Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars

ARTICLE IN PRESS Journal of Plant Physiology 165 (2008) 600—611

www.elsevier.de/jplph

Effects of cadmium on antioxidant enzyme and photosynthetic activities in leaves of two maize cultivars Yasemin Ekmekc-ia,, Deniz Tanyolac-b, Beycan Ayhana a

Hacettepe University, Faculty of Science, Department of Biology, Beytepe Campus, 06800 Ankara, Turkey Hacettepe University, Faculty of Engineering, Department of Chemical Engineering, Beytepe Campus, 06800 Ankara, Turkey

b

Received 23 November 2006; received in revised form 24 January 2007; accepted 24 January 2007

KEYWORDS Antioxidant enzymes; Cadmium (Cd2+); Chlorophyll fluorescence; Maize (Zea mays L.)

Summary Effects of cadmium (Cd2+) on photosynthetic and antioxidant activities of maize (Zea mays L.) cultivars (3223 and 32D99) were investigated. Fourteen-day-old cultivar seedlings were exposed to different Cd concentrations [0, 0.3, 0.6 and 0.9 mM Cd(NO3)2  4H2O] for 8 days. The results of chlorophyll fluorescence indicated that different levels of Cd affected photochemical efficiency in 3223 much more than that in 32D99. In parallel, the level of Cd at 0.9 mM caused oxidative damage but did not indicate cessation of PSII activity of the cultivars; plant death was not observed at highly toxic Cd levels. Additionally, the increase in Cd concentration caused loss of chlorophylls and carotenoid and membrane damage in both cultivars, but greater membrane damage was observed in 3223 than in 32D99. Depending on Cd accumulation, a significant reduction in dry biomass was observed in both cultivars at all Cd concentrations. The accumulation of Cd was higher in roots than in leaves for both cultivars. Nevertheless, cultivar 3223 transferred more Cd from roots to leaves than did 32D99. On the other hand, our results suggest that there were similar responses in SOD, APX and GR activities with increasing Cd concentrations for both cultivars. However, POD activity significantly increased at highly toxic Cd levels in 32D99. This result may be regarded as an indication of better tolerance of the Z. mays L. cultivar 32D99 to Cd contamination. & 2007 Elsevier GmbH. All rights reserved.

Corresponding author. Tel.: +90 312 2976166; fax: +90 312 2992028.

E-mail address: [email protected] (Y. Ekmekc-i). 0176-1617/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2007.01.017

ARTICLE IN PRESS Effects of cadmium in two maize cultivars

Introduction Heavy metal contamination is a serious environmental problem that limits plant productivity and threatens human health. Cadmium (Cd) is one of the most highly toxic environmental pollutants in the atmosphere, soil and water, and in excessive amounts can cause serious problems to all organisms (Benavides et al., 2005). Toxic levels of Cd may be caused by contaminated soil characteristics with abundant Cd or by agricultural manufacturing, mining and other waste disposal practices, or by use of metal-containing pesticides and fertilizers in agricultural soils (Radotic et al., 2000). Cd concentration of uncontaminated soils is usually below 0.5 mg/kg, but can reach up to 3 mg/kg depending on the soil material (Schachtschabel et al., 1984). At low concentrations Cd is not toxic to plants, but it is toxic at higher concentrations and characteristically inhibits root growth and cell division (Liu et al., 2003). In higher plants, roots are the first organs with contact to the toxic metal ions, and roots usually accumulate significantly higher amounts of metal than do shoots (Breckle, 1991). Although Cd is not an essential nutrient for plants, it is readily taken up and both directly and indirectly affects several metabolic activities in different cell compartments, especially chloroplasts. These deleterious effects include inhibition of photosynthesis, such as biosynthesis of chlorophyll (Stobart et al., 1985; Padmaja et al., 1990) and functioning of photochemical reactions (Krupa and Moniak, 1998). Photosystem II (PSII) is extremely sensitive to Cd and its function was inhibited to a much greater extent than that of PSI (Chugh and Sawhney, 1999; Mallick and Mohn, 2003). Recent studies have also indicated that Cd exerts multiple effects on both donor and acceptor sites of PSII. On the donor site, the presence of Cd inhibits the oxygen evolving cycle and, consequently, oxygen evolution; on the acceptor site, it inhibits  electron transfer from Q A to QB (Krupa and Moniak, 1998; Krupa, 1999; Sigfridsson et al., 2004). Plants respond to heavy metal toxicity in a variety of ways. Based on chemical and physical properties of metals, three different molecular mechanisms of heavy metal toxicity can be distinguished: (a) production of active oxygen species (AOS) by autoxidation and Fenton reaction such as for iron or copper, (b) blocking of essential functional groups in biomolecules, such as for Cd and mercury and (c) displacement of essential metal ions from biomolecules (Schu ¨tzendu ¨bel and Polle, 2002). Heavy metals cause oxidative damage to plants, either directly or indirectly through AOS formation (Schu ¨tzendu ¨bel et al., 2001). Redox

