Photosynthetic activity, pigment composition and antioxidative response of two mustard (Brassica juncea) cultivars differing in photosynthetic capacity subjected to cadmium stress

ARTICLE IN PRESS Journal of Plant Physiology 164 (2007) 601—610

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Photosynthetic activity, pigment composition and antioxidative response of two mustard (Brassica juncea) cultivars differing in photosynthetic capacity subjected to cadmium stress Mohammad Mobin, Nafees A. Khan Plant Physiology and Biochemistry Division, Department of Botany, Aligarh Muslim University, Aligarh (U.P.) 202002, India Received 17 November 2005; accepted 14 March 2006

KEYWORDS Anthocyanins; Antioxidant; Brassica; Cadmium; Photosynthesis

Summary Photosynthetic performance, contents of chlorophyll and associated pigments, cellular damage and activities of antioxidative enzymes were investigated in two mustard (Brassica juncea L.) cultivars differing in photosynthetic capacity subjected to cadmium (Cd) stress. Exposure to Cd severely restricted the net photosynthetic rate (PN) of RH-30 compared to Varuna. This corresponded to the reductions in the activities of carbonic anhydrase (CA) and ribulose-1,5-bisphosphate carboxylase (Rubisco) in both the cultivars. Decline in chlorophyll (Chl) (a+b) and Chl a content was observed but decrease in Chl b was more conspicuous in Varuna under Cd treatments, which was responsible for higher Chl a:b ratio. Additionally, the relative amount of anthocyanin remained higher in Varuna compared to RH-30 even in the presence of high Cd concentration, while percent pheophytin content increased in RH-30 at low Cd concentration. A higher concentration of Cd (100 mg Cd kg1 soil) resulted in elevated hydrogen peroxide (H2O2) content in both the cultivars. However, Varuna exhibited lower content of H2O2 in comparison to RH-30. This was reflected in the increased cellular damage in RH-30, expressed by greater thiobarbituric acid reactive substances (TBARS) content and electrolyte leakage. The enhanced activities of antioxidative enzymes, ascorbate peroxidase (APX),

Abbreviations: AOS, active oxygen species; APX, ascorbate peroxidase; CA, carbonic anhydrase; CAT, catalase; Cd, cadmium; Chl, chlorophyll; DMSO, dimethyl sulfoxide; E, transpiration rate; GR, glutathione reductase; gS, stomatal conductance; PN, net photosynthetic rate; Rubisco, ribulose-1, 5-bisphosphate carboxylase; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substance Corresponding author. E-mail address: [email protected] (M. Mobin). 0176-1617/$ - see front matter & 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2006.03.003

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M. Mobin, N.A. Khan catalase (CAT) and glutathione reductase (GR) and also lower activity of superoxide dismutase (SOD) in Varuna alleviated Cd stress and protected the photosynthetic activity. & 2006 Elsevier GmbH. All rights reserved.

