Abstract
A greenhouse pot experiment was conducted to study the protective effect of potassium (K) nutrition against cadmium (Cd) toxicity in mustard (Brassica campestris L.). Cadmium treatment drastically reduced plant growth (plant dry mass, leaf area), photosynthetic traits (net photosynthetic rate, stomatal conductance and internal CO₂ concentration) and the contents of ascorbic acid (AsA), glutathione (GSH) and potassium (K) but significantly increased the contents of thiobarbituric acid reactive substances (TBARS), hydrogen peroxide (H₂O₂) and Cd ions in the leaves. K application was effective in decreasing the Cd-toxicityinduced oxidative burst as evident from the lowering of H₂O₂ and TBARS, increase of AsA and GSH contents as well as enhanced plant growth. These effects of K were associated with a sharp decline in Cd content of leaves. The results are indicative of the ameliorative role of K in mustard against the oxidative stress caused by Cd toxicity.

Key words: Brassica campestris; Cadmium toxicity; Oxidative damage; Potassium nutrition.

Abbreviations: APX, ascorbate peroxidase; AsA, ascorbic acid; CAT, catalase; Cd, cadmium; DAS, days after sowing; GR, glutathione reductase; gs, stomatal conductance; GSH, glutathione reduced; Ci, intercellular CO₂ concentration; PN, net photosynthetic rate; ROS, reactive oxygen species; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substance.

Introduction
Cadmium (Cd), a toxic element, is dispersed in the natural and agricultural environments mainly through human activities and has a long biological half-life (Wagner 1993). It is one of the nonessential heavy metals, toxic to flora and fauna, which is easily taken up by plant roots and translocated to the aerial plant parts (Zhao et al. 2003; Yang et al. 1998). Cadmium accumulation reduces photosynthesis, disturbs plant-water relations and the uptake and translocation of nutrients, and results in visible injury symptoms and/or plant death (Drazkiewicz et al. 2003; Hsu and Kao 2007; Anjum et al. 2008a). Cadmium is known to cause a burst of reactive oxygen species (ROS) in plant tissues, leading to the development of secondary oxidative stress (Qadir et al. 2004; Anjum et al. 2008b, c) that may damage photosynthetic pigments and other bio-molecules such as lipids, proteins and nucleic acids. It causes leakage of electrolytes via lipid peroxidation, a decrease in the AsA and GSH contents and alteration in activities of antioxidant enzymes such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) (Kuo and Kao 2004; Chaoui et al. 1997; Dixit et al. 2001; Chien et al. 2002; Mobin and Khan 2007; Anjum et al. 2008a, b).

Plant nutrients play pivotal roles in protecting plant growth from various environmental stresses including the heavy metal stress (Cakmak and Romheld 1997; Cakmak 2005; Anjana et al., 2006; Anjum et al. 2008c, d). The mineral-nutrient status of plants has a regulatory role with reference to plant resistance to stress factors (Marschner 1995). Potassium (K) is an important macronutrient and the most abundant cation in plant tissues (Zhao et al. 2003; Jordan-Meille and Pellerin 2007). Increasing evidence suggests that raising K-nutrition status of plants can dramatically inhibit the generation of ROS by reducing the activity of NAD(P)H oxidases and maintaining photosynthetic electron transport (Cakmak 2005). In addition, enhanced K nutritional status induces a number of beneficial physiological effects. These include stimulation of root growth, increases of leaf area, chlorophyll content and the net assimilation rate (NAR). Plant water content is also closely regulated by the effects of K on closure and opening of stomata which maintains photosynthetic CO₂ fixation. Additionally K reduces undesirable excess uptake of ions such as Na and Fe as well as benefiting N metabolism as for example by stabilizing nitrate reductase (NR) (Khan 1991; Marschner 1995; Elstner and Osswald 1994; Mengel and Kirkby 2001; Umar 2006). K nutrition has been shown to decrease the uptake of Cd as observed in wheat (Triticum aestivum L.) (Zhao et al. 2004). The present study investigates whether K nutrition may protect plants from Cd-toxicity-induced oxidative damage by reducing the Cd availability to the plant thereby depressing the contents of H₂O₂ and TBARS in the mustard leaves.

