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Pongamia pinnata (L.) Pierre tree seedlings offer a model species for arsenic phytoremediation
Accepted Manuscript Pongamia pinnata (L.) pierre tree seedlings offer a model species for arsenic phytoremediation
Dharmendra Kumar, Durgesh Kumar Tripathi, Shiliang Liu, Vivek Kumar Singh, Shivesh Sharma, Nawal Kishore Dubey, Sheo Mohan Prasad, Devendra Kumar Chauhan PII: DOI: Reference:
S2352-4073(17)30035-5 doi: 10.1016/j.plgene.2017.06.002 PLGENE 111
To appear in:
Plant Gene
Received date: Revised date: Accepted date:
31 December 2016 5 June 2017 8 June 2017
Please cite this article as: Dharmendra Kumar, Durgesh Kumar Tripathi, Shiliang Liu, Vivek Kumar Singh, Shivesh Sharma, Nawal Kishore Dubey, Sheo Mohan Prasad, Devendra Kumar Chauhan , Pongamia pinnata (L.) pierre tree seedlings offer a model species for arsenic phytoremediation, Plant Gene (2017), doi: 10.1016/ j.plgene.2017.06.002
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ACCEPTED MANUSCRIPT Title: Pongamia pinnata (L.) Pierre tree seedlings offer a model species for arsenic phytoremediation Authors: Dharmendra Kumar1, Durgesh Kumar Tripathi2*, Shiliang Liu3, Vivek Kumar Singh4,
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Shivesh Sharma2,5, Nawal Kishore Dubey6, Sheo Mohan Prasad7*, Devendra Kumar Chauhan1*
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Affiliation: 1D D Pant Interdisciplinary Research Laboratory, Department of Botany, University
Centre for Medical Diagnostic and Research, Motilal Nehru National Institute of Technology,
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2
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of Allahabad, Allahabad, 211002, India.
Allahabad, India
Division of Plant Sciences, University of Missouri, Columbia, MO 65211 USA
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Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
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Department of Biotechnology, Motilal Nehru National Institute of Technology, Allahabad, India
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Center of Advanced Study in Botany, Banaras Hindu University, Varanasi, 221005, India
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Ranjan Plant Physiology and Biochemistry Laboratory, Department of Botany, University of
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3
Allahabad, Allahabad 211002, India
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Abstract
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Email: [email protected], [email protected], [email protected]
Hydroponic experiments were conducted to investigate the effect of different concentrations of arsenic (As) on seedlings of Pongamia pinnata (L.). Results revealed that P. pinnata successfully tolerated As up to 0.2-1.0 mM and did not show significant reduction in growth and chlorophyll contents. However, these parameters significantly reduced at 1.5 mM of As exposure. Further, P. pinnata accumulated As about 528-1122 mg kg-1 dry weight in roots,
ACCEPTED MANUSCRIPT and 167-792 mg kg-1 dry weight in shoots, respectively when exposed to 0.2-2.0 mM As. Arsenic exposure at 0.2-1.5 mM significantly enhanced oxidative stress markers [superoxide radical (SOR), hydrogen peroxide (H2O2) and malondialdehyde (MDA)], activities of antioxidants [ascorbate peroxidase (APX) and catalase (CAT)] and nutrient accumulation.
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Increase of antioxidants and nutrient accumulation might have protected plants from As
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exposure. Additionally, bioaccumulation and translocation factors were > 1 which also
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confirmed that test plant had greater ability to accumulate and detoxify greater amount of As.
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Keywords: Arsenic contamination, mineral elements, phytoremediation
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Abbreviations: F0, minimal fluorescence; Fv/Fm, maximum photochemical efficiency of PS II; Fv/F0, the activity of PS II; qP, photochemical quenching; NPQ, nonphotochemical quenching;
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APX, ascorbate peroxidase; CAT, catalase; ROS, reactive oxygen species; SOR, superoxide
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radical; H2O2, hydrogen peroxide; MDA, malondialdehyde.
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1. Introduction
Among toxic elements, arsenic (As) is considered as one of the most toxic metalloids for
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all living organisms caused greater threat to human beings (Nriagu 2002). It is a universal
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metalloid which is commonly present in the environment and released from various anthropogenic and geochemical sources in two forms, i.e. AsIII and AsV (Smith et al. 1998). In relation to plants, it has been well documented that both forms of As are most toxic and adversely affected the growth, yield and quality of plants as well as other internal integrities (Kumar et al. 2015; Tripathi et al., 2016a). On the other hand for human beings it causes various serious diseases like cancer when enters in to the food chain and causes inevitable poisoning. Its exposure also leads to alteration in neuro-transmitter level, thus As toxicity is a serious
ACCEPTED MANUSCRIPT worldwide human concern. In India mostly in southern West Bengal, arsenic contamination has become a serious environmental problem that is posing threat to living beings (Acharyya et al. 2000; Shah 2010). According to WHO guidelines which have been endorsed by the Bureau of Indian Standards, the upper permissible limit of arsenic in ground water was considered as 0.05
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mg/l (WHO 1993; BIS 2003). However, due to higher toxic nature of arsenic, drinking water
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arsenic contamination was reexamined, and the previous level of arsenic in drinking water (0.05
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mg/l) was lowered to 0.01 mg/l. In India, the level of arsenic in drinking water ranges between 0.3-0.5 mg/l in arsenic-contaminated regions which is much above the prescribed limit (Ahamed
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et al. 2006). Studies showed that arsenic contamination in agricultural land of Indo-Gangetic
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flood plain and deltaic regions of West Bengal, and parts of Bihar, Assam, Chhattisgarh and Eastern Uttar Pradesh (Indian Provinces) ranges between 3.34-105 mg/kg soil (Chauhan et al.
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2009; Mallick et al. 2011). In West Bengal, after diagnosis of first case of arsenic poisoning in
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1983, six million people have been affected by arsenic toxicity through drinking water (Bhattacharyya et al. 2004). However, in Bangladesh, arsenic contamination was first reported in
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1993 (Acharyya et al. 2000). Thus, in terms of number of people exposed (30-35 million), it is
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the most serious problem of groundwater arsenic contamination in the world (Acharyya et al. 2000; Bhattacharyya et al. 2004; Ye et al. 2011). Besides, India and Bangladesh, arsenic
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contamination in groundwater has also been reported from different countries of world (Shah 2010). Thus, the problem of arsenic contamination of drinking water and soil is common in entire world including India. In India the regions for arsenic contamination of groundwater is mostly the presence of the Ganges delta and some low-lying areas in the Bengal basin that have been investigated (Acharyya et al. 2000; Shah 2010). Practices of excessive extraction of groundwater for
ACCEPTED MANUSCRIPT irrigation and application of phosphate fertilizers have promoted groundwater flow and phosphate mobilization from fertilizers as well as from decayed organic matter had aided the further release of arsenic. For removal of As contamination from soil, water and environmental field there are several physical and chemical techniques which are being used, are costly and also
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not environmental friendly. Beside this, phytoremediation which is known as green technology
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for the removal of heavy metals is being used from last few decades (Dhir, 2013; Jasrotia et al.,
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2015). In this technique some aquatic and terrestrial trees have been tested for the removal of heavy metal contamination including arsenic and some significant results have been found (Dhir,
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2013; Jasrotia et al., 2015). Thus to establish this technique as a most effective and reliable,
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several studies are being regularly carried out in the renowned laboratories all over the world
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(Dhir, 2013; Kumar et al., 2014; Jasrotia et al., 2015).
The choice of plants is a crucial aspect for the practical use of phytoremediation
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technique because it has been well documented that all species are not proficient to accumulate
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and store the metals in their tissues (Tu and Ma 2005; Kacálková et al. 2015). Most hyperaccumulators plants have small biomass and have slow growing behavior, however, some
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woody species have special significance because of their speedy growth, presence of deep roots
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and rich biomass production and thus showed high tolerance capacity towards heavy metals and fit for phytoremidaition (Huang et al. 2011; Hu et al. 2014). Although, extensive work has been done to examine the responses of plants growing in the As contaminated areas. However, no attempt has been made yet to explore the responses of long lived tree plants of Vindhyan region. Thus, in the present study one woody plant (Pongamia pinnata) frequently grown in the Vindhyan region was selected and its arsenic tolerance
ACCEPTED MANUSCRIPT capacities were studied by analyzing the antioxidant activity, mineral regulation, oxidative stress response and chlorophyll fluorescence. 2. Material and methods 2.1. Seed germination and growth conditions
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Pods of Pongamia pinnata were collected from the forest area of Mirzapur district
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(Vindhyan region), Uttar Pradesh, India during the month of May 2013, and seeds were extracted manually. Healthy and uniform sized seeds were surface sterilized with 10% (v/v) sodium
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hypochlorite solution for 10 min, washed with distilled water several times and soaked in distilled water for 4 h. Further, seeds were wrapped in muslin cloth, and kept in the dark for
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germination. After 12 days, germinated seeds were sown in plastic pots containing fresh half-
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strength Hoagland solution (pH 6.5) (Arditti and Dunn 1969) in such a way that only radicals of germinated seeds were in contact with Hoagland solution. This was achieved by using a
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handmade sieve of thermocol having pore size corresponding to the size of seeds. The thermocol
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sieve containing young seedlings was placed just above the nutrient medium level in plastic pot filled with 50 ml fresh nutrient medium. The seedlings were grown in a growth chamber under a
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photon flux density of 250 µmol photons m-2 s-1 and relative humidity of 50-60% with a
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day/night regime of 12/12 h at 30±2 oC. After the emergence of first two leaves the seedlings were treated with tested doses of arsenic. 2.2. Arsenic treatments Uniform sized seedlings with fully developed first two leaves were exposed to various concentrations (as sodium arsenate; Na2HAsO4. 7H2O: 0, 0.2, 0.5, 1.0, 1.5 and 2.0 mM) of arsenic for 7 days. Three plastic pots were arranged for each concentration of arsenic, and in each plastic pot three uniform sized seedlings were gently placed. During arsenic treatments
ACCEPTED MANUSCRIPT 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. 2.3. Determination of growth parameters Growth parameter such as length, fresh mass, and dry mass of roots and shoots of treated
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and untreated seedlings were measured after harvesting. For dry mass, root and shoot were
balance (Contech- CA 223, India).
