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Effect of Arsenic on Growth, Arsenic Uptake, Distribution of Nutrient Elements and Thiols in Seedlings of Wrightia arborea (Dennst.) Mabb. a
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Dharmendra Kumar , Vijay Pratap Singh , Durgesh Kumar Tripathi , Sheo Mohan Prasad & a
Devendra Kumar Chauhan a
D D Pant Interdisciplinary Research Laboratory, Department of Botany, University of Allahabad, Allahabad, India b
Govt. Ramanuj Pratap Singhdev Post Graduate College, Baikunthpur, Korea, Chhattisgarh, India c
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Ranjan Plant Physiology and Biochemistry Laboratory, Department of Botany, University of Allahabad, Allahabad, India Accepted author version posted online: 30 Jan 2014.
To cite this article: Dharmendra Kumar, Vijay Pratap Singh, Durgesh Kumar Tripathi, Sheo Mohan Prasad & Devendra Kumar Chauhan (2015) Effect of Arsenic on Growth, Arsenic Uptake, Distribution of Nutrient Elements and Thiols in Seedlings of Wrightia arborea (Dennst.) Mabb., International Journal of Phytoremediation, 17:2, 128-134, DOI: 10.1080/15226514.2013.862205 To link to this article: http://dx.doi.org/10.1080/15226514.2013.862205
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International Journal of Phytoremediation, 17: 128–134, 2015 C Taylor & Francis Group, LLC Copyright ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2013.862205
Effect of Arsenic on Growth, Arsenic Uptake, Distribution of Nutrient Elements and Thiols in Seedlings of Wrightia arborea (Dennst.) Mabb. DHARMENDRA KUMAR1, VIJAY PRATAP SINGH2, DURGESH KUMAR TRIPATHI1,#, SHEO MOHAN PRASAD3, and DEVENDRA KUMAR CHAUHAN1 1
D D Pant Interdisciplinary Research Laboratory, Department of Botany, University of Allahabad, Allahabad, India Govt. Ramanuj Pratap Singhdev Post Graduate College, Baikunthpur, Korea, Chhattisgarh, India 3 Ranjan Plant Physiology and Biochemistry Laboratory, Department of Botany, University of Allahabad, Allahabad, India
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Hydroponic experiments were conducted to investigate the effect of arsenic on seedlings of Wrightia arborea and Holoptelea integrifolia. Results revealed that W. arborea could tolerate much higher arsenic concentration than H. integrifolia. Therefore, further investigations were focused on W. arborea using higher arsenic concentrations (0.2–2.0 mM). Seedlings of W. arborea accumulated about 312–2147 and 1048–5688 mg/kg dry weight of arsenic in shoots and roots, respectively, following treatments with 0.2–1.5 mM of arsenic without exhibiting arsenic toxicity signs. However, arsenic at 2.0 mM caused decline in growth. Macronutrients content such as Ca, S (except at 2.0 mM), and K (only in root) increased while Mg, P, and K (shoot) decreased by arsenic treatments. However, the content of micronutrients was enhanced under arsenic treatments. Non-protein thiols (NP-SH) showed positive correlations with arsenic doses up to 0.2–1.5 mM but at 2.0 mM there was a decline in NP-SH thus suggesting important role of NP-SH in imparting arsenic tolerance. This study demonstrated that W. arborea that could tolerate arsenic concentrations up to 0.2–1.5 mM may be useful in arsenic phytoremediation programs. Keywords: arsenic contamination, Holoptelea integrifolia, mineral elements, phytoremediation, Wrightia arborea
Introduction Arsenic contamination of soil and water has become an environmental concern as it poses threat to plants, animals and humans (Acharyya et al. 2000; Chakraborti et al. 2003). Studies have demonstrated that arsenic concentrations in Indian agricultural lands range between 3.34 and 105 mg/kg soils which are much higher than permissible limit (Chakraborti et al. 2003; Patel et al. 2005; Mallick et al. 2011). Besides soil arsenic contamination, level of this metalloid in drinking water is also higher (0.3–0.5 mg/L) than the prescribed limit (0.05 mg/L) (Mazumder et al. 1998; Ahamed et al. 2006). The results of several studies reveal that arsenic adversely affects growth, development, and metabolic process of plants and thus yield (Patel et al. 2005; Li et al. 2006; Mallick et al. 2011). Hence, the problem of arsenic contamination of drinking water and soil has been a global issue. #
Present address: Center of Advance Studies, Department of Botany, Banaras Hindu University, Varanasi-221005, India Address correspondence to Devendra Kumar Chauhan, Department of Botany, University of Allahabad, Allahabad -211002, India. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/bijp.
Vindhyan region (21◦ 29 −25◦ 11 N and 78◦ 15 −84◦ 15 E) covers an area of about 140172 km2 in the central part of India. In this area, the vegetation ranges from herbs to trees. It is one of the largest and the thickest sedimentary successions of the world (Ray 2006) which have been deposited over a very long period of time ranging from Palaeoproterozoic to Neoproterozoic. Das (1977) has shown that Proterozoic shales in the Vindhyan region contain fairly high amount of arsenic in pyrite with a maximum value of 0.26% arsenic at the Amjhore mine. The exposure of pyrite under oxygen and water due to excavation and mining, releases arsenic into acid mines drainage (AMD) (Bednar et al. 2002; Zhu et al. 2008). Natural oxidation of pyrite in Vindhyan region, is continuously adding arsenic but at slower rate due to its limited exposure to oxygen and water. Excessive mining and excavation in the last two decades has resulted in severe problem of arsenic contamination and consequently causes divesting impact on vegetation (Paikaray et al. 2005). Extensive work has been carried out in Gangetic plain and low-lying areas in the Bengal basin of India to investigate the responses of lower vascular plants, crops and vegetables to arsenic. However, to date no attempt has been made to investigate the responses of plants in the Vindhyan region to arsenic despite this region representing the major reserves of arsenic in pyrite bearing shales. Therefore, in the present study we have selected Wrightia
Effect of Arsenic on Seedlings of Wrightia arborea arborea and Holoptelea integrifolia from Vindhyan region and compared their growth behavior under various arsenic concentrations. Further, mineral distribution pattern under various arsenic concentrations and the capability of arsenic accumulation of Wrightia arborea as phytoremediation potential were also studied.