Introduction Cadmium (Cd) can be accumulated to higher levels in the aerial organs (Pence et al., 2000), preferentially in the chloroplasts and disturbs the chloroplast function by inhibiting the activities of enzymes of chlorophyll biosynthesis and CO2 fixation (Bo ¨ddi et al., 1995; Krupa and Baszynski, 1995; Siedlecka et al., 1997) or the aggregation of pigment protein complexes of the photosystems (Horvath et al., 1996). The Cd-induced formation of active oxygen species (AOS), superoxide anion (O2 ), hydroxyl (OH) radicals and H2O2 result in the damage of chloroplast. The presence of H2O2 in the chloroplasts restricts Calvin-cycle enzymes reducing carbon assimilation (Takeda et al., 1995). It induces changes in the functions of membranes by initiating peroxidation of polyunsaturated fatty acids (De Vos et al., 1993), oxidative damage by formation of oxygen free radicals or by the reduction in the status of enzymatic and nonenzymatic antioxidants (Shaw, 1995; Somashekaraiah et al., 1992). Plants appear to possess a wide array of defense strategies to protect the photosynthetic apparatus and cellular membranes from AOS (Foyer and Harbinson, 1994). Production of antioxidative enzymes is one part of the defense system that plants require to protect against stress. Superoxide dismutase (SOD; EC 1.15.1.1) constitutes the primary step of cellular defense. It dismutates O2  to H2O2 and O2. Further, the accumulation of H2O2 is restricted through the action of catalase (CAT; EC 1.11.1.6) or by the ascorbate–glutathione cycle, where ascorbate peroxidase (APX; EC 1.11.1.11) reduces it to H2O. Finally, glutathione reductase (GR; EC 1.6.4.2) catalyzes the NADPH-dependent reduction of oxidized GSSG to the reduced GSH (Noctor et al., 2002). Mustard (Brassica juncea L. Czern & Coss) is recognized as an accumulator of heavy metals. It is, therefore, postulated that the mustard cultivars with diverse photosynthetic capacity detoxifies the AOS and protects the chloroplast functions differently from oxidative damage. In the present investigation, two mustard cultivars, Varuna and RH-30 (Khan, 2004), were used to study carbonic anhydrase (CA), ribulose-1,5-bisphosphate carboxy-

lase (Rubisco), net photosynthetic rate (PN), stomatal conductance (gS), transpiration rate (E) and contents of chlorophyll (Chl), pheophytin and relative amount of anthocyanin, associated changes in the contents of H2O2, thiobarbituric acid reactive substances (TBARS), electrolyte leakage and the capacities of antioxidative enzymes SOD, APX, CAT and GR under Cd stress.

Material and methods Plant material and growth conditions The seeds of mustard (B. juncea L. Czern & Coss) cultivars Varuna (high photosynthetic capacity) and RH-30 (low photosynthetic capacity) were surface sterilized with 0.5% NaOCl for 20 min, rinsed and soaked overnight in sterile water for 12 h at 4 1C for uniform germination. The seeds were transferred to 23-cm-diameter earthen pots filled with 5 kg of reconstituted soil (sand:clay:peat; 70:20:10, by dry weight) in the green house of the Botany Department, Aligarh Muslim University, Aligarh, India, under semi-controlled condition. A polythene plastic film was used to thwart the effects of rainfall, which allowed the transmittance of 90% of visible wavelength (400–700 nm) under natural day and night conditions with a day/night temperature 25/ 2074 1C and relative humidity of 7075%. Cd at a concentration of 0, 25, 50 and 100 mg kg1 was added to the soil as CdCl2  6H2O and thoroughly mixed. The soil pH was 7.5. Plants (4 per pot) were watered every alternate day with half strength of Hoagland nutrient solution. Watering schedule was adjusted throughout the experimental duration in order to avert leaching. The treatments were arranged in a randomized complete block design, and each treatment was replicated five times.

Photosynthetic and gas exchange measurements Photosynthetic parameters, PN, gS and E were recorded on fully expanded leaves of second youngest nodes at 30 days after sowing using an infra-red gas analyzer (IRGA, LICOR, 6200, Lincoln,

ARTICLE IN PRESS Photosynthesis and oxidative stress in cadmium-treated mustard NE, USA) between 11:00 and 13:00 h at light saturation intensity. These observations were recorded on five plants in a treatment.

