This article was downloaded by: [Texas A & M International University] On: 15 August 2015, At: 22:53 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG
International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Involvement of Asada-Halliwell pathway during phytoremediation of Chromium (VI) in Brassica juncea L plants ab
b
c
Mukesh Kumar Kanwar , Poonam , Sikander Pal & Renu Bhardwaj
b
a
Department of Environmental Sciences, Sri Guru Granth Sahib World University, Fatehgarh Sahib-140406, Punjab, India b
Department of Botanical and Environmental Science, Guru Nanak Dev University, Amritsar-143005, Punjab, India c
Department of Botany, University of Jammu, Jammu and Kashmir, India Accepted author version posted online: 19 Jun 2015.
Click for updates To cite this article: Mukesh Kumar Kanwar, Poonam, Sikander Pal & Renu Bhardwaj (2015): Involvement of Asada-Halliwell pathway during phytoremediation of Chromium (VI) in Brassica juncea L plants, International Journal of Phytoremediation, DOI: 10.1080/15226514.2015.1058326 To link to this article: http://dx.doi.org/10.1080/15226514.2015.1058326
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
ACCEPTED MANUSCRIPT Involvement of Asada-Halliwell pathway during phytoremediation of Chromium (VI) in Brassica juncea L plants
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
Mukesh Kumar Kanwara,b, Poonamb, Sikander Palc and Renu Bhardwajb,*
a
Department of Environmental Sciences, Sri Guru Granth Sahib World University, Fatehgarh Sahib-140406, Punjab, India.
b
Department of Botanical and Environmental Science, Guru Nanak Dev University, Amritsar143005, Punjab, India. C
Department of Botany, University of Jammu, Jammu and Kashmir, India.
*
Author for correspondence: [email protected], Phone Number: + 91- 9876214176, Fax Number: +91-0183-2258820.
ABSTRACT Brassica juncea (Indian mustard) L. plants were exposed to different concentrations (0.0, 0.1, 0.3 and 0.5mM) of Chromium (Cr) and harvested after 30 and 60 days of sowing for the analysis of growth parameters, metal uptake and oxidative stress markers. Significant accumulation of Cr (VI) by B. juncea L. plants resulted in the reduced growth and modulations in the pool of various biochemical stress markers. The toxic effects of Cr (VI) on growth and other stress markers (protein content, lipid peroxidation and antioxidative enzymes viz.SOD, CAT, POD, APOX, GR, DHAR and MDHAR) in B. juncea L. were observed to be concentration and time dependent. 1
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Effect of Cr (VI) on biochemical parameters was differential and their maximum activities of SOD, POD, APX, GR, DHAR and lipid peroxidation were recorded at 0.5mM concentration in 30 days old plants. Whereas, trend in the activities of most of the stress markers was reversed in 60 days old plants. The results obtained from the study suggested that Cr (VI) stress inhibited
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
growth of B. juncea L. plants is directly interrelated with its accumulation and resulted in the modulation in activities of various stress markers.
Keywords Chromium, Brassica juncea, pytoremediation, antioxidative enzymes.
2
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT INTRODUCTION Toxic elements released through the burning of fossil fuels, industrialization and industrial effluents are continuously increasing in the environment (Govindasamy et al. 2011). Their elevated concentrations have created an environment which is harmful to humans and plants
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
(Ganesh et al. 2008). Chromium (Cr) is one of the most toxic heavy metal (HM) and in nature is found in two stable forms like Cr (III) and Cr (VI). The hexavalent form of Cr is reported to be most toxic and a strong oxidant as possesses high redox potential which accounts for rapid generation of ROS (Shanker et al. 2005). Surfing of literature revealed that the toxicity of Cr in plants resulted in reduced growth, detrimental to roots, induced chlorosis, disturbed photosynthesis and ultimately leads to plant death (Gill et al. 2015). Earlier studies have reported the uptake, detoxification and stabilization of HMs by the plants from the contaminated soils (Lasat, 2002; Singh and Sinha 2005). The production of reactive oxygen species (ROS) is most common response of HM toxicity in plants (Kanwar et al. 2013), which disrupts the normal metabolism through the initiation of oxidative stress in plants. Plants are well equipped with number of defensive strategies to manage the stress triggers by these toxic elements and maintained homeostasis (Vazquez et al. 2008). The most common adopted defensive mechanism against heavy metals generated ROS is the modulation in the levels of antioxidants and antioxidative enzymes of Asada- Halliwell pathway (Choudhary et al. 2012). In scavenging ROS, SOD plays a first line defense, as it dismutated superoxide radical (O2-) into H2O2. This H2O2 is further scavenged by CAT in peroxisomes; by APOX in the chloroplast or by membrane-bounded POD (Arora et al. 2012). The pool of glutathione in the reduced state is regulated by GR, which reduces dehydro-ascorbate to ascorbate through the 3
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT ascorbate–glutathione cycle (Arora et al. 2012). As a result, their role becomes extremely significant in plants facing various stresses. Members of Brassicaceae family have been reported to be tolerant towards HMs (Kanwar et al. 2012). B. juncea is grown as an oil seed crop and accounts for edible oil production after soya and palm (Sinha et al. 2010). Because of its fast
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
growth, greater biomass, less water requirement and ability to absorb high concentration of HMs (Meng et al. 2009; Gill et al. 2015), it is regarded as a potential plant for phytoremediation of HMs. Therefore, it is imperative to evaluate Cr (VI) accumulation and its effects on morphological and biochemical stress markers like total proteins, antioxidative enzymes (SOD, CAT, POD, APX, GR, MDHAR and DHAR) and MDA content in 30 and 60 days old B. juncea L. plants exposed to different concentrations of Cr (0.0, 0.1, 0.3 & 0.5mM) stress.
MATERIAL AND METHODS PLANT MATERIAL AND EXPERIMENTAL SETUP Certified seeds of B. juncea L., variety PBR 91 were procured from Department of Plant Breeding, Punjab Agriculture University, Punjab, India. Seeds were surface sterilized with 0.01% Sodium hypochlorite and rinsed five times with double distilled water. The seeds were raised in pots containing different concentrations of Chromium metal in the form of K2CrO4 (0, 0.1, 0.3, and 0.5mM) in the Botanical garden of Guru Nanak Dev University, Amritsar, Punjab, India under natural conditions. CHEMICAL AND REAGENTS USED Reduced glutathione, Dehydroascorbate and Ascorbate oxidase were purchased from Sigma Aldrich, St. Louis, USA. Nitric acid, Perchloric acid, FC reagent, NADPH, GSSG, NADH, NBT, 4
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Triton-X and TCA were procured from HIMEDIA laboratories Pvt. Mumbai, Maharashtra, India. Ascorbic acid was obtained from Qualigens Fine Chemicals, Glaxo India Limited, Mumbai; EDTA from SD Fine Chemical Ltd. Mumbai; Guaiacol from Loba chemie, Mumbai, India and H2O2 from RFCL (RANKEM) Pvt. Ltd. New Delhi, India.
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
CHROMIUM ANALYSIS IN B. JUNCEA PLANTS: The leaves and shoots of 30 and 60 days old B. juncea L. plants were harvested. The collected samples were oven dried at 800C for 24 hrs. The dried samples were digested in digestion mixture of concentrated nitric acid (HNO3) and perchloric acid (HClO4) (V/V 3:1). The solution was evaporated to dryness. After digestion the solution was cooled, filtered and made up to 100mL with double distilled water for heavy metal analysis. The heavy metals measurement in samples was performed with Atomic Absorption Spectrophotometer (Shimadzu AA6300, Japan). The metal content was determined by calibration with standard curve made with different concentration of Cr metal ions. STUDIES ON MORPHOLOGICAL PARAMETERS After 30 and 60 days of sowing, plants were harvested and analyzed for morphological parameters like shoot length and number of leaves per plant. Fifteen plants from three replicates of same concentration were selected for analysis.
BIOCHEMICAL PARAMETERS Preparation of Plant Extract These studies were conducted to determine the effect of Cr metal ions on the activities of protein and antioxidative enzyme such as superoxide dismutase, catalase, guaiacol peroxidase, ascorbate 5
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT peroxidase, glutathione reductase, monodehydroascorbate reductase and dehydroascorbate reductase. The leaves were excised for estimation of antioxidative enzyme activities and protein content. 0.5g of leaf tissue was homogenized in 50mM potassium phosphate buffer (pH- 7.0). The homogenate was centrifuged at 40C for 20 min at 15000g. The supernatant was used for
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
determining the activities of antioxidative enzymes by using UV–Visible Double Beam Spectrophotometer (Systronics 2202, India). Protein estimation: Protein content was determined following the method of Lowry et al. (1951). Ascorbate peroxidase (APOX, EC 1.11.1.11): Ascorbate peroxidase activity was determined following the method proposed by Nakano and Asada (1981). Catalase (CAT, EC 1.11.1.6): Catalase activity was calculated as by the method suggested by Aebi (1984). Guaiacol peroxidase (POD, EC 1.11.1.7): The activity of peroxidase was measured according to the method proposed by Putter (1974). Superoxide dismutase (SOD, EC 1.15.1.1): SOD was estimated according to Kono (1978) by noting its potential to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) dye by superoxide radicals, which are produced by the auto-oxidation of hydroxylamine hydrochloride. Glutathione reductase (GR, EC 1.6.4.2): Glutathione reductase was measured by the method proposed by Carlberg and Mannervik (1975). Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4): Monodehydroascorbate reductase activity was determined according to the method proposed by Hossain et al. (1984).