Materials and Methods
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Seed Germination and Growth Conditions Seeds of Wrightia arborea and Holoptelea integrifolia were collected from the forest area of Mirzapur district (Vindhyan region), Uttar Pradesh, India during the period of March 2011 and May 2011, respectively. Before use, uniform sized seeds of both the plants were surface sterilized with 10% (v/v) sodium hypochlorite solution for 10 min, washed with distilled water several times and soaked in distilled water for 4 h. Further, healthy looking uniform sized seeds were wrapped in muslin cloth, and kept in dark for germination. After 5 days, germinated seeds were placed in plastic pots containing fresh half-strength Hoagland solution (pH 6.5) (Arditti and Dunn 1969) in such a way that only radicles of germinated seeds were in contact of Hoagland solution. This was achieved by using a handmade sieve of thermocol having pore size corresponds to the sizes of seeds. The thermocol sieve containing young seedlings was placed just above the nutrient medium level in plastic pot filled with 40 ml fresh medium. The seedlings were grown in a growth chamber under a photon flux density of 250 µmol photons m−2 s−1 and relative humidity of 50–60% with a day/night regime of 12/12 h at 30 ± 2◦ C. After the emergence of first two leaves the seedlings of both the plants were given arsenic treatments. In W. arborea, first two leaves were developed after 25 days of germination, while in H. integrifolia they were emerged after 15 days.
Arsenic Treatments Uniform sized seedlings with fully developed first two leaves were exposed to various concentrations of arsenic (as sodium arsenate; Na2 HAsO4 . 7H2 O: 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1.0, 1.5, and 2.0 mM) for 7 days in a growth chamber. Five plastic pots were arranged for each concentration of arsenic, and in each plastic pot four uniform sized seedlings were gently placed. During arsenic treatments medium of each pot was aerated with sterile air daily to avoid the root anoxia. After 7 days of arsenic treatments seedlings were harvested and various parameters were analyzed.
Determination of Growth and Contents of Arsenic, Nutrient Elements, and Non-protein Thiols For determination of growth (dry weight) arsenic treated and untreated (control) seedlings were harvested, thoroughly washed with distilled water and oven dried at 80◦ C for 24 h and then weighed using digital balance (Contech-CA 223, India).
129 For determination of arsenic, macronutrients (Ca, K, Mg, P, and S) and micronutrients (B, Cu, Fe, Mn, Zn, and Na), 50 mg of each sample of roots and shoot tissues was digested in mixed acid (HNO3 :HClO4 ; 85:15, v/v) until transparent solution was obtained. The volume of digested sample was maintained up to 30 ml with double distilled water. The content of different elements in digested samples was determined by using an inductively coupled argon plasma atomic emission spectrometer. Non-protein thiols (NP-SH) content in root and shoot samples of control and arsenic treated seedlings was estimated according to the method of Ellman (1959). Quality Control The standard reference materials (SRM) were used for the calibration and quality assurance for the heavy metals. Precision and accuracy of heavy metal analysis was ensured with repeated analysis of quality control samples (n = 5) and the results were found within (∼0.5%) of the certified values. The recovery rates for heavy metals were found to be more than 92%. The detection limits for As, Ca, K, Mg, P, S, B, Cu, Fe, Mn, Zn, and Na were found to be 2.33, 0.10, 0.26, 0.14, 0.26, 4.91, 0.32, 0.25, 0.26, 0.03, 0.13, and 0.60 ppb, respectively. Statistical Analysis The results were statistically analyzed by one way ANOVA followed by Duncan’s multiple range test at p < 0.05 significance level to test significance of differences between the control and the treatments. Correlation matrix was also done for sulfur and non-protein thiols content.
Results and Discussion Effect of Arsenic on Growth and Arsenic Accumulation in W. arborea and H. integrifolia Results reveal that W. arborea and H. integrifolia seedlings exhibited differential sensitivity against arsenic stress (Figure 1). W. arborea tolerated arsenic concentration up to 1.5 mM without showing arsenic toxicity signs while H. integrifolia could resist only up to 0.2 mM. The growth of H. integrifolia declined significantly at 0.5 mM arsenic and thereafter arsenic became lethal (Figure 1). Therefore, due to much higher arsenic tolerance capacity we have selected W. arborea for further investigations using higher arsenic concentrations (0.2–2.0 mM). Compare to arsenic treated W. arborea the accumulation of arsenic in roots and shoots of control seedlings was almost in negligible amount (Figure 2a). Seedlings of W. arborea under 0.2, 0.5, 1.0, and 1.5 mM arsenic treatments accumulated about 312, 752, 1521, and 2147 mg arsenic /kg dry weight in shoots, and 1048, 2588, 4765, and 5688 mg arsenic /kg dry weight of roots, respectively, hence exhibited bioconcentration factor >1 without showing arsenic toxicity signs (Figure 2a and b).This suggests that W. arborea which has the ability to grow in the vicinity of arsenic containing shales had developed
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Fig. 1. Effect of different concentrations of arsenic on growth of Wrightia arborea and Holoptelea integrifolia. Each value represents the mean ± SE of three independent experiments. Different letters above bars denote significant differences (P < 0.05) between control and treatments according to the Duncan’s multiple range test. ∗ = plants not survived.