Materials and methods
Plant material and treatments
Seeds of mustard (Brassica campestris L.) cv. Pusa Gold were sown in 40-cm-diameter earthen pots, each filled with 10 kg of soil. The soil was sandy loam in texture with pH 7.8, E.C. 3.8, organic carbon 0.43%, available K and S, 70 and 5 mg/kg soil, respectively. Sufficient amounts of N, P and S at the rate of 120, 30 and 40 mg/kg soil, respectively, were applied at the time of sowing in the form of urea, single super phosphate and gypsum. The soil was mixed with appropriate amounts of CdCl2 to supply a 0, 25, 50 and 100 mg Cd/kg soil, and of KCl to supply a 0 and 60 mg K/kg soil. The treatments were arranged in a randomized block design and each treatment was replicated three times. After the seedling emergence, three plants per pot were maintained and suitably irrigated. The pots were kept in naturally illuminated green house (photosynthetically active radiation, PAR, >960 μmol/m2/s, day/night temperature 25/20 ± 4°C and relative humidity 70 ± 5%) in the Department of Botany, Hamdard University, New Delhi, India (28.38 ′N, 77.11′E and 228 m altitude during a winter season). The measurements were obtained at 30 days after sowing (DAS).

Growth parameters and net photosynthetic traits
Plant dry mass was recorded after drying the plants for 48 h to a constant weight at 80ºC. Leaf area was measured by a leaf area meter (LI-3000A, Nebraska). Net photosynthetic rate (PN) was measured with the fully expanded third leaf from the top, using Infra Red Gas Analyzer (LI-6400, Nebraska), at 9.00-11.00 am at saturating light intensity. Total chlorophyll was extracted using the method of Hiscox and Israelstam (1979) by using dimethyl sulphoxide (DMSO) as an extraction medium, and estimated by the method of Arnon (1949). For Chl extraction fresh leaves (200 mg) were cut into small pieces and collected in test tubes containing 7.0 ml of DMSO. The test tubes were covered with black paper and incubated at 45ºC for 40 min for the extraction. The reaction mixture was transferred to a graduated tube and the final volume was made to 10.0 ml with DMSO. Total Chl content in the leaf samples was calculated according to the following equation given by Arnon (1949).

Total Chlorophyll (mg/g leaf fresh mass) = [20.2 (OD₆₄₅) + 8.02 (OD₆₆₃)] ×, where, V is the volume of the extract, W is the weight of leaf sample taken and OD is optical density taken at 645 and 663 nm wave length.

Estimation of glutathione, ascorbate and K contents
Glutathione was estimated following the method of Anderson (1985). Fresh leaf (0.5 g) was homogenized in 2.0 ml of 5% (w:v) sulphosalicylic acid under cold conditions. The homogenate was centrifuged at 10,000 g for 10 min. To a 0.5 ml of the supernatant, 0.6 ml of 100 mM (pH 7.0) phosphate buffer and 40 μl of 5,5´-dithio-bis-(2-nitro-benzoic acid) (DTNB) were added. Absorbance was read after two minutes at 412 nm on a UV-vis Spectrophotometer (Lambda Bio 20, Perkin Elmer, USA). The ascorbate content was determined by the method of Law et al. (1983). Fresh leaf (0.5 g) was ground in 2.0 ml of 100 mM (pH 7.0) phosphate buffer with 1 mM ethylenediamine tetraacetate (EDTA) and centrifuged at 10,000g for 10 min. The supernatant was collected and added with a 200 μl of 10% (w:v) trichloroacetic acid (TCA). After mixing the solution by vortex, it was allowed to stand for 5 min. A 10 μl of 5M NaOH was then added to the solution and centrifuged for 2 min. To a 200 μl of the supernatant, 200 μl of 150 mM (pH 7.4) phosphate buffer and 100 μl of 10mM 5,5´-dithio-bis-(2-nitro-benzoic acid) (DTNB) were added, mixed thoroughly and left at room temperature for 15 min. 100 μl of 0.5% (w:v) N'N-ethylemaleimide (NEM) was added to it. Samples were mixed by vortex and incubated at 24°C for 30 seconds. To each sample, 400 μl of 10% (w:v) TCA, 400 μl H3PO4, 400 μl of 4% (w:v) bipyridyl dye (N’N-dimethyl bipyridyl) and 200 μl of 3% (w:v) FeCl₃ were added. After vortex mixing, samples were incubated at 37°C for 60 min and absorbance was recorded at 525 nm.

Potassium was determined on the dried ground leaves. Following hot air treatment to remove any residual moisture (65ºC for 20 min) the ground plant material was subject to acid digestion (H₂SO₄, HNO₃; 2:1 v/v) and K then measured using flame photometry after appropriate dilution.