601 metals such as Cu or Fe appear to act directly on the production of AOS (via Fenton or Haber Weiss reactions) (Salin, 1988). In contrast to these heavy metals, Cd is a non-redox metal unable to participate in Fenton-type reactions, but it leads to the formation of AOS (Van Assche and Clijsters, 1990; Dixit et al., 2001; Laspina et al., 2005) such 1 as superoxide radicals (Od 2 ), singlet oxygen ( O2), hydrogen peroxide (H2O2) and hydroxyl radical (OHd). These AOS are highly reactive and damage membrane lipids, proteins, pigments and nucleic acids, resulting in dramatic reductions of growth and productivity, and eventually causing the death of plants (Foyer et al., 1994). AOS are efficiently eliminated by non-enzymatic (glutathione, ascorbate, a-tocopherol and carotenoids) and enzymatic defence systems such as superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD) and glutathione reductase (GR), which protect plants against oxidative damage (Foyer and Mullineaux, 1994). The response of antioxidant enzymes to Cd, and in general to metals, can vary among species and among different tissues (Dixit et al., 2001; Hegedu ¨s et al., 2001; Shah et al., 2001; Vitoria et al., 2001; Wu et al., 2003; Qadir et al., 2004; Sing et al., 2004; Hassan et al., 2005; Tiryakioglu et al., 2006). The risk of Cd uptake by crops, followed by the transfer in the food chain, is an issue of vital concern. Maize is the third most important crop (following wheat and barley) in Turkey and is raised in an approximate area of 800,000 ha (FAOSTAT, 2006). There are a limited number of publications on photosynthesis and protection mechanisms in maize leaves under Cd stress (Wo ´jcik and Tukendorf, 1999; Draz´kiewicz et al., 2003; Draz´kiewicz and Baszyn ´ski, 2005). Nevertheless, there have been no studies to date related to antioxidative and photochemical responses in maize cultivars under Cd stress. The objectives of the present study were, first, to establish and verify Cd accumulation in cultivars, second, to evaluate the effect of Cd toxicity on photosynthetic performance and, finally, to determine the effect of Cd on antioxidant enzymes in tolerance mechanisms of the Zea mays L. cultivars in the range of 300–900 mM Cd concentration.

Materials and methods Plant materials, growth and treatment conditions Maize (Z. mays L.) cultivars 32D99 and 3223 were used in the study due to their tolerant and sensitive characteristics at the early seedling stage, respectively, to Cd stress determined by preliminary experiments

ARTICLE IN PRESS Y. Ekmekc-i et al.

602 (Ayhan et al., 2005). Seeds were germinated under dark conditions at 2372 1C on humidified filter paper with distilled water for 6 days. The seedlings of cultivars were planted on plastic pods containing perlite culture and watered for 8 days with Hewitt’s nutrient solution when required. Plants were grown at a constant temperature regime of 2371 1C, 4075% humidity and at 250 mmol m2 s1 light intensity for a 14 h photoperiod in a controlled growth cabinet. After the 14th day of growth, Cd treatment was initiated by watering Hewitt’s nutrient solution containing 0.3, 0.6 and 0.9 mM Cd(NO3)2  4H2O to seedlings. Plants were grown in the growth cabinet under the same conditions for another 8 days. The second and third leaves of 22-day-old plants were evaluated in the experimental analyses. The biomass of leaves and roots was measured on a dry weight basis after drying at 80 1C for 48 h.

Chlorophyll a fluorescence measurements Chlorophyll a fluorescence measurements were performed in a growth cabinet at 24 1C with a modulated fluorescence monitoring system (FMS-II-Hansatech, UK) on selected leaves of the cultivars. Following 30 min of dark adaptation, the minimum chlorophyll fluorescence (F0) was determined using a measuring beam of 0.2 mmol m2 s1 intensity. A saturation pulse (white light 7500 mmol m2 s1) was used to obtain the maximum fluorescence (FM) in the dark-adapted state and the quantum efficiency of PSII open centers in dark-adapted seedlings (FV/FM) was determined. The leaves were then illuminated with actinic light (300 mmol m2 s1). The steady-stage value of fluorescence (FS) was recorded thereafter and a second saturating pulse at 7500 mmol m2 s1 was imposed to determine maximum fluorescence in the light-saturated stage (F 0 M). The actinic light was removed and the minimum fluorescence in the lightsaturated stage (F 0 o) was determined by illuminating the leaves with far-red light (7 mmol m2 s1). The quantum efficiency of PSII open centers’ light-adapted state, referred to as FPSII (F 0 M–FS/F 0 M), and the quantum efficiency of excitation energy trapping of PSII, (F 0 V/F 0 M), were calculated as done by Genty et al. (1989). The photochemical quenching, (qP) ¼ (F 0 M–FS)/(F 0 M–F 0 o), and non-photochemical quenching, (qNP) ¼ (FM–F 0 M)/ (FM–F 0 o), parameters were calculated according to Schreiber et al. (1986). The electron transport rate (ETR) was also calculated in accordance with Genty et al. (1989).