capacity to accumulate high amount of arsenic in its roots and shoots as its survival strategies. It is needful to mention that the characteristics of arsenic hyper accumulator plants are (i) arsenic content >1000 mg/kg dry weight in shoot (ii) bioaccumulation factor >1, sometimes reaching up to 50–100 (Brooks 1998), (iii) translocation factor is >1 (Wei and Zhou 2004), and (iv) greater ability to detoxify and sequester huge amount of arsenic (Rascio and Navari-Izzo 2011). Hence, test plant W. arborea fulfills following criteria i.e. shoot arsenic content greater than 1000 mg/kg dry weight (at 1.0 and 1.5 mM arsenic), bioaccumulation factor greater than 1 reaching up to 21.4 (Figure 2a and b), and greater detoxification of high amount of arsenic in shoots particularly at 1.0 and 1.5 mM arsenic as evidenced by without arsenic toxicity signs in W. arborea seedlings (Figure 1). Loading of arsenic (III) into the xylem is a key step in arsenic translocation from roots to shoots. Ma et al. (2006) have demonstrated the presence of the gene encoding silicon/arsenite effulxer protein Lsi2 (with no similarity to the silicon influx transporter Lsi1), which is mainly responsible for loading of arsenic into the xylem of rice. Taking together the results of Ma et al. (2006), higher arsenic accumulation in roots of W. arborea despite fulfilling some of the criteria of arsenic hyperaccumulator plants i.e. shoot arsenic content >1000 mg/kg dry weight, bioaccumulation factor >1 and efficient arsenic detoxification (up to 1.5 mM) might be attributed to less-efficient functioning of transporter protein responsible for efflux of arsenic to the xylem for translocation to shoots. This assumption may be supported by the fact that W. arborea which is growing on arsenic containing shales might have been in the process of natural selection for surviving in the situation of arsenic contamination in soil in Vindhyan region which has been resulted due to excess mining and excavation. It has been shown that metal hyperaccumulator plants
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Fig. 2. Effect of different concentrations of arsenic on accumulation of arsenic (a) and bioaccumulation factor (b) in Wrightia arborea seedlings. Each value represents the mean ± SE of three independent experiments. Different letters above bars of same color denote significant differences (P < 0.05) between control and treatments according to the Duncan’s multiple range test.
might have been evolved on metal rich soil as a result of selective forces that acted during their evolution together with the specific alleles that were targets of selection (Jim´enez-Ambriz et al. 2007; Kr¨amer 2010; Meyer et al. 2010; Hanikenne and Nouet 2011). Further, Macnair (1993) has also proposed that the metal tolerance in terrestrial plants has generally been evolved by natural selection processes acting on appropriate genetic variability. Arsenic at 2.0 mM concentration produced significant decline in biomass accumulation suggested that at this dose arsenic became toxic for growth of W. arborea (Figure 1). Similar to our result, Mirza et al. (2010) have also reported reduction in growth of Arundo donax at high concentration of arsenic. Hence, decline in biomass accumulation of W. arborea
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Fig. 3. Effect of different concentrations of arsenic on contents of macronutrients in Wrightia arborea seedlings. Each value represents the mean ± SE of three independent experiments. Different letters above bars of same color denote significant differences (P < 0.05) between control and treatments according to the Duncan’s multiple range test.
might have occurred due to toxic property of arsenic on key metabolic process such as photosynthesis, protein synthesis as reported in cork oak (Quercus suber L.), Pinus massoniana, Cryptomeria fortune, Cunninghamia lanceolata and Populus alba (Gogorcena et al. 2011; Liu et al. 2011; Beritognolo et al. 2011). Effect of Arsenic on Distribution of Nutrient Elements in Roots and Shoots Mineral elements (Mg, S, Ca, P, K, Cu, Zn, B, Fe, Mn, Na etc.) play important role in the accumulation of biomass. Effect of arsenic(V) stress may also occur through its impact on homeostasis of essential elements. Therefore, we have examined the consequences of arsenic on nutrient elements distribution in
roots and shoots of W. arborea. Results reveal that accumulation of macronutrients (Ca, K, Mg, P, and S) exhibited differential responses against arsenic exposures (Figure 3). Arsenic treatments increased calcium (Ca) accumulation both in roots and shoots; however, Ca content in roots was higher than that of shoots. Similar to our results, Li et al. (2006) and Mallick et al. (2011) have also noticed increased Ca content in Pteris vittata and Zea mays, respectively under arsenic stress. Similarly, arsenic exposures also significantly increased potassium (K) content in roots while it did not affect K content in shoots (Figure 3). Potassium is also known to serve as a dominant cation to counterbalance anions in plants (Marschner 1995). Thus, under arsenic exposure a significant increase in K content in roots might have taken place as an arrangement to counterbalance anions which were resulted due to excessive
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Sulfur (S) content in roots and shoots of W. arborea significantly increased by arsenic treatments (0.2–1.5 mM) (Figure 3). However, arsenic at 2.0 mM significantly decreased the accumulation of S in roots and shoots (Figure 3). It is known that sulfur is a major component of glutathione, nonprotein thiols (metabolites antioxidants) and phytochelatins (Sakai et al. 2010), therefore, under 0.2–1.5 mM of arsenic treatments increase in S content could be correlated with enhanced metabolites antioxidants as this fact is supported by data of NP-SH. On the other hand, arsenic at 2.0 mM decreased S content in tissues hence this result is correlated with declined NP-SH contents and growth (Figures 1 and 3). The contents of micronutrients (B, Cu, Fe, Mn, Zn, and Na) in tissues of W. arborea were significantly elevated by arsenic treatments (Figure 4). Thus, this study pointed out that arsenic stress has stimulatory effect on accumulation of micronutrients in W. arborea. Mallick et al. (2011) reported that arsenic
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arsenic uptake as suggested by Tu and Ma (2005). Magnesium (Mg) content increased significantly under 0.2–1.5 mM arsenic treatment, however, it declined significantly at 2.0 mM of arsenic. Magnesium being a central atom of chlorophyll and cofactor in many physiological reactions decreased significantly at 2.0 mM arsenic treatment hence decline in growth of W. arborea. Phosphorus (P) is a constituent of nucleic acids, lipids, energy rich compounds, etc. and also plays important role in energy transfer mechanisms (Marschner 1995). Results of the present study reveal that arsenic exposures produced a decreasing impact on P content in roots and shoots of W. arborea (Figure 3). Decline in P uptake may also be correlated with its competitive inhibition by arsenic as this metal acts as analog of phosphate and enters in roots through high affinity phosphate transporter protein (Verbruggen et al. 2009; Indriolo et al. 2010).