Estimation of thiobarbituric acid reactive substances and H₂O₂
The level of lipid peroxidation products in leaves was determined by estimating thiobarbituric acid reactive substances (TBARS) as described by Dhindsa et al. (1981). Fresh tissue (0.5 g) was ground in 0.25% 2-thiobarbituric acid (TBA) in 10% trichloroacetic acid (TCA) using 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 non-specific turbidity by subtracting the absorbance of the same at 600 nm. The blank was 0.25% TBA in 10% TCA. The TBARS content was calculated using the extinction coefficient (155 mM-1 cm-1).

The H₂O₂ level was measured by the modified colorimetrical method of Okuda et al. (Okuda et al. 1991). Leaf tissue (200 mg, three replications) was homogenized at 4°C for 5 min in 2 ml of 0.2 N HClO₄. The extract was centrifuged at 10,000 g for 7 min. The supernatant was adjusted to pH 7.5 with 2 N NaOH and an aliquot of 100 μl passed through a 0.4 ml column of Dowex AG-1-X2. The column was washed twice with 750 μl 0.375 M phosphate buffer (pH 6.5) and the eluate was used for the assay of H₂O₂. The reaction mixture contained 1.6 ml of the eluate, 600 μl of 12.5 mM 3-(dimethylamino) benzoic acid in phosphate buffer, 20 μl of 3-methyl-2-benzothiazoline hydrazone and 10 μl of peroxidase (13 units). After 15 min, the increase in absorbance at 590 nm was measured using UV-vis spectrophotometer.

Estimation of cadmium content
Cadmium concentration was estimated after digesting the sample in sulphuric-nitric acid (2:1, v/v) in digestion tubes. Digestion tubes were heated in the digestion chamber. Dense fumes were given off at this stage and the content turned black. The tubes were cooled for 15 min. After cooling, 0.5 ml of chemically pure 30% hydrogen peroxide was added with dropping and the solution was heated again till its colour changed from black to light yellow. After heating for about 30-45 min, tubes were cooled for 10 min. Three to four drops of hydrogen peroxide were added to digestion tubes followed by heating for more 30 min to get the extracts clear and almost colourless. The digested sample was diluted by adding Milli Q water. Cd concentration in digested aliquots was determined by atomic absorption spectrophotometry (AAS ZEEnit 65, Analytikjena, Germany).

Statistical analysis
Data were analyzed statistically and given as mean ± SE. Analysis of variance (ANOVA) was performed by Statistical Package for the Social Sciences (SPSS), Version 10.0, Inc, USA. The least significant difference (LSD) was calculated to identify significant differences between treatments. The treatments mean was separated using the Duncan’s multiple range test at P≤ 0.05.

Results
Plant growth parameters
All applications of Cd reduced plant dry mass and leaf area significantly (P≤ 0.05) (Table 1). Application of 100 mg Cd/kg soil caused maximum reduction, compared with the control. Plant dry mass was reduced by 15.90%, 30.78% and 46.67%, and leaf area by 16.84%, 42.16% and 42.44% due to application of 25, 50 and 100 mg Cd/kg soil, respectively, in comparison with control. K application (60 mg/kg soil) alleviated Cd toxicity and lowered the reductions caused by Cd. K alleviation effect was more pronounced with the lowest level of Cd (25 mg/kg soil) followed by 50 and 100 mg Cd/kg soil. With the application of 60 mg K/kg soil, plant dry mass increased by 38.41%, 31.11% and 16.35%, compared to that at 25, 50 and 100 mg Cd/kg soil, respectively. K application increased the leaf area maximally (28.23%) at 25 mg and minimally (3.76%) at 100 mg/Cd kg soil.