6
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Dehydroascorbate reductase (DHAR, EC 1.8.5.1): Dehydroascorbate reductase activity was measured as by the method given by Dalton et al. (1986). Malondialdehyde content: The malondialdehyde (MDA) content was measured using the method described by Heath and Packer (1968).
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
Statistical Analysis All the data were subjected to one way analysis of variance (ANOVA) for scrutinizing the effect of Cr (VI) ions on various morphological and biochemical parameters as mean ± standard error of five replicates. The Fisher LSD post hoc test (p≤ 0.05) was applied for the comparisons against control values using Sigma-Stat Version 3.5.
RESULTS Morphological parameters of 30 and 60 days old plants B. juncea plants In 30 days old plants, Cr-induced phytotoxicity was apparent in terms of reduced shoot length and number of leaves of B. juncea plants facing Cr stress when compared to control plants. Increasing concentrations of Cr ions harmfully affected the growth of 30days old plants. The shoot length and number of leaves decreased with increasing concentrations of Cr ions and a decrease of 2.047 folds in shoot length and 1.675 folds in number of leaves were recorded in 0.5mM of Cr treatment and significantly differ from control plants (Table 1). Similar results were observed in 60 days old plants, as the escalating concentrations of Cr ions revealed distorted growth of B. juncea plants. The decrease in dose dependent manner was noticed for shoot length and number of leaves in all treatments of Cr metal which was statistically different
7
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT from control plants. The shoot length was decreased by 1.74 folds and number of leaves by 2.09 folds at 0.5mM of Cr treatment as compared to control plants (Table 1). Cr ion accumulation In shoots
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
30 days old B. juncea plants revealed significant uptake of Cr ions. It was noted that the uptake effectiveness of Brassica plants increased with increasing concentration of Cr ion. Maximum uptake was noticed at highest concentration of 0.5mM treated plants followed by 0.3mM and 0.1mM dose of Cr ions respectively. Whereas in 60 days old plants the uptake efficiency decreased with increasing doses of Cr ion. Maximum uptake of 119.67μg g-1 DW was noticed in 0.1mM treated plants. A decrease of 1.14 folds and 1.29 folds in metal uptake was observed at 0.3mM and 0.5mM Cr treated plants respectively as compared to lower doses (Fig. 1 & 2). In leaves Similarly, metal uptake was also noticed in the leaves of 30 days old Brassica plants. The metal uptake was lesser than that of shoots, but the drift of uptake was same. 0.5mM Cr treated plants revealed the maximum uptake followed by 0.3mM and 0.1mM treated plants (Fig. 1 & 2). A decreasing trend of Cr metal uptake was noticed in leaves of 60 days and maximum uptake was recorded in 0.2mM treatment (104.173μg g-1 DW). A decrease of 1.07 folds and 1.14 times was noticed in 0.3mM & 0.5mM of Cr ion treatment compared to lower concentration (Fig. 1 & 2). Biochemical Parameter Protein content The protein content was increased with the increasing concentration of Cr ion in 30 days old plants. Maximum protein content was recorded at 0.5mM of Cr ion treatment which was 1.87 8
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT folds higher than control plants. The increase of 1.54 folds and 1.72 folds was assessed in 0.1mM and 0.3mM of Cr treatment when compared to control Brassica plants (Table 2). The decreasing trend in protein content with the increasing concentration of Cr ions in 60 days old B. juncea plants was observed. Maximum decrease of 1.706 folds in protein content was recorded in
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
0.5mM Cr treated plants. Treatments 0.1mM and 0.3mM of Cr ion also showed decrease in protein content which was assessed by 1.106 folds and 1.303 folds respectively than control plants (Table 2). Activities of Antioxidative enzymes Cr ion toxicity had significantly altered activities of antioxidant enzymes in 30 days old B. juncea plants. Activities of SOD, CAT, POD, GR and DHAR were increased with the increasing concentration of Cr stress as compared to control plants except MDHAR where a decrease in its activity was observed (Table 2). Maximum SOD activity was recorded in 0.5mM of Cr dose (12.696UA mg-1protein). CAT activity was decreased with increasing concentration of Cr ion but the activity of treated plants was remained higher than untreated plants. Highest activity of CAT was noticed in 0.1mM (10.133UA mg-1 protein) treated plants. Similarly the activity of POD (16.633UA mg-1 protein) APOX (19.79UA mg-1 protein), GR (25.89UA mg-1 protein) and DHAR (21.20UA mg-1 protein) was increased with increasing concentrations of Cr ion and maximum activity was noticed in 0.5mM of Cr ion treatment (Table 2). In case of MDHAR a decreasing trend was observed and maximum drop in its activity was noticed at 0.5mM Cr stress plants (Table 2). In 60 days old plants, activities of antioxidant enzymes had considerably altered under Cr ion toxicity. Activities of SOD and APOX were increased with the increasing concentration of Cr 9
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT ion stress as compared to control plants and activities of CAT, POD, GR, DHAR and MDHAR were decreased (Table 2). Maximum SOD (45.35UA mg-1 protein) and APOX (42.67UA mg-1 protein) activity was recorded in 0.5mM concentration of Cr ion. Whereas, APOX activity was lowered by 1.54 folds and 1.03 folds in lower concentration of Cr ions. But its activity got
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
increased in 0.5mM of Cr concentration. CAT activity was decreased in dose dependent manner with increasing concentrations of Cr ion and lowest activity was recorded in 0.5mM treatment (10.796UA mg-1 protein). Similarly POD (19.390UA mg-1 protein), GR (12.97 UA mg-1 protein), DHAR (15.63 UA mg-1 protein) and MDHAR (16.46 UA mg-1 protein) showed their lowest activities in 0.5mM of Cr treated plants, respectively. The same decreasing trend was observed for lower concentration of Cr metal ions and POD showed a decrease of 1.18 folds and 1.45 folds for 0.1 and 0.3mM of Cr dose. Similarly, GR activity got decreased by 1.03 folds and 1.67 folds, DHAR by 1.14 folds and 1.45 folds and MDHAR was decreased by 1.38 folds and 1.61 folds in 0.1mM and 0.3mM of Cr ion treated plants as compared to untreated ones (Table 2). MDA content: Cr phytotoxicity significantly increased the levels of lipid peroxidation in 30 days old metal treated B. juncea plants. With the increasing dose of Cr (VI) ions, the levels of MDA content were increased in significantly. Maximum content of MDA (9.70 μmol g-1 FW) were noticed in 0.5mM Cr ion treatment respectively. The increased levels of MDA content was also noticed in other concentrations of Cr ions also and found to be increased by 1.41 folds in 0.1mM and 1.606 folds in 0.3mM concentration respectively (Table 2). In 60 days old Cr treated plants, MDA content got decreased in higher doses of Cr ion and maximum value for MDA content was recorded in 0.1mM (14.20μmol g-1 FW). A sharp decline in MDA content was noticed in 10
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT 0.5mM of Cr treatment and this decline was assessed by 1.15 folds as compared to untreated B. juncea plants (Table 2).
DISCUSSION:
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
Under natural conditions, plants are exposed to variety of environmental stresses. Heavy metal stress is one of the common stresses that limit over all development and growth of plants. A number of studies keep concern with the problem of the relationship between the increased HMs amounts in nature due to industrial environment and their mutagenic and carcinogenic effects (Memon et al. 2001). Members of Cruciferae family like B. juncea and R. sativus are well-reported for their high capacity of accumulation of metals or metalloids (Kanwar et al. 2012; Vamerali et al. 2010). Hexavalent Cr is actively taken up by plants by a metabolic driven process (Shanker et al. 2005). Therefore the study was designed to observe the metal uptake capacity of B. juncea in soil amended with Cr doses (0.1mM, 0.3mM and 0.5mM). The observations made from the study revealed better uptake efficiency of B. juncea plants as Cr (VI) accumulation in different plant parts were found to be dose dependent in 30 days old Brassica plants (Fig. 1). Maximum uptake was noticed at 0.5mM Cr ions treatment in both the cases. Whereas, the trend in uptake of Cr ions was significantly reversed in 60 days old Brassica plants as the maximum uptake was recorded at lower dose (0.1mM; Fig. 2). Singh and Sinha (2005) studied the metal accumulation potential of B. juncea L. for different metals (Cr, Fe, Zn, and Mn) after 30, 60, and 90 days of sowing. Their finding revealed the accumulation of metals in a dose-dependent manner and further got increased with increasing the exposure time. Our findings are in line with the studies conducted by Ganesh et al. (2008) who studied the 11
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT accumulation of chromium in roots and shoots of water lettuce and soya bean. Their finding confirmed that the chromium contents in both tissues significantly increased with the increasing concentrations. Morphological parameters like plant biomass, root length, shoot length andnumber of leaves is a
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
clear indicator of toxicity caused by HMs. In the present investigation, morphological parameters like shoot length and number of leaves were studied to observe the toxic effects of Cr (VI) ions on B. juncea plants. The results obtained clearly depicted that phyto-toxicity of Cr ions that has leads to the significant decrease in the shoot length and number of leaves in all development stages of Brassica plants. The dose dependent decrease was noticed in both shoot length and number of leaves and maximum reduction was observed at 0.5mM concentration of Cr ions and was significantly different from control plants in all the stages of growth (Table 1). Our findings were clearly supported by the study conducted by Gill et al. (2015) who studied the growth of Brassica napus plant in terms of stem and root lengths, number of leaves and leaf area per plant under Cr metal stress and concluded that growth was decreased with the increasing concentration Cr in the solution. Studies conducted earlier confirmed that Cr adversely affected the roots of the plants (Ali et al. 2013). This might have been the cause of decreased nutrient supply to upper parts of B. juncea plants in the present investigation, which resulted in decrease in shoot length and leaf number. A study conducted by Datta et al. (2011) on five cultivares of wheat viz., HD2956, HD2932, DBW14, KO512, WH775 grown under Cr metal stress revealed phytotoxicity of Cr in terms of decrease in rate of germination and reduced root and shoot length as compared to control plants.