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Fig. 4. Effect of different concentrations of arsenic on contents of micronutrients in Wrightia arborea seedlings. Each value represents the mean ± SE of three independent experiments. Different letters above bars of same color denote significant differences (P < 0.05) between control and treatments according to the Duncan’s multiple range test.
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Table 1. Correlation matrix between arsenic treatments, and root and shoot S and NP-SH contents in W. arborea. Arsenic treatments
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exposure to maize seedlings decreased Fe and Mn contents and increased Na content; however, arsenic did not influence on Cu and Zn accumulation. In another finding Tu and Ma (2005) have observed that arsenic exposure to Pteris vittata caused increased B, Zn, Cu, and Mn accumulation, however, with rising arsenic concentration these micronutrients exhibited decreasing trend. This variable response of micronutrient homeostasis against arsenic stress may be attributed to species specific response.
Non-protein Thiols Non-protein thiols (NP-SH) are well known for their property of forming complex with excess metals inside cell and thus protect cell form metal toxicity (Raj et al. 2011). Results reveal that arsenic (0.2–1.5 mM) significantly increased NP-SH content in roots and shoots of W. arborea (Figure 5). However, arsenic exposure at 2.0 mM resulted significant decline in NP-SH content (Figure 5). Correlation matrix also showed positive correlations between arsenic treatments and NP-SH in root (P < 0.01; r = 0.899) and shoot (P < 0.01; r = 0.848) (Figure 1 and Table 1). These results
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The present study is the first effort to investigate arsenic accumulating behavior of plants of Vindhyan region, representing major reserves of arsenic in its shales. Further, this study highlights greater arsenic accumulating potential of W. arborea when grown in hydroponic culture. The results also suggest that status of mineral elements is actively regulated by arsenic stress. Significant increase in S and NP-SH contents during tested doses of arsenic points towards the crucial role of S and NP-SH in imparting tolerance against arsenic stress. Though, roots accumulated higher amount of arsenic than shoots that is why W. arborea does not fulfill one of the criteria (translocation factor) of arsenic hyperaccumulator plants, however, its higher arsenic accumulating capacity may be useful for arsenic phytoremediation program. Further studies at molecular level are needed to verify the higher arsenic accumulating capacity of W. arborea.
Acknowledgments
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clearly show that NP-SH play important role in conferring arsenic tolerance in the range of 0.2–1.5 mM in W. arborea as supported by the data of growth (Figures 1 and 5). However, decrease in growth at 2.0 mM arsenic exposure may be explained on the basis of significant decline in NP-SH content.
Authors are thankful to the Head, Department of Botany, University of Allahabad for providing laboratory facilities. Authors are also thankful to Dr. Dhanwinder Singh, Sr. Soil Scientist, Department of Soil, Punjab Agriculture University, Ludhiana, for providing ICAP-AES facility to determine arsenic and mineral elements.
Funding
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Fig. 5. Effect of different concentrations of arsenic on content of non-protein thiols (NP-SH) in Wrightia arborea seedlings. Each value represents the mean ± SE of three independent experiments. Different letters on line denote significant differences (P < 0.05) between control and treatments according to the Duncan’s multiple range test. Red color line represents NP-SH content in roots while blue color line represents NP-SH content in shoots.
Authors extend their thanks to UGC, New Delhi for financial support.
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D. Kumar et al. Ma JF, Tamai K, Yamaji N, Mitani N, Konishi K, Katsuhara M, Ishiguro M, Murata Y, Yano M. 2006. A silicon transporter in rice. Nature 440:688–691. Macnair MR. 1993. The genetics of metal tolerance in vascular plants. New Phytol 124:541–559. Mallick S, Sinam G, Sinha S. 2011. Study on arsenate tolerant and sensitive cultivars of Zea mays L.: Differential detoxification mechanism and effect on nutrients status. Ecotoxicol Environ Safety 74:1316–1324. Marschner H. 1995. Mineral Nutrition of Higher Plants, second ed. Academic Press Limited, London, UK. Mazumder DNG, Haque R, Ghosh N, De BK, Santra A, Chakraborty D, Smith AH. 1998. Arsenic levels in drinking water and the prevalence of skin lesions in West Bengal, India. Internatl J Epidemiol 27:871–877. Meyer CL, KOstecka AA, Saumitou-Laprade P, Cr´each A, Castric V, Pauwels M, Fr´erot H. 2010. Variability of zinc tolerance among and within populations of the pseudometallophyte species Arabidopsis halleri and possible role of directional selection. New Phytol 185:130–142. Mirza N, Mahmood Q, Pervez A, Ahmad R, Farooq R, Shah MM, Azim, RM. 2010. Phytoremediation potential of Arundo donax in arsenic-contaminated synthetic wastewater. Bioresour Technol 101:5815–5819. Patel KS, Shrivas K, Brandt R, Jakubowski N, Corns W, Hoffmann P. 2005. Arsenic contamination in water, soil, sediment and rice of central India. Environ Geochem Health 27:131–145. Paikaray S, Banerjee S, Mukherji. 2005. Sorption of arsenic onto Vindhyan shales: Role of pyrite and organic carbon. Curr Sci India 88(10):1580–1585. Raj A, Pandey AK, Sharma YK, Khare PB, Srivastava PK, Singh N. 2011. Metabolic adaptation of Pteris vittata L. gametophyte to arsenic induced oxidative stress. Bioresour Technol 102: 9827–9832. Rascio N, Navari-Izzo F. 2011. Heavy metal hyperaccumulating plants: how and why do they do it? And what makes them so interesting? Plant Sci 180:169–181. Ray JS. 2006. Age of the Vindhyan subgroup: a review of recent findings. J Earth Syst Sci 115:149–160. Sakai Y, Watanabe T, Wasaki J, Senoura T, Shinano T, Osaki M. 2010. Influence of arsenic stress on synthesis and localization of low-molecular-weight thiols in Pteris vittata. Environ Pollut 158:3663–3669. Tu C, Ma LQ. 2005. Effects of arsenic on concentration and distribution of nutrients in the fronds of the arsenic hyperaccumulator Pteris vittata L. Environ Pollut 135:333–340. Verbruggen N, Hermans C, Schat H. 2009. Mechanisms to cope with arsenic or cadmium excess in plants. Curr Opin Plant Biol 12:1–9. Wei SH, Zhou QX. 2004. Discussion on basic principles and strengthening measures for phytoremediation of soils contaminated by heavy metals. Chinese J Ecol 23:65–72. Zhu W, Young LY, Yee N, Serfes M, Rhine ED, Reinfelder JR. 2008. Sulfide-driven arsenic mobilization from arsenopyrite and black shale pyrite. Geochimi et Cosmochimi Acta 72:5243–5250.
