Journal of Plant Physiology & Pathology ISSN: 2329-955X

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Research Article, J Plant Physiol Pathol Vol: 2 Issue: 2

Effects of Chromium (VI) on Groundnut (Arachis Hypogeae L.) Metabolism

Rajeev Gopal* and Yogesh K Sharma
Department of Botany, Lucknow University, Lucknow-226007, India
Corresponding author : Rajeev Gopal
Department of Botany, Lucknow University, Lucknow-226007, India
E-mail: [email protected]
Received: March 13, 2014 Accepted: April 07, 2014 Published: April 11, 2014
Citation: Gopal R, Sharma YK (2014) Effects of Chromium (VI) on Groundnut (Arachis Hypogeae L.) Metabolism. J Plant Physiol Pathol 2:2. doi:10.4172/2329-955X.1000120

Abstract

Effects of Chromium (VI) on Groundnut (Arachis Hypogeae L.) Metabolism

Chromium is fairly abundant in earth’s crust and enters in biotic components of ecosystem in various ways. Cr (VI) is highly reactive and influences both plants and animals. A glasshouse experiment was conducted with 0.05, 0.1, 0.2, 0.4, and 0.5 mM of Cr to assess the Cr accumulation pattern and its influence on growth, activities of antioxidant enzymes, relative water content, electrolyte leakage and lipid peroxidation in groundnut (Arachis hypogeae L.) cv. Kaushal plants. In groundnut Cr at 0.5 mM supply caused visible lesions as chlorosis and wilting of mature leaves.

Keywords: Chromium; Oxidative stress; Groundnut; Phytotoxic lesions

Keywords

Chromium; Oxidative stress; Groundnut; Phytotoxic lesions

Introduction

Chromium toxicity is considered as an important growth limiting factor for plants in agricultural soils irrigated mainly through the water discharged from several industries and municipal sewage [1,2]. Though chromium at lower concentration has been proved to be stimulatory for some crops, nevertheless, higher concentrations have always been inhibitory for plant growth and metabolism [3,4]. Chromium exists in nature in a number of oxidation states but the most stable and common forms are Cr (III) and Cr (VI). Chromium (VI), as an anion, can be readily extracted from soil and sediments and is the most toxic form [5]. Once absorbed, Cr absorbed by plants remained primarily in the roots and poorly translocated to leaves [6]. However, Cr distribution in plants had a stable character that did not depend on soil properties and concentration of the element resulting its minimum content in reproductive organs [7]. Concentrations of Cr in plant tissues, definitely associated with toxicity symptoms, are usually in the several hundred ppm range. Accumulation of chromium by plants reduces growth, induces chlorosis in young leaves, alters enzymatic functions, damages root cells and causes ultra structural modifications in chloroplasts and cell membranes [5,8,9]. Excess chromium affects roots of plants causing wilting and plasmolysis in root cells [10]. The uptake of several nutrient elements including Fe by plants was affected by high Cr levels [11,12]. Chromium stress causes lipid peroxidation and degrades biological membranes making them susceptible to oxidative damage [13]. In this paper an attempt has been made to find out the tolerance limit of groundnut since it is an important oil crop, to chromium and the changes in growth parameters and appearance of visible toxicity symptoms. Information has also been generated on the distribution of chromium and iron in different plant parts and antioxidative stress responsive enzymes by the plants in response to variable chromium (VI) supply in refined sand.

