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

© 1998 Kluwer Academic Publishers Printed in the Netherlands.

Cadmium toxicity effects on growth, mineral and chlorophyll contents,

and activities of stress related enzymes in young maize plants (Zea mays

L.)

A Lagriffoul1,3, B Mocquot1, M Mench1and J Vangronsveld2

1Agronomy Unit, INRA Bordeaux Aquitaine Research Center, BP 81, F-33883 Villenave d’Ornon cedex, France and2Limburgs Universitair Centrum, Department SBG, Universitaire Campus, B-3590 Diepenbeek, Belgium.

3Corresponding 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 length and 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

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 both total Cd content in 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: CO2-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 (H2O2), hydroxyl radical (OH·−) and singlet oxygen (1O2)

(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 uptake and partitioning in maize roots and 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 CaSO4, seedlings were transferred to a modified HNS that was continuously aerated and renewed every two days Cd(NO3)2was 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

a growth chamber at the following conditions: 25/20

◦C day/night temperature, 75% relative humidity, 16/8

h photoperiod and 400 µmol m−2s−1photosynthetic

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 concentration of Cd and other

elements was realised per sample All the used

chemi-cals were purchased from Prolabo (Normapur), except

Cd(NO3)2 and HNO3 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

and 1 mM EDTA, 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% H2O2 for 1.5 h at 37 ◦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-ventional analysis of 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−1DM 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−1DM), 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, r2 = 0.99), for L3 (Km = 6.5, cm =

147, r2= 0.98) and for L4 (Km = 4.9, Cm = 82, r2= 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

Figure 1 Cd concentrations (mg kg−1DM) 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−1DM in L3 and 73 mg kg−1DM

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 concentrations studied

(data not shown) Chlorophyll pigments 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−1DM.

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± 0.2 mg g−1FW in roots and 10.2

± 1.3 mg g−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−1DM (Figure

Figure 2 Changes in morphological parameters, i.e (a) shoot

length (cm) and L3 area (cm2) 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−1FW 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)

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−1FW) 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−1DM in L3 and 5 mg kg−1DM

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−1DM for G6PDH (Figure 5e) and 22 mg

Cd kg−1DM 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 errors for 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

Figure 5 Enzyme activities (mU g−1FW) 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 and 14 mg kg−1DM 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

Table 1 Toxic threshold values (mg Cd kg−1DM) 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.

Volga

Plant organs Roots Third leaf (L3) Fourth leaf (L4)

Growth parameters

Chlorophyllous pigments

Enzyme activity

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−1DM) 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

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.

concentration of 562 mg kg−1DM This was higher

that Cd concentration found in the present study (304

mg kg−1DM 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 reported in 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−1DM, 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+2ions

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−1DM in L3 and L4 respectively (Table 1) This

is comparable to the Cd threshold values previously

found for POD in primary bean and soybean leaves

which were reported to be 5.5 mg kg−1DM (Van

Ass-che et al., 1988) and 3 mg kg−1DM (Lee et al., 1976),

respectively Since morphological parameters in 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 H2O2and/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 H2O2 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: RH2+

H2O2⇒ R + 2H2O 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 the shoots of durum wheat 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 concentrations increase significantly in 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 compounds such as hydrogen perox-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

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Section editor: L V Kochian