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Effect of Arsenic on Growth, Arsenic Uptake, Distribution of Nutrient Elements and Thiols in Seedlings of Wrightia arborea (Dennst.) Mabb. a
b
a
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Dharmendra Kumar , Vijay Pratap Singh , Durgesh Kumar Tripathi , Sheo Mohan Prasad & a
Devendra Kumar Chauhan a
D D Pant Interdisciplinary Research Laboratory, Department of Botany, University of Allahabad, Allahabad, India b
Govt. Ramanuj Pratap Singhdev Post Graduate College, Baikunthpur, Korea, Chhattisgarh, India c
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Ranjan Plant Physiology and Biochemistry Laboratory, Department of Botany, University of Allahabad, Allahabad, India Accepted author version posted online: 30 Jan 2014.
To cite this article: Dharmendra Kumar, Vijay Pratap Singh, Durgesh Kumar Tripathi, Sheo Mohan Prasad & Devendra Kumar Chauhan (2015) Effect of Arsenic on Growth, Arsenic Uptake, Distribution of Nutrient Elements and Thiols in Seedlings of Wrightia arborea (Dennst.) Mabb., International Journal of Phytoremediation, 17:2, 128-134, DOI: 10.1080/15226514.2013.862205 To link to this article: http://dx.doi.org/10.1080/15226514.2013.862205
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International Journal of Phytoremediation, 17: 128–134, 2015 C Taylor & Francis Group, LLC Copyright ISSN: 1522-6514 print / 1549-7879 online DOI: 10.1080/15226514.2013.862205
Effect of Arsenic on Growth, Arsenic Uptake, Distribution of Nutrient Elements and Thiols in Seedlings of Wrightia arborea (Dennst.) Mabb. DHARMENDRA KUMAR1, VIJAY PRATAP SINGH2, DURGESH KUMAR TRIPATHI1,#, SHEO MOHAN PRASAD3, and DEVENDRA KUMAR CHAUHAN1 1
D D Pant Interdisciplinary Research Laboratory, Department of Botany, University of Allahabad, Allahabad, India Govt. Ramanuj Pratap Singhdev Post Graduate College, Baikunthpur, Korea, Chhattisgarh, India 3 Ranjan Plant Physiology and Biochemistry Laboratory, Department of Botany, University of Allahabad, Allahabad, India
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Hydroponic experiments were conducted to investigate the effect of arsenic on seedlings of Wrightia arborea and Holoptelea integrifolia. Results revealed that W. arborea could tolerate much higher arsenic concentration than H. integrifolia. Therefore, further investigations were focused on W. arborea using higher arsenic concentrations (0.2–2.0 mM). Seedlings of W. arborea accumulated about 312–2147 and 1048–5688 mg/kg dry weight of arsenic in shoots and roots, respectively, following treatments with 0.2–1.5 mM of arsenic without exhibiting arsenic toxicity signs. However, arsenic at 2.0 mM caused decline in growth. Macronutrients content such as Ca, S (except at 2.0 mM), and K (only in root) increased while Mg, P, and K (shoot) decreased by arsenic treatments. However, the content of micronutrients was enhanced under arsenic treatments. Non-protein thiols (NP-SH) showed positive correlations with arsenic doses up to 0.2–1.5 mM but at 2.0 mM there was a decline in NP-SH thus suggesting important role of NP-SH in imparting arsenic tolerance. This study demonstrated that W. arborea that could tolerate arsenic concentrations up to 0.2–1.5 mM may be useful in arsenic phytoremediation programs. Keywords: arsenic contamination, Holoptelea integrifolia, mineral elements, phytoremediation, Wrightia arborea
Introduction Arsenic contamination of soil and water has become an environmental concern as it poses threat to plants, animals and humans (Acharyya et al. 2000; Chakraborti et al. 2003). Studies have demonstrated that arsenic concentrations in Indian agricultural lands range between 3.34 and 105 mg/kg soils which are much higher than permissible limit (Chakraborti et al. 2003; Patel et al. 2005; Mallick et al. 2011). Besides soil arsenic contamination, level of this metalloid in drinking water is also higher (0.3–0.5 mg/L) than the prescribed limit (0.05 mg/L) (Mazumder et al. 1998; Ahamed et al. 2006). The results of several studies reveal that arsenic adversely affects growth, development, and metabolic process of plants and thus yield (Patel et al. 2005; Li et al. 2006; Mallick et al. 2011). Hence, the problem of arsenic contamination of drinking water and soil has been a global issue. #
Present address: Center of Advance Studies, Department of Botany, Banaras Hindu University, Varanasi-221005, India Address correspondence to Devendra Kumar Chauhan, Department of Botany, University of Allahabad, Allahabad -211002, India. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/bijp.