Electrolyte leakage The injury to membranes of selected leaf tissues was measured and calculated using the method of Sairam et al. (1997), with minor modifications. Cd accumulation After harvesting, leaves and roots of plants were extensively washed with distilled water. Samples were then oven-dried and ground to powder and were ashed in a muffle furnace at 550 1C for 4 h; the residue was brought to a standard volume with 1 M HNO3. Cd concentration was determined by a flame atomic absorption spectrophotometer (Unicam, 929 AAS). Assays of antioxidant enzyme activities Fresh leaf samples (0.5 g) of plants were ground in a mortar after treating with liquid nitrogen and suspended in a specific buffer for each enzyme extraction (Rao et al., 1995). The homogenates were centrifuged at 14,000 rpm for 20 min at 4 1C and the resulting supernatant was used for enzyme assays. The protein concentration of leaf crude extract was determined according to Bradford (1976). Total SOD (EC 1.15.1.1) activity was assayed according to Beyer and Fridovich (1987). One unit of SOD was defined as the amount of enzyme that caused a 50% decrease of the SOD-inhibited NBT reduction. APX (EC 1.11.1.11) activity was determined according to the method of Wang et al. (1991). The enzyme activity was calculated from the initial rate of the reaction using the extinction coefficient, e, of ascorbate (e ¼ 2.8 mM cm1) at 290 nm. GR (EC 1.6.4.2) activity was determined according to the method of Sgherri et al. (1994). The enzyme activity was calculated from the initial rate of the reaction after subtracting the non-enzymatic initial oxidation rate using the extinction coefficient of NADPH (e ¼ 6.2 mM cm1) at 340 nm. Guaiacol POD (EC 1.11.1.7) activity was based on the determination of guaiacol oxidation (e ¼ 26.6 mM cm1) at 470 nm by H2O2 (Bergmeyer, 1974). Statistics The experiments were arranged in a randomized design. Differences among Cd concentrations and cultivars, as well as interactions between these variables, were tested using the SPSS statistical program. Statistical variance analysis of the data with three replicates was performed using ANOVA and compared with least significant differences (LSD) at the 5% level.

Pigment analysis

Results

Photosynthetic pigments were extracted from selected leaf segments in 1 ml of 100% acetone. Extraction was performed at +4 1C for 96 h. The concentrations of chlorophyll (a+b) and carotenoids (x+c) were calculated using adjusted extinction coefficients (Lichtenthaler, 1987).

Effect of Cd on growth parameters of maize cultivars Increased concentrations of Cd produced significant growth inhibition in maize cultivars, as

ARTICLE IN PRESS Effects of cadmium in two maize cultivars

603

observed in dry weight of leaves and roots (Table 1). At all Cd concentrations, a significant reduction in biomass was observed for both cultivars compared with their controls. Although Cd content in the roots of 32D99 was 0.868 mg Cd g DW1 at 0.6 mM and 1.349 mg Cd g DW1 at 0.9 mM (Table 2), Cd-induced biomass reduction of 32D99 was lower than that of cultivar 3223 (Table 1).

Effect of Cd on chlorophyll a fluorescence parameters Exposing maize cultivars to different levels of Cd resulted in changes of the chlorophyll fluorescence parameters, F0 and FM, as shown in Figures 1A and B. F0 values increased with increasing Cd concentrations compared with the control, but this effect was significant in the 32D99 cultivar only at the highly toxic Cd level (0.9 mM). In contrast, FM values decreased significantly in both cultivars for Table 1. Cultivars

the whole range of Cd concentrations. In control leaves, FV/FM values were measured at approximately 0.83 (Figure 1C). FV/FM ratios for cultivars 3223 and 32D99 decreased by 24% and 16%, respectively, compared with that of the controls at the highly toxic Cd level. Examination of (F 0 V/ F 0 M) and FPSII values clearly showed that these parameters had a high correlation with FV/FM and yielded similar responses (Figure 1D and E). FPSII and F 0 V/F 0 M diminished at all Cd concentrations in both cultivars (Figures 1D and E). The Cd concentration of 0.9 mM caused the maximum decrease in these fluorescence parameters compared with those of the control. Cd induced qP determined by the redox state of QA, the primary electron acceptor of PSII. A significant decrease for qP was observed in 3223 than in 32D99 at all Cd levels compared with that of the control (Figure 1F). On the other hand, variation of qNP, reflecting the nonradioactive energy dissipation in both cultivars, was non-significant for all Cd concentrations (Figure 1G).

