Phytochemistry 108 (2014) 57–66

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Exogenous application of methyl jasmonate lowers the effect of cadmium-induced oxidative injury in rice seedlings Indra Singh a, Kavita Shah b,⇑ a b

Department of Bioinformatics, MMV, Banaras Hindu University, Varanasi 221 005, India Institute of Environment and Sustainable Development, Banaras Hindu University, Varanasi 221 005, India

a r t i c l e

i n f o

Article history: Received 18 June 2014 Received in revised form 7 September 2014 Available online 6 October 2014 Keywords: Antioxidant Cadmium Free radical Methyl jasmonate Rice

a b s t r a c t Rice seedlings grown under 50 lM cadmium alone or in combination with 5 lM methyl jasmonate were investigated for Cd-induced oxidative injury at 3, 7 and 10 days of treatment. MeJA treatments alone did not have any significant change in antioxidant enzyme activities or levels of H2O2 and O 2 in roots/shoots, as compared to controls during 3–10 days. The Cd-stressed plants When supplemented with exogenous MeJA revealed significant and consistent changes in activities of antioxidant enzymes CAT, SOD, POD and GR paralleled with an increased GSH-pools than that in plants subjected to Cd-stress alone. Synthesis of GSH driven by increasing demand for GSH in response to Cd-induced oxidative stress in rice was evident. Increased activity of LOX under Cd-stress was noted. Results suggest enhanced Cd-tolerance, lowered Cd2+ uptake, an improved membrane integrity and ‘switching on’ of the JA-biosynthesis by LOX in the Cd-stressed rice roots/shoots exposed to MeJA. Exposure to MeJA improved antioxidant response and accumulation of antioxidants which perhaps lowered the Cd-induced oxidative stress in rice. It is this switching on/off of the JA-biosynthesis and ROS mediated signal transduction pathway involving glutathione homeostasis via GR which helps MeJA to mitigate Cd-induced oxidative injury in rice. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Cadmium, a non-essential toxic heavy metal, affects cellular processes via membrane damage, altered electron transport, enzyme activation/inhibition and DNA alteration (Smeets et al., 2005). Oxygen is essential for the existence of aerobic life, but toxic reactive oxygen species (ROS), which include the superoxide anion  (O 2 ), hydroxyl radical (OH ), and hydrogen peroxide (H2O2), are generated in all aerobic cells during metabolic processes (Noctor and Foyer, 1998). Injury caused by these ROS, known as oxidative stress, is one of the major damaging factors in plants exposed to environmental stresses. An increased production of ROS which are potentially harmful for the cell components, is a common outcome of cadmium exposure (Shah et al., 2001; Sanità diToppi et al., 2007; Sharma and Dietz, 2009; Rai et al., 2013). ROS are produced in a controlled manner through normal metabolic processes in aerobic organisms (Gratão et al., 2005), as signaling molecules in pathogen, programmed cell death and abiotic stress responses (Desikan et al., 2001; Mittler, 2002). Stressful conditions cause an imbalance in the steady-state level of ROS in plants (Foyer and Noctor, 2005; Sharma and Dietz, 2009). The over-accumulation of ROS induces ⇑ Corresponding author. Tel.: +91 542 6701086, mobile: +91 9450955423. E-mail address: [email protected] (K. Shah). http://dx.doi.org/10.1016/j.phytochem.2014.09.007 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

oxidative processes like membrane lipid peroxidation, protein oxidation, enzyme inhibition, DNA and RNA damage that lead to cell damage (Gratão et al., 2005; Møller et al., 2007). In order to avoid the deleterious effects of ROS, several efficient antioxidant mechanisms comprising both of enzymatic components such as ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD) or guaiacol peroxidase (POD) and non-enzymatic components such as ascorbic acid (AA) or glutathione (GSH) come to rescue of plant cells (Gratão et al., 2005; Shah et al., 2013; Singh and Shah, 2014). Jasmonates (JA) are a class of plant hormones that mediate various aspects of gene and metabolic regulation, stress responses, reproduction, defense and cell communication (Soares et al., 2010). Jasmonic acid and its volatile methyl ester, methyl jasmonate (MeJA), are a class of cyclopentanone compounds, regarded as endogenous regulators that play important roles for regulating the stress response, plant growth and development (Creelman and Mullet, 1997). In recent research, MeJA has been reported to reduce the development of chilling injury symptoms in a number of horticultural crops, including mango, sweet pepper and tomato fruit. These phytohormones induce the production of a wide array of direct and indirect chemicals such as pathogenesis-related and cellular protection molecules, including proteins involved in detoxification and redox balance, proteinase inhibitors, antimicrobial secondary metabolites, antioxidants and toxins (Farmer and

Leaf

Fresh weight (g) Electrolyte leakage (millisiemens cm1) Cell viability (%) Cd content (lg(g FW)1)

Indicates statistically significant change in treatments relative to control plants (P 6 0.05). *

2.96 ± 0.43 8.24 ± 0.028 3.66 ± 0.04 6.50 ± 0.40 0.19 ± 0.006 0.15 ± 0.005 37.97* ± 1.53 24.27 ± 0.54 2.91 ± 0.31 3.06 ± 0.33 4.60 ± 0.80 5.23 ± 0.35 0.042 ± 0.0029 0.042 ± 0.0070 21.03 ± 0.83 47.57* ± 1.79 3.41 ± 0.34 5.96 ± 0.20 0.06 ± 0.003 22.2 ± 1.99 Shoot Root Length (cm)

Amount of jasmonate derivatives (ng(g FW)1)

12.91* ± 0.50 7.95 ± 0.55 0.17 ± 0.002 33.02 ± 1.80 5.23 ± 0.36 4.73 ± 0.25 0.14* ± 0.004 23.31 ± 0.68 5.88 ± 0.70 6.40* ± 0.67 4.80 ± 0.38 05.70 ± 0.35 0.13 ± 0.004 0.11 ± 0.0034 22.10 ± 0.67 33.70 ± 0.61