Determination of enzymes of carbon assimilation and reduction pathway Activity of CA in leaf was estimated according to the method described by Makino et al. (1992). Sampled leaves were homogenized in 10 mL of buffer containing 50 mM HEPES-NaOH (pH 7.5), 10 mM DTT, 0.5 mM EDTA and 10% (v/v) glycerol. Triton X-100 was added to the homogenate to a final concentration of 0.1% (v/v). The homogenate was centrifuged (15,000g, 10 min), and the supernatant was used for the determination of CA. Activity of CA was determined in Wilbur–Anderson unit following time-dependent reduction in pH from 8.25 to 7.45 at 0–3 1C. The Unit [U] of enzyme activity was calculated according to the formula U ¼ 10ðT  T 0 Þ=T 0 , where T and T0 represent the time required to change the pH from 8.25 to 7.45, with and without the extract of crude enzyme, respectively. The enzyme activity was presented as U per mg protein. Rubisco activity was determined spectrophotometrically by monitoring NADH oxidation at 30 1C and A340 (Usuda, 1985). Leaf samples were homogenized in a chilled mortar with ice-cold extraction buffer solution containing 0.25 M Tris–HCl (pH 7.8), 0.05 M MgCl2, 0.0025 M EDTA and 37.5 mg DTT. The homogenate was centrifuged at 4 1C for 10 min at 10,000g. The resulting supernatant was used for assay of the enzyme. The reaction mixture contained 100 mM Tris–HCl (pH 8.0), 40 mM NaHCO3, 10 mM MgCl2, 0.2 mM NADH, 4 mM ATP, 0.2 mM EDTA, 5 mM DTT, 1 U of glyceraldehyde 3-phosphodehydrogenase and 1 U of 3-phosphoglycerate kinase. The activity was estimated after the addition of enzyme extract and 0.2 mM ribulose-1,5-bisphosphate (RuBP). Enzyme activity was expressed as mmol CO2 fixed min1 mg1 protein. Estimation of protein was done according to Bradford (1976) using bovine serum albumin as standard.

Pigment analysis Determination of chlorophyll content Chlorophyll was extracted from freshly sampled leaves using the DMSO method based on Barnes et al. (1992). Chl absorption in the extract was measured using UV-VIS spectrophotometer. Content of the Chl was calculated from the following formulae taken from Barnes et al. (1992): Chl a ¼ 14:85A664:9  5:14A648:2 ,

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Chl b ¼ 25:48A648:2  7:36A664:9 , Chl a þ b ¼ 7:49A664:9 þ 20:3A648:2 .

Determination of anthocyanin and pheophytin contents The relative amount of anthocyanin was estimated spectrophotometrically after extraction in acidified methanol (methanol:water:HCl: 79:20:1) as described by Mancinelli (1984) using A530– 0.25A657 to correct for Chl and non-specific degradation products. For determination of pheophytin, leaf samples were extracted in 80% acetone and the percentage of Chl converted to pheophytin was estimated by an increase in absorbance at A553 relative to the absorbance at A665 (Bowler et al., 1991). Determination of TBARS content, electrolyte leakage and H2O2 content Contents of TBARS were measured according to Cakmak and Horst (1991) by recording absorbance at A532 and corrected for non-specific turbidity by subtracting the absorbance at A600. The TBARS content was calculated using its extinction coefficient of 155 mM1 cm1. For measuring electrolyte leakage, samples were thoroughly washed with sterile water to get rid of surface adhered electrolyte and then kept in closed vials containing 10 mL of deionized water and incubated at 25 1C for 6 h on a shaker and consequently electrical conductivity was determined (C1). Samples were then kept at 90 1C for 2 h and the electrical conductivity was obtained after attaining equilibrium at 25 1C (C2). Electrolyte leakage was calculated using the following equation: Percent electrolyte leakage ð%Þ ¼ ðC1 =C2 Þ  100. The assay of H2O2 was made following Okuda et al. (1991). Leaves (0.5 g) were ground in ice-cold 200 mM perchloric acid. After centrifugation at 1200g for 10 min, perchloric acid of the supernatant was neutralized with 4 M KOH. The insoluble potassium perchlorate was eliminated by centrifugation at 500g for 3 min. The reaction was started by the addition of peroxidase and increase in the absorbance was recorded at A590 for 3 min. Extraction and estimation of antioxidative enzymes The overall procedures were carried out at 4 1C unless otherwise mentioned. Leaf tissue was ground in chilled mortar using different specific buffers and pH values for each enzyme.