Dry biomass of roots and leaves in maize cultivars exposed to different Cd concentrations Cd concentrations (mM)

Root

Leaf

Dry biomass (g plant1)

Percent of control

Dry biomass (g plant1)

Percent of control

32D99

0(control) 0.3 0.6 0.9

0.16170.011 0.14570.006 0.10770.009 0.09370.005

100 90.0 66.5 57.8

0.16070.008 0.14370.006 0.11770.010 0.09570.004

100 89.4 73.1 59.4

3223

0(control) 0.3 0.6 0.9

0.20370.015 0.14970.006 0.10870.014 0.09170.005 0.03

100 73.4 53.2 44.8

0.22770.011 0.18670.003 0.13070.006 0.12570.006 0.02

100 81.9 57.3 55.1

LSD 5%

Table 2. Cultivars

Cd uptake and accumulation of roots and leaves in maize cultivars exposed to different Cd concentrations Cd concentration (mM)

Cd accumulation (mg Cd g DW1)

Root

Leaf

Total amount of Cd accumulation (mg Cd g DW1)

Cd accumulation (%)

Root

Leaf

32D99

0 (Control) 0.3 0.6 0.9

070.0 0.43970.002 0.86870.009 1.34970.014

070.0 0.13670.001 0.32970.004 0.42970.004

070.0 0.57570.003 1.19770.013 1.77870.018

0 76.3 72.5 75.8

0 23.7 27.5 24.1

3223

0 (Control) 0.3 0.6 0.9

070.0 0.42670.003 0.63270.005 0.91170.011 0.019

070.0 0.15570.002 0.27570.004 0.42570.006 0.009

070.0 0.58170.005 0.90770.009 1.33670.017 0.028

0 73.3 69.6 68.2

0 26.7 30.3 31.8

LSD 5%

ARTICLE IN PRESS Y. Ekmekc-i et al.

604 400

A

350

LSD 5%

300

*

200

1200

LSD 5%

*

* *

800

150

600

100

400

50

200

0

*

*

0 3223

32D99

3223

C

1

LSD 5%

*

0.8

*

*

*

0.6

D

LSD 5%

0.8 FV'/FM'

1

0.4

*

*

*

0.6 0.4 0.2 0

0.2 3223 1

32D99

1.2

1.2

FV/FM

B

1000

FM

Fo

250

1400

0mM Cd 0.3mM Cd 0.6mM Cd 0.9mM Cd

3223

32D99

32D99

1.1

E

0.8

F

LSD 5%

*

*

* *

1

qP

0.6

LSD 5%

1.05

0.4

*

0.95

* *

0.9

0.2

0.85

0

0.8 3223

3223

32D99

32D99

0.4

G

LSD 5%

110

H

LSD 5%

90

0.2

*

ETR

q NP

0.3

*

*

*

70

0.1

0

50 3223

32D99

3223

32D99

Cultivars Figure 1. Effect of Cd on photochemical parameters of maize cultivars. (A) Minimum fluorescence, (B) maximum fluorescence, (C) maximum quantum efficiency of PSII, (D) quantum efficiency of excitation energy trapping of PSII, (E) quantum efficiency of PSII, (F) photochemical quenching, (G) non-photochemical quenching and (H) electron transport rate; significant differences from controls (Pp0.05) are marked with an asterisk.

ARTICLE IN PRESS Effects of cadmium in two maize cultivars

605

Chlorophyll a content (mg ml-1 gfwt-1)

The ETR of both cultivars decreased significantly with increasing Cd concentration (Figure 1H). However, this effect was not significant at moder4

3

0mM Cd 0.3mM Cd 0.6mM Cd 0.9mM Cd

LSD 5%

2

*

*

*

1

*

*

Chlorophyll b content (mg ml-1 gfwt-1)