Materials and Methods

Plant material and growth conditions
Groundnut (Arachis hypogeae L.) cv. Kaushal plants were grown in refined sand contained in polyethylene trays (30 cm diameter) having a central drainage hole in a glasshouse at ambient temperature (15-32°C). Plants were given a daily supply of nutrient solution [14] containing: 4 mM KNO3; 4 mM Ca(NO3)2; 2 mM MgSO4; 1.5 mM NaH2PO4; 0.1 mM Fe-EDTA; 0.1 mM NaCl; 30 μM H3BO3 ; 10 μM MnSO4; 1 μM CuSO4; 1 μM ZnSO4; 0.2 μM Na2MoO4; 0.1 μM CoSO4 and 0.1 μM NiSO4. The stock solutions of all nutrients were prepared with Analytical Reagent grade salts. Before supply all ingredients were mixed in required proportion and the pH of the nutrient solution was maintained at 6.7 ± 0.2. Initially for 40 days groundnut plants were supplied complete nutrient solution. On 41st day each tray contained four plants and the pots were divided into six lots. One lot was allowed to grow as such and received full nutrient solution without Cr and served as control. To the remaining five lots Cr was supplied as potassium dichromate at 0.05, 0.1, 0.2, 0.4 and 0.5mM along with the basal nutrient solution. On Sundays, the trays were flushed with deionised water to remove the accumulated salts and root exudates. Plants were analysed for various parameters at different time intervals after Cr supply as indicated in the tables and figures. The glasshouse conditions during the experiment were: maximum photosynthetic photon flux density (12:00 noon) 1155-1315 μmol m-2 s-1, daily maximum and minimum temperatures 33.3-36.4°C and 24.4-28.6°C, respectively, and RH (9:00 am) 70-80%. The average photoperiod was 12:00 ± 0:20 h.
Visual observation, dry matter yield and Cr concentration
The visual symptoms were recorded daily. Plants were sampled for dry matter at d 55 (15 days after metal supply).The concentration of chromium and iron was estimated in oven dried plant samples after di-acid digestion (HNO3: HClO4, 10:1) [15] by Atomic Absorption Spectrophotometer.
Chloroplastic pigments, Relative water content and lipid peroxidation
Chlorophyll and carotenoids were extracted from leaves in 80% (v/v) acetone and estimated spectrophotometrically [16]. Relative water content (RWC) was determined in freshly harvested comparable middle leaves [17]. All measurements for RWC were made between 9 and 10 a.m when the sand of the trays was still saturated with nutrient solution. The temperature was 35-40oC and humidity was 60-70%. Lipid peroxidation was determined in terms of malondialdehyde (MDA) content by thiobarbituric acid (TBA) reaction [18]. The amount of TBA reactive substance (TBARS) was calculated from the difference in absorbance at 532 and 600 nm using an extinction coefficient of 155 mm-1 cm-1.
Enzyme extraction and protein determination
Fourth fully expanded fresh leaf tissue (2.5 g) was homogenized in 10.0 ml chilled 50 mm potassium phosphate buffer (pH 7.0) containing 0.5% (w/v) insoluble polyvinylpolypyrrolidone and 1.0 mm phenylmethylsulfonylfluoride in a chilled pestle and mortar kept in ice bath. The homogenate was filtered through twofold muslin cloth and centrifuged at 20000 g for 10 min at 2°C. The supernatant was stored at 2°C and used for enzyme assays within 4 h. For the assay of APX 5.0 mm AsA was also included in the extraction medium. The protein concentration in the homogenate was determined in the TCA precipitate [19].
Assays of antioxidative enzymes
Superoxide dismutase (SOD) (EC 1.15.1.1) activity was assayed by measuring its ability to inhibit the photochemical reduction of NBT at 560 nm as described previously (Giannopolitis and Reis) [20]. The activity is expressed as units’ min-1 mg-1 protein. Ascorbate peroxidase (APX) (EC 1.11.1.11) was measured in 3 ml reaction mixture containing 50 mm phosphate buffer pH 7.0, 0.5 mm AsA, 0.1 mm H2O2, 0.1 mm EDTA and suitable quantity of enzyme extract as described by Nakano and Asada [21]. The activity of APX was calculated in terms of μmol ascorbate oxidized min-1 mg-1 protein. Catalase (CAT) (EC 1.11.1.6) activity was estimated in a reaction mixture containing 500 μmol H2O2 in 10 ml 100 mm phosphate buffer (pH 7.0) and 1.0 ml tissue extract as described earlier by Euler and Josephson [22]. CAT activity is expressed as μmol H2O2 decomposed min-1 mg-1 protein. Peroxidase (POD) (EC 1.11.1.7) activity was estimated after Luck [23] in a reaction mixture containing 5.0 ml 100 mm phosphate buffer pH 6.5, 1.0 ml 0.5% p-phenylenediamine, 1.0 ml 0.01% H2O2 and 1.0 ml tissue extract. The enzyme activity is expressed as units min-1 mg-1 protein. The enzyme unit is defined as ΔA485 of 0.01 between the blank and the sample per minute of reaction time.
Statistical analysis
All determinations were made in duplicate and the data have been tested statistically for significance at 5% level of probability (Fisher LSD method) using SigmaStat (ver. 2.03) software.