12
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Lipid peroxidation is taken as a marker of oxidative stress as they are prone to attack by free radicals. Lipid peroxidation is commonly measured as MDA content and taken as an index of oxidative stress (Choudhary et al. 2012). In the present study, MDA level rose significantly when plants were subjected to high levels of Cr. This suggests that high level of Cr lead to
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
production of superoxide radicals, further leading to increased lipid peroxidation. Enhanced lipid peroxidation content is a direct evident that chromium induces oxidative stress (Diwan et al. 2008). Diwan et al. (2010) has also discovered increase in the MDA content under various concentrations of Cr (0, 50, 100, 200 M Cr) in B. juncea and V. radiate. It was observed by Liu et al. (2008) that the leaves of A. viridis when exposed of 1 µM Cr resulted in reduced MDA content, but higher concentrations of 10 and 100 µM lead to increase in MDA content. HMs ions are reported to generate reactive oxygen species (ROS) in plants cells, which cause the molecular damage and disrupts their normal homeostasis (Khan et al. 2009). The treatment of B. juncea plants with Cr ions in the present finding revealed that metal stress escalated the pool of protein content and antioxidant enzymes and it was found that activities of protein content got statistically increased with increasing concentration of metal in 30 days old plants (Table 2). This is in agreement with other reports in literature in which an increase in protein content with increased metal stress has been reported. In a study conducted on Lemna gibba subjected to Mn and Ni, a significant increase in total protein content in plants stressed with excess Mn was observed as compared to the control plants (Doganlar et al. 2012). However, in 60 days old plants, protein content along with the enzyme activity got decreased in all the concentration of all the metals (Table 2). Both decrease and increase in total protein content have been reported in plants under heavy metal stress (Vajpayee et al. 2000; Lei et al. 2007). 13
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT In this study, the decrease in amount of total soluble protein in the 60 days old plants may be attributed to protein degradation due to oxidative damage. However, the increase in total protein content in 30 days old plants might have been due to the increase of specific stressrelated proteins were involved in antioxidant metabolism and phytochelatin biosynthesis (Lei et
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
al., 2007). Changes in the total soluble protein contents are taken as indicators of the physiological status as well as of the reversible or irreversible metabolic changes of the plant (Piotrowska et al. 2010; Doganlar and Atmaca, 2011). In present study, activities of almost all the enzymes were statistically increased in 30 days old plants treated Cr (VI) metal ions when compared with control plants. Whereas, in 60 days old Brassica plants activities of all the enzymes were decreased except SOD and APOX (Table 2). The expressions of enzymatic activities largely dependent on metal, time of exposure and may also depend on types of plant species under study. Findings of the present studies are fully supported by the studies of Pandey et al. (2005). They observed the differential response in antioxidative defense system of Brassica juncea cv. pusa jaikisan grown in hydroponic culture supplemented with 0.2, 2 and 20 µM of hexavalent Cr for 15 days. The activity of SOD and CAT was higher at lower dose Cr (VI) whereas the activities of GST and GR were increased with the increased dose of Cr (VI) stress. Similarly, Gill et al. (2015) reported that the activities of SOD, POD and CAT were enhanced under different concentrations of Cr ions i.e. 100 and 400µM, except APX activity which was reduced under higher concentration of Cr. Another study conducted by Liu et al. (2008) in leaves of A. viridis L. reported that the activities of SOD and POD were activated by Cr (VI), while CAT activity was inhibited. Ali et al. (2013) has also recorded the increase in various enzymes (SOD, POD, APX, GR and CAT) under Cr stress in Barley plants. Decrease in the CAT, GR and 14
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT DHAR activity in roots and shoots of P. sativum was observed after 11 days of Cr stress (Gangwar et al. 2011).
CONCLUSION:
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
It can be inferred from the study under taken that the effect of Cr (VI) ions on various stress parameters was concentrations and day dependent. Significant accumulation and translocation of Cr (VI) ions in the shoots and leaves of Brassica juncea plants revealed its potential for phytoremediation of Cr metal. Furthermore, the effect of Cr ions on the pool of antioxidative enzymes in Brassica juncea accounts for the enhanced tolerance in combating the Cr induced toxicity.
ACKNOWLEDGEMENTS Authors are thankful to Department of Biotechnology and Council of Scientific and Industrial Research, Government of India for providing financial assistance.
15
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT REFERENCES
Aebi H. 1984. Catalase in Vitro. Methods Enzymol 105:121-126.
Ali S, Farooq MA, Jahangir MM, Abbas F, Bharwana SA, Zhang GP. 2013. Effect of chromium
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
and nitrogen form on photosynthesis and anti-oxidative system in barley. Biologia Plantarum 57: 758–763.
Arora P, Bhardwaj R, Kanwar MK. 2012. Effect of 24-epibrassinolide on growth, protein content and antioxidative defense system of Brassica juncea L. subjected to cobalt ion toxicity. Acta Physiol Plant 34: 2007-2017.
Carlberg I, Mannervik B. 1975. Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J Biol Chem 250: 5475-5480.
Choudhary SP, Kanwar M, Bhardwaj R, Yu JQ, Tran LSP. 2012. Chromium stress mitigation by polyamine-brassinosteroid application involves phytohormonal and physiological strategies in Raphanus sativus L. PloS one 7 (3), e33210
Dalton DA, Russell SA, Hanus FJ, Pascoe GA, Evans HJ. 1986.
Enzymatic reactions of
ascorbate and glutathione that prevent peroxide damage in soybean root nodules. Proc Natl Acad Sci USA 83: 3811–3815.
Datta JK, Bandhyopadhyay A, Banerjee A, Mondal NK. 2011 Phytotoxic effect of chromium on the germination, seedling growth of some wheat (Triticum aestivum L.) cultivars under laboratory condition. J Agri Technol 7: 395-402. 16
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Diwan, H, Ahmad A, Iqbal M. 2008. Genotypic variation in the phytoremediation potential of Indian mustard for chromium. Environ Manage 41: 734-741.
Diwan, H, Khan, I, Ahmad, A, Iqbal, M. 2010. Induction of phytochelatins and antioxidant defence system in Brassica juncea and Vigna radiata in response to chromium
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
treatments. Plant Growth Regul.. 61:97-107.
Doganlar ZB, Atmaca M. 2011. Influence of Airborne Pollution on Cd, Zn, Pb, Cu, and Al accumulation and physiological parameters of plant leaves in Antakya (Turkey). Water, Air and Soil Pollution 214: 509-523.
Donganlar BZ, Seher C, Telat Y. 2012. Metal uptake and physiological changes in Lemna gibba exposed to manganese and nickel. Inter J Biol 4: 148-151.
Ganesh KS, Baskaran L, Rajasekaran S, Sumathi K, Chidambaram ALA, Sundaramoorthy P. 2008. Chromium stress induced alterations in biochemical and enzyme metabolism in aquatic and terrestrial plants. Colliods Surf B: Biointerfaces 63: 159-163.
Gangwar S, Singh VP, Prasad SM, Maurya JN. 2011. Differential responses of pea seedlings to indole acetic acid under manganese toxicity. Acta Physiol Plant 33: 451–462.
Gill RA, Zang L, Ali B, Farooq MA, Cui P, Yang S, Ali S,Zhou W. 2015. Chromium-induced physio-chemical and ultrastructural changes in four cultivars of Brassica napus L. Chemosphere 120: 154–164.
17
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Govindasamy C, Arulpriya M, Ruban P, Francisca LJ, Ilayaraja A. 2011. Concentration of heavy metals in seagrasses tissue of the Palk Strait, Bay of Bengal”. Int J Environl Sci 2 (1): 145-153.