32D99

1 0.8

LSD 5%

0.6 0.4 0.2

*

*

0

*

*

3223 Chlorophyll a+b content (mg ml-1 gfw-1)

*

0 3223

*

32D99

5 4 LSD 5% 3 2

*

* *

1

*

*

*

0 3223

The leaves of both cultivars were significantly affected by Cd treatment, which resulted in the destruction of chlorophyll (bleaching) (Figures 2A–C). Simultaneously, with enhanced Cd accumulation in leaves, as characteristic visual symptoms, the reduction of chlorophyll a and b contents of leaves could be detected. In both cultivars, light-induced chlorophyll accumulation was inhibited by increasing Cd concentration. Chlorophyll a, b and (a+b) contents of control leaves of cultivars did not show remarkable variations (Figures 2A–C). Consequently, Cd treatment induced the loss of chlorophyll in both cultivars. Under Cd stress, the chlorophyll b content of cultivars was more affected than the chlorophyll a content (Figure 2B). The chlorophyll (a+b) content of the leaves of both cultivars decreased significantly with increasing Cd concentration. Total chlorophyll content of cultivar 3223 decreased approximately by 62% at the lowest Cd concentration (0.3 mM), while for 32D99, this decrease was around 49%. However, the changes in chlorophyll contents of both cultivars were similar at the highly toxic Cd level. The highest carotenoid content was measured in control plants for both cultivars, and it decreased with increasing Cd concentration (Figure 2D). Nevertheless, variation in carotenoid contents of 32D99 was not significant at any of the Cd concentrations. The carotenoid content of 3223 was slightly less than that of 32D99 at the highly toxic Cd level.

Effect of Cd on membrane damage

0.8

LSD 5%

0.6 0.4

Effect of Cd on pigment contents

32D99

1 Caroteniod content (mg ml-1 gfw.-1)

ate Cd treatments (0.3 and 0.6 mM) for 32D99. Increasing Cd concentration to 0.9 mM caused only a 17% reduction in ETR for 32D99, but a considerable reduction for 3223 (24%).

*

*

*

*

*

*

0.2 0 3223

32D99

Membrane damage was evaluated indirectly with conductivity measurements of solute leakage from cells. The leaf tissues of 3223 exhibited a significantly higher electrolyte leakage under Cd treatments than that of 32D99, whereas no significant change was observed for the control of either of the two cultivars (Figure 3). Based on the electrolyte leakage results, a greater extent of membrane damage was observed for 3223 at the highly toxic Cd level than that of 32D99.

Cultivars

Figure 2. Effects of Cd on chlorophyll a (A), chlorophyll b (B), total chlorophyll (C) and carotenoid contents (D) of maize cultivars; significant differences from controls (Pp0.05) are marked with an asterisk.

Cd accumulation Results indicated an increase in Cd accumulation in leaves and roots with increasing Cd

ARTICLE IN PRESS Y. Ekmekc-i et al.

606

Electrolyte leakage (%)

50 0mM Cd 0.3mM Cd 0.6mM Cd 0.9mM Cd

LSD 5%

40 30 20 10 0

3223

32D99 Cultivars

Figure 3. Electrolyte leakage (%) in leaves of maize cultivars at different Cd concentrations.

600 0mM Cd 0.3mM Cd 0.6mM Cd 0.9mM Cd

Total SOD activity (units mg protein-1)

500

*

400

*

300 200

*

LSD 5%

*

*

100 0 3223

Cd level. The proportion of Cd in the roots of 3223 decreased with an increase in Cd concentration, while the proportion of Cd in the leaves increased. For 32D99, the proportion of Cd in the root decreased with an increase in Cd concentration, except at the 0.9 mM Cd concentration.

Effect of Cd on antioxidant enzyme activity Significant changes in SOD activity were observed in both maize cultivars with the increase in level of Cd treatment when compared with controls (Figure 4A). With the increase in level of Cd treatment in situ, a gradual increase in SOD activity was observed for both cultivars. However, 32D99 showed a significant increase in SOD activity relative to the control at a 0.9 mM Cd concentration. The activities of APX increased with increasing Cd concentration for both cultivars compared with those of controls (Figure 4B). 32D99 also showed a significant increase in APX activity at moderate Cd treatments relative to the control. At the highly toxic Cd level, a marked decline in APX activity was observed in both cultivars, but moderate Cd

Total APX activity (µmol ascorbate min-1 mg protein-1)

concentration in the growth medium for both maize cultivars (Table 2). Cd accumulated primarily in the roots, and small amounts were transferred to leaves. The roots of 32D99 were found to contain higher Cd concentration than the roots of 3223 in all Cd treatments, but a significantly higher percent of Cd was translocated to the leaves of 3223 compared with 32D99. Approximately 50% of the Cd was translocated to the leaves of 3223, while for 32D99, this value was around 30% at the highly toxic

2.5

100

*

LSD 5%

*

*

80 60 40 20

*

1

0 3223

*

*

0.5

32D99

*

*

*

*

Total GR activity (nmolNADPH min-1 mg protein-1)

Total POD activity (nmolH2O2 min-1 mg protein-1)

120

LSD 5%

1.5

160 140

*

2

32D99

80 LSD 5%

*

60

* *

40

*

20

0

0 3223

3223

32D99

32D99

Cultivars

Figure 4. Changes in antioxidant enzyme activities of maize cultivars exposed to Cd. (A) Total SOD activity, (B) total APX activity, (C) total POD activity and (D) total GR activity; significant differences from controls (Pp0.05) are marked with an asterisk.