Results

Plant growth and visible symptoms
The growth and metabolism of groundnut was adversely affected when plants were exposed to Cr (VI) at different levels (0.05 to 0.5 mM) in refined sand. After 5 days of metal exposure (d 45) depression in growth was apparent at 0.4 and 0.5 mM Cr and plants exhibited characteristic visible symptoms of Cr toxicity at levels >0.2 mM. The symptoms of Cr excess (0.5 mM) appeared on older leaves as chlorosis, which gradually spread to the upper middle leaves. In another few days these affected old and middle leaves turned golden yellow, became wilted and due to loss of turgor hung down from petioles. With continued supply of Cr the lamina of affected old leaves became permanently wilted, necrotic, dry and shed.
The biomass of groundnut decreased with increase in Cr supply from 0.05 to 0.5 mM. The decrease in dry matter of roots was more than that of the above ground parts (Figure 1).
Figure 1: Dry matter (A), concentration of total chlorophyll (B) and carotenoids (C), relative water content (D), lipid peroxidation (E) and electrolyte leakage (F) in groundnut leaves at variable chromium at d 55 (15 days after metal exposure). Vertical lines represent ±SE.
Tissue Cr and Fe
Increasing Cr supply increased tissue concentration of chromium in different plant parts (Table 1). The roots had a maximum tissue concentration of Cr (950 μg g-1). However, the concentration of Fe was decreased in above ground plant parts (leaves and stem) and increased in roots with increasing supply of Cr from 0.05 to 0.5 mM (Table 2).
Table 1: Specific activities of some anti-oxidant enzymes in groundnut leaves at variable chromium at d 55 (15 days after metal exposure).
Table 2: Concentration of Cr and Fe in different parts of groundnut plants at variable chromium at d 55 (15 days after metal exposure).
Photosynthetic pigments
The concentrations of chlorophyll a and chlorophyll b and carotenoids decreased with increases in Cr supply from 0.05 to 0.5 mM. The reduction in chlorophyll a and chlorophyll b concentrations was found to be 34 and 53% respectively at 0.5 mM Cr (Figure 1).
Relative water content, electrolyte leakage and lipid peroxidation
Relative water content decreased progressively in the leaf tissue with increase in Cr supply. A gradual increase in the concentration of lipid peroxidation and electrolyte leakage was observed with increasing supply of Cr with a maximum of 15% and 347% at 0.5 mM Cr (Figure 1).
Activities of oxidative stress-responsive enzymes
The activity of CAT was decreased and that of SOD increased significantly in leaves of Cr treated groundnut plants (Table 1). The decrease in the activity of CAT in leaves in response to excess Cr was 50% at 0.5 mM.
Significant and progressive stimulation in the activity of POD was found with increase in Cr supply from 0.05 to 0.5 mM. The effect was particularly marked at higher Cr supply (Table 1). Activities of both APX and POD increased with increase in Cr supply and the effect was more marked on APX than on POD (Table 1).