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Cadmium determination Root and leaf samples were immersed in 5 mM CaCl2 solution for 5 min, rinsed in distilled water, desorbed and blotted (Meuwly and Rauser, 1992) for Cd determination. Cd concentration was estimated after digesting the sample in nitric:perchloric acid (3:1, v/v). Cd concentration was determined by atomic absorption spectrophotometer (Perkin-Elmer A, Analyst, 300).

Data analysis Analysis of variance (ANOVA) for all the measured variables was performed by SPSS Ver. 10, Inc., Chicago, USA. The treatment means were separated using Duncan’s multiple range test (DMRT) taking Po0:05 as significant.

Results The Cd concentration in roots and leaves was greater in RH-30 than Varuna at all Cd treatments (Fig. 1A and B). Significant reductions were found in photosynthetic parameters with all Cd treatments in both the cultivars (Table 1). PN was 3.5%, 30.9% and 35.5% less in Varuna and 4.7%, 35.0% and 50.0% less in RH-30 with 25, 50 and 100 mg Cd kg1 soil, respectively, compared to the control. gS and E were significantly enhanced at 50 and 100 mg Cd kg1 soil in RH-30, but in Varuna the changes in gS and E were non-significant (Table 1). Increasing Cd concentration depressed the activities of CA and Rubisco in general, but the

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Homogenates were squeezed through four layers of cheesecloth and centrifuged at 15,000g in a cooling centrifuge at 4 1C for 15 min. Supernatants obtained were used for enzyme determinations. SOD activity was assayed by monitoring the inhibition of photochemical reduction of nitroblue tetrazolium (NBT) according to Giannopolitis and Ries (1977). The extraction buffer consisted of 50 mM phosphate buffer (pH 7.8) containing 0.1% (w/v) BSA, 0.1% (w/ v) as ascorbate and 0.05% (w/v) b-mercaptoethanol. The photoreduction of NBT (production of blue formazan) was measured at A560 and an inhibition curve was made against different volumes of the extract. One unit of SOD was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT at A560. CAT activity was assayed following the degradation of H2O2 (extinction coefficient 39.4 mM1 cm1) at 25 1C according to Chaparro-Giraldo et al. (2000) with minor modifications. Leaf samples were homogenized in a buffer composed of 100 mM potassium phosphate buffer (pH 7.5), containing 1 mM EDTA, 3 mM DTT and 5% insoluble PVP (w/v). The reaction was initiated by the addition of leaf extract and the activity was measured by monitoring degradation of H2O2 at A240 over 1 min, against a leaf extract free blank. For determination of APX activity, leaf samples were extracted in the buffer containing 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA and 1% PVP (w/v) with the addition of 1 mM ascorbic acid. APX activity was estimated according to Nakano and Asada (1981) by following the reduction in a reaction mixture at A290 (extinction coefficient 2.8 mM1 cm1). The reaction was started by the addition of enzyme extract. For non-enzymatic oxidation of ascorbate, correction was made by H2O2. The leaf samples for estimation of GR were extracted in 100 mM potassium phosphate buffer (pH 7.0) containing 1 mM Na2-EDTA and 4% polyclar AT (w/v). Activity of GR was determined following the method of Sgherri et al. (1994) by measuring decrease in absorbance at A340 and using extinction coefficient of 6.2 mM1 cm1. The reaction was started with the addition of NADPH. This method does not require correction for GSSH-independent NADH oxidation.

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Figure 1. Cadmium contents of root (A) and leaf (B) of Varuna (high photosynthetic capacity) and RH-30 (low photosynthetic capacity) cultivars of mustard (Brassica juncea L.) treated with cadmium concentrations. Results are means of five replications7SE. The data followed by different letters are significantly different at Pp0:05.

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Each value represents mean of five replicates7SE. Means were compared using ANOVA. Data followed by different letters in a row are significantly different at Pp0:05.