Heath RL, Packer L. 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125: 189–198.
Hossain MA, Nakano Y, Asada K. 1984.
Monodehydroascorbate reductase in spinach
chloroplasts and its participation in the regeneration of ascorbate for scavenging Hydrogen Peroxide. Plant Cell Physiol 25: 385-395.
Kanwar MK, Bhardwaj R, Arora P, Chowdhary SP, Sharma P, Kumar S. 2012. Plant steroid hormones produced under Ni stress are involved in the regulation of metal uptake and oxidative stress in Brassica juncea L. Chemosphere 86: 41-49.
Kanwar MK, Bhardwaj R, Chowdhary SP, Arora P, Sharma P, Kumar S. 2013. Isolation and characterization of 24-Epibrassinolide from Brassica juncea L. and its effects on growth, Ni uptake and antioxidant defense of Brassica plants and In vitro cytotoxicity. Acta Physiol Plant 35: 1351-1362.
Khan NA, Nazar R, Anjum NA. 2009. Growth, photosynthesis and antioxidant metabolism in mustard (Brassica juncea L.) cultivars differing in ATP-sulfurylase activity under salinity stress. Scientia Horticulturae 122(3): 455-460.
18
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Kono Y. 1978. Generation of superoxide radical during autooxidation of hydroxylamine and an assay for superoxide dismutase. Arch Biochem Biophys 186: 189-195.
Lasat MM. 2002. Phytoextraction of toxic metals: A review of biological mechanisms. J Environ
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
Qual 3: 109-120.
Lei Y, Korpelainen H, Li C. 2007. Physiological and biochemical responses to high Mn concentrations in two contrasting Populus cathayana populations. Chemosphere 68: 686694.
Liu JN, Zhou QX, Sun T, Ma LQ, Wang S. 2008. Hexavalent Cr uptake and its effects on mineral uptake, antioxidant defence system and uptake and photosynthesis in Amaranthus viridis L. Biosource Tachnol 99: 2628-2636.
Lowry OH, Resebrough NJ, Farr AL, Randall RJ. 1951. Protein determination with folin reagent. J Biol Chem 193: 265-275.
Memon AR, Aktoprakligil D, Ozdemir A, Vertii A. 2001. Heavy metal accumulation and detoxification mechanisms in plants. Turk J Bot 25: 111-121.
Meng H, Hua S, Shamsi I, Jilani G, Li Y, Jiang L. 2009. Cadmium-induced stress on the seed germination and seedling growth of Brassica napus L. and its alleviation through exogenous plant growth regulators. Plant Growth Regul 58: 47–59.
Nakano Y, Asada K. 1981. Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22: 867-880. 19
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Pandey V, Dixit V, Shyam R. 2005. Antioxidative responses in relation to growth of mustard (Brassica juncea cv. Pusa Jaikisan) plants exposed to hexavalent chromium. Chemosphere 61:40-47.
Piotrowska A, Bajguz A, Czerpak R, Kot K. 2010. Changes in the growth, chemical
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
composition, and antioxidant activity in the aquatic plant Wolffia arrhiza (L.) Wimm. (Lemnaceae) exposed to jasmonic acid. J Plant Growth Regul 29: 53-62.
Putter J, 1974. Peroxidase. In: Bergemeyer HU, ed. Methods of Enzymatic Analysis, London: Academic Press p. 685-690.
Shanker AK, Cervantes C, Tavera HL, Avudainayagam. 2005. Chromium toxicity in plants. Environ Int. 31: 739-753. Singh S, Sinha S. 2005. Accumulation of metals and its effects in Brassica juncea (L.) Czern. (cv. Rohini) grown on various amendments of tannery waste. Ecotox Environ Safety 62: 118–127.
Sinha S, Sina G, Mishra RK, Mallick S. 2010. Metal accumulation, growth,antioxidants and oil yield of Brassica juncea L. exposed to different metals. Ecotoxicol Environ Safety 73: 1352-1361.
Vajpayee P, Tripati RD, Rai UN, Ali MB, Singh SN. 2000. Chromium accumulation reduces chlorophyll biosynthesis, nitrate reductase activity and protein content of Nymphaea alba. Chemosphere 41: 1075-1082. 20
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Vamerali T, Marianna B, Mosca G. 2010. Field crops for phytoremediation of metalcontaminated of metal-contaminated land: A review. Environ Chem Lett 8: 1-17.
Vazquez S, Esteban E, Carpena RO. 2008. Evolution of arsenate toxicity in nodulated white
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
lupine in a long-term culture. J Agric Food Chem 56: 8580-8587.
21
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Table1. Effect of Cr ions stress on shoot length and number of leaves in 30 and 60 days old B. juncea plants.
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
Days of Harvest ing 30 days
60 days
Treatments Shoot Length (cm) 0.0mM
0.1mM
0.3mM
0.5mM
0.0M m
Number of Leaves 0.1m 0.3mM M
14.333±1. 201 a
13.666±1. 452 a
8.333±1. 201 b
7.000±1. 154 b
6.7±0. 67 a
8.0±0. 58 b
5.33±0. 333 c
48.67± 1.452 c
23.00 ± 1.154
19.00 ± 0.577
14.67± 1.452 c
85.00± 1.732 a
72.67± 3.929 b
57.67± 4.807 c
a
b
0.5m M 4.0±0. 58 d 11.00 ± 1.00 d
Data presented in Mean ± SE. Different letters (a, b, c & d) within various concentrations of Cr (0, 0.1, 0.3 and 0.5mM) are significantly different (Fisher LSD post hoc test, p≤0.05) and signify the effect of Cr metal ions on morphological parameters.
22
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Table2. Effect of Cr ions stress on protein content, specific activities of antioxidative enzymes
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
and MDA content in 30 and 60 days old B. juncea plants
Treatm Protein SOD CAT POD APX GR DHAR MDHA MDA ents content (UA (UA (UA (UA (UA (UAmgR (μmol -1 -1 -1 -1 -1 -1 1 (mM) (mg g-1 (UA mg mg mg mg mg g -1 FW) protein protein protein protein protein) protein mg FW) ) ) ) ) protein ) ) 0. 11.412± 4.316±0 5.566±0 6.8001± 13.233± 13.733± 11.500± 17.666± 4.815± a a a a a a a 0 0.495 a .547 a .371 0.264 1.713 1.505 0.832 0.894 1.309 0. 17.618± 7.643±0 10.133± 10.767± 14.566± 16.466± 13.966± 15.466± 6.820± 1 0.129 b .489 b 1.097 b 0.775 b 1.594 ab 1.377 ab 1.697 a 2.544 a 0.636 ab
30 day 9.509±0 6.666±0 12.700± 18.733± 21.866± 20.233± 10.500± 7.737± s 0. 19.635± c c a b b bc b b 0.581 3 0.089 .422 .440 0.945 2.162 2.215 1.260 0.461 bc 0. 21.383± 12.696± 9.610±0 16.633± 19.790± 25.896± 21.200± 5.0333± 9.70±0. 5 0.063 d 0.702 d .713 b 0.865 c 0.839 b 2.357 c 1.154 b 0.726 c 658 c 0. 16.608± 25.380± 34.706± 36.600± 35.503± 25.900± 38.833± 35.800± 8.47±0. 0 1.793 a 0.280 a 0.609 a 1.616 a 1.809 a 2.569 a 4.998 a 2.635 a 634 ab 0. 15.003± 32.513± 18.166± 30.933± 23.033± 25.066± 33.800± 25.933± 14.20± 1 1.338 a 0.863 ab 0.260 a 2.540 b 1.996 b 1.906 a 2.223 a 1.865 b 1.325 c 60 day 0. 12.739± 41.993± 12.666± 25.200± 34.266± 15.466± 23.666± 22.133± 10.98± a bc c c a b b bc a s 3 1.060 1.804 0.735 0.833 0.693 1.501 0.578 3.575 0.762 0. 7.996±1 45.350± 10.796± 19.390± 42.666± 12.966± 15.633± 16.466± 7.35±0. 5 .156 b 7.353 c 0.665 c 0.792 d 2.140 c 1.128 b 2.028 b 1.398 c 521 b
23
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Data presented in Mean ± SE. Different letters (a, b, c & d) within various concentrations of Cr (0, 0.1, 0.3 and 0.5mM) are significantly different (Fisher LSD post hoc test, p≤0.05) and signify
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
the effect of Cr metal on various biochemical parameters.
24
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
150
Shoots Leaves
Cr ions upatke ( mg g
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
-1
DW)
30 days old Plants
100
50
0 0.0
0.1 0.3 Cr tretaments (mM )
0.5
Fig. 1 Chromium uptake (μg g-1 DW) in leaves and shoots of 30 days old B. juncea plants.
25
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
150
Shoots Leaves
Cr ions upatke ( mg g
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
-1
DW)
60 days old Plants
100
50
0 0.0
0.1 0.3 Cr tretaments (mM )
0.5
Fig. 2 Chromium uptake (μg g-1 DW) in leaves and shoots of 60 days old B. juncea plants.