+JA JA +JA JA +JA JA +JA JA

Leaf 0.270 ± 0.0036 0.28 ± 0.0062 0.20 ± 0.001 0.21 ±.001 0.22 ± 0.001 0.37 ± 0.016 0.12 ± 0.0006 0.18 ± 0.009 0.25 ± 0.12 0.26 ± 0.012 0.14 ± 0.006 0.19 ± 0.045 Shoot – – 46.32* ± 1.90 29.95 ± 1.20 – – 55.00* ± 2.60 36.32 ± 1.20 – – 61.95* ± 2.91 40.97* ± 1.90 Root – – 74.40* ± 3.20 53.85 ± 2.50 – – 89.45* ± 4.3 60.32 ± 3.01 – – 173.00* ± 7.9 69.40* ± 3.31 Shoot 240.00 ± 11.70 1111.00* ± 49.55 865.00* ± 41.2 1210.00 ± 57.5 484.00 ± 24.2 2352.00* ± 96.95 961.00 ± 45.05 2423.00* ± 118.05 1764.00 ± 76.20 1974.00* ± 94.71 3957.00 ± 184.7 4010.00* ± 156.5 Root 110.00 ± 4.20 1095.00* ± 49.75 657.00* ± 32.05 660.00 ± 29.2 346.00 ± 14.3 1760.00* ± 71.88 815.00 ± 37.75 1490.00 ± 62.5 952.00 ± 32.65 1911.00* ± 82.55 1614.00* ± 69.70 1984.00* ± 86.20

+JA 9.87 ± 0.61 6.00 ± 0.77 0.14 ± 0.002 69.00* ± 3.10 11.70 ± 0.44 5.30 ± 0.45 0.20 ± 0.004 30.08 ± 1.40

50 lM Cd

JA +JA JA

10

Control 50 lM Cd Control

7

50 lM Cd Control

;

The amount or uptake of Cd2+ present in roots and shoots of rice seedlings as estimated by Atomic Absorption Spectrophotometer (AAS) is represented in Table 1. Amount of Cd2+ increased in both roots and shoots with increasing exposure, the values being higher

Parameter

2.2. Effect of exogenous MeJA on cadmium content

3

Exposure of 50 lM Cd2+ resulted in decrease in shoot/root length and fresh weight of rice throughout growth period as seen in Table 1. Cadmium stress caused reduction in length by 10%, 22% and 24% at 3, 7 and 10 days respectively in shoots and 13%25% in roots of rice seedlings (Table 1). Application of exogenous MeJA alone caused a slight decrease in the root/shoot length of rice plants with increasing days of treatment with almost 100% restoration in growth of rice seedlings at day 10 as reflected by the length and weight of rice seedlings exposed to a combination of Cd2+ + MeJA (Table 1). Cadmium led to a loss of 46% cell viability in rice plants at day 7 of the growth period. Application of MeJA alone did not induce significant change in cell viability as compared to controls whereas Cd2+ + MeJA treatments resulted in only 27% loss in cell viability that accounted for almost 45% restoration of cells in rice seedlings in the latter at day 7 (Table 1). Control or MeJA treated plants had less electrolyte leakage (EL) than Cd-treatments alone. Plants exposed to cadmium had a significantly increased EL at 3 and 10 days of exposure as compared to control plants. Cd2+ + MeJA treatments had significant lowering of EL values by 43%76% during 3–10 days by growth (Table 1).

?

2.1. Evaluation of growth and physiological parameters of rice seedlings exposed to Cd2+ and/or MeJA in the growth medium

Treatments

2. Results

Days of growth

Ryan 1990; Farmer, 2007; Cao et al., 2009; Soares et al., 2010; Guo et al., 2013; Zhou et al., 2013; Gill et al., 2013). Plants continuously sense and assess the level of ROS and reprogram their gene expression to respond to the changing conditions in the environment (Xiang and Oliver, 1998; Soares et al., 2010). The variations in antioxidant activity within the cell can modify ROS content leading into death or acclimatization response. Soares et al. (2010) reported a 33% increase in ROS 1 h after treatment with MeJA in Ricinus communis with decreased activities of CAT and POD in these plants. Accumulation and synthesis of stress-specific defense related protein in Cd-stressed plants are well established (Shah and Dubey, 1998). Increased level of heatshock protein, defensin, etc. is reported widely under stress conditions in plants (Gill and Tuteja, 2012; Gill et al., 2013). Recently plant annexins (Ca2+ and phospholipid binding proteins) have been reported to be induced by heavy metals and jasmonic acid in Zea mays (Zhou et al., 2013). The annexin proteins are implicated in plant growth, development and stress responses and these were observed to be regulated by jasmonic acid. Expression of lipoxygenase (LOX) genes in plants is regulated throughout development (Bell et al., 1995). JA biosynthesis gene LOX2 and few other genes of the pathway are activated upon wounding and water deficit resulting in increased jasmonate levels under abiotic stress (Sasaki et al., 2001; Stenzel et al., 2003). Therefore, this study aims to explore the effect of exogenously applied methyl jasmonate on Cd-induced oxidative injury in rice plants. A detailed study of the relationship between ROS accumulation and antioxidant enzyme activities in roots and shoots of Cd-stressed rice (Oryza sativa L.) plants in absence or presence of methyl jasmonate for different time durations is carried out. The activity of LOX, a JA-biosynthetic enzyme is also studied.

9.90* ± 11.65 5.90* ± 0.65 0.19* ± 0.005 43.51 ± 1.90

I. Singh, K. Shah / Phytochemistry 108 (2014) 57–66

Table 1 Effect of exogenous application of 5 lM methyl jasmonate (MeJA) on cadmium (Cd)-stressed rice seedlings at increasing days of growth. Values are mean of three replicates ± SE.

58

0.175* ± 0.008 0.092 ± 0.004 0.069 ± 0.0036* 0.041 ± 0.0010* 0.158* ± 0.0056 0.196 ± 0.0078 3.66* ± 0.165 2.30* ± 0.099 * 2.12 ± 0.98 1.91* ± 0.085 0.089* ± 0.0053 0.064* ± 0.0035 0.074* ± 0.0031 0.063* ± 0.30

2.3. Levels of JA derivatives in rice exposed to cadmium stress Table 1 shows the level of jasmonic acid derivatives in rice plants treated with Cd and/or methyl jasmonate at increasing days of treatment. Level of MeJA increased in controls as well as under Cd-stress in rice roots/shoots with increasing days of treatment. Exposure to Cd2+ alone caused a 2–4 times increase in MeJA levels in shoots and 2–6 times in roots as compared to their corresponding control plants. Roots were associated with lower levels of MeJA than shoots in all the treatments. Rice plants exposed to MeJA alone had 5-time increased MeJA levels in shoots and 2–10 times in roots during 3–7 days of treatment as compared to controls (no exposure to MeJA). Combined application of Cd2+ + MeJA raised jasmonate levels in both roots and shoots by only 0.5–1.0 times when compared to corresponding controls suggesting that it is the exogenous and not the endogenous jasmonate that plays a significant role in management of Cd-stress in rice in this study.