Vindhyan region (21◦ 29 −25◦ 11 N and 78◦ 15 −84◦ 15 E) covers an area of about 140172 km2 in the central part of India. In this area, the vegetation ranges from herbs to trees. It is one of the largest and the thickest sedimentary successions of the world (Ray 2006) which have been deposited over a very long period of time ranging from Palaeoproterozoic to Neoproterozoic. Das (1977) has shown that Proterozoic shales in the Vindhyan region contain fairly high amount of arsenic in pyrite with a maximum value of 0.26% arsenic at the Amjhore mine. The exposure of pyrite under oxygen and water due to excavation and mining, releases arsenic into acid mines drainage (AMD) (Bednar et al. 2002; Zhu et al. 2008). Natural oxidation of pyrite in Vindhyan region, is continuously adding arsenic but at slower rate due to its limited exposure to oxygen and water. Excessive mining and excavation in the last two decades has resulted in severe problem of arsenic contamination and consequently causes divesting impact on vegetation (Paikaray et al. 2005). Extensive work has been carried out in Gangetic plain and low-lying areas in the Bengal basin of India to investigate the responses of lower vascular plants, crops and vegetables to arsenic. However, to date no attempt has been made to investigate the responses of plants in the Vindhyan region to arsenic despite this region representing the major reserves of arsenic in pyrite bearing shales. Therefore, in the present study we have selected Wrightia
Effect of Arsenic on Seedlings of Wrightia arborea arborea and Holoptelea integrifolia from Vindhyan region and compared their growth behavior under various arsenic concentrations. Further, mineral distribution pattern under various arsenic concentrations and the capability of arsenic accumulation of Wrightia arborea as phytoremediation potential were also studied.
Materials and Methods
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Seed Germination and Growth Conditions Seeds of Wrightia arborea and Holoptelea integrifolia were collected from the forest area of Mirzapur district (Vindhyan region), Uttar Pradesh, India during the period of March 2011 and May 2011, respectively. Before use, uniform sized seeds of both the plants were surface sterilized with 10% (v/v) sodium hypochlorite solution for 10 min, washed with distilled water several times and soaked in distilled water for 4 h. Further, healthy looking uniform sized seeds were wrapped in muslin cloth, and kept in dark for germination. After 5 days, germinated seeds were placed in plastic pots containing fresh half-strength Hoagland solution (pH 6.5) (Arditti and Dunn 1969) in such a way that only radicles of germinated seeds were in contact of Hoagland solution. This was achieved by using a handmade sieve of thermocol having pore size corresponds to the sizes of seeds. The thermocol sieve containing young seedlings was placed just above the nutrient medium level in plastic pot filled with 40 ml fresh medium. The seedlings were grown in a growth chamber under a photon flux density of 250 µmol photons m−2 s−1 and relative humidity of 50–60% with a day/night regime of 12/12 h at 30 ± 2◦ C. After the emergence of first two leaves the seedlings of both the plants were given arsenic treatments. In W. arborea, first two leaves were developed after 25 days of germination, while in H. integrifolia they were emerged after 15 days.
Arsenic Treatments Uniform sized seedlings with fully developed first two leaves were exposed to various concentrations of arsenic (as sodium arsenate; Na2 HAsO4 . 7H2 O: 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1.0, 1.5, and 2.0 mM) for 7 days in a growth chamber. Five plastic pots were arranged for each concentration of arsenic, and in each plastic pot four uniform sized seedlings were gently placed. During arsenic treatments medium of each pot was aerated with sterile air daily to avoid the root anoxia. After 7 days of arsenic treatments seedlings were harvested and various parameters were analyzed.
Determination of Growth and Contents of Arsenic, Nutrient Elements, and Non-protein Thiols For determination of growth (dry weight) arsenic treated and untreated (control) seedlings were harvested, thoroughly washed with distilled water and oven dried at 80◦ C for 24 h and then weighed using digital balance (Contech-CA 223, India).
129 For determination of arsenic, macronutrients (Ca, K, Mg, P, and S) and micronutrients (B, Cu, Fe, Mn, Zn, and Na), 50 mg of each sample of roots and shoot tissues was digested in mixed acid (HNO3 :HClO4 ; 85:15, v/v) until transparent solution was obtained. The volume of digested sample was maintained up to 30 ml with double distilled water. The content of different elements in digested samples was determined by using an inductively coupled argon plasma atomic emission spectrometer. Non-protein thiols (NP-SH) content in root and shoot samples of control and arsenic treated seedlings was estimated according to the method of Ellman (1959). Quality Control The standard reference materials (SRM) were used for the calibration and quality assurance for the heavy metals. Precision and accuracy of heavy metal analysis was ensured with repeated analysis of quality control samples (n = 5) and the results were found within (∼0.5%) of the certified values. The recovery rates for heavy metals were found to be more than 92%. The detection limits for As, Ca, K, Mg, P, S, B, Cu, Fe, Mn, Zn, and Na were found to be 2.33, 0.10, 0.26, 0.14, 0.26, 4.91, 0.32, 0.25, 0.26, 0.03, 0.13, and 0.60 ppb, respectively. Statistical Analysis The results were statistically analyzed by one way ANOVA followed by Duncan’s multiple range test at p < 0.05 significance level to test significance of differences between the control and the treatments. Correlation matrix was also done for sulfur and non-protein thiols content.