26
ACCEPTED MANUSCRIPT
International Journal of Phytoremediation Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bijp20
Involvement of Asada-Halliwell pathway during phytoremediation of Chromium (VI) in Brassica juncea L plants ab
b
c
Mukesh Kumar Kanwar , Poonam , Sikander Pal & Renu Bhardwaj
b
a
Department of Environmental Sciences, Sri Guru Granth Sahib World University, Fatehgarh Sahib-140406, Punjab, India b
Department of Botanical and Environmental Science, Guru Nanak Dev University, Amritsar-143005, Punjab, India c
Department of Botany, University of Jammu, Jammu and Kashmir, India Accepted author version posted online: 19 Jun 2015.
Click for updates To cite this article: Mukesh Kumar Kanwar, Poonam, Sikander Pal & Renu Bhardwaj (2015): Involvement of Asada-Halliwell pathway during phytoremediation of Chromium (VI) in Brassica juncea L plants, International Journal of Phytoremediation, DOI: 10.1080/15226514.2015.1058326 To link to this article: http://dx.doi.org/10.1080/15226514.2015.1058326
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
ACCEPTED MANUSCRIPT Involvement of Asada-Halliwell pathway during phytoremediation of Chromium (VI) in Brassica juncea L plants
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
Mukesh Kumar Kanwara,b, Poonamb, Sikander Palc and Renu Bhardwajb,*
a
Department of Environmental Sciences, Sri Guru Granth Sahib World University, Fatehgarh Sahib-140406, Punjab, India.
b
Department of Botanical and Environmental Science, Guru Nanak Dev University, Amritsar143005, Punjab, India. C
Department of Botany, University of Jammu, Jammu and Kashmir, India.
*
Author for correspondence: [email protected], Phone Number: + 91- 9876214176, Fax Number: +91-0183-2258820.
ABSTRACT Brassica juncea (Indian mustard) L. plants were exposed to different concentrations (0.0, 0.1, 0.3 and 0.5mM) of Chromium (Cr) and harvested after 30 and 60 days of sowing for the analysis of growth parameters, metal uptake and oxidative stress markers. Significant accumulation of Cr (VI) by B. juncea L. plants resulted in the reduced growth and modulations in the pool of various biochemical stress markers. The toxic effects of Cr (VI) on growth and other stress markers (protein content, lipid peroxidation and antioxidative enzymes viz.SOD, CAT, POD, APOX, GR, DHAR and MDHAR) in B. juncea L. were observed to be concentration and time dependent. 1
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Effect of Cr (VI) on biochemical parameters was differential and their maximum activities of SOD, POD, APX, GR, DHAR and lipid peroxidation were recorded at 0.5mM concentration in 30 days old plants. Whereas, trend in the activities of most of the stress markers was reversed in 60 days old plants. The results obtained from the study suggested that Cr (VI) stress inhibited
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
growth of B. juncea L. plants is directly interrelated with its accumulation and resulted in the modulation in activities of various stress markers.
Keywords Chromium, Brassica juncea, pytoremediation, antioxidative enzymes.
2
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT INTRODUCTION Toxic elements released through the burning of fossil fuels, industrialization and industrial effluents are continuously increasing in the environment (Govindasamy et al. 2011). Their elevated concentrations have created an environment which is harmful to humans and plants
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
(Ganesh et al. 2008). Chromium (Cr) is one of the most toxic heavy metal (HM) and in nature is found in two stable forms like Cr (III) and Cr (VI). The hexavalent form of Cr is reported to be most toxic and a strong oxidant as possesses high redox potential which accounts for rapid generation of ROS (Shanker et al. 2005). Surfing of literature revealed that the toxicity of Cr in plants resulted in reduced growth, detrimental to roots, induced chlorosis, disturbed photosynthesis and ultimately leads to plant death (Gill et al. 2015). Earlier studies have reported the uptake, detoxification and stabilization of HMs by the plants from the contaminated soils (Lasat, 2002; Singh and Sinha 2005). The production of reactive oxygen species (ROS) is most common response of HM toxicity in plants (Kanwar et al. 2013), which disrupts the normal metabolism through the initiation of oxidative stress in plants. Plants are well equipped with number of defensive strategies to manage the stress triggers by these toxic elements and maintained homeostasis (Vazquez et al. 2008). The most common adopted defensive mechanism against heavy metals generated ROS is the modulation in the levels of antioxidants and antioxidative enzymes of Asada- Halliwell pathway (Choudhary et al. 2012). In scavenging ROS, SOD plays a first line defense, as it dismutated superoxide radical (O2-) into H2O2. This H2O2 is further scavenged by CAT in peroxisomes; by APOX in the chloroplast or by membrane-bounded POD (Arora et al. 2012). The pool of glutathione in the reduced state is regulated by GR, which reduces dehydro-ascorbate to ascorbate through the 3
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT ascorbate–glutathione cycle (Arora et al. 2012). As a result, their role becomes extremely significant in plants facing various stresses. Members of Brassicaceae family have been reported to be tolerant towards HMs (Kanwar et al. 2012). B. juncea is grown as an oil seed crop and accounts for edible oil production after soya and palm (Sinha et al. 2010). Because of its fast
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
growth, greater biomass, less water requirement and ability to absorb high concentration of HMs (Meng et al. 2009; Gill et al. 2015), it is regarded as a potential plant for phytoremediation of HMs. Therefore, it is imperative to evaluate Cr (VI) accumulation and its effects on morphological and biochemical stress markers like total proteins, antioxidative enzymes (SOD, CAT, POD, APX, GR, MDHAR and DHAR) and MDA content in 30 and 60 days old B. juncea L. plants exposed to different concentrations of Cr (0.0, 0.1, 0.3 & 0.5mM) stress.
MATERIAL AND METHODS PLANT MATERIAL AND EXPERIMENTAL SETUP Certified seeds of B. juncea L., variety PBR 91 were procured from Department of Plant Breeding, Punjab Agriculture University, Punjab, India. Seeds were surface sterilized with 0.01% Sodium hypochlorite and rinsed five times with double distilled water. The seeds were raised in pots containing different concentrations of Chromium metal in the form of K2CrO4 (0, 0.1, 0.3, and 0.5mM) in the Botanical garden of Guru Nanak Dev University, Amritsar, Punjab, India under natural conditions. CHEMICAL AND REAGENTS USED Reduced glutathione, Dehydroascorbate and Ascorbate oxidase were purchased from Sigma Aldrich, St. Louis, USA. Nitric acid, Perchloric acid, FC reagent, NADPH, GSSG, NADH, NBT, 4
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Triton-X and TCA were procured from HIMEDIA laboratories Pvt. Mumbai, Maharashtra, India. Ascorbic acid was obtained from Qualigens Fine Chemicals, Glaxo India Limited, Mumbai; EDTA from SD Fine Chemical Ltd. Mumbai; Guaiacol from Loba chemie, Mumbai, India and H2O2 from RFCL (RANKEM) Pvt. Ltd. New Delhi, India.
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
CHROMIUM ANALYSIS IN B. JUNCEA PLANTS: The leaves and shoots of 30 and 60 days old B. juncea L. plants were harvested. The collected samples were oven dried at 800C for 24 hrs. The dried samples were digested in digestion mixture of concentrated nitric acid (HNO3) and perchloric acid (HClO4) (V/V 3:1). The solution was evaporated to dryness. After digestion the solution was cooled, filtered and made up to 100mL with double distilled water for heavy metal analysis. The heavy metals measurement in samples was performed with Atomic Absorption Spectrophotometer (Shimadzu AA6300, Japan). The metal content was determined by calibration with standard curve made with different concentration of Cr metal ions. STUDIES ON MORPHOLOGICAL PARAMETERS After 30 and 60 days of sowing, plants were harvested and analyzed for morphological parameters like shoot length and number of leaves per plant. Fifteen plants from three replicates of same concentration were selected for analysis.
BIOCHEMICAL PARAMETERS Preparation of Plant Extract These studies were conducted to determine the effect of Cr metal ions on the activities of protein and antioxidative enzyme such as superoxide dismutase, catalase, guaiacol peroxidase, ascorbate 5
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT peroxidase, glutathione reductase, monodehydroascorbate reductase and dehydroascorbate reductase. The leaves were excised for estimation of antioxidative enzyme activities and protein content. 0.5g of leaf tissue was homogenized in 50mM potassium phosphate buffer (pH- 7.0). The homogenate was centrifuged at 40C for 20 min at 15000g. The supernatant was used for
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
determining the activities of antioxidative enzymes by using UV–Visible Double Beam Spectrophotometer (Systronics 2202, India). Protein estimation: Protein content was determined following the method of Lowry et al. (1951). Ascorbate peroxidase (APOX, EC 1.11.1.11): Ascorbate peroxidase activity was determined following the method proposed by Nakano and Asada (1981). Catalase (CAT, EC 1.11.1.6): Catalase activity was calculated as by the method suggested by Aebi (1984). Guaiacol peroxidase (POD, EC 1.11.1.7): The activity of peroxidase was measured according to the method proposed by Putter (1974). Superoxide dismutase (SOD, EC 1.15.1.1): SOD was estimated according to Kono (1978) by noting its potential to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) dye by superoxide radicals, which are produced by the auto-oxidation of hydroxylamine hydrochloride. Glutathione reductase (GR, EC 1.6.4.2): Glutathione reductase was measured by the method proposed by Carlberg and Mannervik (1975). Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4): Monodehydroascorbate reductase activity was determined according to the method proposed by Hossain et al. (1984).