0.054 ± 0.0021 0.040 ± 0.0013 0.272 ± 0.011 2.01 ± 0.09 1.08 ± 0.004 0.060 ± 0.0048 0.061 ± 0.0025

2.4. Effect of Cd2+ and/or MeJA on level of reactive oxygen species and assessment of oxidative damage in rice

Malondialdehydecontent: MDA, Chlorophyll content: CC, Hydrogen peroxide: H2O2, Superoxide anion: O 2 . * Indicates statistically significant change in treatments relative to control plants (P 6 0.05).

+JA

59

in shoots than in roots. Rice plants treated with Cd2+ + MeJA showed 0.5 times decreased uptake and accumulation of Cd2+ in shoots and 2.5 times in roots as compared to the corresponding Cd2+ treatments alone (Table 1).

0.050 ± 0.0019 0.069* ± 0.0028 0.052* ± 0.0023 0.079 ± 0.0031 0.046 ± 0.0018 0.050 ± 0.0023* 0.040 ± 0.0012* 0.042 ± 0.0011 0.016 ± 0.0005 0.130 ± 0.0035 0.140* ± 0.0061 0.265* ± 0.0125 2.55 ± 0.117 3.71* ± 0.162 3.00* ± 0.13 2.10 ± 0.105 2.04* ± 0.098 1.13 ± 0.046 1.68 ± 0.081 2.19* ± 0.099 * 0.090 ± 0.0061 0.060 ± 0.0049 0.076 ± 0.0046 0.093 ± 0.0059 0.068 ± 0.0032 0.089 ± 0.0044 0.073 ± 0.0033 0.061 ± 0.0030 0.052 ± 0.0022 0.047 ± 0.0015 0.015 ± 0.0007 2.86 ± 0.135 1.74 ± 0.074 0.078 ± 0.0032 0.069 ± 0.0028 0.026 ± 0.0013 0.045* ± 0.0019 0.041 ± 0.0016 0.028 ± 0.0011 0.046 ± 0.0013 0.047 ± 0.0012 0.009 ± 0.0003 0.005* ± 0.0002 0.006 ± 0.0003 0.44 ± 0.014 0.35 ± 0.12 0.56* ± 0.019 0.17 ± 0.0078 0.24 ± 0.0009 0.20 ± 0.009 0.076 ± 0.0048 0.089 ± 0.0056 0.085 ± 0.0054 0.054 ± 0.0024 0.070 ± 0.0029 0.064 ± 0.0031 0.026 ± 0.0011 0.023 ± 0.001 0.008 ± 0.0004 0.35 ± 0.015 0.17 ± 0.0065 0.075 ± 0.0041 0.053 ± 0.0021 Shoot Root CC (mg g1 FW) Leaf H2O2 (lmol g1 FW) Shoot Root 1 1  O2 (nmol min g FW) Shoot Root

MDA (nmol g1 FW)

50 lM Cd

JA +JA JA JA Parameter

;

+JA

JA

+JA

JA

+JA

JA

+JA

10

Control 50 lM Cd Control

7

50 lM Cd Control

3

? Treatment

Days of growth

Table 2 Effect of exogenous application of 5 lM methyl jasmonate (JA) on MDA levels, chlorophyll content, formation of reactive oxygen species (ROS) and oxidative damage in shoots and roots of Cd-stressed rice seedlings at increasing days of growth. Values are mean of three replicates ±SE.

I. Singh, K. Shah / Phytochemistry 108 (2014) 57–66

The levels of malondialdehyde (TBARS derivative), leaf chlorophyll, hydrogen peroxide and superoxide anion in Cd-stressed rice seedlings exposed to exogenous MeJA is listed as Table 2. MDA levels in Cd-stressed plants were 20–50% higher in shoots and 30–40% in roots than control plants throughout the 3–7 days of exposure. The lipid peroxidation as indicated by MDA levels almost doubled in roots and shoots of cadmium exposed rice plants compared to control (non-stressed) plants. Cd2+ + MeJA treated seedlings showed less increase in MDA levels as compared to the Cd-exposed counterparts. Chlorophyll content (CC) is a measure of the capacity of plants to perform optimum photosynthetic activity (Arnon, 1949). The level of chlorophyll increased in leaves with increasing days of growth in controls and all treatments. MeJA alone did not alter the level of chlorophyll at 3, 7 or 10 days when compared with controls (Table 2). Exposure to 50 lM Cd-alone significantly decreased the CC by 50% in leaves of 10 day old rice seedlings as compared to controls. Cd2+ + MeJA treatments however, increased the levels of chlorophyll in rice leaves by 25% in 10 day Cd-stressed rice plants. Cadmium induced oxidative damage has been linked to the enhanced production of ROS including H2O2. Addition of 50 lM cadmium almost doubled H2O2 levels in shoots and roots of rice as compared to control plants. Combination of Cd2+ + MeJA however, decreased the elevated H2O2 levels progressively during 3–10 days of exposure. Similarly, exposure to Cd-alone increased level of superoxide anion in shoots/roots of rice seedlings during 3–10 days of treatment. Exogenous JA significantly lowered the Cd toxicity in shoots and roots of rice through significant decrease in the level of superoxide anion in shoots/roots at 7 and 10 day of growth (Table 2) indicating lowered formation of H2O2 or O 2 upon addition of MeJA in rice. 2.5. Effect of Cd2+ and/or MeJA on level of glutathione in rice exposed to cadmium Table 3 shows the level of glutathione (reduced and oxidized) in roots/shoots of rice seedlings exposed to Cd2+and/or MeJA in the growth medium at increasing days of treatment. The total glutathione levels (reduced (GSH) + oxidized (GSSG)) varied in shoots/roots of rice seedlings under Cd2+/MeJA alone or