ARTICLE IN PRESS Effects of cadmium in two maize cultivars treatments induced an increase in APX activity in both cultivars compared with those of controls. The decline of APX activity was higher in 3223 than in 32D99. The activity of POD increased gradually with increasing Cd concentration up to the highly toxic Cd level for both cultivars, but a sharp decrease in POD activity was observed for 3223 at the highly toxic Cd level, while POD activity in 32D99 was still being enhanced by Cd treatment (Figure 4C). Cd at the lowest concentration (0.3 mM) induced a slight increase in GR activity for both cultivars. The effect of Cd on GR activity in 32D99 was similiar to that of 3223 at low and high Cd concentrations. At the 0.6 mM Cd concentration, both cultivars showed a significant increase in the GR activity relative to the control (Figure 4D). GR activity in 3223 significantly decreased at the 0.9 mM Cd concentration compared with that at the 0.6 mM Cd concentration.

Discussion Physiological processes such as photosynthesis have been shown to be very sensitive to heavy metals in higher plants (Lu and Zhang, 2000; Lu et al., 2000; Tanyolac- et al., 2007). Our research has clearly illustrated that Cd inhibited the photoactivation of PSII. F0 increased slightly in 3223, whereas it did not change significantly in 32D99. On the other hand, FM decreased progressively with increasing Cd concentration for both cultivars (Figures 1A and B). The increase in F0 with Cd treatment can be attributed to an impact on the PSII reaction centre, or a reduction in the energy transfer from the antennae to the reaction center (Ralph and Burchett, 1998). It is generally accepted that the FM intensity expresses the state of PSII when all QA molecules are in the reduced stage (Mallick and Mohn, 2003). The decline in the FM suggests a change in the ultrastructure of thylakoid membrane, affecting the ETR. Both F0 and FM caused a decrease in FV/FM when exposed to increasing Cd concentrations for both cultivars. The ratio of FV/FM is often used as a stress indicator and describes the potential yield of the photochemical reaction (Bjoerkman and Demming, 1987; Mallick and Mohn, 2003). Mallick and Mohn (2003) reported that FV/FM may decrease if reoxidation of QA was limited by the decrease or partial block of electron transport from PSII to PSI. These results showed that a highly toxic Cd level more strongly affected the photochemical efficiency in 3223 than that in 32D99 (Figure 1C). In parallel, photochemi-

607 cal parameters showed that Cd treatment likely caused more oxidative damage and did not indicate cessation of PSII activity of both cultivars even though plant death was not observed at the highly toxic Cd level. Also, decreased FV/FM resulted in a decrease in the quantum efficiency of excitation energy trapping of the open PSII reaction center (F 0 V/F 0 M) and in the quantum efficiency of PSII open centers in a light-saturated state, FPSII, for both cultivars. However, the 0.9 mM Cd concentration caused the maximum decrease in FV/FM, F 0 V/ F 0 M and FPSII compared with those of controls (Figure 1D and E). Similar results were observed by Sko ´rzyn ´ska-Polit and Baszyn ´ski (1997). An increase in qNP, indicating an increase in the thermal dissipation in PSII antennae, often results in a decrease in F 0 V/F 0 M (Lu and Zhang, 2000; Lu et al., 2000). F 0 V/F 0 M decreased in the presence of Cd; however, qNP did not change considerably with increasing Cd concentration (Figure 1G). It was suggested that the Cd-induced decrease in F 0 V/F 0 M was probably not involved in the process of nonphotochemical quenching. On the other hand, qP decreased with increasing Cd concentration for both cultivars, but this effect was not significant in 32D99. The decreased qP suggested a down regulation of the open PSII reaction centers (Genty et al., 1989). Cultivar 3223 was likely subject to a higher excess of excitation energy, which could potentially increase the probability of generating AOS, resulting in possible membrane damage and deterioration of membrane integrity. This effect was verified by the electrolyte leakage results (Figure 3). Similar to qP and F 0 V/F 0 M, a higher decline in ETR was observed for 3223 compared with 32D99 on exposure to Cd stress (Figure 1H). Leaf chlorosis is one of the most commonly observed consequences of Cd toxicity (Sko ´rzyn ´skaPolit and Baszyn ´ski, 1997). Chlorophyll contents (a, b and a+b) of cultivars abruptly declined with increasing Cd concentrations (Figures 2A, B and C). Similar results have been reported previously by several authors (Padmaja et al., 1990; Sko ´rzyn ´skaPolit et al., 1995; Wu et al., 2003). A decrease in the photosynthetic activity may be partly due to the decreased chlorophyll content. Additionally, the decrease in photosynthetic activity significantly reduced dry biomass of leaves for both cultivars grown at different Cd concentrations (Table 1). Carotenoids act as light-harvesting pigments as well, and can protect chlorophyll and membrane destruction by quenching triplet chlorophyll and removing oxygen from the excited chlorophyll– oxygen complex (Young, 1991). In this work, carotenoid content of cultivars decreased with increasing Cd concentrations (Figure 2D). As a