Discussion

Cr in excess concentration (>0.05 mM) appeared quite for the growth of groundnut plants causing an 83% decrease at 0.5 mM Cr. The symptoms of Cr toxicity observed here are somewhat similar to those observed in cabbage [4] and green gram [24]. In excess Cr chlorosis and necrosis of leaves interfered with photosynthetic reactions [25] and ultimately retarded the plant growth. The decrease in biomass at excess Cr might be due to accumulation of Cr in different parts of groundnut [26-28]. Groundnut responded to Cr stress by decreasing the relative water content and osmotic potential. Reduction in the rate of leaf elongation under the metal stress is attributed to the change in leaf water status [29].
The decrease in chlorophyll and carotenoids contents in the present study at excess Cr has resemblance with the previous results obtained in barley [30] and radish [27]. The decrease in pigment content might be due to either the inhibitory effect of the metal on chlorophyll biosynthesis or a metal and plant specific effect as reported for other metals in different plants [30,31]. Cr exposure brought down the levels of chlorophyll and carotenoids with a marked decrease in the activity of catalase in groundnut leaves. These effects of excess Cr might be attributed to complexation of the heavy metals to sulphydryl sites of the enzyme involved in dehydrogenation of δ-aminolevulinic acid, a common precursor of both chlorophylls and heme [32]. An increase in carotenoids to chlorophyll ratio in plants showing decreased concentrations of chlorophyll and carotenoids might be of protective values as carotenoids are known to be potent quenchers of ROS, particularly singlet oxygen [33]. Cr exposure in groundnut caused imbalance in the level of other nutrients affecting their availability for different anabolic pathways, ultimately arresting the normal developmental process. Membrane damage caused by high oxidation power of Cr (VI) as has been reported by Panda [13] might be the reason behind arrest of the normal development.
The increased activities of antioxidative enzymes in response to excess Cr are suggestive of strong induction of oxidative stress. Excess Cr increased SOD activity, the enzyme responsible for the dismutation of O2 [9]. A further indication of increased generation of ROS due to excess Cr was provided by the increases in the activities of POD and APOD that take care of the H2O2 resulting from SOD activity [34]. Peroxidases are involved not only in scavenging H2O2 produced in chloroplasts but also in growth and developmental processes. The increased activity of peroxidase observed in Cr stress might result in increase in H2O2 level or low protein content [30]. An increase in SOD activity associated with high build up of .O2 and H2O2 levels in response to Cr (VI) exposure seem well correlated with the decrease in catalase and increase in peroxidase activity as has also been reported for other metals including Cr [28,35,36]. Excess Cr also led to marked increase in ascorbate peroxidase another H2O2 scavenging enzyme is in agreement with the findings of other workers [9] suggesting implication of Mahler-peroxidase reaction. An increase in the activity of APX in Cr supplied groundnut plants appears to be in response to increased accumulation of H2O2. The increased build-up of H2O2 in the plants might have triggered the up regulation of APX, a key enzyme for H2O2 detoxification in both chloroplasts and cytosol [37]. However, the specific activity of catalase decreased may be due to inhibition of enzyme synthesis and denaturation of enzyme protein. The decrease may also be associated with degradation caused by induced peroxisomal proteases or may be due to photo inactivation of enzyme.
Membrane destabilization is generally attributed to lipid peroxidation, due to an increased production of free radicals under heavy metal stresses. These reactive oxygen species induced lipid peroxidation due to removal of hydrogen from unsaturated fatty acids leading to formation of lipid radicals and reactive aldehydes. This results in cyclic cascade of reactions causing distortion of lipid bilayer and membrane proteins [38]. In the present study, there was gradual increase in malondialdehyde (MDA) content and electrical conductance which was positively correlated with accumulated metal at all growth period, suggesting damage done to the membrane and resulting ion leakage. Cr like other metals has strong affinity towards N and S containing ligands and proteins. It thus forms disulfide bridges within proteins leading to distorted membrane ion channels and leakage of ions.
Concentration of iron in leaves and stem decreased with an increase in Cr supply from 0.05 to 0.5 mM, but it showed a reverse trend in roots. The observed effect of excess Cr on iron concentration is in consonance with the findings of Barcelo and Poschenrieder [26]. Cr exposure to plants significantly restricted the translocation of Fe from root to shoot suggesting that Cr can displace Fe from physiologically active sites thus producing induced Fe deficiency [39]. Concentration of chromium in leaves (young and old), stem and roots increased with an increase in Cr supply from 0.05 to 0.5 mM. Compared to the above ground parts Cr concentration was higher in roots irrespective of Cr supply. Different species and different plant parts differ in Cr accumulation [40]. This resembles partially with the present study. Significant quantity of Cr was present in roots particularly at greater Cr exposure levels. In the present study Cr accumulation was in the order: root>stem>old leaves>young leaves. Though comparatively less amount of Cr was translocated to young and old leaves, even this concentration was sufficient to produce phytotoxic lesions in groundnut. The tissue Cr concentration in leaves, stem and roots of groundnut corresponding to 90% relative dry weight were 9, 12 and 40 μg g−1 and 66% relative dry weight were 32, 44 and 150 μg g−1, respectively.

Conclusion

Visible and metabolic lesions observed in this study clearly indicate the inhibitory effect of excess Cr (VI) on growth and development of groundnut. Moreover, the present study showed a Cr (VI) mediated free radical reaction as manifested by an increase in the activities of POD and SOD and CAT to help in detoxifying the H2O2 produced in response to Cr (VI) treatments.

Acknowledgments

The authors are grateful to ICAR for providing financial assistance during the course of the study.

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