Results and Discussion Effect of Arsenic on Growth and Arsenic Accumulation in W. arborea and H. integrifolia Results reveal that W. arborea and H. integrifolia seedlings exhibited differential sensitivity against arsenic stress (Figure 1). W. arborea tolerated arsenic concentration up to 1.5 mM without showing arsenic toxicity signs while H. integrifolia could resist only up to 0.2 mM. The growth of H. integrifolia declined significantly at 0.5 mM arsenic and thereafter arsenic became lethal (Figure 1). Therefore, due to much higher arsenic tolerance capacity we have selected W. arborea for further investigations using higher arsenic concentrations (0.2–2.0 mM). Compare to arsenic treated W. arborea the accumulation of arsenic in roots and shoots of control seedlings was almost in negligible amount (Figure 2a). Seedlings of W. arborea under 0.2, 0.5, 1.0, and 1.5 mM arsenic treatments accumulated about 312, 752, 1521, and 2147 mg arsenic /kg dry weight in shoots, and 1048, 2588, 4765, and 5688 mg arsenic /kg dry weight of roots, respectively, hence exhibited bioconcentration factor >1 without showing arsenic toxicity signs (Figure 2a and b).This suggests that W. arborea which has the ability to grow in the vicinity of arsenic containing shales had developed
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Fig. 1. Effect of different concentrations of arsenic on growth of Wrightia arborea and Holoptelea integrifolia. Each value represents the mean ± SE of three independent experiments. Different letters above bars denote significant differences (P < 0.05) between control and treatments according to the Duncan’s multiple range test. ∗ = plants not survived.
capacity to accumulate high amount of arsenic in its roots and shoots as its survival strategies. It is needful to mention that the characteristics of arsenic hyper accumulator plants are (i) arsenic content >1000 mg/kg dry weight in shoot (ii) bioaccumulation factor >1, sometimes reaching up to 50–100 (Brooks 1998), (iii) translocation factor is >1 (Wei and Zhou 2004), and (iv) greater ability to detoxify and sequester huge amount of arsenic (Rascio and Navari-Izzo 2011). Hence, test plant W. arborea fulfills following criteria i.e. shoot arsenic content greater than 1000 mg/kg dry weight (at 1.0 and 1.5 mM arsenic), bioaccumulation factor greater than 1 reaching up to 21.4 (Figure 2a and b), and greater detoxification of high amount of arsenic in shoots particularly at 1.0 and 1.5 mM arsenic as evidenced by without arsenic toxicity signs in W. arborea seedlings (Figure 1). Loading of arsenic (III) into the xylem is a key step in arsenic translocation from roots to shoots. Ma et al. (2006) have demonstrated the presence of the gene encoding silicon/arsenite effulxer protein Lsi2 (with no similarity to the silicon influx transporter Lsi1), which is mainly responsible for loading of arsenic into the xylem of rice. Taking together the results of Ma et al. (2006), higher arsenic accumulation in roots of W. arborea despite fulfilling some of the criteria of arsenic hyperaccumulator plants i.e. shoot arsenic content >1000 mg/kg dry weight, bioaccumulation factor >1 and efficient arsenic detoxification (up to 1.5 mM) might be attributed to less-efficient functioning of transporter protein responsible for efflux of arsenic to the xylem for translocation to shoots. This assumption may be supported by the fact that W. arborea which is growing on arsenic containing shales might have been in the process of natural selection for surviving in the situation of arsenic contamination in soil in Vindhyan region which has been resulted due to excess mining and excavation. It has been shown that metal hyperaccumulator plants
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Fig. 2. Effect of different concentrations of arsenic on accumulation of arsenic (a) and bioaccumulation factor (b) in Wrightia arborea seedlings. Each value represents the mean ± SE of three independent experiments. Different letters above bars of same color denote significant differences (P < 0.05) between control and treatments according to the Duncan’s multiple range test.
might have been evolved on metal rich soil as a result of selective forces that acted during their evolution together with the specific alleles that were targets of selection (Jim´enez-Ambriz et al. 2007; Kr¨amer 2010; Meyer et al. 2010; Hanikenne and Nouet 2011). Further, Macnair (1993) has also proposed that the metal tolerance in terrestrial plants has generally been evolved by natural selection processes acting on appropriate genetic variability. Arsenic at 2.0 mM concentration produced significant decline in biomass accumulation suggested that at this dose arsenic became toxic for growth of W. arborea (Figure 1). Similar to our result, Mirza et al. (2010) have also reported reduction in growth of Arundo donax at high concentration of arsenic. Hence, decline in biomass accumulation of W. arborea
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might have occurred due to toxic property of arsenic on key metabolic process such as photosynthesis, protein synthesis as reported in cork oak (Quercus suber L.), Pinus massoniana, Cryptomeria fortune, Cunninghamia lanceolata and Populus alba (Gogorcena et al. 2011; Liu et al. 2011; Beritognolo et al. 2011). Effect of Arsenic on Distribution of Nutrient Elements in Roots and Shoots Mineral elements (Mg, S, Ca, P, K, Cu, Zn, B, Fe, Mn, Na etc.) play important role in the accumulation of biomass. Effect of arsenic(V) stress may also occur through its impact on homeostasis of essential elements. Therefore, we have examined the consequences of arsenic on nutrient elements distribution in
roots and shoots of W. arborea. Results reveal that accumulation of macronutrients (Ca, K, Mg, P, and S) exhibited differential responses against arsenic exposures (Figure 3). Arsenic treatments increased calcium (Ca) accumulation both in roots and shoots; however, Ca content in roots was higher than that of shoots. Similar to our results, Li et al. (2006) and Mallick et al. (2011) have also noticed increased Ca content in Pteris vittata and Zea mays, respectively under arsenic stress. Similarly, arsenic exposures also significantly increased potassium (K) content in roots while it did not affect K content in shoots (Figure 3). Potassium is also known to serve as a dominant cation to counterbalance anions in plants (Marschner 1995). Thus, under arsenic exposure a significant increase in K content in roots might have taken place as an arrangement to counterbalance anions which were resulted due to excessive
