycle SH enzymes in SO 2 - fumigated spinach leaves. Plant Cell Physiol. 23, 1009–1018. Van Assche F, Cardinaels C and Clijsters H 1988 Induction of enzyme capacity in plants as a result of heavy metal toxicity; dose-reponse relations in Phaseolus vulgaris L., treated with zinc and cadmium. Environ. Pollut. 52, 103–115. Van Assche F and Clijsters C 1990a Effects of metals on enzyme activity in plants. Plant Cell Environ. 13, 195–206. Van Assche F and Clijsters C 1990b A biological test system for the evaluation of the phytotoxicity of metal contaminated soils. Environ. Pollut. 66, 157–172. Vangronsveld J and Clijsters H 1994 Toxic effects of metals. In Plants and the Chemical Elements. Biochemistry, Uptake, Tolerance and Toxicity. Ed. M E Farago. pp 149–177. VCH Verlagsgesellschaft, Weinheim, Germany. Wagner G J 1993 Accumulation of cadmium in crop plants and its consequences to human health. Adv. Agron. 51, 173–205. Weigel H J and Jäger H J 1980 Subcellular distribution and chemical form of Cd in bean plants. Plant Physiol. 65, 480–482. Section editor: L V Kochian . Cd treatment. Data concerning mean concentrations and standard errorsfor these elements are given in Table 2. The effects of Cd on the concentrations of. 281, Community Bureau of Reference, Commission of the European Communities) treated in the same way. Only one determination of the concentrationof Cd andother elements