0.5670.04c 0.1470.01d 4.070.22c 0.9570.03d 33.7971.7c 3.3370.23d 0.2670.01c 8.6170.44c 44979d 4.6970.25c 0.7470.05b 0.2070.01c 3.6670.2c 1.1670.03c 29.6972.27c 5.0770.29c 0.3870.03c 11.2670.54b 39079c 4.3370.23b 1.1170.05a 0.5370.02a 2.0970.03a 0.4970.01a 0.4970.009a 9.9670.54a 0.7770.05a 17.3570.29a 31376a 3.6270.16a 0.7970.03c 0.177.02d 4.6270.32c 1.4570.04c 35.8372.88c 4.6470.31d 0.2770.01c 12.6370.39b 3327 9c 4.0670.30c 0.9070.04c 0.3170.02c 2.9770.22b 1.3570.07c 16.1671.11b 7.0270.29c 0.4170.04c 13.5370.43b 30078b 3.8270.27b Chl a (mg g1) Chl b (mg g1) Chl a:b Anthocyanin (A530–0.25A667 g1) Percent pheophytin CA (U mg1 protein) Rubisco (mmol CO2 mg1 protein min1) PN (mmol m2 s1) gs (mmol m2 s1) E (mmol m2 s1)

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0.8470.04b 0.287.02b 2.9970.08b 0.6870.01b 9.17371.22b 6.6170.43b 0.5670.04b 16.5270.42a 35477b 3.8470.22a 1.0870.04b 0.4070.03b 2.717.1ab 0.8570.03b 10.5070.79b 8.0970.39b 0.6270.06b 18.8870.26a 27278a 3.4470.30a 1.2770.02a 0.5970.01a 2.157.04a 0.5270.01a 0.5270.01a 11.2870.45a 0.8070.04a 19.5870.48a 27679a 3.1670.29a

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Table 1. Chlorophyll (Chl) a, Chl b, Chl a:b, anthocyanin, percent pheophytin, carbonic anhydrase (CA) activity, ribulose-1,5-bisphosphate carboxylase (Rubisco), rate of photosynthesis (PN), stomatal conductance (gS) and transpiration rate (E) of two cultivars of mustard (Brassica juncea L.) exposed to cadmium stress

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reduction in the activity of Rubisco was greater in RH-30 at 50 and 100 mg Cd kg1 soil compared to Varuna. Reduction in the activity of CA was 11.9%, 34.9% and 45.0% in Varuna, while 18.9%, 38.1% and 62.9% in RH-30 with 25, 50 and 100 mg Cd kg1 soil, respectively, compared to the control. The inhibition in the activity of Rubisco was 22.7%, 48.6% and 55.0% in Varuna and 30.7%, 50.0% and 72.0% in RH30 as the Cd concentration increased from 25 to 100 mg Cd kg1 soil in comparison to the control (Table 1). Total Chl decreased with increasing Cd concentrations in the soil. The reduction in Chl content was 20.0%, 35.0% and 50.0% in Varuna and 31.0%, 43.0% and 57.0% in RH-30 at 25, 50 and 100 mg Cd kg1 soil, respectively, compared to the control. Decrease in Chl a content was almost similar in both the cultivars. The reduction in Chl a was 15.0%, 29.0% and 56.0% in Varuna and 24.0%, 33.0% and 49.0% in RH-30 with 25, 50 and 100 mg Cd kg1 soil, respectively, in comparison to the control. The decline in Chl b was more than Chl a. It decreased to 32.0%, 48.0% and 70.0% in Varuna, and 46.0%, 62.0% and 74.0% in RH-30 with 25, 50 and 100 mg Cd kg1 soil, respectively, in comparison to the control. Cd treatment affected Chl b more than Chl a, and therefore Chl a:b ratio was higher at higher Cd concentration in the soil. Chl a:b ratio in Varuna was 2.1, 2.7, 2.9 and 4.6, while in RH-30, the ratio was 2.9, 2.0, 3.6 and 4.1 with 0, 25, 50 and 100 mg Cd kg1 soil (Table 1). The relative amount of anthocyanin increased significantly up to 50 mg Cd kg1 soil in both the cultivars. The absolute value was equal when there was no Cd but the relative amount of anthocyanin increased by 38.0%, 136.0% and 53.0% in Varuna and 44.0%, 82.0% and 28.0% in RH-30 with 25, 50 and 100 mg Cd kg1 soil in comparison to the control. An increase in pheophytin was observed in both the cultivars with increasing Cd concentration. However, no significant increase was noted beyond 50 mg Cd kg1 soil in RH-30 (Table 1). The TBARS content increased by 19.7%, 217.0% and 230.0% in comparison to the control in Varuna with 25, 50 and 100 mg Cd kg1 soil. However, the increase in the TBARS in RH-30 was 53.0%, 282.0% and 307.0% in the respective Cd treatments in comparison to the control (Fig. 2A). The degree of membrane disruption as represented by electrolyte leakage increased by 187.0% and 372.0% in Varuna and 83.0% and 308.0% in RH-30 with 25 and 50 mg Cd kg1 soil in comparison to the control, respectively. However, the release of electrolytes was less at 100 mg Cd kg1 soil, which was 47.0% in Varuna and 96.0% in RH-30 (Fig. 2B). The production of H2O2 in the Varuna increased by 120.0%,