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Sulfur (S) content in roots and shoots of W. arborea significantly increased by arsenic treatments (0.2–1.5 mM) (Figure 3). However, arsenic at 2.0 mM significantly decreased the accumulation of S in roots and shoots (Figure 3). It is known that sulfur is a major component of glutathione, nonprotein thiols (metabolites antioxidants) and phytochelatins (Sakai et al. 2010), therefore, under 0.2–1.5 mM of arsenic treatments increase in S content could be correlated with enhanced metabolites antioxidants as this fact is supported by data of NP-SH. On the other hand, arsenic at 2.0 mM decreased S content in tissues hence this result is correlated with declined NP-SH contents and growth (Figures 1 and 3). The contents of micronutrients (B, Cu, Fe, Mn, Zn, and Na) in tissues of W. arborea were significantly elevated by arsenic treatments (Figure 4). Thus, this study pointed out that arsenic stress has stimulatory effect on accumulation of micronutrients in W. arborea. Mallick et al. (2011) reported that arsenic
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arsenic uptake as suggested by Tu and Ma (2005). Magnesium (Mg) content increased significantly under 0.2–1.5 mM arsenic treatment, however, it declined significantly at 2.0 mM of arsenic. Magnesium being a central atom of chlorophyll and cofactor in many physiological reactions decreased significantly at 2.0 mM arsenic treatment hence decline in growth of W. arborea. Phosphorus (P) is a constituent of nucleic acids, lipids, energy rich compounds, etc. and also plays important role in energy transfer mechanisms (Marschner 1995). Results of the present study reveal that arsenic exposures produced a decreasing impact on P content in roots and shoots of W. arborea (Figure 3). Decline in P uptake may also be correlated with its competitive inhibition by arsenic as this metal acts as analog of phosphate and enters in roots through high affinity phosphate transporter protein (Verbruggen et al. 2009; Indriolo et al. 2010).
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Fig. 4. Effect of different concentrations of arsenic on contents of micronutrients in Wrightia arborea seedlings. Each value represents the mean ± SE of three independent experiments. Different letters above bars of same color denote significant differences (P < 0.05) between control and treatments according to the Duncan’s multiple range test.
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Table 1. Correlation matrix between arsenic treatments, and root and shoot S and NP-SH contents in W. arborea. Arsenic treatments
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1 0.977∗∗ 0.864∗∗ 0.848∗∗ 0.899∗∗
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(r) is significant at p < 0.01 significance level.
exposure to maize seedlings decreased Fe and Mn contents and increased Na content; however, arsenic did not influence on Cu and Zn accumulation. In another finding Tu and Ma (2005) have observed that arsenic exposure to Pteris vittata caused increased B, Zn, Cu, and Mn accumulation, however, with rising arsenic concentration these micronutrients exhibited decreasing trend. This variable response of micronutrient homeostasis against arsenic stress may be attributed to species specific response.
Non-protein Thiols Non-protein thiols (NP-SH) are well known for their property of forming complex with excess metals inside cell and thus protect cell form metal toxicity (Raj et al. 2011). Results reveal that arsenic (0.2–1.5 mM) significantly increased NP-SH content in roots and shoots of W. arborea (Figure 5). However, arsenic exposure at 2.0 mM resulted significant decline in NP-SH content (Figure 5). Correlation matrix also showed positive correlations between arsenic treatments and NP-SH in root (P < 0.01; r = 0.899) and shoot (P < 0.01; r = 0.848) (Figure 1 and Table 1). These results
NP-SH content (µmol g -1 dry weight)
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∗∗ correlation
220 Root Shoot b
200 180 160
a a c
c d
60
The present study is the first effort to investigate arsenic accumulating behavior of plants of Vindhyan region, representing major reserves of arsenic in its shales. Further, this study highlights greater arsenic accumulating potential of W. arborea when grown in hydroponic culture. The results also suggest that status of mineral elements is actively regulated by arsenic stress. Significant increase in S and NP-SH contents during tested doses of arsenic points towards the crucial role of S and NP-SH in imparting tolerance against arsenic stress. Though, roots accumulated higher amount of arsenic than shoots that is why W. arborea does not fulfill one of the criteria (translocation factor) of arsenic hyperaccumulator plants, however, its higher arsenic accumulating capacity may be useful for arsenic phytoremediation program. Further studies at molecular level are needed to verify the higher arsenic accumulating capacity of W. arborea.
Acknowledgments
b
b
100 80
b
a
120
Conclusion
a
b
140
clearly show that NP-SH play important role in conferring arsenic tolerance in the range of 0.2–1.5 mM in W. arborea as supported by the data of growth (Figures 1 and 5). However, decrease in growth at 2.0 mM arsenic exposure may be explained on the basis of significant decline in NP-SH content.
Authors are thankful to the Head, Department of Botany, University of Allahabad for providing laboratory facilities. Authors are also thankful to Dr. Dhanwinder Singh, Sr. Soil Scientist, Department of Soil, Punjab Agriculture University, Ludhiana, for providing ICAP-AES facility to determine arsenic and mineral elements.
Funding
40 0
0.2 0.5 1.0 1.5 2.0 As concentrations (mM)
Fig. 5. Effect of different concentrations of arsenic on content of non-protein thiols (NP-SH) in Wrightia arborea seedlings. Each value represents the mean ± SE of three independent experiments. Different letters on line denote significant differences (P < 0.05) between control and treatments according to the Duncan’s multiple range test. Red color line represents NP-SH content in roots while blue color line represents NP-SH content in shoots.
Authors extend their thanks to UGC, New Delhi for financial support.
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