1.69 ± 0.079 1.02 ± 0.048 1.46 ± 0.069 0.530* ± 0.0199

2.6. Effect of Cd2+ and/or MeJA on antioxidant enzymes in rice

1.91 ± 0.091 1.27 ± 0.0599

1.70 ± 0.079 1.30 ± 0.061

1.88 ± 0.089 1.83 ± 0.088

Regulation of ROS levels are achieved by the antioxidant system comprising of enzymatic scavengers like SOD, CAT, GR and POD enzymes. Exposure to 50 lM cadmium had harmful effect on rice seedlings as noted by the suppressed CAT activity throughout the growth period (Fig. 1). Cadmium decreased the CAT activity in shoots by 17% at day 7 and 11% at day 10 as compared to controls whereas roots had 16%, 13% and 12% lowered CAT activity at 3, 7 and 10th day of growth of rice plants, respectively as compared to controls (Fig. 1A, B). Addition of Cd2+ + MeJA led to an increase in the CAT activity by 20% at day 3 in roots of rice plants when compared with Cd-treatments alone (Fig. 1B). A reverse trend than that of CAT was noted for SOD activity in rice plants under Cd-stress alone or in combination with MeJA. The Cd-stressed plants when supplemented with MeJA showed an increased SOD activity at day 7 in shoots as compared to the SOD activity in control (non-stressed) plants (Fig. 2A). No significant changes could however be observed in roots under similar conditions (Fig. 2B). Application of Cd2+ + MeJA increased POD activity by 4–8% at day

*

Indicates statistically significant change in treatments relative to control plants (P 6 0.05).

2.26 ± 0.110 1.82 ± 0.087 1.60 ± 0.069 2.74* ± 0.126 1.21 ± 0.060 1.76 ± 0.69 2.31* ± 0.105 2.25 ± 0.1.7 1.68 ± 0.079 1.64 ± 0.81 Shoot Root

Shoot 0.074 ± 0.0035 0.062 ± 0.0030 Root 0.073 ± 0.0032 0.054 ± 0.0020

in combination, however the GSH/GSSG ratio was reduced under 50 lM Cd2+ treatments as compared to control (Table 3). MeJA treatment alone did not alter GSH levels in both roots and shoots in rice plants. Rice plants grown in presence of Cd2+ + MeJA exhibited an increase in GSH/GSSG ratio suggesting less depletion of cellular GSH levels upon addition of MeJA as compared to Cd-stressed plants.

2.43 ± 0.120 1.20* ± 0.052 1.99 ± 0.0873 0.730* ± 0.041

0.160 ± 0.007 0.120 ± 0.004

0.146 ± 0.0071 0.096* ± 0.0041 0.151 ± 0.0073 0.120 ± 0.0053 0.075 ± 0.0037 0.069 ± 0.0032 0.110 ± 0.0052 0.105 ± 0.0041 0.146 ± 0.0068 0.110 ± 0.0046 0.110 ± 0.0049 0.113 ± 0.0049 0.085 ± 0.0048 0.056 ± 0.0027 0.121 ± 0.0051 0.114 ± 0.0043 0.373* ± 0.0085 0.125 ± 0.006 0.120 ± 0.004 0.095 ± 0.0041

JA

0.186 ± 0.0087 0.213 ± 0.0098 0.067* ± 0.0029 0.161 ± 0.0078 0.175 ± 0.0086 0.080* ± 0.003 Shoot 0.124 ± 0.0058 0.142 ± 0.0053 0.093* ± 0.0039 0.111 ± 0.0042 0.249 ± 0.0118 0.256 ± 0.125 Root 0.120 ± 0.0051 0.119 ± 0.00489 0.065* ± 0.0028 0.098 ± 0.0037 0.222 ± 0.010 0.220 ± 0.009

Reduced glutathione (GSH) (lmol g1 FW) Oxidized glutathione (GSSG) (lmol g1 FW) GSH/GSSG ratio

JA +JA JA JA

+JA

50 lM Cd Control

3

0.176* ± 0.0078 0.220 ± 0.008 0.110* ± 0.0049 0.160 ± 0.003

Control

JA JA ; Parameters

Control

7

+JA ? Treatments

50 lM Cd

+JA

10

+JA

50 lM Cd

+JA

I. Singh, K. Shah / Phytochemistry 108 (2014) 57–66

Days of growth

Table 3 Effect of exogenous application of 5 lM methyl jasmonate (MeJA) on level of antioxidants in shoots and roots of rice seedlings exposed to Cd-stress at increasing days of growth. Values are mean of three replicates ±SE.

60

Fig. 1. Effect of 50 lM Cd-stress in absence or presence of 5 lM exogenous methyl jasmonate on activity of catalase (CAT) in (A) shoots and (B) roots of rice seedlings at increasing days of treatment. Values are mean of three independent replicates ± SE and (⁄) indicates values significant at P 6 0.05.

I. Singh, K. Shah / Phytochemistry 108 (2014) 57–66

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Fig. 2. Effect of 50 lM Cd-stress in absence or presence of 5 lM exogenous methyl jasmonate on activity of superoxide dismutase (SOD) in (A) shoots, (B) roots and peroxidase (POD) in (C) shoots and (D) roots of rice seedlings at increasing days of treatment. Values are mean of three independent replicates ± SE and (⁄) indicates values significant at P 6 0.05.

3 which declined thereafter at day 7 in both shoots and roots of rice seedlings (Fig. 2C, D). Treatment of 50 lM cadmium decreased GR activity in shoots and roots by 19% at day 3, 15% at day 7 and 11% at day 10 in shoots and 16%, 10% and 15% in roots at day 3, 7 and 10 of growth respectively. A positive effect of MeJA on cadmium stressed plants was revealed by the increased activity of GR in shoots and roots of rice seedlings exposed to Cd2+ + MeJA at 3, 7 and 10 days of exposure (Fig. 3A, B). The activity of lipoxygenase (LOX), a biosynthetic enzyme of JA pathway was found to be highest in cadmium stressed plants however plants treated with MeJA had a suppressed activity of LOX as compared to control (Fig. 3C, D). The increase in LOX activity under Cd-stress and lowering of its activity in presence of MeJA could possibly be a consequence of altered regulation of LOX gene in rice, however this needs further investigations. 3. Discussion Abiotic stresses including salinity, drought, chilling, low nutrient availability and heavy metal are the major limiting factors for crop productivity. Jasmonates are known to mediate and regulate major stress responses. The role of jasmonates and its derivatives in plants has received considerable attention and various modes of action have been discussed (Farmer and Ryan, 1990; Farmer, 2007; Cao et al., 2009; Soares et al., 2010; Guo et al., 2013; Zhou et al., 2013; Gill et al., 2013). Oxidative stress stimulates synthesis of antioxidant metabolites and enhances antioxidant enzyme activities that could protect plant tissues. This study aimed at evaluating the ability of methyl jasmonate, exogenously applied