Photosynthetic traits
Relative to the control, the chlorophyll content, net photosynthetic rate, stomatal conductance and concentration of internal CO₂ decreased significantly at P≤ 0.05 with increase in Cd level in the soil (Fig. 1a-d). K supplementation to Cd-exposed plants improved the content of chlorophyll by 37.04%, 24.47% and 6.78% at 25, 50 and 100 mg Cd/kg soil, respectively (Fig. 1a). The net photosynthetic rate increased with K application by 21.20%, 6.19% and 5.04%, with 25, 50 and 100 mg Cd/kg soil, respectively (Fig. 1b), whereas stomatal conductance decreased by 17.50%, 47.50% and 72.50% with 25, 50 and 100 mg Cd/kg soil (without K), respectively. Data in Fig. 1c reveal that the stomatal conductance increased due to supply of K (60 mg/kg soil) to Cdexposed mustard plants, the extent of increase varying with treatments. Stomatal conductance increased by 48.48%, 23.81% and 27.27% when 25, 50 and 100 mg Cd/kg soil was added (Fig. 1c). Concentration of internal CO₂ decreased maximally with 100 mg Cd/kg soil (43.79%) followed by 50 mg Cd/kg soil (18.31%) and 25 mg Cd/kg soil (6.37%). K supplementation to Cdexposed plants improved the CO₂ level by 20.59%, 16.39% and 7.73% at 25, 50 and 100 mg Cd/kg soil, respectively (Fig. 1d).

TBARS and H₂O₂ contents
To evaluate the Cd-induced oxidative damage to membranes, contents of TBARS and H₂O₂ were determined. The presence of Cd in the soil caused a significant (P≤ 0.05) increase in TBARS content in mustard leaves (Fig. 2a-c). With application of Cd alone (without K), it was 613% (highest) at 100 mg, followed by 244% at 50 mg and 74.84% (lowest) at 25 mg Cd/kg soil. The application of K to Cd-exposed plants decreased the content of TBARS maximally (49.26%) at 25 mg, followed by 18.91% at 50 mg and 8.45% at 100 mg Cd/kg soil (Fig. 2a). The H₂O₂ content in mustard leaves with supply of Cd alone was also maximum (269%) at 100 mg, followed 110% at 50 mg and 38.04% at 25 mg Cd/kg soil. Application of K to Cd-exposed plants decreased the H₂O₂ content maximally (43.17%) at 25 mg, followed by 22.53% at 50 mg and 9.76% at 100 mg Cd/kg soil (Fig. 2b).

Cadmium content
Addition of Cd to the soil caused an increase in Cd content of mustard leaves, as expected (Fig. 2c). Plants grown in the soil without Cd also contained some Cd, although the contents were very low, resulting probably from soil contamination caused by agricultural chemicals. Significant differences in leaf Cd content (P≤ 0.05) were found in plants grown with 100, 50 and 25 mg Cd/kg soil (without K). With application of K (60 mg/kg soil) to the soil, Cd content of leaf decreased significantly, indicating an antagonistic effect of K nutrition on Cd uptake by plants. The leaf Cd content decreased by 25.53%, 12.21% and 5.25% at 25, 50 and 100 mg Cd/kg soil, respectively (Fig. 2c).

Contents of ascorbate, glutathione and potassium
The content of ascorbate decreased by 14.74%, 37.89% and 63.63% at 25, 50 and 100 mg Cd/kg soil (without K), respectively. As shown in Table 2, the ascorbate content increased with addition of K (60 mg/kg soil) to the Cd-exposed plants, the increase being treatment dependent. The content of leaf ascorbate increased with K application by 33.33%, 24.58% and 8.45% on application of 25, 50 and 100 mg Cd/kg soil, respectively (Table 2). The influence of K and Cd treatments on glutathione content of leaves is shown in Fig. 1c. It decreased significantly (P≤ 0.05) at 100 mg Cd (68.29%), 50 mg Cd (41.16%) and 25 mg Cd/kg soil (21.04%). Application of K (60 mg/kg soil) reduced the decline in glutathione content substantially; the effect being more pronounced with the lowest Cd treatment (25 mg Cd/kg soil). Thus, maximum content of glutathione occurred with 25 mg Cd (25.48%), followed by the value of 15.54% with 50 mg Cd and 8.65% with 100 mg Cd/kg soil when supplemented with 60 mg K/kg soil (Table 2). The content of potassium decreased by 5.80%, 14.36% and 21.82% at 25, 50 and 100 mg Cd/kg soil (without K), respectively. As shown in Table 2, potassium content increased with addition of K (60 mg/kg soil) to the Cd-exposed plants, the increase being treatment dependent. The content of leaf K increased with K application by 5.28%, 3.87% and 2.12% on application of 25, 50 and 100 mg Cd/kg soil, respectively (Table 2).

Discussion
Cadmium treatment causes oxidative stress in plants through increase in the production of H₂O₂ (Kuo and Kao 2004; Schutzendubel et al. 2001; Olmos et al. 2003) and induction of lipid peroxidation (Chien et al. 2002; Gallego et al. 1996a, b; Kuo and Kao 2004; Anjum et al. 2008a; Singh et al. 2008). Our results have shown not only that Cd increased the content of H₂O₂ and TBARS (Fig. 2a-b.) but also that it lowered the GSH and AsA contents (Table 2). Pigment loss (Fig. 1) and lipid peroxidation (Fig. 2a-b) were also prominent in Cd-treated mustard leaves. All these observations suggest that the Cd-induced toxicity in mustard leaves is mediated through oxidative stress. Glutathione (GSH) functions as a direct antioxidant of ROS and is involved in the generation of AsA, which is utilized as a substrate for APX (Noctor and Foyer 1998). Our results indicate that the decrease in GSH content is one of the earliest steps in the Cd-induced oxidative stress in mustard leaves; it was maximum at the highest Cd-level (Table 2). It may be supposed that the decrease in GSH may favour the accumulation of ROS in the form of H₂O₂ and TBARS in Cd-treated mustard leaves. Previous studies by Qadir et al. (2004) and Anjum et al. (2008a) and a review by Schutzendubel and Polle (2002) suggest that the depletion of GSH is apparently a critical step in Cd toxicity. Cd induced a significant accumulation of H₂O₂ in mustard leaves (Fig. 2). H₂O₂ accumulation has also been observed in Cd-treated pine and pea roots, pea leaves, and tobacco cells (Olmos et al. 2003; Romero-Puertas et al. 2003; 2004; Schutzendubel et al. 2001; Hsu and Kao 2007). There are reports showing that NADPH oxidase is possibly involved in Cd-induced H₂O₂ production in pea leaves and tobacco cells (Olmos et al. 2003; Romero-Puertas et al. 2004).

That the Cd-induced oxidative damage in mustard leaves is reduced by K nutrition can be inferred from observations that K supplementation prevented Cd-induced depressions in PN, gs, Ci and in the contents of chlorophyll (Fig. 1a-d), AsA and GSH (Table 2) and increases in the contents of H₂O₂ and TBARS (Fig. 2a-b). An adequate K supply plays a central role in the maintenance of PN and the related processes (Bendnarz and Oosterhuis 1999; Umar 2006). As evidenced in the present study, Cd-induced decrease in leaf K content may also contribute Cd-induced changes in mustard plants (Table 2) which accord with the findings of Anjum et al. (2008b). The decrease in PN and gs appears to be related to K-deficiency, which is in agreement with the findings of Cakmak and Engels (1999) and Zhao et al. (2001). ROS are highly toxic causing degradation of Chl. It is generally accepted that K supply strongly controls the process of photosynthetic CO₂ fixation as well as utilization of photoassimilates (Cakmak 1994). The role of GSH and AsA in scavenging processes against heavy metals and other stress conditions has been extensively investigated (Gallego et al. 1996a, b; Noctor and Foyer 1998). AsA contributes directly to ROS scavenging and by means of ascorbate peroxidase (APX). The decrease in AsA and GSH contents in mustard leaves treated with Cd suggests that AsA and GSH contents may be regulated by the synthesis and oxidation. GSH is the precursor of phytochelatins, cysteinerich peptides synthesized via phytochelatin synthase (Cobbett and Goldsbrough 2002). GSH is severely depleted in response to Cd due to its increased consumption in phytochelatin production (Schutzendubel and Polle 2002).

The present results indicate that K nutrition decreased the Cd-induced decline in the AsA and GSH contents (Table 2). The capacity of K to scavenge H₂O₂ in mustard leaves was at a maximum with 60 mg K/kg soil plus Cd (Fig. 2b). In considering a possible mechanism for the depression of Cd-induced oxidative damage by K nutrition, it may be supposed that K might inhibit Cd uptake from the medium i.e. an antagonistic effect between Cd and K uptake (Zhao et al. 2004). This is supported by our findings which have shown that the Cd content in mustard leaves treated with K60 + Cd levels was lower than in those treated with Cd alone (Fig. 2c). Our findings suggest that increase in the K nutrient status of plants may prevent, though slightly (27%), the uptake of Cd. K60 + Cd levels treatment inhibited, almost completely, the Cd-induced generation of H₂O₂ and lipid peroxidation (TBARS) in mustard leaves (Fig. 2a-b). It may thus be concluded that K nutrition by depressing Cd uptake is able to protect plants from Cd-induced oxidative burst thereby avoiding H₂O₂ and TBARS generation.

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