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TBARS content (nmolg-1 FW)

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100 mg Cd kg1 soil, respectively, in comparison to the control plants (Fig. 3B). The activity of APX in the control plants was higher in Varuna compared to RH-30. APX activity was enhanced by 215.0%, 369.0% and 328.0% in Varuna and 161.0%, 230.0% and 303.0% in RH-30 in comparison to the control with 25, 50, 100 mg Cd kg1 soil (Fig. 3C). GR activity was higher in Varuna than RH-30 even without Cd treatment. The increase in GR activity was 122.0%, 144.0% and 136.0% with 25, 50 and 100 mg Cd kg1 soil for Varuna, and 124.0%, 132.0% and 79.0% for RH-30, respectively, in comparison to the control (Fig. 3D).

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Figure 2. TBARS content (A), % electrolyte leakage (B) and H2O2 content (C) in leaves of Varuna (high photosynthetic capacity) and RH-30 (low photosynthetic capacity) cultivars of mustard (Brassica juncea L.) treated with cadmium concentrations. Results are means of five replications7SE. The data followed by different letters are significantly different at Pp0:05.

131.0% and 146.0% in 25, 50 and 100 mg Cd kg1 soil, while the increase in H2O2 in RH-30 was 134.0%, 248.0% and 250.0%, respectively, in comparison to the control (Fig. 2C). Cd treatment of plants enhanced the SOD activity in Varuna as well as in RH-30. The enzyme activity was increased by 118.0%, 145.0% and 142.0% in Varuna, whereas the increase was 169.0%, 190.0%, and 193.0% in RH-30 with 25, 50 and 100 mg Cd kg1 soil, respectively, in comparison to the control (Fig. 3A). CAT and APX mediate in scavenging high levels of H2O2. The level of induction in CAT activity was 141.0%, 142.0% and 134.0% in Varuna and 104.0%, 128.0% and 176.0% in RH-30 with 25, 50 and

Plant species and genotypes significantly differ in the uptake of Cd and its subsequent translocation from roots into shoots (Metwally et al., 2005; Salt et al., 1995). In our study, Varuna accumulated less Cd in both roots and leaves than RH-30 (Fig. 1A and B). The accumulation of Cd in roots and shoots depends on binding to extracellular matrix (Horst, 1995), complexing inside the cell (Cobbett et al., 1998) and on the transport efficiency (Marchiol et al., 1996). Further, the transport efficiency relies on transpiration rate and thereby on stomatal conductance. It was observed that exposure to Cd influenced the stomatal conductance and transpiration rate in both the cultivars, but to a significantly greater extent in RH-30. Likewise, net photosynthetic rate was affected by Cd stress. It has been shown that light and dark reactions of photosynthesis are suppressed by heavy metals at different target sites (Krupa and Baszynski, 1995). In RH-30, the decrease in the rate of photosynthesis in response to Cd treatment was accompanied by an increase in the stomatal conductance but in Varuna it remains unaltered (Table 1). The present result indicates that activities of CA and Rubisco declined in both the cultivars, but Varuna maintained higher levels of these enzymes than RH-30 at all Cd treatments. Hence, the mechanism of photosynthetic response involved both stomatal and nonstomatal effects under Cd stress. Siedlecka et al. (1997) reported that Cd affects photosynthesis by inhibiting different reaction steps of Calvin-cycle. However, Di Cagno et al. (2001) observed significant reduction in CO2-assimilation rate and Rubisco activity while stomatal conductance and Fv:Fm ratio remain unchanged when sunflower plants were grown in the presence of Cd. The decrease in total Chl, Chl a and Chl b was in a dose-dependent manner in both the cultivars. The