to Cd-stressed plants, to counteract Cd-stress-induced responses like oxidative stress, formation of ROS and altered activities of antioxidant enzymes. Cytotoxicity caused by ROS is dangerous, as they also act as intermediate signaling molecules and regulate gene-expression associated with antioxidant defense mechanisms (Neill et al., 2002; Rai et al., 2012). In order to cope with the oxidative damage, plants have evolved complex antioxidant enzymatic systems like SOD, CAT, APX, POD, GR and nonenzymatic antioxidants like ascorbate and glutathione (Chien et al., 2001; Choudhury and Panda, 2004; Singh and Shah, 2014). It is reported that MeJA alters the activity of these enzymes and causes the alleviation of the oxidative stress in plants (Li et al., 1998; Jung, 2004; Singh and Shah, 2014). Superoxide dismutase catalyses the dismutation of superoxide anion into H2O2. Catalase together with peroxidases that can use guaiacol as substrate destroys H2O2. Ascorbate peroxidase reacts and scavenges H2O2 in presence of ascorbate (Shah et al., 2001; Shah and Nahakpam, 2013). Glutathione reductase, an NAD(P)H-dependent enzymatic antioxidant efficiently maintains the reduced pool of GSH. It is the differential modulation of both GR and GSH in plants that has major significance in plant defense operations under stressful conditions (Gill et al., 2013). Reduced cell viability, increased formation of H2O2 and lipid peroxidation, low chlorophyll content together with lowered levels of glutathione resulted due to Cd-induced toxicity in rice. This was complemented with altered antioxidant enzyme activities in rice roots/shoots as seen in this work. Results thus indicate a differential and tissue-specific expression of these molecules in rice plants subjected to Cd-stress. Cd2+ + MeJA treatments

62

I. Singh, K. Shah / Phytochemistry 108 (2014) 57–66

Fig. 3. Effect of 50 lM Cd-stress in absence or presence of 5 lM exogenous methyl jasmonate on activity of glutathione reductase (GR) in (A) shoots, (B) roots and lipoxygenase (LOX) in (C) shoots and (D) roots of rice seedlings at increasing days of treatment. Values are mean of three independent replicates ± SE and (⁄) indicates values significant at P 6 0.05.

however, increased the levels of chlorophyll in rice leaves by 25% suggesting a restoration of photosynthetic apparatus by MeJA in 10 day Cd-stressed rice plants. Information on chlorophyll content is not always sufficient to define photosynthetic activity. Although we did not observe any decrease in chlorophyll content, however possible restoration of chlorophyll levels by other cellular protection machinery cannot be denied. Lowered EL in Cd2+ + MeJA treatments suggest that MeJA may help to preserve membrane integrity in rice enabling the seedlings to resist and withstand cadmium stress. The lowering of MDA levels in rice plants exposed to a combination of Cd2+ + MeJA indicate low lipid peroxidation and improved tolerance of the rice plants exposed to Cd. This suggests that application of MeJA in Cd-stressed rice lowers the formation of H2O2 or O 2 and gears the rice plants for enhanced tolerance towards Cd-induced injuries. Application of MeJA together with Cd2+ seems to have an opposite effect on Cdinduced oxidative cell death which is in accordance with the reports in Arabidopsis (Overmyer et al., 2000). Some data indicate that accumulation of JA-derivatives in plants occurs after the first few hours of the action of stress-factors (Rakwal et al., 2002), which represents the endogenous levels of JA-derivatives. During 0–3 days the levels of JA-derivatives in shoots of rice plants exposed to Cd2+ alone increased by four times and declined thereafter suggesting that the endogenous JA levels increase with increasing Cd2+ exposure. It is possible that H2O2 stimulate the accumulation of JA-derivatives as also reported in ginseng (Hu et al., 2003). In accordance with the finding of decreased GSH/GSSG

ratio in rice plants grown under high concentrations of Al, Ni, Mn and As (Srivastava and Dubey, 2011) a decrease in GSH/GSSG ratio under Cd-stress was also noted in this study. Elevated GSH pools in presence of MeJA as observed herein in Cd-stressed rice are important for Cd tolerance. The depletion of GSH is regarded as a critical step in Cd-sensitivity, since plants with improved capacity for GSH synthesis display higher Cd-tolerance (Schutzendubel and Polle, 2002). Studies by Xiang and Oliver (1998) show that glutathione metabolic genes coordinately respond to heavy metal cadmium. These workers also showed that jasmonic acid activated the signal transduction pathway which controls the glutathione concentration at multiple levels. Activity of LOX enzyme increased in Cd-stressed plants. Similar reports of enhanced expression of LOX-genes and corresponding products is reported under other abiotic stresses like water deficit and wounding in Arabidopsis (Bell et al.,1995). It is also reported that salicylates act as an antagonist in jasmonate pathway and suppresses the expression of LOX, however stress–induced defense genes PR-1 is synergistically affected by both jasmonate and salicylate treatments in plants (Stenzel et al., 2003). The significant elevation in the activities of SOD, APX and GR under Cd-stress in rice seedlings and their altered behavior under Cd2+ + MeJA applications suggest an important role of these antioxidant enzymes in maintaining cellular homeostasis and in regulation of intracellular level of ROS (Singh and Shah, 2014). The activity of SOD is reported to increase under diverse stress situations including Cd-metal toxicity in rice (Shah and Nongkynrih, 2007), wheat (Khan et al.,