6
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Dehydroascorbate reductase (DHAR, EC 1.8.5.1): Dehydroascorbate reductase activity was measured as by the method given by Dalton et al. (1986). Malondialdehyde content: The malondialdehyde (MDA) content was measured using the method described by Heath and Packer (1968).
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
Statistical Analysis All the data were subjected to one way analysis of variance (ANOVA) for scrutinizing the effect of Cr (VI) ions on various morphological and biochemical parameters as mean ± standard error of five replicates. The Fisher LSD post hoc test (p≤ 0.05) was applied for the comparisons against control values using Sigma-Stat Version 3.5.
RESULTS Morphological parameters of 30 and 60 days old plants B. juncea plants In 30 days old plants, Cr-induced phytotoxicity was apparent in terms of reduced shoot length and number of leaves of B. juncea plants facing Cr stress when compared to control plants. Increasing concentrations of Cr ions harmfully affected the growth of 30days old plants. The shoot length and number of leaves decreased with increasing concentrations of Cr ions and a decrease of 2.047 folds in shoot length and 1.675 folds in number of leaves were recorded in 0.5mM of Cr treatment and significantly differ from control plants (Table 1). Similar results were observed in 60 days old plants, as the escalating concentrations of Cr ions revealed distorted growth of B. juncea plants. The decrease in dose dependent manner was noticed for shoot length and number of leaves in all treatments of Cr metal which was statistically different
7
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT from control plants. The shoot length was decreased by 1.74 folds and number of leaves by 2.09 folds at 0.5mM of Cr treatment as compared to control plants (Table 1). Cr ion accumulation In shoots
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
30 days old B. juncea plants revealed significant uptake of Cr ions. It was noted that the uptake effectiveness of Brassica plants increased with increasing concentration of Cr ion. Maximum uptake was noticed at highest concentration of 0.5mM treated plants followed by 0.3mM and 0.1mM dose of Cr ions respectively. Whereas in 60 days old plants the uptake efficiency decreased with increasing doses of Cr ion. Maximum uptake of 119.67μg g-1 DW was noticed in 0.1mM treated plants. A decrease of 1.14 folds and 1.29 folds in metal uptake was observed at 0.3mM and 0.5mM Cr treated plants respectively as compared to lower doses (Fig. 1 & 2). In leaves Similarly, metal uptake was also noticed in the leaves of 30 days old Brassica plants. The metal uptake was lesser than that of shoots, but the drift of uptake was same. 0.5mM Cr treated plants revealed the maximum uptake followed by 0.3mM and 0.1mM treated plants (Fig. 1 & 2). A decreasing trend of Cr metal uptake was noticed in leaves of 60 days and maximum uptake was recorded in 0.2mM treatment (104.173μg g-1 DW). A decrease of 1.07 folds and 1.14 times was noticed in 0.3mM & 0.5mM of Cr ion treatment compared to lower concentration (Fig. 1 & 2). Biochemical Parameter Protein content The protein content was increased with the increasing concentration of Cr ion in 30 days old plants. Maximum protein content was recorded at 0.5mM of Cr ion treatment which was 1.87 8
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT folds higher than control plants. The increase of 1.54 folds and 1.72 folds was assessed in 0.1mM and 0.3mM of Cr treatment when compared to control Brassica plants (Table 2). The decreasing trend in protein content with the increasing concentration of Cr ions in 60 days old B. juncea plants was observed. Maximum decrease of 1.706 folds in protein content was recorded in
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
0.5mM Cr treated plants. Treatments 0.1mM and 0.3mM of Cr ion also showed decrease in protein content which was assessed by 1.106 folds and 1.303 folds respectively than control plants (Table 2). Activities of Antioxidative enzymes Cr ion toxicity had significantly altered activities of antioxidant enzymes in 30 days old B. juncea plants. Activities of SOD, CAT, POD, GR and DHAR were increased with the increasing concentration of Cr stress as compared to control plants except MDHAR where a decrease in its activity was observed (Table 2). Maximum SOD activity was recorded in 0.5mM of Cr dose (12.696UA mg-1protein). CAT activity was decreased with increasing concentration of Cr ion but the activity of treated plants was remained higher than untreated plants. Highest activity of CAT was noticed in 0.1mM (10.133UA mg-1 protein) treated plants. Similarly the activity of POD (16.633UA mg-1 protein) APOX (19.79UA mg-1 protein), GR (25.89UA mg-1 protein) and DHAR (21.20UA mg-1 protein) was increased with increasing concentrations of Cr ion and maximum activity was noticed in 0.5mM of Cr ion treatment (Table 2). In case of MDHAR a decreasing trend was observed and maximum drop in its activity was noticed at 0.5mM Cr stress plants (Table 2). In 60 days old plants, activities of antioxidant enzymes had considerably altered under Cr ion toxicity. Activities of SOD and APOX were increased with the increasing concentration of Cr 9
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT ion stress as compared to control plants and activities of CAT, POD, GR, DHAR and MDHAR were decreased (Table 2). Maximum SOD (45.35UA mg-1 protein) and APOX (42.67UA mg-1 protein) activity was recorded in 0.5mM concentration of Cr ion. Whereas, APOX activity was lowered by 1.54 folds and 1.03 folds in lower concentration of Cr ions. But its activity got
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
increased in 0.5mM of Cr concentration. CAT activity was decreased in dose dependent manner with increasing concentrations of Cr ion and lowest activity was recorded in 0.5mM treatment (10.796UA mg-1 protein). Similarly POD (19.390UA mg-1 protein), GR (12.97 UA mg-1 protein), DHAR (15.63 UA mg-1 protein) and MDHAR (16.46 UA mg-1 protein) showed their lowest activities in 0.5mM of Cr treated plants, respectively. The same decreasing trend was observed for lower concentration of Cr metal ions and POD showed a decrease of 1.18 folds and 1.45 folds for 0.1 and 0.3mM of Cr dose. Similarly, GR activity got decreased by 1.03 folds and 1.67 folds, DHAR by 1.14 folds and 1.45 folds and MDHAR was decreased by 1.38 folds and 1.61 folds in 0.1mM and 0.3mM of Cr ion treated plants as compared to untreated ones (Table 2). MDA content: Cr phytotoxicity significantly increased the levels of lipid peroxidation in 30 days old metal treated B. juncea plants. With the increasing dose of Cr (VI) ions, the levels of MDA content were increased in significantly. Maximum content of MDA (9.70 μmol g-1 FW) were noticed in 0.5mM Cr ion treatment respectively. The increased levels of MDA content was also noticed in other concentrations of Cr ions also and found to be increased by 1.41 folds in 0.1mM and 1.606 folds in 0.3mM concentration respectively (Table 2). In 60 days old Cr treated plants, MDA content got decreased in higher doses of Cr ion and maximum value for MDA content was recorded in 0.1mM (14.20μmol g-1 FW). A sharp decline in MDA content was noticed in 10
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT 0.5mM of Cr treatment and this decline was assessed by 1.15 folds as compared to untreated B. juncea plants (Table 2).
DISCUSSION:
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
Under natural conditions, plants are exposed to variety of environmental stresses. Heavy metal stress is one of the common stresses that limit over all development and growth of plants. A number of studies keep concern with the problem of the relationship between the increased HMs amounts in nature due to industrial environment and their mutagenic and carcinogenic effects (Memon et al. 2001). Members of Cruciferae family like B. juncea and R. sativus are well-reported for their high capacity of accumulation of metals or metalloids (Kanwar et al. 2012; Vamerali et al. 2010). Hexavalent Cr is actively taken up by plants by a metabolic driven process (Shanker et al. 2005). Therefore the study was designed to observe the metal uptake capacity of B. juncea in soil amended with Cr doses (0.1mM, 0.3mM and 0.5mM). The observations made from the study revealed better uptake efficiency of B. juncea plants as Cr (VI) accumulation in different plant parts were found to be dose dependent in 30 days old Brassica plants (Fig. 1). Maximum uptake was noticed at 0.5mM Cr ions treatment in both the cases. Whereas, the trend in uptake of Cr ions was significantly reversed in 60 days old Brassica plants as the maximum uptake was recorded at lower dose (0.1mM; Fig. 2). Singh and Sinha (2005) studied the metal accumulation potential of B. juncea L. for different metals (Cr, Fe, Zn, and Mn) after 30, 60, and 90 days of sowing. Their finding revealed the accumulation of metals in a dose-dependent manner and further got increased with increasing the exposure time. Our findings are in line with the studies conducted by Ganesh et al. (2008) who studied the 11
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT accumulation of chromium in roots and shoots of water lettuce and soya bean. Their finding confirmed that the chromium contents in both tissues significantly increased with the increasing concentrations. Morphological parameters like plant biomass, root length, shoot length andnumber of leaves is a
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
clear indicator of toxicity caused by HMs. In the present investigation, morphological parameters like shoot length and number of leaves were studied to observe the toxic effects of Cr (VI) ions on B. juncea plants. The results obtained clearly depicted that phyto-toxicity of Cr ions that has leads to the significant decrease in the shoot length and number of leaves in all development stages of Brassica plants. The dose dependent decrease was noticed in both shoot length and number of leaves and maximum reduction was observed at 0.5mM concentration of Cr ions and was significantly different from control plants in all the stages of growth (Table 1). Our findings were clearly supported by the study conducted by Gill et al. (2015) who studied the growth of Brassica napus plant in terms of stem and root lengths, number of leaves and leaf area per plant under Cr metal stress and concluded that growth was decreased with the increasing concentration Cr in the solution. Studies conducted earlier confirmed that Cr adversely affected the roots of the plants (Ali et al. 2013). This might have been the cause of decreased nutrient supply to upper parts of B. juncea plants in the present investigation, which resulted in decrease in shoot length and leaf number. A study conducted by Datta et al. (2011) on five cultivares of wheat viz., HD2956, HD2932, DBW14, KO512, WH775 grown under Cr metal stress revealed phytotoxicity of Cr in terms of decrease in rate of germination and reduced root and shoot length as compared to control plants.