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Figure 3. SOD (A), CAT (B), APX (C) and GR (D) activities in leaves of Varuna (high photosynthetic capacity) and RH-30 (low photosynthetic capacity) cultivars of mustard (Brassica juncea L.) treated with cadmium concentrations. Results are means of five replications7SE. The data followed by different letters are significantly different at Pp0:05.

reduction in Chl b was extremely sharp in Varuna which resulted in higher Chl a:b ratio as the concentration of Cd increased in the soil (Table 1). The increases in the ratio of Chl a:b have been linked with the change in pigment composition of photosynthetic apparatus which possesses lower level of light harvesting chlorophyll proteins (LHCPs) (Loggini et al., 1999). The reduction in LHCPs content is an adaptive defense mechanism of chloroplasts, leaves and plants which allows them to endure the adverse conditions (Asada et al., 1998). Induction of higher level of relative amount of anthocyanin in response to Cd stress was observed in both the cultivars (Table 1), although it increased greatly in Varuna in comparison to RH-30. Several attempts have been made to explain the accumulation of anthocyanin in leaves under stress (Gould

et al., 1995, 2002; Krupa et al., 1996). The anthocyanin synthesis takes place in cytoplasm, rapidly transported into the cell vacuoles (Marrs et al., 1995) and is not in direct contact with the stress-generated activated oxygen species. In fact, cytosolic and organelle-bound antioxidants act as a primary cellular defense system rather than the vacuolar anthocyanins. Our findings indicate that accumulation of anthocyanins may be only of secondary importance in living cells. Nonetheless, induction of anthocyanin accumulation might help in the protection of photosynthetic apparatus by screening it from deleterious effects of stressgenerated superoxide radicals without limiting photosynthesis. Percent pheophytin remained relatively much higher in RH-30 than Varuna at higher Cd concentration in the soil, but at lower Cd level percent conversion of chlorophyll to pheophytin