I. Singh, K. Shah / Phytochemistry 108 (2014) 57–66

2007), Brassica juncea (Mobin and Khan, 2007) and Vigna mungo (Singh et al., 2008). Enhanced expression of APX in plants under Cd-stress has been demonstrated earlier in our lab (Singh and Shah, 2014). Increased leaf APX activity under Cd-stress is also reported in Ceratophyllu mdemersum (Arvind and Prasad, 2003), Triticum aestivum (Khan et al., 2007), B. juncea (Mobin and Khan, 2007), V. mungo (Singh et al., 2008) and O. sativa (Singh and Shah, 2014). The overproduction of APX by MeJA increases POD activity which strengthens the ROS-scavenging system leading thereby into oxidative stress tolerance in rice plants. Increased expression of APX isoforms has been observed in rice plants grown under Cd and heat stress (Shah and Nahakpam, 2012). Evidences for structural basis of altered APX activity in Cd stressed rice plants exposed to jasmonates have been recently reported by us wherein the interaction between heme and ascorbate was noted to be modulated differently in presence of Cd/ JA in rice. (Singh and Shah, 2014). Glutathione reductase, an important enzyme of Halliwell– Asada pathway showed decrease in activity under Cd-stress. Application of MeJA, however elevated the GR activity in the same plants. This could be attributed to the depletion of GSH pools under Cd-stress conditions which in turn inhibits the activity of GR in rice tissues. GSH reductase and the GSH synthesis enzymes were reported to increase in Arabidopsis plants subjected to metal-stress by de novo protein synthesis at the post-translational level (Xiang and Oliver, 1998). Under normal conditions, a steady state GSH level is maintained in plant cells via a low level of transcription, translation and optimal enzyme activity. When plants are challenged by stress, the homeostasis is perturbed and GSH pool is consumed to combat stress. In response, plant cells have a requirement to replenish GSH by increasing its synthesis so as to maintain a high GSH/GSSG ratio. Consequently more GR enzyme is required which is supplemented via de novo protein synthesis. Xiang and Oliver (1998) also correlated that H2O2 and the concentrations of GSH and GSSG or GSH/GSSG ratio are not directly involved in the signal transduction pathway, instead it is the JA-induced up-regulation of GSH metabolic genes that perhaps provide answers to JA-induced cross tolerance in plants. 4. Conclusions In conclusion, rice plants exposed to 50 lM-Cd2+ alone and/or 5 lM-MeJA when investigated for oxidative injury during 3– 10 day exposure revealed exogenous-MeJA to effectively mitigate Cd-induced oxidative injury. Exposure to MeJA improved antioxidant response and accumulation of antioxidants. Synthesis of GSH driven by increasing demand for GSH in response to Cdinduced oxidative stress in rice was evident. Exogenous-MeJA mitigates Cd-induced oxidative injury in rice possibly by lowering Cd2+-uptake that contributes to improved membrane-integrity, ‘switches on’ the JA-biosynthetic pathway involving LOX, alters ROS-mediated signaling via GR and all these confer tolerance to rice plants towards Cd-stress. There appears existence of elegant regulatory controls to switch ‘on or off’ the GSH homeostasis mechanisms in accordance with the changing status of the stress. 5. Experimental 5.1. Plant material and stress conditions Seeds of rice (O. sativa L.) cv. HUR3022 were surface sterilized with 0.1% sodium hypochlorite solution and imbibed in water for 24 h. Seedlings were raised for 10 days in sand cultures, saturated either with Hoagland nutrient solution (Hoagland and Arnon, 1938) that served as control or nutrient solution supplemented

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with either 50 lM Cd(NO3)2 or 5 lM methyl jasmonate (MeJA) or in combination (50 lM Cd(NO3)2 + 5 lM MeJA) as treatments. Pots were maintained at field saturation capacity (pH 7.0) and irrigation was done as required. Seedlings were maintained in a growth chamber for 10 days at 28 ± 1 °C, 80% relative humidity and 12 h light (irradiance 40–50 lM m2 s1) followed by a dark period. Seedlings from control and treatments were uprooted at 3, 7 and 10 days intervals, roots and shoots separated and used for experiments. All the estimations were carried out in triplicate. 5.2. Evaluation of physiological parameters 5.2.1. Determination of seedling growth Growth of rice seedlings in terms of fresh weight as well as root and shoot lengths were measured at 3, 7 and 10 days in control, 50 lM Cd2+/5 lM MeJA treatments alone or in combination of 50 lM Cd2+ + 5 lM MeJA. 5.2.2. Determination of cell viability The loss of cell viability was evaluated using Evans blue staining method (Baker and Mock, 1994). Freshly harvested roots and shoots were stained with 0.25% (v/v) aqueous solution of Evans blue for 15 min. After washing with distilled water for 15 min, 20 root segments (2 mm) were excised and treated with 200 ll of N,N-dimethyl formamide for 1 h at room temperature. The optical density of released Evans blue was measured spectrophotometrically at 600 nm and expressed as CV% (i.e. no. of viable cells as compared with control). 5.2.3. Measurement of electrolyte leakage Electrolyte leakage was measured using a conductivity meter (CM-180, ELICO, India) following McNabb and Takahashi (2000). Ten leaf discs were placed in 25 ml water and conductivity measured (i) after 15-min of vacuum filtration and/or (ii) after autoclaving at 121 °C for 30 min. Values were calculated using the equation EL (%) = value of a/value of b  100 where a = conductivity at 15 min after vacuum filtration, b = conductivity at 30 min after autoclaving (Khare et al., 2010). 5.3. Determination of cadmium Extraction and estimation of cadmium in root/shoot samples were performed according to Shah and Dubey (1998) with certain modifications. Seedlings were surface sterilized with 1 mol/L HCl followed by 1 mol/L Na2EDTA to resolve excess surface bound Cd2+ and then samples were dried in an oven at 70 °C for 4 days. Completely oven dried samples were weighed and estimation of Cd2+ was carried out in triplicate. The samples were grinded and digested in concentrated HNO3 followed by its dissolution in dilute perchloric acid (PCA) which led to the release of bound Cd2+ in ionic form in the solution. Cd2+content was then measured using Atomic Absorption Spectrophotometer (AAS) fitted with a PerkinElmer-2380, and expressed in lg g1 fresh weight (FW). 5.4. Extraction and quantification of jasmonate-derivatives 100 mg plant sample was grinded under liquid nitrogen and extracted in isopropanol, acetonitrile and water in the ratio 3:3:2 and cooled at 20 °C. Ribitol (60 ll) was added as internal standard and centrifuged at 12,000 rpm for 10 min. Supernatant was dried under speed vacuum and derivatized by adding 50 ll of methoxy amine hydrochloride (20 mg/ml) in pyridine with shaking at 30 °C for 90 min followed by silylation of samples with 100 ll of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) at 37 °C and incubated for 30 min according to Engelberth et al. (2003) with modifications. A 1 ll of prepared sample was then injected to

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GC–MS (Shimadzu, GCMS-QP2010 Plus, Japan) fitted with fused silica Rtx-5 MS low bleed GC–MS column (Restek, USA) for 60 min at a split ratio of 1:25 using ramp temperature 230– 280 °C with full scan mode of 40 m/z to 750 m/z acquisition. The JA-derivatives were assigned using manufacturer supplied WILEY8 library. Values obtained for estimation of MeJA and its derivatives are expressed as ng g1 fresh weight (FW). 5.5. Assessment of oxidative damage The oxidative damage in leaves was assessed by measuring the lipid peroxidation and altered chlorophyll content in plant samples. 5.5.1. Measurement of lipid peroxides as malondialdehyde content The malondialdehyde (MDA) content was measured as described by Heath and Packer (1969) in 200 mg fresh root/shoot tissues. Samples were homogenized in 0.25% 2-thiobarbituric acid (TBA) in 10% TCA using a mortar and pestle. After heating at 95 °C for 30 min, the mixture was quickly cooled in an ice bath and centrifuged at 10,000  g for 10 min. The absorbance of the supernatant was read at 532 nm and corrected for unspecific turbidity by subtracting the absorbance of the same at 600 nm. The blank was 0.25% TBA in 10% TCA. The concentration of lipid peroxides together with oxidatively modified proteins of root/shoot tissue were thus quantified in terms of MDA content using an extinction coefficient () of 155 mM1 cm1 and expressed as nmol g1 fresh weight (FW). 5.5.2. Estimation of chlorophyll Extraction and determination of chlorophyll was performed according to the method of Arnon (1949). A 100 mg of fresh leaf material was homogenized with 10 ml of 80% acetone at 4 °C and centrifuged at 2500 rpm for 10 min at 4 °C. This procedure was repeated until the residue became colourless. The extract was transferred to a graduated tube and made up to 10 ml with 80% acetone and assayed immediately. Three millilitre aliquots of the extract were transferred to a cuvette and the absorbance was read at 645 nm and 663 nm with a spectrophotometer against 80% acetone as blank. Chlorophyll content was calculated using the formula of Arnon (1949) and expressed in mg g1 fresh weight (FW). 5.5.3. Determination of hydrogen peroxide (H2O2) The H2O2 levels in roots/shoots from rice seedlings were measured as given by Jana and Choudhuri (1982). About 200 mg tissue was homogenized in 5 ml of 50 mM phosphate buffer (pH 6.5) and centrifuged. To 3 ml of the supernatant, 1 ml of 0.1% titanium sulphate in 20% H2SO4 was added. The mixture was then centrifuged at 7000  g for 15 min. The intensity of the yellow color developed was measured at 410 nm. The amount of H2O2 was calculated using an extinction coefficient of 0.2 lM1 cm1 and expressed as lmol g1 fresh weight (FW). 5.5.4. Determination of superoxide anion (O 2 ) The rate of superoxide anion (O 2 ) formed was measured according to Misra and Fridovich (1971). About 200 mg fresh plant samples were homogenized using chilled mortar and pestle in cold (0–4 °C), in 50 mM potassium phosphate buffer (pH 7.5) containing 250 mM sucrose, 10 mM MgCl2 and 1 mM diethyl dithiocarbamate to inhibit SOD activity. After centrifugation the O 2 was measured in the supernatant, as its capacity to reduce epinephrine. The assay mixture in total volume of 3 ml contained 3 mM epinephrine in phosphate buffer (pH 7.5), 0.3 mM NADH and the supernatant. The absorbance was recorded at 480 nm in an ELICO SL-159 (India) UV–Vis spectrophotometer and NADH dependent adrenochrome formation was recorded for 7–8 min. The amount of superoxide

anion was calculated using extinction coefficient of 4 mM1 cm1 and expressed as nmol g1 fresh weight (FW). All the experiments were carried out in sealed tube under N2 atmosphere to minimize oxidation and generation of ROS. 5.6. Determination of level of glutathione (GSH and GSSG) Total glutathione, i.e. GSSG (oxidized) and GSH (reduced) were determined using spectrophotometer as given by Griffith (1980) and Hodges et al. (1996). About 7.5–10 g plant sample was homogenized in a mortar and pestle in cold, along with 0.5 g inert sand and 15 ml of ice-cold freshly-made 5% (w/v) 5-sulphosalicylic acid and centrifuged. Two separate solutions: A (pH 7.2) containing 100 mM Na2HPO4.7H2O, 40 mM NaH2PO4.H2O, 15 mM EDTA, 1.8 mM 5,50 -dithio-bis-(2-nitrobenzoic acid) and 0.04% BSA and B (pH 7.2) containing 1.0 mM EDTA, 50 mM imidazole, 0.2% BSA and 2 units ml1 glutathione reductase enzyme (Sigma Aldrich) were prepared. Total glutathione was measured in a reaction mixture containing 400 ll solution A, 320 ll solution B, 400 ll 1:25 dilution of supernatant in KH2PO4 (0.5 M, pH 7.0), and 80 ll NAPDH (3.0 mM). Total glutathione was measured by following the change in absorbance at 412 nm for 5 min. For GSSG estimation similar procedure was followed except that 1.0 ml of 1:10 diluted supernatant in KH2PO4 (0.5 M, pH 6.5) was first incubated with 20 ll 2-vinylpyridine at 25 °C for 1 h to derivatize GSH. GSH and GSSG standards were between 0 and 18 lM in 5% (w/v) 5-sulphosalicylic acid diluted appropriately with KH2PO4 (0.5 M, pH 7.0). For each sample, GSH was estimated from the difference between total glutathione and GSSG and expressed as lmol g1 fresh weight (FW). 5.7. Enzyme preparation and assays All enzyme assays were carried out using a UV–Vis spectrophotometer (ELICO, SL-159, India). About 200 mg of fresh root/shoot samples were homogenized in specific extraction buffers as given in literature. 5.7.1. Catalase (CAT) assay Catalase (CAT, EC 1.11.1.6) was extracted and assayed as given by Beers and Sizers (1952). Assay mixture in a total volume of 1.5 ml contained l000 ll of 100 mM KH2PO4 buffer (pH 7.0), 400 ll of 200 mM H2O2 and 100 ll enzyme. Decrease in H2O2 was monitored at 240 nm (extinction coefficient of 0.036 mM1 cm1). Enzyme specific activity is expressed as lmol of H2O2 oxidized mg1 (protein) min1. 5.7.2. Superoxide dismutase (SOD) assay The superoxide dismutase (SOD, EC 1.15.1.1) activity was extracted and assayed according to Misra and Fridovich (1972). The assay mixture contained 50 mM sodium carbonate-bicarbonate buffer (pH 9.8), containing 0.1 mM EDTA, 0.6 mM epinephrine and enzyme extract in a total volume of 3 ml. Epinephrine was the last component to be added. The adrenochrome formation during the next 5 min was recorded at 470 nm. One unit of SOD activity is defined as the amount of enzyme required to cause 50% inhibition of epinephrine oxidation under the experimental conditions. 5.7.3. Guaiacol peroxidase (POD) assay The activity of guaiacol peroxidase (POD, EC 1.11.1.7) was extracted and assayed according to Egley et al. (1983). Assay mixture in a final volume of 2 ml contained 50 ll enzyme, 200 ll guaiacol and 50 ll H2O2 in 1.7 ml of buffer. The increase in absorbance was measured at 470 nm (extinction coefficient 26.6 mM1 cm1).

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Enzyme specific activity is expressed as lmol H2O2 reduced mg1 (protein) min1. 5.7.4. Glutathione reductase (GR) assay Glutathione reductase (GR, EC 1.6.4.2) was extracted and assayed according to Dalton et al. (1986) with modifications. The assay mixture contained 0.25 mM GSSG, 0.125 mM NADPH, 50 mM tricine (pH 7.8), 0.5 mM EDTA and 50 ll of extract in a final volume of 2 ml. The decrease in absorbance due to NADPH oxidation was recorded at 340 nm (extinction coefficient of 6.22 mM1 cm1) and expressed in terms of nmol NADPH oxidized mg1 (protein) min1. 5.8. Assay of JA biosynthetic enzyme lipoxygenase Lipoxygenase (LOX, EC 1.13.11.12) was extracted by homogenising 100 mg shoot/root tissue with 3 ml of 50 mM phosphate buffer (pH 6.0) (Surrey, 1963). The homogenate was centrifuged at 2000  g for 15 min at 4 °C and supernatant was used for the enzyme assay. The assay mixture in a total volume of 1.5 ml contained 200 mM borate buffer (pH 6.0), 0.25% linoleic acid, 0.25% Tween-20 and 50 ll of enzyme extract. The react ion was carried out at 25 °C for 5 min and immediately stopped by addition of 2 ml of absolute alcohol. The reaction mixture was centrifuged to make the solution optically clear. The absorbance of solution was measured at 234 nm. Enzyme activity is expressed as units mg1 (protein) min1. 5.9. Protein determination In all enzyme preparations protein was determined by the method of Lowry et al. (1951) using bovine serum albumin (BSA, Himedia) as standard. 5.10. Statistical analysis The data were analyzed by a simple variance analysis (ANOVA) and significant difference was compared by t-test. Acknowledgements Financial support to I.S. by Department of Science and Technology, New Delhi under Women scientist scheme is gratefully acknowledged. Authors are thankful to Bioinformatics division, Mahila Mahavidyalaya and School of Biotechnology for providing infrastructure facilities. English language editing by Dr Gerard Charmsson is gratefully acknowledged. References Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1–15. Arvind, P., Prasad, M.N.V., 2003. Zinc alleviates cadmium-induced oxidative stress in Ceratophyllum demersum L: a free floating fresh water macrophyte. Plant Physiol. Biochem. 41, 391–397. Baker, C.J., Mock, N.M., 1994. An improved method for monitoring cell death in cell suspension and leaf disc assays using Evans blue. Plant Cell Tissue Organ Culture 39, 7–12. Beers Jr., R.F., Sizer, I.W., 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133–140. Bell, E., Creelman, R.A., Mullet, J.E., 1995. A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis. Proc. Nat. Acad. Sci. U.S.A. 92, 8675–8679. Cao, S., Zheng, Y., Wang, K., Jin, P., Rui, H., 2009. Methyl jasmonate reduces chilling injury and enhances antioxidant enzyme activity in postharvest loquat fruit. Food Chem. 115 (4), 1458–1463. Chien, H.F., Wang, J.W., Lin, C.C., Kao, C.H., 2001. Cadmium toxicity of rice leaves is mediated through lipid peroxidation. J. Plant Growth Regul. 33, 205–213.

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Shah, K., Kumar, R.G., Verma, S., Dubey, R.S., 2001. Effect of cadmium on lipid peroxidation, superoxide anion generation and activities of antioxidant enzymes in growing rice seedlings. Plant Sci. 161, 1135–1144. Shah, K., Singh, P., Nahakpam, S., 2013. Effect of cadmium uptake and heat stress on root ultrastructure, membrane damage and antioxidative response in rice seedlings. J. Plant Biochem. Biotechnol. 22, 103–112. Sharma, S.S., Dietz, K.J., 2009. The relationship between metal toxicity and cellular redox imbalance. Trends Plant Sci. 14 (1), 43–50. Singh, I., Shah, K., 2014. Evidences for structural basis of altered ascorbate peroxidase activity in cadmium-stressed rice plants exposed to jasmonate. Biometals 27, 247–263. Singh, S., Khan, N.A., Nazar, R., Anjum, N.A., 2008. Photosynthetic traits and activities of antioxidant enzymes in black gram (Vigna mungo L. Hepper) under cadmium stress. Am. J. Plant Physiol. 3, 25–32. Smeets, K., Cuypers, A., Lambrechts, A., Semane, B., Hoet, P., Van Laere, A., Vangronsveld, J., 2005. Induction of oxidative stress and antioxidative mechanisms in Phaseolus vulgaris after Cd2+ application. Plant Physiol. Biochem. 43, 437–444. Soares, A.M.S., deSouza, T.F., Jacinto, T., Machado, O.L.T., 2010. Effect of methyl jasmonate on antioxidative enzyme activities and on the contents of ROS and H2O2 in Ricinus communis leaves. Braz. J. Plant Physiol. 22 (3), 151–158. Srivastava, S., Dubey, R.S., 2011. Manganese excess induces oxidative stress, lowers the pool of antioxidants and elevated activities of key antioxidative enzymes in rice seedlings. Plant Growth Reg. 64, 1–16. Stenzel, I., Hause, B., Miersch, O., Kurz, T., Maucher, H., Weichert, H., Ziegler, J., Feussner, I., Wasternack, C., 2003. Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana. Plant Mol. Biol. 51, 895–911. Surrey, K., 1963. Spectrophotometric method for determination of lipoxidase activity. Plant Physiol. 39, 65–70. Xiang, C., Oliver, D.J., 1998. Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. Plant Cell 10, 1539–1550. Zhou, M.L., Yang, X.B., Zhang, Q., Zhou, M., Zhao, E.Z., Tang, Y.X., Zhu, X.M., Shao, J.R., Wu, Y.M., 2013. Induction of annexin by heavy metals and jasmonic acid in Zea mays. Funct. Integr. Genomics 13 (2), 241–251.