12
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Lipid peroxidation is taken as a marker of oxidative stress as they are prone to attack by free radicals. Lipid peroxidation is commonly measured as MDA content and taken as an index of oxidative stress (Choudhary et al. 2012). In the present study, MDA level rose significantly when plants were subjected to high levels of Cr. This suggests that high level of Cr lead to
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
production of superoxide radicals, further leading to increased lipid peroxidation. Enhanced lipid peroxidation content is a direct evident that chromium induces oxidative stress (Diwan et al. 2008). Diwan et al. (2010) has also discovered increase in the MDA content under various concentrations of Cr (0, 50, 100, 200 M Cr) in B. juncea and V. radiate. It was observed by Liu et al. (2008) that the leaves of A. viridis when exposed of 1 µM Cr resulted in reduced MDA content, but higher concentrations of 10 and 100 µM lead to increase in MDA content. HMs ions are reported to generate reactive oxygen species (ROS) in plants cells, which cause the molecular damage and disrupts their normal homeostasis (Khan et al. 2009). The treatment of B. juncea plants with Cr ions in the present finding revealed that metal stress escalated the pool of protein content and antioxidant enzymes and it was found that activities of protein content got statistically increased with increasing concentration of metal in 30 days old plants (Table 2). This is in agreement with other reports in literature in which an increase in protein content with increased metal stress has been reported. In a study conducted on Lemna gibba subjected to Mn and Ni, a significant increase in total protein content in plants stressed with excess Mn was observed as compared to the control plants (Doganlar et al. 2012). However, in 60 days old plants, protein content along with the enzyme activity got decreased in all the concentration of all the metals (Table 2). Both decrease and increase in total protein content have been reported in plants under heavy metal stress (Vajpayee et al. 2000; Lei et al. 2007). 13
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT In this study, the decrease in amount of total soluble protein in the 60 days old plants may be attributed to protein degradation due to oxidative damage. However, the increase in total protein content in 30 days old plants might have been due to the increase of specific stressrelated proteins were involved in antioxidant metabolism and phytochelatin biosynthesis (Lei et
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
al., 2007). Changes in the total soluble protein contents are taken as indicators of the physiological status as well as of the reversible or irreversible metabolic changes of the plant (Piotrowska et al. 2010; Doganlar and Atmaca, 2011). In present study, activities of almost all the enzymes were statistically increased in 30 days old plants treated Cr (VI) metal ions when compared with control plants. Whereas, in 60 days old Brassica plants activities of all the enzymes were decreased except SOD and APOX (Table 2). The expressions of enzymatic activities largely dependent on metal, time of exposure and may also depend on types of plant species under study. Findings of the present studies are fully supported by the studies of Pandey et al. (2005). They observed the differential response in antioxidative defense system of Brassica juncea cv. pusa jaikisan grown in hydroponic culture supplemented with 0.2, 2 and 20 µM of hexavalent Cr for 15 days. The activity of SOD and CAT was higher at lower dose Cr (VI) whereas the activities of GST and GR were increased with the increased dose of Cr (VI) stress. Similarly, Gill et al. (2015) reported that the activities of SOD, POD and CAT were enhanced under different concentrations of Cr ions i.e. 100 and 400µM, except APX activity which was reduced under higher concentration of Cr. Another study conducted by Liu et al. (2008) in leaves of A. viridis L. reported that the activities of SOD and POD were activated by Cr (VI), while CAT activity was inhibited. Ali et al. (2013) has also recorded the increase in various enzymes (SOD, POD, APX, GR and CAT) under Cr stress in Barley plants. Decrease in the CAT, GR and 14
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT DHAR activity in roots and shoots of P. sativum was observed after 11 days of Cr stress (Gangwar et al. 2011).
CONCLUSION:
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
It can be inferred from the study under taken that the effect of Cr (VI) ions on various stress parameters was concentrations and day dependent. Significant accumulation and translocation of Cr (VI) ions in the shoots and leaves of Brassica juncea plants revealed its potential for phytoremediation of Cr metal. Furthermore, the effect of Cr ions on the pool of antioxidative enzymes in Brassica juncea accounts for the enhanced tolerance in combating the Cr induced toxicity.
ACKNOWLEDGEMENTS Authors are thankful to Department of Biotechnology and Council of Scientific and Industrial Research, Government of India for providing financial assistance.
15
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT REFERENCES
Aebi H. 1984. Catalase in Vitro. Methods Enzymol 105:121-126.
Ali S, Farooq MA, Jahangir MM, Abbas F, Bharwana SA, Zhang GP. 2013. Effect of chromium
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
and nitrogen form on photosynthesis and anti-oxidative system in barley. Biologia Plantarum 57: 758–763.
Arora P, Bhardwaj R, Kanwar MK. 2012. Effect of 24-epibrassinolide on growth, protein content and antioxidative defense system of Brassica juncea L. subjected to cobalt ion toxicity. Acta Physiol Plant 34: 2007-2017.
Carlberg I, Mannervik B. 1975. Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J Biol Chem 250: 5475-5480.
Choudhary SP, Kanwar M, Bhardwaj R, Yu JQ, Tran LSP. 2012. Chromium stress mitigation by polyamine-brassinosteroid application involves phytohormonal and physiological strategies in Raphanus sativus L. PloS one 7 (3), e33210
Dalton DA, Russell SA, Hanus FJ, Pascoe GA, Evans HJ. 1986.
Enzymatic reactions of
ascorbate and glutathione that prevent peroxide damage in soybean root nodules. Proc Natl Acad Sci USA 83: 3811–3815.
Datta JK, Bandhyopadhyay A, Banerjee A, Mondal NK. 2011 Phytotoxic effect of chromium on the germination, seedling growth of some wheat (Triticum aestivum L.) cultivars under laboratory condition. J Agri Technol 7: 395-402. 16
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Diwan, H, Ahmad A, Iqbal M. 2008. Genotypic variation in the phytoremediation potential of Indian mustard for chromium. Environ Manage 41: 734-741.
Diwan, H, Khan, I, Ahmad, A, Iqbal, M. 2010. Induction of phytochelatins and antioxidant defence system in Brassica juncea and Vigna radiata in response to chromium
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
treatments. Plant Growth Regul.. 61:97-107.
Doganlar ZB, Atmaca M. 2011. Influence of Airborne Pollution on Cd, Zn, Pb, Cu, and Al accumulation and physiological parameters of plant leaves in Antakya (Turkey). Water, Air and Soil Pollution 214: 509-523.
Donganlar BZ, Seher C, Telat Y. 2012. Metal uptake and physiological changes in Lemna gibba exposed to manganese and nickel. Inter J Biol 4: 148-151.
Ganesh KS, Baskaran L, Rajasekaran S, Sumathi K, Chidambaram ALA, Sundaramoorthy P. 2008. Chromium stress induced alterations in biochemical and enzyme metabolism in aquatic and terrestrial plants. Colliods Surf B: Biointerfaces 63: 159-163.
Gangwar S, Singh VP, Prasad SM, Maurya JN. 2011. Differential responses of pea seedlings to indole acetic acid under manganese toxicity. Acta Physiol Plant 33: 451–462.
Gill RA, Zang L, Ali B, Farooq MA, Cui P, Yang S, Ali S,Zhou W. 2015. Chromium-induced physio-chemical and ultrastructural changes in four cultivars of Brassica napus L. Chemosphere 120: 154–164.
17
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Govindasamy C, Arulpriya M, Ruban P, Francisca LJ, Ilayaraja A. 2011. Concentration of heavy metals in seagrasses tissue of the Palk Strait, Bay of Bengal”. Int J Environl Sci 2 (1): 145-153.
Heath RL, Packer L. 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125: 189–198.