ARTICLE IN PRESS 608 result, the lowest carotenoid content of cultivars could not facilitate detoxification of toxic oxidation radicals formed in response to Cd treatments. The results of this study also demonstrated a marked increase in electrolyte leakage due to increasing Cd treatments in leaves of 3223 compared with that of 32D99 (Figure 3). Cd applications probably resulted in membrane damage and deterioration of membrane integrity. Based on this parameter, a higher extent of membrane damage was observed in 3223. Nevertheless, the lower membrane damage of 32D99 may be correlated with elevated protective mechanisms of the plant. Our results showed that the accumulation of Cd in roots was higher than in leaves for both cultivars (Table 2). The roots of 32D99 were found to contain higher amounts of Cd than the roots of 3223 in all Cd treatments. Most of the Cd that entered the plant system accumulated in the roots. A first barrier against Cd stress, operating mainly at the root level, can be immobilization of Cd by means of the cell wall and extracellular carbohydrates (Wagner, 1993). However, Cd was translocated in significantly greater quantities to the leaves in 3223 compared with 32D99. These results indicated that 32D99 has a greater ability to accumulate Cd, primarily in roots, and to prevent the transfer of excess Cd to the leaves. Similar results were reported by Wang et al. (2007). On the other hand, 3223 transferred more Cd from roots to leaves than did 32D99. The reduced translocation of Cd to leaves in 32D99 sensitive to Cd stress generally have poorer growth in comparison with the tolerant ones (Sanita´ Di Toppi and Gabbrielli, 1999). Some researchers have also found higher heavy metal concentrations in the shoots of sensitive plants (Ouzounidou et al., 1994; Liu et al., 2004). In this study, increasing Cd concentration in growth medium enhanced Cd accumulation in leaves. On the other hand, the increase of Cd concentration induced a decrease in dry mass, more pronounced in the cultivar 3223 than the cultivar 32D99 compared with their corresponding controls (Table 1). In this study, it was also found that dry biomass of root and leaves of 32D99 in the control was lower than that of cultivar 3223 (Table 1). This observation might be explained as a consequence of additional energy cost of metal-tolerant mechanisms (Lefebvre and Vernet, 1990; Liu et al., 2004). The presence of toxic metals in the cell leads to the formation of AOS, which cause further severe oxidative damage to different cell organelles and biomolecules (Radotic et al., 2000). AOS scavenging can be achieved by antioxidant enzymes such as SOD and APX. A constitutively high antioxidant capacity or increase in antioxidant level could

Y. Ekmekc-i et al. prevent oxidative damage and improve tolerance to the oxidative stress established. Responses of SOD, APX, POD and GR enzymes activate the essential component of the plant antioxidative defense system as they dismutases two O 2 to water and oxygen (C-akmak and Horst, 1991). Our results verified an enhancement in the activity of SOD in two maize cultivars (Figure 4A). The increase in SOD activity has been reported previously for certain plant species exposed to toxic Cd concentrations (Shah et al., 2001; Vitoria et al., 2001; Iannelli et al., 2002; Qadir et al., 2004 ). In our study, at the highly toxic Cd level, increased SOD activity in leaves was more conspicuous in 32D99 than that in 3223 relative to the controls. It was shown that Cd stress at the highly toxic level increased SOD activity in 3223, which proved to have greater O2 radical-scavenging ability (Herna ´ndez et al., 2000; Azevedo Neto et al., 2006). APX appeared to play an essential protective role in the scavenging process when co-ordinated with SOD activity (Massacci et al., 1995). Apparently, SOD leads to the over-production of H2O2 to eliminate the toxicity of Od 2 . In this study, the activities of APX increased with elevated Cd concentrations for both cultivars at moderate Cd concentrations (Figure 4B). 32D99 also showed a significant increase in APX activity at moderate Cd concentrations relative to the control, but high concentrations of Cd inhibited APX activity for both cultivars. This is more likely due to the harmful effect of over-production of H2O2 or its poisonous AOS (Hegedu ¨s et al., 2001). POD activity reflects the modified mechanical properties of the cell wall and cell membrane integrity of plant leaves under stress conditions. The activity of POD increased with increasing Cd concentration for both cultivars when compared with those of controls, but POD activity was inhibited at a highly toxic Cd level in only 3223 (Figure 4C). The increase in SOD, POD and APX activities indicated that these cultivars had the capacity to adapt to moderate Cd concentrations by developing an antioxidative defence system. However, at very high Cd concentrations, Cd toxicity likely caused damage to tissue development and function in 3223 compared with 32D99. A similar result was obtained by Wu et al. (2003), who observed the increase in SOD, POD and CAT activities for barley genotypes at moderate Cd stress, and the decrease in enzymes activities at very high Cd concentrations. In this study, Cd at low concentrations induced a slight increase in GR activity, but an activity decrease was observed at the highly toxic Cd level for both cultivars (Figure 4D). The GR activity pattern in Allysum plants was similar to that in our result (Leo ´n et al.,

ARTICLE IN PRESS Effects of cadmium in two maize cultivars 2002). However, in pea plants, no significant change in GR activities was observed (Dixit et al., 2001; Sandalio et al., 2001). The observation of Cd-induced depletion of antioxidative defences has been further supported by the result that GR was inhibited at a highly toxic level of Cd in this study. In many reactions involving reduced glutathione (GSH), the cys thiol group is oxidized into glutathione (GSSG), and the reverse reaction is catalyzed by GR using NADPH. The GSH pool maintained by GR is necessary for active protein function, and millimolar concentration of GSH acts as a key redox buffer, forming a barrier between thiol groups and AOS (Ranieri et al., 2005). It is well established that GSH increases the thiol concentration in the cytoplasm and chloroplast. Enhancement of GR activities likely caused an increase of the thiols and helped to preserve the activity of key photosynthetic enzymes and photosynthesis up to the highly toxic level of Cd concentration (Pietrini et al., 2003, 2005). Also, GR contains a highly conserved disulfide bridge between Cys76 and Cys81 (Lee et al., 1998), which may undergo cleavage by heavy metals. At the highly toxic Cd level, the activities of SOD and POD increased significantly, whereas APX and GR activities diminished in 32D99. Peroxidase induction has been observed as a general response of higher plants to toxic amounts of heavy metals (Van Assche and Clijsters, 1990; Demirevska-Kepova et al., 2004), and our investigation is specifically correlated with the high level of Cd in leaves. The sensitivity of APX to Cd excess could be explained by the fact that APX was rapidly inactivated in the absence of ascorbate. The change in activity of antioxidant enzymes was dependent on the plant species, age, duration of treatment and experimental conditions. Our results suggested that there were no different responses in SOD, APX and GR enzyme activities with increasing Cd concentrations among the two cultivars. On the other hand, 32D99 and 3223 exhibited similar behaviors in SOD, APX and GR enzyme activities. In this study, POD activity significantly increased at highly toxic Cd levels in the 32D99 cultivar. Similar results were also reported by Dong et al. (2006). H2O2 produced during oxidative stress in plants is scavenged by POD, APX and CAT enzymes. The probable greater activity of CAT in C3 plants suggests its larger role in C3 plants for the removal of photorespiratory H2O2 (Nocter et al., 2002), while in maize (C4 plants) the removal of H2O2 is probably achieved by POD or APX enzymes. POD participating in lignin biosynthesis could build up a physiological barrier against toxic heavy metals (Radotic et al., 2000; Hegedu ¨s et al., 2001). A remarkable increase in the

609 activity of POD at the highly toxic Cd level is usually regarded as an indicator of better tolerance in the Z. mays L. cultivar 32D99. In conclusion, variation of chlorophyll fluorescence and antioxidant enzyme activities induced by Cd treatments revealed that 32D99 was more tolerant to Cd stress compared with 3223. Cd stress resulted in a reduction in photosynthetic efficiency, with no apparent ultimate irreversible damage, as verified by the electrolyte leakage. Tolerance of 32D99 could be associated with scavenging capacity of AOS. Some protective mechanisms, such as activity of antioxidant enzymes, and especially of POD, may be protected from oxidative damage. In the case of 3223, Cd treatments possibly caused more oxidative damage than they did to 32D99. Protective mechanisms of 3223 likely prevent oxidative damage, but are less efficient than in 32D99. Analysis and evaluation of all parameters allowed classification of cultivars as tolerant (32D99) and less tolerant (3223). Wang et al. (2007) concluded that it is important to select a suitable photoremediation species with high Cd uptake and accumulation capabilities without severe damage to the plant. Research must be expanded to prevent the risk of Cd uptake by crops in the food chain before the growth of 32D99 in Cdpolluted regions.

Acknowledgments We would like to thank Hacettepe University, Scientific Research Unit (Project no. 02 02 602 013) for the financial support. We are also grateful to ¨ nalan, Chemical Engineering Department, S- eniz U for her assistance in experiments.

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