ARTICLE IN PRESS 608 was almost identical in both the cultivars (Table 1). Ku ¨pper et al. (1996) carried out experiments with submerged water plants and observed that the substitution of Mg2+ ion in the chlorophyll molecule by toxic heavy metals, such as Cu, Zn, Cd or Hg, resulted in an abrupt cessation of photosynthesis. Although Cd is not a transition metal like Fe and Cu, and therefore, it is not capable of generating AOS by catalyzing Haber–Weiss or Fenton type reactions (Deckert, 2005). Nevertheless, Cd toxicity results from the alteration of oxidant levels in plants (Foyer and Noctor, 2005). Accumulation of Cd was correlated with the generation of AOS in sensitive clones of Holcus lanatus (Hendry et al., 1992). Cd has been shown to elevate lipid peroxidation via AOS formation in plants (Halliwell and Gutteridge, 1989). Cd-dependent induction of TBARS was observed in the leaves of both the cultivars (Fig. 2A), which is an index of lipid peroxidation and thereby oxidative stress. It has been observed that exposure to Cd increased lipid peroxidation in Pisum sativum (Chaoui et al., 1997; Metwally et al., 2005), rice (Chien et al., 2001) and sunflower seedlings (Gallego et al., 1996). The production of lipid peroxides was lower in Varuna compared to RH-30 at all Cd concentrations. This could be explained in terms of higher degree of protection against oxidative damage in Varuna by fast removal of H2O2 or by other scavenging systems. The enhancement in the activities of antioxidative enzymes was negatively correlated with the level of TBARS content. As a sequel to Cd-catalyzed AOS generation, disruption of membrane stability, enhanced permeability and inactivation of proteins take place (De Vos et al., 1993). The increase in TBARS content in the present study induced electrolyte leakage in a dose-dependent manner (Fig. 2B). The enhanced cellular damage in RH-30 seems to reflect deterioration on the equilibrium between generation of AOS and defense mechanisms towards removal of AOS. The electrolyte leakage through plasmalemma has been associated with depressed photosynthetic and mitochondrial activity (Ristic et al., 1996). H2O2 generation is induced in plants following exposure to a wide variety of abiotic and biotic stimuli (Karpinski et al., 1999; Lamb and Dixon, 1997). The level of H2O2 was lower in Varuna than RH-30 even in the presence of Cd, which implies that the generation of H2O2 was quenched by the efficient antioxidative mechanism of Varuna. In RH30, the level of H2O2 increased in a dose-dependent manner (Fig. 2C), which is indicative of higher oxidative stress imposed by Cd in soil. It is most likely that the presence of Cd inhibited the H2O2-

M. Mobin, N.A. Khan scavenging system, i.e. GR, CAT and APX and increased activity of SOD (Fig. 3A–D), which catalyzes the conversion of superoxide anion to O2 and H2O2 (Sudhakar et al., 2001). There was more pronounced increase in the SOD activity in RH-30 with the increase in Cd levels, which possibly generated higher level of superoxide radicals and resulted in higher cellular damage in comparison to Varuna. Gossett et al. (1994) reported that higher SOD activity without complementary increase in the ability to scavenge the formed H2O2 can result in the increased cellular damage. The detoxification of H2O2 takes place by involvement of ascorbate–glutathione cycle, where H2O2 sends a systemic signal for the induction of APX (Karpinski et al., 1999). In the present study, the comparison of the absolute enzymatic values in the Cd stressed RH-30 plants indicated that APX level kept on increasing with the increasing levels of Cd while in Varuna the sub-lethal concentration of Cd in the soil induced the maximum level of APX. The increase in CAT activity is considered as an indirect evidence of an enhanced oxidative damage (Smirnoff, 1995). We found dose-dependent increase of CAT activity, which further indicated the oxidative damage from Cd treatments in both the cultivars. Varuna was found to possess higher level of CAT activity both constitutively and inductively. However, the CAT activity was found to be induced nearly 1.5 times more in RH-30 than in Varuna at 100 mg Cd kg1 soil, which further pointed towards increased oxidative stress experienced by RH-30. GR catalyzes the last rate-limiting step of ascorbate–glutathione cycle. This enzyme maintained high ratio of GSH/GSSG which is required for the regeneration of ascorbate and for the activation of several CO2-fixing enzymes (Noctor and Foyer, 1998). Contrary to APX gene expression, which is activated by H2O2, GR is stimulated by different stimuli (Schu ¨tzendu ¨bel et al., 2001). This is well correlated in the present study, where the constitutive and Cd-induced GR activity was remarkably higher in Varuna. The reduction in GR activity of RH-30 was prominent at 100 mg Cd kg1 soil. The cooperative action of APX and GR in Varuna suggested the presence of more efficient ascorbate–glutathione cycle, which resulted in the development of higher tolerance to Cd. It is concluded that deleterious effect of Cdgenerated AOS on photosynthetic characteristics was more conspicuous in RH-30 than Varuna. The greater tolerance in Varuna to Cd was due to better synergies between the antioxidative enzymes which help to protect the photosynthetic apparatus.

ARTICLE IN PRESS Photosynthesis and oxidative stress in cadmium-treated mustard

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