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2.4. Determination of photosynthetic pigment contents
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wrapped in butter paper and oven dried for 48 h at 65–75 °C and then weighed using digital
The amounts of chlorophyll a, b (Chl a and Chl b) and carotenoids (Car) were determined
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2.5. Determination of total protein content
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according to the method of Lichtenthaler (1987).
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For the measurement of total protein, the method of Lowry et al. (1951) was adopted using bovine serum albumin as standard.
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assessment
of
photosynthetic
performance,
chlorophyll
fluorescence
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For
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2.6. Chlorophyll fluorescence measurements
measurements were taken in dark adapted leaves of control and treated seedlings using hand held
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leaf fluorometer (FluorPen FP 100, Photos System Instrument, Czech Republic). 2.7. Determination of As and mineral nutrient contents in tissues For determination of arsenic, macronutrients (Ca, K, Mg, P and S) and micronutrients (B, Cu, Fe, Mn, Zn and Na), 50 mg of root and shoot tissues of each sample 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
ACCEPTED MANUSCRIPT in digested samples was determined by using an Inductively Coupled Argon Plasma - Atomic Emission Spectrometer (ICAP-AES). 2.7. Determination of superoxide radical and hydrogen peroxide contents Superoxide radical (SOR; O2•¯) in each sample was determined following the method of
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Elstner and Heupel (1976). This assay is based on formation of NO2 from hydroxylamine in the
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presence of O2•¯. The absorbance of the colored aqueous phase was recorded at 530 nm. A
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standard curve was prepared with NaNO2 and used to calculate the amount of O2•¯ in each
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sample.
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Determination of H2O2 was done according to the method of Velikova et al. (2000). Absorbance of reaction mixture was recorded at 390 nm. The hydrogen peroxide concentration
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was calculated by using a standard curve prepared with graded solution of H2O2.
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2.8. Determination of lipid peroxidation
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The lipid peroxidation as malondialdehyde (MDA) content was estimated according to the method of Heath and Packer (1968). The content of MDA was determined by using an
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extinction coefficient of 155 mM-1 cm-1.
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2.9. Determination of CAT and APX activity and NP-SH content Catalase (CAT; EC 1.11.3.6) activity was determined in terms of decrease in absorbance due to dissociation of H2O2 according to the method of Aebi (1984). Ascorbate peroxidase (APX; EC 1.11.1.11) activity was deter-mined according to the method of Nakano and Asada (1981). The non-protein thiols (NP-SH) content was measured following the method of Ellman (1959).
ACCEPTED MANUSCRIPT 2.10. 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. Linear correlation matrix was also done among mineral elements
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contents in roots and shoots of P. pinnata seedlings, under different As concentrations.
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3. Results
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3.1. Impact of arsenic on growth and photosynthetic pigments (chlorophylls and carotenoids) and protein contents
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Growth parameters of P. pinnata were measured in terms of length, fresh mass and dry
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mass of root and shoot. Data pertaining to the growth parameters showed that the lower concentration (0.2 and 0.5 mM) of arsenic affected them marginally as length of root was
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lowered by 4 and 5% and shoot by 4 and 7% at 0.2 and 0.5 mM As, respectively (Fig.1 A).
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Likewise, at 0.2 and 0.5 mM of As fresh mass was also lowered by 3 and 4% in root and by 3 and 1% in shoot, and corresponding decrease in dry mass was 3 and 7% in root and 1, and 2% in
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shoot, respectively (Fig.1 B, C). The higher doses (1.0, 1.5 and 2.0 mM) of As significantly
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declined the length by 9, 24, 66% in root and 13, 17 and 62% in shoot, respectively. Under similar condition, fresh mass was declined by 5, 14 and 49% in root and by 2, 7 and 70% in
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shoot, respectively, and a similar pattern in decrease in dry mass was also recorded in root and shoot of tested seedlings (Fig.1 A-C) Further, correlation coefficient analyses between different As treatments and growth parameters were also performed to statistically authenticate the data set which shows the negative correlation like length (root, r = -0.987, p = 0.002; shoot, r = -0.951, p = 0.013), fresh mass (root, r = -0.989, p = 0.001; shoot, r = -0.962, p = 0.009), and dry mass (root, r = -0.993, p
ACCEPTED MANUSCRIPT = 0.001; shoot, r = -0.993, p = 0.001) (Table 1). The results of the growth parameters clearly confirmed that P. pinnata may survive under 0.2–1.0 mM As stress without showing toxicity symptoms when compared to the control (Fig. 1A-C). However, at 2.0 mM concentration of arsenic plants were not able to survive, thus we have examined further only 0.1-1.5 mM As
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treatments. Therefore, due to much higher arsenic tolerance capability we have selected
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seedlings of P. pinnata for further investigations under different parameters using arsenic
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concentrations of 0.2-1.5 mM.
Data pertaining to the photosynthetic pigments, i.e. Chl a and b and Car are depicted in
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Table 2. Similar to growth, the data of photosynthetic pigments also suggested that at the lower
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concentration (0.2 and 0.5 mM) of As, seedlings showed marginal impact, however higher doses (1.0 and 1.5 mM) of As significantly declined content of Chl a by 19 and 31% and Chl b by 17
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and 33%, respectively, hence resulted into increased Chl a to Chl b ratio (Table 2). Under similar
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As treatments (1.0 and 1.5 mM), Car contents were reduced by 13 and 20% while Chl (a+b)/ Car ratios were declined by 17 and 24%, respectively (Table 2).
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Further, upon exposure of seedlings with 1.0 and 1.5 mM As total protein content was
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declined by 15 and 23% in root and 17 and 29% in shoot, respectively over the values of respective control while at 0.2 and 0.5 mM the effect on protein was marginal (Table 2).
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Correlation coefficient analysis between different As treatments and photosynthetic pigments; Chl a (r = -0.996, p = 0.001), Chl b (r = -0.993, p = 0.001) and Car (r = -0.990, p = 0.001), and total protein content in root (r = -0.991, p = 0.001) and shoot (r = -0.976, p = 0.004) showed negative correlation (Table 1). 3.2. Arsenic accumulation in P. pinnata
ACCEPTED MANUSCRIPT The seedlings of P. pinnata accumulated 1129, 1442, 1789, and 3322 mg As kg−1 dry weight in root and in shoot it was 345, 537, 1677, and 3662 mg kg−1 dry weight, respectively, under 0.2, 0.5, 1.0, and 1.5 mM As treatments (Fig. 1D). In addition to this, correlation coefficient analysis between different As treatments and As content in root (r = 0.955, p = 0.012) and shoot (r =
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0.968, p = 0.007)) showed positive correlation, hence exhibited bioaccumulation factor (BF) and
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translocation factor (TF) >1 without showing arsenic toxicity signs (Fig. 1E, F; Table 1).
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3.3. Impact of arsenic on photochemistry of PS II
The results pertaining to changes in photochemistry of PS II measured as chlorophyll
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fluorescence parameters such as Fv/Fm, Fv/F0, Fm/F0, qP and NPQ in the presence of arsenic
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treatments are depicted in Fig. 2A-B. Arsenic at 0.2, 0.5, 1.0, and 1.5 mM declined (p < 0.05) Fv/Fm by 3, 5, 41 and 63%, Fv/F0 by 6, 9, 55 and 72%, Fm/F0 by 8, 11, 53 and 62% and qP by 2,
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4, 50 and 56%, respectively (Fig. 2A, B). In contrast, NPQ was raised (p < 0.05) by 6, 12, 76,
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and 109% under 0.2, 0.5, 1.0, and 1.5 mM As exposure, respectively as compared to respective control (Fig. 2B). Further, correlation coefficient analysis among As treatments and
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photosynthetic performance showed negative correlation; Fv/Fm (r = -0.986, p = 0.002), Fv/F0 (r
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= -0.959, p = 0.010), Fm/F0 (r = -0.962, p = 0.009) and qP (r = -0.919, p = 0.027) except NPQ that exhibited positive correlation i.e. r = 0.992, p = 0.001 (Table 1).
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3.4. Impact of arsenic on oxidative stress markers Superoxide radical (SOR), hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents in root and shoot were analyzed to record the level of oxidative stress in P. pinnata under different As treatments (Fig. 3A-C). Data revealed that under 0.2, 0.5, 1.0, and 1.5 mM As treatments SOR content was significantly raised (p < 0.05) in root by 15, 41, 111 and 148% and in shoot by 24, 52, 91 and 115% (Fig.3A) and under similar exposure, H2O2 content in root was
ACCEPTED MANUSCRIPT increased by 11, 22, 32 and 44% and in shoot by 3, 23, 30 and 44%, respectively (Fig. 3B). Lipid peroxidation measured as MDA content was increased by 15, 50, 82, and 120% in root and by 31, 58, 125 and 175% in shoot, respectively over the value of control (Fig.3C). The correlation coefficient analysis between As treatments and oxidative stress markers; SOR (root, r = 0.993, p
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= 0.001; shoot, r = 0.985, p = 0.002), H2O2 (root, r = 0.983, p = 0.003; shoot, r = 0.977, p =
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0.004) and MDA (root, r = 0.996, p = 0.001; shoot, r = 0.999, p = 0.001) showed positive
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correlation (Table 1).
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3.5. Effect of arsenic on distribution of nutrients in roots and shoots
Results showed that arsenic treatments increased the contents of macronutrients Ca, Mg,
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K and S in root from 58183–230477; 37866–69417; 12697-32751; 6899573–9786445 mg kg−1
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dry weight and in shoot from 20567–28642; 8731–20778; 34971-50924; 891870–1999114 mg kg−1 dry weight respectively when the concentration of arsenic in nutrient solution was raised
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from 0.2-1.5 mM (Fig. 4A-D). Further correlations analysis between the macronutrient contents
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and different As treatments showed positive correlation: Ca (root, r = 0.894, p = 0.041; shoot, r = 0.926, p = 0.024), Mg (root, r = 0.779, p = 0.120; shoot, r = 0.870, p = 0.055), and S (root, r =
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0.882, p = 0.048; shoot, r = 0.975, p = 0.005), and K (root, r = 0.974, p = 0.005) contents showed
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positive correlation but in shoot K presented negative correlation (r = -0.905, p = 0.034) under different As treatments. The levels of micronutrients regulation under different As treatments (0.2-1.5 mM) in tested plants were also analyzed which showed that the levels of Fe, Mn, Zn, and Na were increased from 2304–12066, 1661–6014, 473–2686, and 382435–1539142 in roots and 910– 1088, 279–407, 139–315, and 99394–144393 mg kg−1 in shoots, respectively (Fig. 4E-H). The systematic correlation analysis between the micronutrient contents and different As treatments
ACCEPTED MANUSCRIPT suggest that there was positive correlation: Mn (root, r = 0.904, p = 0.035; shoot, r = 0.956, p = 0.011), Zn (root, r = 0.977, p = 0.004; shoot, r = 0.973, p = 0.005), Na (root, r = 0.872, p = 0.054; shoot, r = 0.923, p = 0.025) and Fe (root, r = 0.919, p = 0.027; shoot, r = 0.958, p = 0.010), Table 3 also represents the relationship among nutrient contents affected by As stress and
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how they manage themselves to protect the plant from As toxicity.
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3.6. Impact of arsenic on antioxidant activity and non-protein thiols
The results pertaining to antioxidants (APX and CAT) showed that due to different
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arsenic treatments the activity of APX was increased by 2, 6, 14 and 25% in shoot and 3, 7, 15
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and 28% in root, respectively (Fig. 5A and B). Similarly, at the similar concentrations of As CAT activity was increased by 3, 8, 18 and 53% in root and 2, 6, 12 and 18 % in shoots
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respectively (Fig. 5B).
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Further, content of NP-SH was also influenced by different As treatments (0.2, 0.5 and
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1.0 mM) and results showed that NP-SH in shoots was increased by only 3, 8, 1% in shoots and 2, 6, 1% in root while at higher concentration (1.5 Mm) of arsenic NP-SH content was
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significantly decreased as evidenced by 7% reduction in shoot and by 5% in root respectively (Fig. 5C). Correlation analysis among the antioxidants and different As treatments showed that
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there was positive correlation for APX (root, r = 0.990, p = 0.001; shoot, r = 0.991, p = 0.001) and CAT (root, r = 0.942, p = 0.017; shoot, r = 1.00, p = 0.000) (Table 1) while a negative correlation was recorded for NP-SH (root, r = -0.595, p = 0.290 and in shoot, r = -0.582, p = 0.303) (Table 1). 4. Discussion
ACCEPTED MANUSCRIPT Among environmental stresses, heavy metal contamination is regarded as one of the most serious threats, occurred due to several anthropogenic activities such as industrial activities, agricultural practices, and also due to natural sources (Sharma and Pandey 2014). There are several studies which demonstrated the negative impacts of numerous heavy metals not only on
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plants growth and their metabolisms but they also caused serious risk to human health by
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entering in to the food chain (Adrees et al. 2015a; Ali et al. 2015a; Keller et al. 2015; Tripathi et
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al., 2014; Tripathi et al. 2015, 2016a,b; Tauqeer et al. 2016). Further, it has been clear that the toxic behavior of heavy metals generated oxidative stress in plants by producing reactive oxygen
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species (ROS) which ultimately alter plant metabolic system and thus resulted into the plant
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death (Ali et al. 2015b; Habiba et al. 2015; Tauqeer et al. 2016). Studies also suggested that although toxicity of heavy metals appears on entire plant system but the photosynthetic apparatus
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and pigments are basically the main targets and thus affected significantly (Adreeset al. 2015b;
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Habiba et al. 2015; Keller et al. 2015; Tauqeer et al. 2016). Several techniques have been developed and frequently used to remediate soil contaminated with metals and thus are useful in
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metal immobilization, soil washing and phytoremediation (Wuana and Okieimen 2011).
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Phytoremediation is regarded as a revolutionary technique with the incorporation of indigenous plants for the remediation of heavily metal contaminated soil (Ehsan et al. 2014; Kamran et al.
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2014; Amna et al. 2015). Thereby, numerous species of plants have been utilized to remediate soil contaminated with metal (Raziuddin et al. 2011; Kamran et al. 2014; Habiba et al. 2015). However, the idea of using native plant species in phytoremediation technique has significant consequence since these plants could potentially endure their own environmental conditions in contrast to any other plant species (Kamran et al. 2014).
ACCEPTED MANUSCRIPT Thus, in the present study P. pinnata, a woody plant, has been used to test the capability of its phytoremediation potential against arsenic contamination through the morphophysiological and biochemical investigation. Arsenic treatments at higher concentration (1.5 mM) caused significant reduction in growth, chlorophylls (a and b), carotenoids and protein
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contents. Chlorophyll fluorescence parameters of P. pinnata seedlings were also affected by As
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(Kumar et al. 2014; Kumar et al. 2015). Reduction in morphological and physiological
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parameters at higher concentration of As was accompanied with enhanced level of As accumulation in root and shoot of P. pinnata which caused oxidative stress in plants (Fig. 1 and
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3). These results are in consistence with earlier findings where As stress negatively affected
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morphological and physiological traits of plants (Keller et al. 2015; Tripathi et al. 2016a; Rizwan et al. 2016). Mirza et al. (2010) have studied and documented about decline in growth of Arundo
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donax under elevated arsenic concentration. Hence, decline in biomass accumulation of P.
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pinnata might have occurred due to toxic property of arsenic on key metabolic processes such as photosynthesis and protein synthesis as reported in cork oak (Quercus suber L.), Pinus
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massoniana, Cryptomeria fortune, Cunninghamia lanceolata and Populus alba (Gogorcena et al.
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2011; Liu et al. 2011; Beritognolo et al. 2011).
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It has been well documented that characteristics of arsenic hyper accumulator plants are; arsenic content >1000 mg/kg dry weight in shoot, bioaccumulation factor >1, sometimes reaching up to 50–100 (Brooks 1998), translocation factor is >1 (Wei and Zhou 2004), and greater ability to detoxify and sequester huge amount of arsenic (Rascio and Navari-Izzo 2011). Hence, in the present study it has been well observed that P. pinnata successfully accomplished subsequent 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 (Fig. 2a and b), and
ACCEPTED MANUSCRIPT 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 P. pinnata seedlings (Fig. 1). Further, results of antioxidant enzymes showed that activities of key antioxidant enzymes like APX and CAT were significantly increased in root and shoot of P. pinnata under different
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As treatments. Similar to this, enhanced levels of antioxidant enzymes under heavy metal stress
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in plants have been reported in earlier studies which suggest that this phenomenon might be a powerful strategy for the survival of metal accumulating plants (Afshan et al.2015; Habiba et al.
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2015; Tripathi et al. 2016a). Thus, results of the present study supported one of the possible mechanisms of metal tolerance by enhancing the activities of antioxidant enzymes even under
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lower As concentration (Fig. 5) which reduced oxidative stress. It has been well reported that
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mineral elements play significant role in the accumulation of biomass and protection of plants against different stress conditions (Tripathi et al. 2014; Kumar et al. 2015; Singh et al. 2015).
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Hence, we have investigated consequences of arsenic treatments on nutrient elements
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distribution in roots and shoots of P. pinnata. Similar to the results of antioxidants, levels of mineral elements (Ca, Mg, Fe, Zn, K, Na, S and Mn) were also increased significantly under
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each treatment of As. The results further suggested that mineral elements like Ca, Fe, Zn, Mn
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and Na contents in root and shoot were significantly increased as compared to control (except K in root). In Pteris vittata and Zea mays, Ca contents enhanced significantly under arsenic stress (Li et al. 2006; Mallick et al. 2011). In agreement with our results Marschner (1995) had also demonstrated enhanced level of K contents under As exposure while in other studies Na, Zn, and Mn contents have also been reported to increase under As exposure in different plants (Tu and Ma 2005; Sakai et al. 2010; Mallick et al. 2011). Therefore, significant increase of nutrient elements under As treatments clearly supported the mechanisms of nutrient regulation under
ACCEPTED MANUSCRIPT metal stress condition in plants (Tu and Ma 2005; Sakai et al. 2010; Mallick et al. 2011). Similar results have also been observed by Kumar et al. (2015) in Wrightia arborea seedlings treated with As. Furthermore, Sakai et al. (2010) have proposed that sulfur (S) is a major component of
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glutathione, non protein thiols (metabolites antioxidants) and phytochelatins. NP-SH form
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complex with excess metals inside the cell and thus protect cell form metal toxicity (Raj et al. 2011). The outcome of present study suggests that similar to sulfur content NP-SH content was
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significantly greater in root and shoot of P. pinnata which was exposed to 0.2-1.0 mM As (Fig.4C). However, at 1.5 mM As, NP-SH content was declined significantly. These results
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clearly demonstrate the positive role of NP-SH in conferring the arsenic tolerance in P. pinnata
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at the range of 0.2–1.5 mM As concentration as supported by data of growth, pigments etc.
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5. Conclusion
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This study highlights greater arsenic accumulating potential of P. pinnata when grown in hydroponic culture. The results also suggest that status of antioxidant enzymes and mineral
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elements are actively regulated under arsenic stress. Under tested concentrations of arsenic, S and NP-SH contents have significantly increased, thereby suggesting their essential role in
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upholding tolerance against arsenic stress. Translocation factor and bioaccumulation factors also suggested that P. pinnata fulfills the criteria of arsenic hyper accumulator plants and 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 P. pinnata. Acknowledgments
ACCEPTED MANUSCRIPT Authors are thankful to the Head, Department of Botany, University of Allahabad for providing laboratory facilities. Authors are also thankful to Dr. Dhanvinder Singh, Sr. Soil Scientist, Department of Soil, Punjab Agriculture University, Ludhiana, for providing ICAPAES facility to determine As and mineral elements. Authors extend their thanks to the UGC,
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New Delhi for financial support.
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References
Acharyya, S. K., Lahiri, S., Raymahashay, B. C., Bhowmik, A., 2000. Arsenic toxicity of ground
US
water in parts of the Bengal basin in India and Bangladesh: the role of Quaternary
AN
stratigraphy and Holocene sea-level fluctuation. Environ Geol 39, 1127-1137. Adrees, M., Ali, S., Rizwan, M., Ibrahim, M., Abbas, F., Farid, M., Rehman, M. Z., Irshad, M.
M
K., Bharwana, S. A., 2015b. The effect of excess copper on growth and physiology of
ED
important food crops: a review. Environ. Sci. Pollut. Res. 22, 8148–8162.
PT
Adrees, M., Ali, S., Rizwan, M., Rehman, M. Z., Ibrahim, M., Abbas, F., Farid, M., Qayyum, M. K., Irshad, M. K., 2015a. Mechanisms of silicon-mediated alleviation of heavy metal
CE
toxicity in plants: a review. Ecotoxicol. Environ. Saf. 119, 186–197.
AC
Aebi, I.I,, 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Afshan, S., Ali, S., Bharwana, S. A., Rizwan, M., Farid, M., Abbas, F., Ibrahim, M., Mehmood, M. A., Abbasi, G. H., 2015. Citric acid enhances the phytoextraction of chromium, plant growth, and photosynthesis by alleviating the oxidative damages in Brassica napus L. Environmental Science and Pollution Research 22(15), 11679-11689.
ACCEPTED MANUSCRIPT Ahamed, S., Sengupta, M. K., Mukherjee, A., Hossain, M. A., Das, B., Nayak, B., Pal, A., Chakraborti, D., 2006. Arsenic groundwater contamination and its health effects in the state of Uttar Pradesh (UP) in upper and middle Ganga plain, India: a severe danger. Sci Total Environ 370, 310-322.
IP
T
Ali, S., Bharwana, S. A., Rizwan, M., Farid, M., Kanwal, S., Ali, Q., Ibrahim, M., Gill, R. A.,
CR
Khan, M. D., 2015b. Fulvic acid mediates chromium (Cr) tolerance in wheat (Triticum aestivum L.) through lowering of Cr uptake and improved antioxidant defense system.
US
Environ. Sci. Pollut. Res. 22, 10601–10609.
AN
Ali, S., Chaudhary, A., Rizwan, M., Anwar, H. T., Adrees, M., Farid, M., Irshad, M. K., Hayat, T., Anjum, S. A., 2015a. Alleviation of chromium toxicity by glycinebetaine is related to
M
elevated antioxidant enzymes and suppressed chromium uptake and oxidative stress in
ED
wheat (Triticum aestivum L.). Environ. Sci. Pollut. Res. 22, 10669–10678.
PT
Amna, Ali, N., Masood, S., Mukhtar, T., Kamran, M. A., Rafique, M., Munis, M. F. H., Chaudhary, H. J., 2015. Differential effects of cadmium and chromium on growth,
CE
photosynthetic activity, and metal uptake of Linum usitatissimum in association with
AC
Glomus intraradices. Environ. Monit. Assess. 187, 1–11. Arditti, J., Dunn, A., 1969. Environmental Plant Physiology-Experiments in Cellular and Plant Physiology. New York: Holt, Rinehart and Winston Inc., pp. 312. Beritognolo, I., Harfouche, A., Brilli, F., Prosperini, G., Gaudet, M., Brosche´, M., Salani, F., Kuzminsky, E., Auvinen, P., Paulin, L., Kangasjarvi, J., Loreto, F., Valentini, R., Mugnozza, G. S., Sabatti, M., 2011. Comparative study of transcriptional and physiological
ACCEPTED MANUSCRIPT responses to salinity stress in two contrasting Populusalba L. genotypes. Tree Physiol 31, 1335–1355. Bhattacharyya, D., Mukherjee, P. K., Ray, A. K., Sengupta, S., 2004. Arsenic-polluted
T
groundwater in West Bengal: A cost-effective remedy. Current Science 86(9), 1206-1209.
IP
Brooks, R. R., 1998. Plants that Hyperaccumulate Heavy Metals: Their Role in
CR
Phytoremediation, Microbiology, Archaeology, Mineral Exploration and Phytomining. CAB
US
Internationsl, Oxford, UK.
Bureau of Indian Standards (BIS), 2003. Indian Standard: Drinking Water. Specification (first
AN
revision), Amendment No. 2, New Delhi, September 2003.
M
Chauhan, V. S., Nickson, R. T., Chauhan, D., Iyengar, L., Sankararamakrishnan, N., 2009. Ground water geochemistry of Ballia district, Uttar Pradesh, India and mechanism of arsenic
ED
release. Chemosphere 75, 83-91.
PT
Ehsan, S., Ali, S., Noureen, S., Mahmood, K., Farid, M., Ishaque, W., Shakoor, M.B., Rizwan,
CE
M., 2014. Citric acid assisted phytoremediation of cadmium by Brassica napus L.
AC
Ecotoxicol. Environ. Saf. 106, 164–172. Ellmann, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Elstner, E. F., Heupel, A., 1976. Inhibition of nitrite formation from hydroxyl ammonium chloride: a simple assay for superoxide dismutase. Anal Biochem 70, 616-620. Gogorcena, Y., Larbi, A., Andaluz, S., Carpena, R. O., Abadía, A., Abadía, J., 2011. Effects of cadmium on cork oak (Quercus suber L.) plants grown in hydroponics. Tree physiology 31(12), 1401-1412.
ACCEPTED MANUSCRIPT Habiba, U., Ali, S., Farid, M., Shakoor, M. B., Rizwan, M., Ibrahim, M., Abbasi, G. H., Hayat, T., Ali, B., 2015. EDTA enhanced plant growth, antioxidant defense system, and phytoextraction of copper by Brassica napus L. Environmental Science and Pollution Research 22(2), 1534-1544.
IP
T
Heath, R. L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts I. Kinetics and
CR
stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125, 189-198. Hu, Y., Nan, Z., Jin, C., Wang, N., Luo, H., 2014. Phytoextraction potential of poplar (Populus
AN
cadmium. Int J Phytoremed 16(5), 482-495.
US
alba L. var. pyramidalis Bunge) from calcareous agricultural soils contaminated by
Huang, H., Yu, N., Wang, L., Gupta, D. K., He, Z., Wang, K., Yang, X. E., 2011. The
M
phytoremediation potential of bioenergy crop Ricinus communis for DDTs and cadmium co-
ED
contaminated soil. Biores Technol 102(23), 11034-11038.
PT
Kacálková, L., Tlustoš, P., Száková, J., 2015. Phytoextraction of risk elements by willow and
CE
poplar trees. Int J Phytoremed, 17(5), 414-421. Kamran, M. A., Mufti, R., Mubariz, N., Syed, J. H., Bano, A., Javed, M. T., Chaudhary, H. J.,
AC
2014. The potential of the flora from different regions of Pakistan in phytoremediation: areview. Environ. Sci. Pollut. Res. 21, 801–812. Keller, C., Rizwan, M., Davidian, J. C., Pokrovsky, O. S., Bovet, N., Chaurand, P., Meunier, J. D., 2015. Effect of Silicon on wheat seedlings (Triticum turgidum L.) grown in hydroponics under Cu stress. Planta 241, 847–860.
ACCEPTED MANUSCRIPT Kumar, D., Singh, V. P., Tripathi, D. K., Prasad, S. M., Chauhan, D. K., 2015. Effect of arsenic on growth, arsenic uptake, distribution of nutrient elements and thiols in seedlings of Wrightia arborea (Dennst.) Mabb. Int J Phytoremed17, 128-134. Kumar, D., Tripathi, D. K., Chauhan, D. K., 2014. Phytoremediation potential and nutrient status
IP
T
of Barringtonia acutangula gaerth. Tree seedlings grown under different chromium (CrVI)
CR
treatments. Biological trace element research 157(2), 164-174.
Li, W., Chen, T., Huang, Z., Lei, M., Liao, X., 2006. Effect of arsenic on chloroplast
US
ultrastructure and calcium distribution in arsenic hyperaccumulator Pteris vittata L.
AN
Chemosphere 62, 803–809.
Lichtenthaler, H. K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic
ED
M
biomembranes, Methods Enzymol 148, 350-382.
Liu, T., Wu, F., Wang, W., Chen, J., Li, Z., Dong, X., Patton, J., Pei, Z., Zheng, H., 2011. Effects
PT
of calcium on seed germination, seedling growth and photosynthesis of six forest tree
CE
species under simulated acid rain. Tree Physiol 31, 402–413. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J., 1951. Protein measurement with the
AC
Folinphenol reagent. J Biol Chem 193, 265–275. 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.
ACCEPTED MANUSCRIPT Mirza, N., Mahmood, Q., Pervez, A., Ahmad, R., Farooq, R., Shah, M. M., Azim, R. M., 2010. Phytoremediation potential of Arundo donax in arsenic-contaminated synthetic wastewater. Bioresour Technol 101, 5815–5819. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate specific
IP
T
peroxidasein spinach chloroplasts. Plant Cell Physiol. 22, 867–280.
CR
Nriagu, J. O., 2002. Arsenic poisoning through the ages. Marcel Dekker, Inc.: New York,pp. 1-
US
26.
Raj, A., Pandey, A. K., Sharma, Y. K., Khare, P. B., Srivastava, P. K., Singh, N., 2011.
M
Bioresour Technol 102, 9827–9832.
AN
Metabolic adaptation of Pteris vittata L. gametophyte to arsenic induced oxidative stress.
ED
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.
PT
Raziuddin, Farhatullah, Hassan, G., Akmal, M., Shah, S. S., Mohammad, F., Shafi, M., Bakht, J.,
CE
Zhou, W., 2011. Effects of cadmium and salinity on growth and photosynthesis parameters
AC
of brassica species. Pak. J. Bot. 43, 333–340. Rizwan, M., Meunier, J. D., Davidian, J. C., Pokrovsky, O. S., Bovet, N., Keller, C., 2016. Silicon alleviates Cd stress of wheat seedlings (Triticum turgidum L. cv. Claudio) grown in hydroponics. Environmental Science and Pollution Research 23(2), 1414-1427. 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.
ACCEPTED MANUSCRIPT Shah, B. A., 2010. Arsenic-contaminated groundwater in Holocene sediments from parts of Middle Ganga Plain, Uttar Pradesh, India. Curr Sci 98(10), 1359-1365. Sharma, P., Pandey, S., 2014. Status of phytoremediation in world scenario. International Journal
T
of Environmental Bioremediation & Biodegradation 2(4), 178-191.
IP
Singh, S., Srivastava, P. K., Kumar, D., Tripathi, D. K., Chauhan, D. K., Prasad, S. M., 2015.
CR
Morpho-anatomical and biochemical adapting strategies of maize (Zea mays L.) seedlings against lead and chromium stresses. Biocatalysis and Agricultural Biotechnology 4(3), 286-
US
295.
AN
Smith, A. H., Goycolea, M., Haque, R., Biggs, M. L., 1998. Marked increase in bladder and lung cancer mortality in a region of northern Chile due to arsenic in drinking water. Amr J
ED
M
Epidemiol 147, 660-669.
Tauqeer, H. M., Ali, S., Rizwan, M., Ali, Q., Saeed, R., Iftikhar, U., Ahmad, R., Farid, M.,
PT
Abbasi, G. H., 2016. Phytoremediation of heavy metals by Alternanthera bettzickiana:
CE
growth and physiological response. Ecotoxicology and environmental safety 126, 138-146. Tripathi, D. K., Singh, S., Singh, V. P., Prasad, S. M., Chauhan, D. K., Dubey, N. K., 2016a.
AC
Silicon Nanoparticles More Efficiently Alleviate Arsenate Toxicity than Silicon in Maize Cultiver and Hybrid Differing in Arsenate Tolerance. Frontiers in Environmental Science, 4, p.46. Tripathi, D. K., Singh, V. P., Chauhan, D. K., Prasad, S. M., Dubey, N. K., 2014. Role of macronutrients in plant growth and acclimation: recent advances and future prospective. In Improvement of Crops in the Era of Climatic Changes. Springer New York, pp. 197-216
ACCEPTED MANUSCRIPT Tripathi, D. K., Singh, V. P., Prasad, S. M., Chauhan, D. K., Dubey, N. K., Rai, A. K., 2015. Silicon-mediated alleviation of Cr(VI) toxicity in wheat seedlings as evidenced by chlorophyll fluorescence, laser induced break down spectroscopy and anatomical changes. Ecotoxicol. Environ. Saf. 113, 133–144.
IP
T
Tripathi, D. K., Singh, V. P., Prasad, S. M., Dubey, N. K., Chauhan, D. K., Rai, A. K., 2016b.
CR
LIB spectroscopic and biochemical analysis to characterize lead toxicity alleviative nature of silicon in wheat (Triticum aestivum L.) seedlings. Journal of Photochemistry and
US
Photobiology B: Biology 154, 89-98.
AN
Tu, C., Ma, L. Q., 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.
M
Velikova, V., Yordanov, I., Edreva, A., 2000. Oxidative stress and some antioxidant system in
ED
acid rain-treated bean plants, Plant Sci151, 59-66.
PT
Wei, S. H., Zhou, Q. X., 2004. Discussion on basic principles and strengthening measures for
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phytoremediation of soils contaminated by heavy metals. Chinese J Ecol 23, 65–72.
edn.
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WHO, 1993. Guideline for drinking water quality. Recommendations, Geneva, 1993, vol. 1, 2nd
Wuana, R. A., Okieimen, F. E., 2011. Heavy metals in contaminated soils: a review of sources, chemistry, risks and best available strategies for remediation. Isrn Ecology, 2011. Ye, N., Zhu, G., Liu, Y., Li, Y., Zhang, J., 2011. ABA Controls H2O2 Accumulation Through the Induction of OsCATB in Rice Leaves Under Water Stress. Plant Cell Physiol 52, 689-698.
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Table 1: Correlation (r) between arsenic treatments (0.2-2.0 mM), and root, shoot and leaf with different parameters in seedlings of Pongamia pinnata Investigated parameter
Arsenic Shoot
Root
Leaf
AC
CE
PT
ED
M
AN
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CR
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r P r p R p Length -0.987** 0.002 -0.951* 0.013 Fresh mass -0.989** 0.001 -0.962** 0.009 Dry mass -0.993** 0.001 -0.993** 0.001 SOR 0.993** 0.001 0.985** 0.002 H2O2 0.983** 0.003 0.977** 0.004 MDA 0.996** 0.001 0.999** 0.001 Total protein -0.991** 0.001 -0.976** 0.004 CAT 0.942* 0.017 1.000** 0.000 APX 0.990** 0.001 0.991** 0.001 NP-SH -0.595 0.290 -0.582 0.303 Chlorophyll a -0.996** 0.001 Chlorophyll b -0.993** 0.001 Carotenoids -0.990** 0.001 Fv/Fm -0.986** 0.002 Fv/F0 -0.959* 0.010 Fm/F0 -0.962** 0.009 qP -0.919* 0.027 NPQ 0.992** 0.001 **correlation (r) is significant at p<0.01 significance level, and *correlation (r) is significant at p<0.05 significance level
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Table 2: Effect of different concentrations of As on chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll a/ chlorophyll b (Chl a/Chlb ratio), carotenoids (Car), chlorophyll/carotenoids ratio (Chl (a+b)/Car ratio), and total protein; root and shoot in seedlings of Pongamia pinnata
0.8 03 0.7 22 0.6 49 0.5 63 0.4 31
2.7 52 2.8 12 2.8 20 2.8 24 2.8 31
0.6 43 0.6 11 0.5 87 0.5 59 0.5 13
4.6 86 4.5 04 4.2 23 3.8 52 3.2 18
±0.02 6a ±0.02 7a ±0.02 8ab ±0.02 2b ±0.02 3c
±0.1 6a ±0.1 8a ±0.1 9ab ±0.1 7ab ±0.1 4c
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Root ±0.2 7a ±0.3 5a ±0.3 3a ±0.2 5ab ±0.2 0c
Shoot 16. ±0.4 74 2a 15. ±0.6 38 0a 14. ±0.6 47 9ab 13. ±0.6 81 1ab 11. ±0.4 79 6c
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Table 3: Correlation (r) matrix among nutrient contents in roots and shoots of Pongamia pinnata seedlings, under different As concentrations (0.2-2.0 mM) FeS
FeR Mn
Mn
Zn
Zn
Na
Na
Ca
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R
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R
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R
.87 2 .88 3* .88 1* .96 1** .84 0 .97 5** .88 8* .94 8* .86 7
.94 3* .86 4 .72 3 .99 7** .91 6* .99 2** .81 9 .85 6
.96 9** .81 2 .92 2* .86 9 .91 2* .81 4 .97 6**
.85 6 .95 9* .95 6* .96 5** .93 4* .90 9* .85 6 .94 6* .98 5** .97 9** .94 0* .88 4*
M gS
.77 9
.91 8*
.77 9
.77 1
.92 8*
.78 8
M gR
.61 1
.76 5
.66 4
.57 4
.90 1*
SS
.99 5**
.93 3*
.96 4**
.95 0*
SR
.74 3
.79 9
.80 6
.67 1
S
K R
M gS
M gR
SS
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.83 6 .84 5 .80 6 .94 8* .99 3** .85 3 .76 5 .79 8 .77 9 .82 2 .99 3**
.87 0 .99 7** .96 2** .88 7* .73 2 .99 2** .90 2* .98 0** .80 7 .88 4*
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M nS M nR Z nS Z nR N aS N aR C aS C aR K
KR
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R
.86 0 .82 9 .85 5 .84 6 .86 5 .99 3**
.67 6 .88 9* .73 8 .91 1* .85 2
.89 4* .98 7** .78 3 .82 1
.94 7* .96 .86 7** 5 .82 .82 3 4
.76 9
.82 6
.89 1*
.75 2
.95 5*
.84 3
.58 5
.61 7
.68 6
.94 6*
.53 8
.84 9
.63 7
.87 9
.93 1*
.99 7**
.98 3**
.81 6
.90 7*
.86 8
.90 4*
.93 5*
.66 0
.76 0
.80 7
.99 3**
.61 2
.84 2
.67 2
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Fe
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S
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A sR Fe
KS
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.94 6* .93 4* .97 5** .88 4* .95 9* .99 8** .96 2** .79 9 .94 1* .88 1* .93 2* .82 2 .96 5**
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R
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As
M
AsS
.81 6 .99 7** .89 7* .83 3 .86 1
.77 0 .65 4
.90 0*
.98 4**
.79 .62 0 5
.87 0
.84 .94 0 8*
.7 6 0
ACCEPTED MANUSCRIPT *p<0.05 probability level, **p<0.01 probability level, S= Shoot, and R = Root
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P. pinnata successfully tolerate the arsenic concentration upto 0.2-1.0 mM without
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Research Highlights
showing As toxicity while at 1.5 mM of As shows toxicity symptoms. Mineral nutrients and antioxidant activity were increased significantly due to increased As concentration
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Bioaccumulation and translocation factor was >1 and also had greater ability to detoxify
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and sequester huge amount of arsenic
Thus, P. pinnata may be useful for arsenic phytoremediation program
Further, non-protein thiols played important role in imparting arsenic tolerance
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Dharmendra Kumar, Durgesh Kumar Tripathi, Shiliang Liu, Vivek Kumar Singh, Shivesh Sharma, Nawal Kishore Dubey, Sheo Mohan Prasad, Devendra Kumar Chauhan PII: DOI: Reference:
S2352-4073(17)30035-5 doi: 10.1016/j.plgene.2017.06.002 PLGENE 111
To appear in:
Plant Gene
Received date: Revised date: Accepted date:
31 December 2016 5 June 2017 8 June 2017
Please cite this article as: Dharmendra Kumar, Durgesh Kumar Tripathi, Shiliang Liu, Vivek Kumar Singh, Shivesh Sharma, Nawal Kishore Dubey, Sheo Mohan Prasad, Devendra Kumar Chauhan , Pongamia pinnata (L.) pierre tree seedlings offer a model species for arsenic phytoremediation, Plant Gene (2017), doi: 10.1016/ j.plgene.2017.06.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Title: Pongamia pinnata (L.) Pierre tree seedlings offer a model species for arsenic phytoremediation Authors: Dharmendra Kumar1, Durgesh Kumar Tripathi2*, Shiliang Liu3, Vivek Kumar Singh4,
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Shivesh Sharma2,5, Nawal Kishore Dubey6, Sheo Mohan Prasad7*, Devendra Kumar Chauhan1*
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Affiliation: 1D D Pant Interdisciplinary Research Laboratory, Department of Botany, University
Centre for Medical Diagnostic and Research, Motilal Nehru National Institute of Technology,
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2
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of Allahabad, Allahabad, 211002, India.
Allahabad, India
Division of Plant Sciences, University of Missouri, Columbia, MO 65211 USA
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Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA
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Department of Biotechnology, Motilal Nehru National Institute of Technology, Allahabad, India
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Center of Advanced Study in Botany, Banaras Hindu University, Varanasi, 221005, India
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Ranjan Plant Physiology and Biochemistry Laboratory, Department of Botany, University of
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3
Allahabad, Allahabad 211002, India
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Abstract
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Email: [email protected], [email protected], [email protected]
Hydroponic experiments were conducted to investigate the effect of different concentrations of arsenic (As) on seedlings of Pongamia pinnata (L.). Results revealed that P. pinnata successfully tolerated As up to 0.2-1.0 mM and did not show significant reduction in growth and chlorophyll contents. However, these parameters significantly reduced at 1.5 mM of As exposure. Further, P. pinnata accumulated As about 528-1122 mg kg-1 dry weight in roots,
ACCEPTED MANUSCRIPT and 167-792 mg kg-1 dry weight in shoots, respectively when exposed to 0.2-2.0 mM As. Arsenic exposure at 0.2-1.5 mM significantly enhanced oxidative stress markers [superoxide radical (SOR), hydrogen peroxide (H2O2) and malondialdehyde (MDA)], activities of antioxidants [ascorbate peroxidase (APX) and catalase (CAT)] and nutrient accumulation.
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Increase of antioxidants and nutrient accumulation might have protected plants from As
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exposure. Additionally, bioaccumulation and translocation factors were > 1 which also
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confirmed that test plant had greater ability to accumulate and detoxify greater amount of As.
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Keywords: Arsenic contamination, mineral elements, phytoremediation
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Abbreviations: F0, minimal fluorescence; Fv/Fm, maximum photochemical efficiency of PS II; Fv/F0, the activity of PS II; qP, photochemical quenching; NPQ, nonphotochemical quenching;
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APX, ascorbate peroxidase; CAT, catalase; ROS, reactive oxygen species; SOR, superoxide
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radical; H2O2, hydrogen peroxide; MDA, malondialdehyde.
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1. Introduction
Among toxic elements, arsenic (As) is considered as one of the most toxic metalloids for
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all living organisms caused greater threat to human beings (Nriagu 2002). It is a universal
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metalloid which is commonly present in the environment and released from various anthropogenic and geochemical sources in two forms, i.e. AsIII and AsV (Smith et al. 1998). In relation to plants, it has been well documented that both forms of As are most toxic and adversely affected the growth, yield and quality of plants as well as other internal integrities (Kumar et al. 2015; Tripathi et al., 2016a). On the other hand for human beings it causes various serious diseases like cancer when enters in to the food chain and causes inevitable poisoning. Its exposure also leads to alteration in neuro-transmitter level, thus As toxicity is a serious
ACCEPTED MANUSCRIPT worldwide human concern. In India mostly in southern West Bengal, arsenic contamination has become a serious environmental problem that is posing threat to living beings (Acharyya et al. 2000; Shah 2010). According to WHO guidelines which have been endorsed by the Bureau of Indian Standards, the upper permissible limit of arsenic in ground water was considered as 0.05
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mg/l (WHO 1993; BIS 2003). However, due to higher toxic nature of arsenic, drinking water
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arsenic contamination was reexamined, and the previous level of arsenic in drinking water (0.05
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mg/l) was lowered to 0.01 mg/l. In India, the level of arsenic in drinking water ranges between 0.3-0.5 mg/l in arsenic-contaminated regions which is much above the prescribed limit (Ahamed
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et al. 2006). Studies showed that arsenic contamination in agricultural land of Indo-Gangetic
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flood plain and deltaic regions of West Bengal, and parts of Bihar, Assam, Chhattisgarh and Eastern Uttar Pradesh (Indian Provinces) ranges between 3.34-105 mg/kg soil (Chauhan et al.
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2009; Mallick et al. 2011). In West Bengal, after diagnosis of first case of arsenic poisoning in
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1983, six million people have been affected by arsenic toxicity through drinking water (Bhattacharyya et al. 2004). However, in Bangladesh, arsenic contamination was first reported in
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1993 (Acharyya et al. 2000). Thus, in terms of number of people exposed (30-35 million), it is
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the most serious problem of groundwater arsenic contamination in the world (Acharyya et al. 2000; Bhattacharyya et al. 2004; Ye et al. 2011). Besides, India and Bangladesh, arsenic
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contamination in groundwater has also been reported from different countries of world (Shah 2010). Thus, the problem of arsenic contamination of drinking water and soil is common in entire world including India. In India the regions for arsenic contamination of groundwater is mostly the presence of the Ganges delta and some low-lying areas in the Bengal basin that have been investigated (Acharyya et al. 2000; Shah 2010). Practices of excessive extraction of groundwater for
ACCEPTED MANUSCRIPT irrigation and application of phosphate fertilizers have promoted groundwater flow and phosphate mobilization from fertilizers as well as from decayed organic matter had aided the further release of arsenic. For removal of As contamination from soil, water and environmental field there are several physical and chemical techniques which are being used, are costly and also
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not environmental friendly. Beside this, phytoremediation which is known as green technology
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for the removal of heavy metals is being used from last few decades (Dhir, 2013; Jasrotia et al.,
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2015). In this technique some aquatic and terrestrial trees have been tested for the removal of heavy metal contamination including arsenic and some significant results have been found (Dhir,
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2013; Jasrotia et al., 2015). Thus to establish this technique as a most effective and reliable,
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several studies are being regularly carried out in the renowned laboratories all over the world
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(Dhir, 2013; Kumar et al., 2014; Jasrotia et al., 2015).
The choice of plants is a crucial aspect for the practical use of phytoremediation
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technique because it has been well documented that all species are not proficient to accumulate
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and store the metals in their tissues (Tu and Ma 2005; Kacálková et al. 2015). Most hyperaccumulators plants have small biomass and have slow growing behavior, however, some
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woody species have special significance because of their speedy growth, presence of deep roots
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and rich biomass production and thus showed high tolerance capacity towards heavy metals and fit for phytoremidaition (Huang et al. 2011; Hu et al. 2014). Although, extensive work has been done to examine the responses of plants growing in the As contaminated areas. However, no attempt has been made yet to explore the responses of long lived tree plants of Vindhyan region. Thus, in the present study one woody plant (Pongamia pinnata) frequently grown in the Vindhyan region was selected and its arsenic tolerance
ACCEPTED MANUSCRIPT capacities were studied by analyzing the antioxidant activity, mineral regulation, oxidative stress response and chlorophyll fluorescence. 2. Material and methods 2.1. Seed germination and growth conditions
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Pods of Pongamia pinnata were collected from the forest area of Mirzapur district
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(Vindhyan region), Uttar Pradesh, India during the month of May 2013, and seeds were extracted manually. Healthy and uniform sized seeds were surface sterilized with 10% (v/v) sodium
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hypochlorite solution for 10 min, washed with distilled water several times and soaked in distilled water for 4 h. Further, seeds were wrapped in muslin cloth, and kept in the dark for
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germination. After 12 days, germinated seeds were sown in plastic pots containing fresh half-
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strength Hoagland solution (pH 6.5) (Arditti and Dunn 1969) in such a way that only radicals of germinated seeds were in contact with Hoagland solution. This was achieved by using a
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handmade sieve of thermocol having pore size corresponding to the size of seeds. The thermocol
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sieve containing young seedlings was placed just above the nutrient medium level in plastic pot filled with 50 ml fresh nutrient medium. The seedlings were grown in a growth chamber under a
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photon flux density of 250 µmol photons m-2 s-1 and relative humidity of 50-60% with a
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day/night regime of 12/12 h at 30±2 oC. After the emergence of first two leaves the seedlings were treated with tested doses of arsenic. 2.2. Arsenic treatments Uniform sized seedlings with fully developed first two leaves were exposed to various concentrations (as sodium arsenate; Na2HAsO4. 7H2O: 0, 0.2, 0.5, 1.0, 1.5 and 2.0 mM) of arsenic for 7 days. Three plastic pots were arranged for each concentration of arsenic, and in each plastic pot three uniform sized seedlings were gently placed. During arsenic treatments
ACCEPTED MANUSCRIPT 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. 2.3. Determination of growth parameters Growth parameter such as length, fresh mass, and dry mass of roots and shoots of treated
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and untreated seedlings were measured after harvesting. For dry mass, root and shoot were
balance (Contech- CA 223, India).
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2.4. Determination of photosynthetic pigment contents
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wrapped in butter paper and oven dried for 48 h at 65–75 °C and then weighed using digital
The amounts of chlorophyll a, b (Chl a and Chl b) and carotenoids (Car) were determined
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2.5. Determination of total protein content
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according to the method of Lichtenthaler (1987).
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For the measurement of total protein, the method of Lowry et al. (1951) was adopted using bovine serum albumin as standard.
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assessment
of
photosynthetic
performance,
chlorophyll
fluorescence
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For
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2.6. Chlorophyll fluorescence measurements
measurements were taken in dark adapted leaves of control and treated seedlings using hand held
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leaf fluorometer (FluorPen FP 100, Photos System Instrument, Czech Republic). 2.7. Determination of As and mineral nutrient contents in tissues For determination of arsenic, macronutrients (Ca, K, Mg, P and S) and micronutrients (B, Cu, Fe, Mn, Zn and Na), 50 mg of root and shoot tissues of each sample 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
ACCEPTED MANUSCRIPT in digested samples was determined by using an Inductively Coupled Argon Plasma - Atomic Emission Spectrometer (ICAP-AES). 2.7. Determination of superoxide radical and hydrogen peroxide contents Superoxide radical (SOR; O2•¯) in each sample was determined following the method of
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Elstner and Heupel (1976). This assay is based on formation of NO2 from hydroxylamine in the
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presence of O2•¯. The absorbance of the colored aqueous phase was recorded at 530 nm. A
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standard curve was prepared with NaNO2 and used to calculate the amount of O2•¯ in each
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sample.
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Determination of H2O2 was done according to the method of Velikova et al. (2000). Absorbance of reaction mixture was recorded at 390 nm. The hydrogen peroxide concentration
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was calculated by using a standard curve prepared with graded solution of H2O2.
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2.8. Determination of lipid peroxidation
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The lipid peroxidation as malondialdehyde (MDA) content was estimated according to the method of Heath and Packer (1968). The content of MDA was determined by using an
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extinction coefficient of 155 mM-1 cm-1.
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2.9. Determination of CAT and APX activity and NP-SH content Catalase (CAT; EC 1.11.3.6) activity was determined in terms of decrease in absorbance due to dissociation of H2O2 according to the method of Aebi (1984). Ascorbate peroxidase (APX; EC 1.11.1.11) activity was deter-mined according to the method of Nakano and Asada (1981). The non-protein thiols (NP-SH) content was measured following the method of Ellman (1959).
ACCEPTED MANUSCRIPT 2.10. 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. Linear correlation matrix was also done among mineral elements
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contents in roots and shoots of P. pinnata seedlings, under different As concentrations.
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3. Results
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3.1. Impact of arsenic on growth and photosynthetic pigments (chlorophylls and carotenoids) and protein contents
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Growth parameters of P. pinnata were measured in terms of length, fresh mass and dry
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mass of root and shoot. Data pertaining to the growth parameters showed that the lower concentration (0.2 and 0.5 mM) of arsenic affected them marginally as length of root was
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lowered by 4 and 5% and shoot by 4 and 7% at 0.2 and 0.5 mM As, respectively (Fig.1 A).
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Likewise, at 0.2 and 0.5 mM of As fresh mass was also lowered by 3 and 4% in root and by 3 and 1% in shoot, and corresponding decrease in dry mass was 3 and 7% in root and 1, and 2% in
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shoot, respectively (Fig.1 B, C). The higher doses (1.0, 1.5 and 2.0 mM) of As significantly
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declined the length by 9, 24, 66% in root and 13, 17 and 62% in shoot, respectively. Under similar condition, fresh mass was declined by 5, 14 and 49% in root and by 2, 7 and 70% in
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shoot, respectively, and a similar pattern in decrease in dry mass was also recorded in root and shoot of tested seedlings (Fig.1 A-C) Further, correlation coefficient analyses between different As treatments and growth parameters were also performed to statistically authenticate the data set which shows the negative correlation like length (root, r = -0.987, p = 0.002; shoot, r = -0.951, p = 0.013), fresh mass (root, r = -0.989, p = 0.001; shoot, r = -0.962, p = 0.009), and dry mass (root, r = -0.993, p
ACCEPTED MANUSCRIPT = 0.001; shoot, r = -0.993, p = 0.001) (Table 1). The results of the growth parameters clearly confirmed that P. pinnata may survive under 0.2–1.0 mM As stress without showing toxicity symptoms when compared to the control (Fig. 1A-C). However, at 2.0 mM concentration of arsenic plants were not able to survive, thus we have examined further only 0.1-1.5 mM As
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treatments. Therefore, due to much higher arsenic tolerance capability we have selected
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seedlings of P. pinnata for further investigations under different parameters using arsenic
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concentrations of 0.2-1.5 mM.
Data pertaining to the photosynthetic pigments, i.e. Chl a and b and Car are depicted in
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Table 2. Similar to growth, the data of photosynthetic pigments also suggested that at the lower
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concentration (0.2 and 0.5 mM) of As, seedlings showed marginal impact, however higher doses (1.0 and 1.5 mM) of As significantly declined content of Chl a by 19 and 31% and Chl b by 17
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and 33%, respectively, hence resulted into increased Chl a to Chl b ratio (Table 2). Under similar
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As treatments (1.0 and 1.5 mM), Car contents were reduced by 13 and 20% while Chl (a+b)/ Car ratios were declined by 17 and 24%, respectively (Table 2).
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Further, upon exposure of seedlings with 1.0 and 1.5 mM As total protein content was
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declined by 15 and 23% in root and 17 and 29% in shoot, respectively over the values of respective control while at 0.2 and 0.5 mM the effect on protein was marginal (Table 2).
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Correlation coefficient analysis between different As treatments and photosynthetic pigments; Chl a (r = -0.996, p = 0.001), Chl b (r = -0.993, p = 0.001) and Car (r = -0.990, p = 0.001), and total protein content in root (r = -0.991, p = 0.001) and shoot (r = -0.976, p = 0.004) showed negative correlation (Table 1). 3.2. Arsenic accumulation in P. pinnata
ACCEPTED MANUSCRIPT The seedlings of P. pinnata accumulated 1129, 1442, 1789, and 3322 mg As kg−1 dry weight in root and in shoot it was 345, 537, 1677, and 3662 mg kg−1 dry weight, respectively, under 0.2, 0.5, 1.0, and 1.5 mM As treatments (Fig. 1D). In addition to this, correlation coefficient analysis between different As treatments and As content in root (r = 0.955, p = 0.012) and shoot (r =
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0.968, p = 0.007)) showed positive correlation, hence exhibited bioaccumulation factor (BF) and
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translocation factor (TF) >1 without showing arsenic toxicity signs (Fig. 1E, F; Table 1).
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3.3. Impact of arsenic on photochemistry of PS II
The results pertaining to changes in photochemistry of PS II measured as chlorophyll
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fluorescence parameters such as Fv/Fm, Fv/F0, Fm/F0, qP and NPQ in the presence of arsenic
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treatments are depicted in Fig. 2A-B. Arsenic at 0.2, 0.5, 1.0, and 1.5 mM declined (p < 0.05) Fv/Fm by 3, 5, 41 and 63%, Fv/F0 by 6, 9, 55 and 72%, Fm/F0 by 8, 11, 53 and 62% and qP by 2,
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4, 50 and 56%, respectively (Fig. 2A, B). In contrast, NPQ was raised (p < 0.05) by 6, 12, 76,
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and 109% under 0.2, 0.5, 1.0, and 1.5 mM As exposure, respectively as compared to respective control (Fig. 2B). Further, correlation coefficient analysis among As treatments and
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photosynthetic performance showed negative correlation; Fv/Fm (r = -0.986, p = 0.002), Fv/F0 (r
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= -0.959, p = 0.010), Fm/F0 (r = -0.962, p = 0.009) and qP (r = -0.919, p = 0.027) except NPQ that exhibited positive correlation i.e. r = 0.992, p = 0.001 (Table 1).
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3.4. Impact of arsenic on oxidative stress markers Superoxide radical (SOR), hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents in root and shoot were analyzed to record the level of oxidative stress in P. pinnata under different As treatments (Fig. 3A-C). Data revealed that under 0.2, 0.5, 1.0, and 1.5 mM As treatments SOR content was significantly raised (p < 0.05) in root by 15, 41, 111 and 148% and in shoot by 24, 52, 91 and 115% (Fig.3A) and under similar exposure, H2O2 content in root was
ACCEPTED MANUSCRIPT increased by 11, 22, 32 and 44% and in shoot by 3, 23, 30 and 44%, respectively (Fig. 3B). Lipid peroxidation measured as MDA content was increased by 15, 50, 82, and 120% in root and by 31, 58, 125 and 175% in shoot, respectively over the value of control (Fig.3C). The correlation coefficient analysis between As treatments and oxidative stress markers; SOR (root, r = 0.993, p
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= 0.001; shoot, r = 0.985, p = 0.002), H2O2 (root, r = 0.983, p = 0.003; shoot, r = 0.977, p =
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0.004) and MDA (root, r = 0.996, p = 0.001; shoot, r = 0.999, p = 0.001) showed positive
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correlation (Table 1).
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3.5. Effect of arsenic on distribution of nutrients in roots and shoots
Results showed that arsenic treatments increased the contents of macronutrients Ca, Mg,
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K and S in root from 58183–230477; 37866–69417; 12697-32751; 6899573–9786445 mg kg−1
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dry weight and in shoot from 20567–28642; 8731–20778; 34971-50924; 891870–1999114 mg kg−1 dry weight respectively when the concentration of arsenic in nutrient solution was raised
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from 0.2-1.5 mM (Fig. 4A-D). Further correlations analysis between the macronutrient contents
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and different As treatments showed positive correlation: Ca (root, r = 0.894, p = 0.041; shoot, r = 0.926, p = 0.024), Mg (root, r = 0.779, p = 0.120; shoot, r = 0.870, p = 0.055), and S (root, r =
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0.882, p = 0.048; shoot, r = 0.975, p = 0.005), and K (root, r = 0.974, p = 0.005) contents showed
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positive correlation but in shoot K presented negative correlation (r = -0.905, p = 0.034) under different As treatments. The levels of micronutrients regulation under different As treatments (0.2-1.5 mM) in tested plants were also analyzed which showed that the levels of Fe, Mn, Zn, and Na were increased from 2304–12066, 1661–6014, 473–2686, and 382435–1539142 in roots and 910– 1088, 279–407, 139–315, and 99394–144393 mg kg−1 in shoots, respectively (Fig. 4E-H). The systematic correlation analysis between the micronutrient contents and different As treatments
ACCEPTED MANUSCRIPT suggest that there was positive correlation: Mn (root, r = 0.904, p = 0.035; shoot, r = 0.956, p = 0.011), Zn (root, r = 0.977, p = 0.004; shoot, r = 0.973, p = 0.005), Na (root, r = 0.872, p = 0.054; shoot, r = 0.923, p = 0.025) and Fe (root, r = 0.919, p = 0.027; shoot, r = 0.958, p = 0.010), Table 3 also represents the relationship among nutrient contents affected by As stress and
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how they manage themselves to protect the plant from As toxicity.
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3.6. Impact of arsenic on antioxidant activity and non-protein thiols
The results pertaining to antioxidants (APX and CAT) showed that due to different
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arsenic treatments the activity of APX was increased by 2, 6, 14 and 25% in shoot and 3, 7, 15
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and 28% in root, respectively (Fig. 5A and B). Similarly, at the similar concentrations of As CAT activity was increased by 3, 8, 18 and 53% in root and 2, 6, 12 and 18 % in shoots
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respectively (Fig. 5B).
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Further, content of NP-SH was also influenced by different As treatments (0.2, 0.5 and
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1.0 mM) and results showed that NP-SH in shoots was increased by only 3, 8, 1% in shoots and 2, 6, 1% in root while at higher concentration (1.5 Mm) of arsenic NP-SH content was
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significantly decreased as evidenced by 7% reduction in shoot and by 5% in root respectively (Fig. 5C). Correlation analysis among the antioxidants and different As treatments showed that
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there was positive correlation for APX (root, r = 0.990, p = 0.001; shoot, r = 0.991, p = 0.001) and CAT (root, r = 0.942, p = 0.017; shoot, r = 1.00, p = 0.000) (Table 1) while a negative correlation was recorded for NP-SH (root, r = -0.595, p = 0.290 and in shoot, r = -0.582, p = 0.303) (Table 1). 4. Discussion
ACCEPTED MANUSCRIPT Among environmental stresses, heavy metal contamination is regarded as one of the most serious threats, occurred due to several anthropogenic activities such as industrial activities, agricultural practices, and also due to natural sources (Sharma and Pandey 2014). There are several studies which demonstrated the negative impacts of numerous heavy metals not only on
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plants growth and their metabolisms but they also caused serious risk to human health by
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entering in to the food chain (Adrees et al. 2015a; Ali et al. 2015a; Keller et al. 2015; Tripathi et
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al., 2014; Tripathi et al. 2015, 2016a,b; Tauqeer et al. 2016). Further, it has been clear that the toxic behavior of heavy metals generated oxidative stress in plants by producing reactive oxygen
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species (ROS) which ultimately alter plant metabolic system and thus resulted into the plant
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death (Ali et al. 2015b; Habiba et al. 2015; Tauqeer et al. 2016). Studies also suggested that although toxicity of heavy metals appears on entire plant system but the photosynthetic apparatus
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and pigments are basically the main targets and thus affected significantly (Adreeset al. 2015b;
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Habiba et al. 2015; Keller et al. 2015; Tauqeer et al. 2016). Several techniques have been developed and frequently used to remediate soil contaminated with metals and thus are useful in
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metal immobilization, soil washing and phytoremediation (Wuana and Okieimen 2011).
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Phytoremediation is regarded as a revolutionary technique with the incorporation of indigenous plants for the remediation of heavily metal contaminated soil (Ehsan et al. 2014; Kamran et al.
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2014; