The effect of zinc stress combined with high irradiance stress on membrane damage and antioxidative response in bean seedlings

Environmental and Experimental Botany 74 (2011) 171–177

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The effect of zinc stress combined with high irradiance stress on membrane damage and antioxidative response in bean seedlings Prabhu Inbaraj Michael, Muthuchelian Krishnaswamy ∗ Department of Bioenergy, School of Energy, Environment and Natural Resources, Madurai Kamaraj University, Madurai, India

a r t i c l e

i n f o

Article history: Received 16 June 2010 Received in revised form 18 May 2011 Accepted 23 May 2011 Keywords: Antioxidant enzymes Bean High irradiance Membrane damage Proline Zinc

a b s t r a c t Zinc (Zn) is a necessary element for plants, but excess Zn can be detrimental. The effect of Zn and high irradiance (HI) stress on the growth, lipid peroxidation (MDA), membrane permeability (EC), hydrogen peroxide (H2 O2 ) accumulation, non-enzymatic antioxidants like proline accumulation and ascorbic acid (AsA) and the activities of major antioxidant enzymes (superoxide dismutase, SOD; peroxidase, POX; polyphenol oxidase, PPO) of bean leaves were investigated under controlled growth conditions. The root length was not reduced at excess Zn level. Application of Zn significantly increased Zn concentration in the leaves of bean plants. Under Zn and HI stress, the Zn-deficient and Zn-excess conditions significantly increased the EC, MDA and H2 O2 content of excised leaves of bean. The SOD activity was found to be increased significantly in both Zn-deficiency and Zn-excess leaves under Zn and HI stress. Under both Zn and HI stress conditions, the antioxidant enzyme activities; POX, PPO and the non-enzymatic antioxidants, AsA and proline accumulation were found to be significantly increased in the Zn-excess leaves which showed that the bean plant had the ability to tolerate the excess level of Zn and HI stress. A significant increase in MDA, H2 O2 , and EC with a simultaneous decrease in the antioxidant enzyme activities under Zn-deficiency compared to Zn-sufficient condition shows the inefficiency of the bean plant in response to Zn deficiency. To the best of our knowledge, this is the first report on the effect of Zn stress combined with HI stress in bean plant. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Although zinc (Zn) is an essential micronutrient for normal growth and development of plants, it becomes phytotoxic and inhibits cell growth in plants at excessive concentrations (Vaillant et al., 2005; Muschitz et al., 2009). Zn is the second most abundant transition metal after iron (Fe) and is involved in various biological processes in organisms (Broadley et al., 2007). The effects of Zn on plants have been reviewed by many (Cakmak, 2000; Hacisalihoglu and Kochian, 2003; Rout and Das, 2003). Like other heavy metals, excess Zn can have negative effects on plants. At organism level, excess Zn inhibits seed germination, plant growth (Mrozek and Funicelli, 1982) and root development (Lingua et al., 2008), and causes leaf chlorosis (Ebbs and Kochian, 1997). At the cellular level, excess Zn can significantly alter mitotic activity (Rout and Das, 2003), affect membrane integrity and permeability (Stoyanova and Doncheva, 2002), and even kill cells (Chang et al., 2005).

Abbreviations: AsA, ascorbic acid; EC, electrolyte leakage; HI, high irradiance; H2 O2 , hydrogen peroxide; MDA, malondialdehyde; POX, peroxidase; PPO, polyphenol oxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; Zn, zinc. ∗ Corresponding author. Tel.: +91 452 2458020; fax: +91 452 2459181. E-mail address: [email protected] (M. Krishnaswamy). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.05.016

Accumulated data has shown that excess Zn induces oxidative stress by promoting the generation of reactive oxygen species (ROS) and lipid peroxidation (Prasad et al., 1999; Madhava Rao and Srestry, 2000). Irrespective of the production pathways, ROS are highly cytotoxic and their level within plant cells must be controlled by antioxidant defense systems. Plants have defensive mechanisms and utilize several biochemical strategies to avoid damage caused by ROS (Mittler et al., 2004; Foyer and Noctor, 2005). Plant enzymatic defenses include antioxidant enzymes like peroxidases (POX; EC 1.11.1.7) and superoxide dismutases (SOD; EC 1.15.1.1), which, together with other enzymes, promote the scavenging of ROS (Alscher et al., 2002; Veljovic-Jovanovic et al., 2006). SOD catalyzes the dismutation of O2 ·− to H2 O2 and molecular oxygen. POX is widely distributed in all higher plants and protects cells against the destructive influence of H2 O2 by catalyzing its decomposition through oxidation of phenolic and endiolic co-substrates. The biochemical defense system also includes the amino acid proline, an osmolyte and cellular protector largely accumulated in several plant species in response to abiotic stress, which might act as a ROS scavenger (Sharma and Dietz, 2006; Ashraf and Foolad, 2007). Light is essential for plant growth and development, but when plants are subjected to excessive light, active oxygen generation is increased (Asada, 2006), often resulting in photo-oxidative damages; thus light can also be one of the most deleterious envi-

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ronmental factor. The absorption of excess light can be deleterious since it can potentially result in the production of singlet oxygen and reduced reactive oxygen species such as superoxide and H2 O2 . Excess light causes the production of ROS that lead to membrane lipid peroxidation (Parkin et al., 1989). There are many reports detailing changes in the activities of enzymes involved in antioxidant metabolism in response to high light stress (Mishra et al., 1995; Grace and Logan, 1996; Logan et al., 1998). The common bean, Phaseolus vulgaris L., is a herbaceous annual plant grown worldwide as an edible bean. It is a predominantly selfpollinated crop that originated mainly in Latin America. From Latin America, it spread to other parts of world and now it is widely cultivated in the tropics and subtropics as well as in temperate regions of the world (Gepts and Bliss, 1988; Zeven et al., 1999). Bean plant is widely grown in the tropics where possibility of high light is inevitable leading to HI stress. Moreover, environment contamination and plant exposure to heavy metals is a growing problem throughout the world. Even though much supporting evidence on the role of antioxidant enzymes to Zn stress is available (Madhava Rao and Srestry, 2000; Cuypers et al., 2001, 2002; Wójcik et al., 2006; Gupta et al., 2011), so far there is no report on the antioxidant enzyme activities under combined effects of Zn together with HI stress. Because exposure to Zn stress in combination with HI stress might promote oxidative damage, it is important to characterize the role of non-enzymatic and enzymatic antioxidative defense mechanism in the bean plants exposed to Zn and HI stress. The question of what makes the growth of bean plants tolerant to the high light in the tropics made us to carryout this investigation. So, the aim of this work was to investigate the growth responses, the level of oxidative damage, non-enzymatic (Proline, AsA) and enzymatic (SOD, PPO, POX) antioxidative activities and to clarify some aspects of the bean plant’s tolerance mechanism under Zn stress combined with HI stress. 2. Materials and methods 2.1. Plant culture and zinc treatments Bean seeds (Phaseolus vulgaris L. Sel 9) were rinsed in distilled water and surface sterilized with 1% sodium hypochlorite for 20 min, rinsed again, imbibed overnight in distilled water and germinated on moistened filter paper in trays for 3 days in darkness at 23 ◦ C. After 3 days, uniformly germinated seedlings were transferred to plastic cups and grown hydroponically in half-strength Hoagland’s nutrient solution (Hoagland and Arnon, 1950) for 7 days. From the 8th day onwards Zn treatments were given in the concentrations of 0 ppm (Zn-deficient), 5 ppm (Znsufficient) and 50 ppm (Zn-excess) as ZnSO4 ·7H2 O. The growth solutions were adjusted to pH 5.6 ± 0.2 and were replaced every 2 days. Plants were grown in a growth chamber with the following conditions: day/night temperature, 22 ± 2 ◦ C/18 ± 2 ◦ C; relative humidity, 60–70%; 16-h light:8-h dark photo-cycle; light intensity, 150 ␮mol m−2 s−1 . Two weeks after germination, the plants were collected and analyzed.

2.3. Plant growth After 14 days of Zn treatment, 10 plants from each group (Zndeficient, Zn-sufficient and Zn-excess) were taken at random and divided into shoot and root fractions. Shoot lengths and root lengths were measured. Weight measurements were conducted from base to the tip of primary leaves, shoot and entire roots of the seedlings. After measuring the fresh weights of seedlings the same tissues were let to dry in an oven at 70 ◦ C for 48 h, and then the dry weights of the samples were measured. 2.4. Determination of zinc content The Zn content was determined by atomic absorption spectrophotometer (Piper, 1942) after wet digestion of 1 g of dried and powdered leaf material in 5 mL of ternary mixture of HNO3 :H2 SO4 :HClO4 in the ratio of 10:1:4 (v:v:v). The total Zn concentration was expressed as ␮g g−1 DW. 2.5. Membrane permeability Electrolyte leakage was used to assess membrane permeability. This procedure was based on Lutts et al. (1996). Leaf discs (1 cm in diameter) from two randomly chosen plants per replicate were taken from the middle portion of fully developed leaf, washed three times with deionized water to remove surface contamination and were then placed in individual stoppered vials containing 10 mL of deionized water. These samples were incubated at room temperature (ca. 25 ◦ C) on a shaker (100 rpm) for 24 h. Electrical conductivity (EC) of bathing solution (EC1 ) was read after incubation. The same samples were then placed in an autoclave at 120 ◦ C for 20 min and the second reading (EC2 ) was determined after cooling to room temperature. The electrolyte leakage (EC) was expressed following the formula EC = (EC1 /EC2 ) × 100. 2.6. Hydrogen peroxide and lipid peroxidation assay Concentration of H2 O2 was determined following Velikova et al. (2000). Fresh leaf tissue (0.5 g) was homogenized with 5 mL of 0.1% (w/v) trichloroacetic acid (TCA) in a pre-chilled pestle and mortar and the homogenate was then centrifuged at 12,000 × g for 15 min. To 0.5 mL of the supernatant, 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of potassium iodide (1 M) were added. The mixture was vortexed and its absorbance was read at 390 nm. Lipid peroxidation was determined by measuring the amount of malondialdehyde (MDA) content according to the method of Davenport et al. (2003). Fresh leaf (0.2 g) was homogenized with 2 mL of 5% (w/v) trichloroacetic acid in an ice bath, and centrifuged at 10,000 × g for 10 min at 4 ◦ C. About 2 mL supernatant mixed with 2 mL of 0.67% (w/v) thiobarbituric acid was incubated in boiling water for 30 min, then cooled and centrifuged. The absorbance of reaction supernatant was assayed at 450, 532, and 600 nm. The MDA content was calculated based on the following formula, MDA (mol g−1 ) = [6.45 × (A532 − A600 ) − (0.56 × A450 )] ×

2.2. Photoinhibition and recovery under controlled conditions

where Vt = 0.002l; W = 0.2 g

Detached leaves which were already subjected to Zn stress were placed in a controlled environment chamber equipped with a 24 V/250 W metal-halide lamp. The upper leaf surface was exposed to a photosynthetic photon flux density (PPFD) of 1900 ␮mol m−2 s−1 for up to 60 min. Air temperature was 20 ◦ C and relative humidity was 65%. After this period, some leaves exposed to HI were returned to normal condition (Rec-recovery) by adapting dark recovery for 60 min before sampling and analyzed.

2.7. Proline and ascorbic acid

Vt W

The amount of proline was measured according to the method of Bates et al. (1973). 0.5 g of fresh leaf was cut into small pieces and homogenized in 10 mL of 3% aqueous sulfosalicylic acid and filtered through Whatman #2 paper. Then, 2 mL of the filtrate was mixed with 2 mL of acid-ninhydrin and 2 mL of glacial acetic acid and heated at 100 ◦ C for 1 h. The reaction was terminated in an

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ice bath and 4 mL of toluene was added to the mixture and contents of tubes were stirred for 15–20 s. Absorbance of the pink red upper phase was recorded at 520 nm against toluene blank. A standard curve for proline was constructed to determine the proline concentration in each sample. Ascorbic acid (AsA) content was assayed as described by Omaye et al. (1979). The extract was prepared by grinding 1 g of fresh leaf material with 5 mL of 10% trichloroacetic acid (TCA), centrifuged at 1235 × g for 20 min, re-extracted twice and supernatant made up to 10 mL and used for assay. To 0.5 mL of extract, 1 mL of 6 mM 2,4dinitrophenylhydrazine-thiourea-CuSO4 (DTC) reagent was added, incubated at 37 ◦ C for 3 h and then 0.75 mL of ice-cold 65% H2 SO4 was added, allowed to stand at 30 ◦ C for 30 min and the resulting colour was read at 520 nm. The ascorbic acid content was determined using a standard curve prepared with ascorbic acid.

2.8. Enzyme extraction and protein determination Fresh leaves (0.3 g) were homogenized with 3 mL of ice-cold 0.05 M potassium phosphate buffer (pH 7.0) containing 1% (w/v) PVP in an ice bath. The homogenized slurry was centrifuged at 10,000 × g for 15 min at 4 ◦ C and the supernatant was collected. Protein concentration in the supernatant was determined according to Lowry et al. (1951) using bovine serum albumin as standard.

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2.10. Statistical analysis The data compiled were submitted to one-way analysis of variance (ANOVA) by using SigmaPlot 11.0. Each data point was the mean of five replicates (n = 5) and comparisons with P-values <0.01 were considered significantly different by Tukey’s test.

3. Results 3.1. Plant growth Effect of Zn stress on the growth of bean seedlings is shown in Table 1. A significant decrease in the fresh and dry weights of leaf, shoot and root was observed in Zn-deficient and Zn-excess plants compared to Zn-sufficient plants. The shoot length was reduced significantly in Zn-excess plants while the decrease was insignificant in Zn-deficient plants compared to Zn-sufficient plants. But the root length was found to be reduced in Zn-deficient while it was increased insignificantly in Zn-excess plants when compared to Znsufficient plants. Visual Zn deficiency and Zn toxicity symptoms such as inhibition of shoot elongation and development of chlorotic areas appeared on plants grown in Zn-deficient and Zn-excess conditions. Altogether, the Zn-sufficient plants showed better growth and dry biomass accumulation than the plants grown under deficiency or excess of Zn.

3.2. Zinc accumulation

Superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed by its ability to inhibit the photochemical reduction of nitro blue tetrazolium chloride (NBT) at 560 nm (Beauchamp and Fridovich, 1971). The assay was carried out at 25 ◦ C in a reaction mixture (3 mL) containing 33 ␮M NBT, 10 mM l-methionine, 0.66 mM EDTA and 0.0033 mM riboflavin in 50 mM sodium phosphate buffer (pH 7.8). Riboflavin was added last and the test tubes containing the reaction mixture were incubated for 10 min under 300 ␮mol m−2 s−1 irradiance at 25 ◦ C. The reaction mixture with no enzyme developed maximum colour due to the maximum rate of reduction of NBT. The non-irradiated reaction mixture did not develop colour and was used as the control. One unit of SOD activity was defined as the quantity of SOD required to produce a 50% inhibition of NBT, and the specific enzyme activity was expressed as Units mg−1 protein. Peroxidase (POX; EC 1.11.1.7) activity was assayed by the method of Kumar and Khan (1982). Assay mixture of POX contained 2 mL of 0.1 M sodium phosphate buffer (pH 6.8), 1 mL of 0.01 M pyrogallol, 1 mL of 0.005 M H2 O2 and 0.5 mL of enzyme extract. The solution was incubated at 25 ◦ C for 5 min and the reaction was terminated by adding 1 mL of 2.5 N H2 SO4 . The amount of purpurogallin formed was determined by measuring the absorbance at 420 nm against a blank prepared by adding the extract after the addition of 2.5 N H2 SO4 at zero time. The POX activity was expressed as Units mg−1 protein. One unit is defined as the change in the absorbance by 0.1 min−1 mg−1 protein. Polyphenol oxidase (PPO; EC 1.10.3.1) activity was assayed by the method of Kumar and Khan (1982). Assay mixture for PPO contained 2 mL of 0.1 M sodium phosphate buffer (pH 6.0), 1 mL of 0.1 M catechol and 0.5 mL of enzyme extract. The assay mixture was incubated at 25 ◦ C for 5 min and the reaction was stopped by adding 1 mL of 2.5 N H2 SO4 . The absorbance of the purpurogallin formed was read at 495 nm. To the blank, 2.5 N H2 SO4 was added at the zero time of the same assay mixture. PPO activity is expressed in Units mg−1 protein. One unit is defined as the change in the absorbance by 0.1 min−1 mg−1 protein.

Zinc accumulation increased significantly in the leaves of bean plants even at the lowest Zn treatment (Fig. 1). Seedlings without Zn treatment contained some Zn, which may be related to the prior growth of bean plants in the half strength nutrient solution for 7 days without any stress.

3.3. Membrane permeability Electrolyte leakage (Fig. 2A) reflected the plasmalemma damage caused by the Zn and HI stress and it increased insignificantly by 19% in Zn-deficient and significantly by 28% in Zn-excess leaves when compared to Zn-sufficient leaves. Upon HI stress, the Zndeficient and Zn-excess leaves showed a further significant increase in EC. The rate of recovery from HI stress was higher in Zn-sufficient (98%) leaves than in the Zn-deficient and Zn-excess (90%) leaves.

c

450

Zn content in leaf (µg g -1 DW)

2.9. Antioxidant enzyme assays

300

b

150

a 0 0 ppm

5 ppm

50 ppm

Zn treatment Fig. 1. Zn concentrations of the leaves of bean plants grown with 0, 5 and 50 ppm of Zn. Values are means ± S.E. (n = 5). Bars carrying different letters are significantly different compared to the sufficient level (5 ppm Zn) at P ≤ 0.01 as determined by Tukey’s test.

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Table 1 Growth parameters of Zn-stressed bean plant (Phaseolus vulgaris L. Sel 9). Values are means ± S.E. of 10 replicates. Different letters indicate that the mean value is significantly different between treatments within each column (P ≤ 0.01). Fresh weight (g plant−1 )

Zn treatment

0 ppm 5 ppm 50 ppm

Dry weight (g plant−1 )

Leaf

Shoot

Root

Leaf

Shoot

Root

1.02 ± 0.048b 1.59 ± 0.053a 0.68 ± 0.045c

1.10 ± 0.054b 1.56 ± 0.064a 1.08 ± 0.047c

0.78 ± 0.031b 1.24 ± 0.042a 0.72 ± 0.031c

0.10 ± 0.007b 0.18 ± 0.008a 0.07 ± 0.005c

0.12 ± 0.007b 0.21 ± 0.010a 0.10 ± 0.007c

0.05 ± 0.003b 0.10 ± 0.008a 0.04 ± 0.004c

3.4. Hydrogen peroxide and lipid peroxidation

A

50

EC (% Ion Leakage)

d

112.5 ± 0.629a 124.2 ± 0.615a 132.7 ± 0.548a

139.5 ± 0.535a 160.3 ± 0.674a 129.1 ± 0.551b

3.5. Proline and ascorbic acid Under Zn stress, accumulation of proline and AsA content was significantly low in Zn-deficient leaves and increased significantly in Zn-excess leaves when compared to Zn-sufficient (Fig. 3A and B) ones. After further HI stress and upon recovery from HI stress, the Zn-sufficient and Zn-excess leaves showed a significant increase in proline accumulation and AsA content whereas the Zn-deficient leaves showed an insignificant increase which was in level with the Zn-sufficient leaves under Zn stress alone.

e

c

3.6. Antioxidant enzymes

a

a

0 ppm

a

a

25

Shoot length (mm)

stress while the Zn-sufficient leaves recovered by 95% for both H2 O2 and MDA contents.

The H2 O2 and MDA content (Fig. 2B and C) increased insignificantly in Zn-deficient leaves while it increased significantly in Zn-excess leaves compared to Zn-sufficient leaves. Under further HI stress, there was a significant increase in both H2 O2 and MDA content in Zn-deficient and Zn-excess leaves. The Zn-deficient and Zn-excess leaves showed a recovery rate of nearly 90% from HI

b

Root length (mm)

a

5 ppm 50 ppm

Fig. 4 shows the changes in the activities of SOD, POX and PPO enzymes in bean plants subjected to Zn, HI stress and Rec. Under Zn stress, SOD activity (Fig. 4A) increased insignificantly in Zndeficient leaves while it increased significantly in Zn-excess leaves when compared to Zn-sufficient leaves. The SOD activity further

0 Zn

Zn + HI

60

Zn + HI + Rec

H2O2 (mM g -1 FW)

30

B

d e

c

b

a

a

a

a

a

15

0 ppm 5 ppm 50 ppm

Proline (µmol g -1 FW)

e

g

A

d

c

b

30

f

0 ppm

b

5 ppm

b a

50 ppm

0

0

Zn

Zn

Zn + HI

Zn + HI

Zn + HI + Rec

Zn + HI + Rec 160

0.06

d

B

f

c

a

a a

g e

a

a

0.03

0 ppm 5 ppm 50 ppm

AsA (mg g -1 FW)

MDA (µmol g -1 FW)

b

e

C c

d

b b

80

b

0 ppm 5 ppm

a

50 ppm

0

0

Zn

Zn

Zn + HI

Zn + HI

Zn + HI + Rec

Zn + HI + Rec

Fig. 2. Changes in EC (A), H2 O2 (B) and MDA (C) contents in the leaves of Phaseolus vulgaris L. Sel 9 subjected to Zn, Zn + HI stress and recovery. Values are means ± S.E. (n = 5). Bars carrying different letters are significantly different compared to the sufficient level (5 ppm Zn) at P ≤ 0.01 as determined by Tukey’s test.

Fig. 3. Changes in proline accumulation (A), and ascorbic acid content (AsA) (B) in the leaves of Phaseolus vulgaris L. Sel 9 subjected to Zn, Zn + HI stress and recovery. Values are means ± S.E. (n = 5). Bars carrying different letters are significantly different compared to the sufficient level (5 ppm Zn) at P ≤ 0.01 as determined by Tukey’s test.

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A

SOD (Units mg -1 Protein)

1.2

d f c

b

e

a

0 ppm

a

0.6

a

a

5 ppm 50 ppm

0 Zn

Zn + HI

Zn + HI + Rec

f

0.34

POX (Units mg -1 Protein)

d b

c a

a 0.17

0 ppm

a

a

B

e

5 ppm 50 ppm

0 Zn

Zn + HI

Zn + HI + Rec

0.28

f

PPO (Units mg -1 Protein)

d c

b a 0.14

C

e

a

a

0 ppm 5 ppm

a

50 ppm

0 Zn

Zn + HI

Zn + HI + Rec

Fig. 4. Changes in the enzyme activities of SOD (A), POX (B) and PPO (C) in the leaves of Phaseolus vulgaris L. Sel 9 subjected to Zn, Zn + HI stress and recovery. Values are means ± S.E. (n = 5). Bars carrying different letters are significantly different compared to the sufficient level (5 ppm Zn) at P ≤ 0.01 as determined by Tukey’s test.

increased significantly in Zn-deficient and Zn-excess leaves under HI stress. Upon recovery from HI stress, the Zn-sufficient leaves showed a maximum rate of recovery. POX and PPO activities decreased insignificantly in Zn-deficient leaves whereas it increased significantly in Zn-excess leaves when compared to the Zn-sufficient leaves (Fig. 4B and C). After further HI stress and upon recovery from HI stress, the Zn-sufficient and Zn-excess leaves showed a significant increase in POX and PPO activities whereas the level of increase in Zn-deficient leaves was in parallel with that of the Zn-sufficient leaves under Zn stress alone. 4. Discussion Although Zn is essential for plant growth, its excess is inhibitive as was reported for a number of leguminous plants (Khudsar et al., 2004). In our study, the Zn-deficient and Zn-excess leaves showed signs of chlorosis in the tip and margins and all the plants were all of smaller size than the Zn-sufficient plants. Our results are in good agreement with Yang et al. (2011) and Gupta et al. (2011) who

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have reported similar results in grape and black gram respectively under Zn-deficiency and Zn-excess conditions. Similarly, reduced shoot and root biomass under Zn-deficiency and Zn-excess has been reported in tomato plants (Cherif et al., 2010). Reduced shoot growth at higher concentration of Zn could be a consequence of its interference with certain essential metabolic events (Van Assche and Clijsters, 1990; Alia et al., 1995). Reduced leaf growth under Zn stress has been reported in Artemisia annua (Khudsar et al., 2004) and Phaseolus vulgaris (Polson and Adams, 1970). This may involve inhibition of cell division as well as cell elongation (Arduini et al., 1994). In the present study, Zn treatment did not have a significant toxic effect on the root growth of the bean plant. Rengel (2000) reported that the response of the plants to high Zn concentration was related to their tolerance capacity to Zn. It has already been established that root growth of the plants sensitive to Zn was severely inhibited when subjected to high Zn concentration but root growth of the Zn tolerant plants was not affected (Souza et al., 2005). In our study, though there was an accumulation of Zn in the leaves at excess Zn level, it did not inhibit the root growth and this result indicated that the bean seedlings might have the ability to tolerate or withstand the toxic effect of Zn which is in agreement to the recent report by Ozdener and Aydin (2010). The accumulation of Zn in bean leaves increased significantly with the increase of Zn concentrations in the nutrient medium in our study which is similar to the results obtained by Cherif et al. (2010) in tomato plants and by Gupta et al. (2011) in black gram under Zn stress. Under Zn-deficiency and Zn-excess conditions, there was an increase in membrane permeability, which is an expression of oxidative stress brought about by higher generation of oxygen radicals in bean plants. Moreover, there was a significant increase in EC when the Zn stressed bean plants were further subjected to HI stress. The increase in EC under Zn-deficiency in our study is in agreement with that of the recent report by Chen et al. (2009). Our results are in good agreement with that of Kaya and Higgs (2000) who have observed similar results in three tomato cultivars grown under low and high Zn. Moreover, the increase in membrane permeability under HI stress is in concurrence with the results reported by Xu et al. (2010). Induction of oxidative stress in the Zn-deficient and Zn-excess conditions in our study was indicated by an increased accumulation of MDA and H2 O2 content. The most distinctive indication of oxidative stress is lipid peroxidation. Under heavy metal stress, H2 O2 and O2 •− , via the Haber–Weiss reaction, are converted into highly reactive OH• radical and this causes lipid peroxidation (Apel and Hirt, 2004). In the present study, MDA content increased significantly in Zn-excess condition under both Zn and HI stress and this has been suggested earlier by Cherif et al. (2010) and Ghnaya et al. (2011). Similarly, the increase in H2 O2 content in Zn-deficient and Zn-excess condition in our study has been reported recently by Gupta et al. (2011) in black gram under Zn stress. It is widely accepted that oxidative damage to critical cell compounds resulting from attack by ROS is the basis of disturbances in plant growth caused by Zn deficiency (Cakmak, 2000). Similar to our results an increase in MDA and H2 O2 content under excess zinc has been reported in sugarcane (Jain et al., 2010) and under Zn deficiency in rice (Chen et al., 2009). Proline accumulation under stress conditions may either be caused by induction or activation of enzymes of proline biosynthesis or a decreased proline oxidation to glutamate, decreased utilization of proline in protein synthesis, and enhanced protein turnover (Delauney and Verna, 1993). Proline has been shown to alleviate metal-induced oxidative stress by scavenging harmful ROS (Siripornadulsil et al., 2002; Tripathi and Gaur, 2004). In accordance to our study, Alia et al. (1995) reported an increase in proline content in Brassica and Cajanus under Zn-toxicity. Therefore, being a hydroxyl and singlet oxygen scavenger, proline has efficiently

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reduced the threat of ROS in the Zn-excess bean leaves both under Zn and HI stress. A role for increased AsA content in amelioration of oxidative stress has been reported by Panda and Upadhyay (2003). In addition to playing roles in enzymatic antioxidant metabolism, AsA can directly scavenge ROS (Asada, 1994). Sharma et al. (2004) reported a decline in AsA content under Zn deficiency which is similar to our results. An increase in AsA content has been reported by Cuypers et al. (2001) in bean plant and Yuan et al. (2009) in Alternanthera philoxeroides under Zn stress. Thus an increase in the accumulation of proline and AsA under excess Zn condition helped the bean plant to withstand the toxicity of Zn along with HI stress which might have protected the Zn-excess plants from the imposed oxidative damage. The induction of certain enzymes that detoxify ROS is considered to play an important role in the defense against oxidative stress caused by toxic metal concentrations (Van Assche and Clijsters, 1990). SOD catalyzes dismutation of superoxide anion (O2 •− ) into hydrogen peroxide (H2 O2 ), while POX detoxifies H2 O2 . Increase in SOD activity similar to that of our study has been reported by Wójcik et al. (2006) in Thlaspi caerulescens and Madhava Rao and Srestry (2000) in pigeon pea under Zn stress. But the enhancement of SOD activity alone cannot alleviate the burden of ROS. H2 O2 is a highly toxic ROS and must be sequestered by the action of POX. In our study, Zn treatment resulted in a significant rise in the SOD activity which can be considered as a circumstantial evidence for enhanced production of free radicals under Zn stress, particularly under Zn-deficiency and Zn-excess conditions. Thus, the increase in the activities of SOD activity under Zn-deficient and Zn-excess conditions suggested an increased production of H2 O2 . POX is known to play a significant role in oxidative stress conditions (Karataglis et al., 1991). POX activity was stimulated in the sugarcane seedlings treated with different concentrations of Zn (Jain et al., 2010) and also in duckweed (Razinger et al., 2007) which is similar to our study. It has often been reported that the activity of the antioxidant enzymes and leaf antioxidant content increase as a result of HI (Grace and Logan, 1996; Logan et al., 1998) in order to prevent damage caused by active oxygen species. Significant increase in the activities of PPO and POX in the Zn-excess conditions in the present investigation suggested their role in constant detoxification of H2 O2 in bean seedlings under Zn and Zn + HI stress. Since PPO has a main proteolytic activity (Kuwabara and Katoh, 1999), its increase allows the plants to remove the proteins damaged by ROS during stress conditions and repair the cell wall by means of cross-linking of matrix polymers (Fry, 1986). PPO activity was stimulated in seedlings of grape (Yang et al., 2011) with various concentrations of Zn which is similar to our study. Thus, the extent of increase in PPO and POX activity induced by Zn-excess condition indicated that, these enzymes might have sufficiently protected the bean plants from oxidative damage thus making them tolerant to excess Zn and HI stress. Therefore it appears that under Zn and HI stress conditions, an increase in the AsA, proline and antioxidant enzymes like PPO, POX, under excess Zn level could be important in keeping ROS at low levels and controlling the cellular redox status of the Zn-excess plants.

5. Conclusions In conclusion, our results demonstrate that Zn-deficiency and Zn-excess conditions mediates oxidative damage in the bean leaves under Zn and HI stress conditions. The increase in the root length and activities of non-enzymatic and enzymatic antioxidants in the Zn-excess condition under Zn and HI stress confirms that the bean plant has the ability to withstand or tolerate the excess level of Zn and HI stress. The data also suggests that non-enzymatic

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