Hossain MA, Nakano Y, Asada K. 1984.
Monodehydroascorbate reductase in spinach
chloroplasts and its participation in the regeneration of ascorbate for scavenging Hydrogen Peroxide. Plant Cell Physiol 25: 385-395.
Kanwar MK, Bhardwaj R, Arora P, Chowdhary SP, Sharma P, Kumar S. 2012. Plant steroid hormones produced under Ni stress are involved in the regulation of metal uptake and oxidative stress in Brassica juncea L. Chemosphere 86: 41-49.
Kanwar MK, Bhardwaj R, Chowdhary SP, Arora P, Sharma P, Kumar S. 2013. Isolation and characterization of 24-Epibrassinolide from Brassica juncea L. and its effects on growth, Ni uptake and antioxidant defense of Brassica plants and In vitro cytotoxicity. Acta Physiol Plant 35: 1351-1362.
Khan NA, Nazar R, Anjum NA. 2009. Growth, photosynthesis and antioxidant metabolism in mustard (Brassica juncea L.) cultivars differing in ATP-sulfurylase activity under salinity stress. Scientia Horticulturae 122(3): 455-460.
18
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Kono Y. 1978. Generation of superoxide radical during autooxidation of hydroxylamine and an assay for superoxide dismutase. Arch Biochem Biophys 186: 189-195.
Lasat MM. 2002. Phytoextraction of toxic metals: A review of biological mechanisms. J Environ
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
Qual 3: 109-120.
Lei Y, Korpelainen H, Li C. 2007. Physiological and biochemical responses to high Mn concentrations in two contrasting Populus cathayana populations. Chemosphere 68: 686694.
Liu JN, Zhou QX, Sun T, Ma LQ, Wang S. 2008. Hexavalent Cr uptake and its effects on mineral uptake, antioxidant defence system and uptake and photosynthesis in Amaranthus viridis L. Biosource Tachnol 99: 2628-2636.
Lowry OH, Resebrough NJ, Farr AL, Randall RJ. 1951. Protein determination with folin reagent. J Biol Chem 193: 265-275.
Memon AR, Aktoprakligil D, Ozdemir A, Vertii A. 2001. Heavy metal accumulation and detoxification mechanisms in plants. Turk J Bot 25: 111-121.
Meng H, Hua S, Shamsi I, Jilani G, Li Y, Jiang L. 2009. Cadmium-induced stress on the seed germination and seedling growth of Brassica napus L. and its alleviation through exogenous plant growth regulators. Plant Growth Regul 58: 47–59.
Nakano Y, Asada K. 1981. Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22: 867-880. 19
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Pandey V, Dixit V, Shyam R. 2005. Antioxidative responses in relation to growth of mustard (Brassica juncea cv. Pusa Jaikisan) plants exposed to hexavalent chromium. Chemosphere 61:40-47.
Piotrowska A, Bajguz A, Czerpak R, Kot K. 2010. Changes in the growth, chemical
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
composition, and antioxidant activity in the aquatic plant Wolffia arrhiza (L.) Wimm. (Lemnaceae) exposed to jasmonic acid. J Plant Growth Regul 29: 53-62.
Putter J, 1974. Peroxidase. In: Bergemeyer HU, ed. Methods of Enzymatic Analysis, London: Academic Press p. 685-690.
Shanker AK, Cervantes C, Tavera HL, Avudainayagam. 2005. Chromium toxicity in plants. Environ Int. 31: 739-753. Singh S, Sinha S. 2005. Accumulation of metals and its effects in Brassica juncea (L.) Czern. (cv. Rohini) grown on various amendments of tannery waste. Ecotox Environ Safety 62: 118–127.
Sinha S, Sina G, Mishra RK, Mallick S. 2010. Metal accumulation, growth,antioxidants and oil yield of Brassica juncea L. exposed to different metals. Ecotoxicol Environ Safety 73: 1352-1361.
Vajpayee P, Tripati RD, Rai UN, Ali MB, Singh SN. 2000. Chromium accumulation reduces chlorophyll biosynthesis, nitrate reductase activity and protein content of Nymphaea alba. Chemosphere 41: 1075-1082. 20
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Vamerali T, Marianna B, Mosca G. 2010. Field crops for phytoremediation of metalcontaminated of metal-contaminated land: A review. Environ Chem Lett 8: 1-17.
Vazquez S, Esteban E, Carpena RO. 2008. Evolution of arsenate toxicity in nodulated white
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
lupine in a long-term culture. J Agric Food Chem 56: 8580-8587.
21
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Table1. Effect of Cr ions stress on shoot length and number of leaves in 30 and 60 days old B. juncea plants.
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
Days of Harvest ing 30 days
60 days
Treatments Shoot Length (cm) 0.0mM
0.1mM
0.3mM
0.5mM
0.0M m
Number of Leaves 0.1m 0.3mM M
14.333±1. 201 a
13.666±1. 452 a
8.333±1. 201 b
7.000±1. 154 b
6.7±0. 67 a
8.0±0. 58 b
5.33±0. 333 c
48.67± 1.452 c
23.00 ± 1.154
19.00 ± 0.577
14.67± 1.452 c
85.00± 1.732 a
72.67± 3.929 b
57.67± 4.807 c
a
b
0.5m M 4.0±0. 58 d 11.00 ± 1.00 d
Data presented in Mean ± SE. Different letters (a, b, c & d) within various concentrations of Cr (0, 0.1, 0.3 and 0.5mM) are significantly different (Fisher LSD post hoc test, p≤0.05) and signify the effect of Cr metal ions on morphological parameters.
22
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Table2. Effect of Cr ions stress on protein content, specific activities of antioxidative enzymes
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
and MDA content in 30 and 60 days old B. juncea plants
Treatm Protein SOD CAT POD APX GR DHAR MDHA MDA ents content (UA (UA (UA (UA (UA (UAmgR (μmol -1 -1 -1 -1 -1 -1 1 (mM) (mg g-1 (UA mg mg mg mg mg g -1 FW) protein protein protein protein protein) protein mg FW) ) ) ) ) protein ) ) 0. 11.412± 4.316±0 5.566±0 6.8001± 13.233± 13.733± 11.500± 17.666± 4.815± a a a a a a a 0 0.495 a .547 a .371 0.264 1.713 1.505 0.832 0.894 1.309 0. 17.618± 7.643±0 10.133± 10.767± 14.566± 16.466± 13.966± 15.466± 6.820± 1 0.129 b .489 b 1.097 b 0.775 b 1.594 ab 1.377 ab 1.697 a 2.544 a 0.636 ab
30 day 9.509±0 6.666±0 12.700± 18.733± 21.866± 20.233± 10.500± 7.737± s 0. 19.635± c c a b b bc b b 0.581 3 0.089 .422 .440 0.945 2.162 2.215 1.260 0.461 bc 0. 21.383± 12.696± 9.610±0 16.633± 19.790± 25.896± 21.200± 5.0333± 9.70±0. 5 0.063 d 0.702 d .713 b 0.865 c 0.839 b 2.357 c 1.154 b 0.726 c 658 c 0. 16.608± 25.380± 34.706± 36.600± 35.503± 25.900± 38.833± 35.800± 8.47±0. 0 1.793 a 0.280 a 0.609 a 1.616 a 1.809 a 2.569 a 4.998 a 2.635 a 634 ab 0. 15.003± 32.513± 18.166± 30.933± 23.033± 25.066± 33.800± 25.933± 14.20± 1 1.338 a 0.863 ab 0.260 a 2.540 b 1.996 b 1.906 a 2.223 a 1.865 b 1.325 c 60 day 0. 12.739± 41.993± 12.666± 25.200± 34.266± 15.466± 23.666± 22.133± 10.98± a bc c c a b b bc a s 3 1.060 1.804 0.735 0.833 0.693 1.501 0.578 3.575 0.762 0. 7.996±1 45.350± 10.796± 19.390± 42.666± 12.966± 15.633± 16.466± 7.35±0. 5 .156 b 7.353 c 0.665 c 0.792 d 2.140 c 1.128 b 2.028 b 1.398 c 521 b
23
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Data presented in Mean ± SE. Different letters (a, b, c & d) within various concentrations of Cr (0, 0.1, 0.3 and 0.5mM) are significantly different (Fisher LSD post hoc test, p≤0.05) and signify
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
the effect of Cr metal on various biochemical parameters.
24
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
150
Shoots Leaves
Cr ions upatke ( mg g
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
-1
DW)
30 days old Plants
100
50
0 0.0
0.1 0.3 Cr tretaments (mM )
0.5
Fig. 1 Chromium uptake (μg g-1 DW) in leaves and shoots of 30 days old B. juncea plants.
25
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
150
Shoots Leaves
Cr ions upatke ( mg g
Downloaded by [Texas A & M International University] at 22:53 15 August 2015
-1
DW)
60 days old Plants
100
50
0 0.0
0.1 0.3 Cr tretaments (mM )
0.5
Fig. 2 Chromium uptake (μg g-1 DW) in leaves and shoots of 60 days old B. juncea plants.
26
ACCEPTED MANUSCRIPT
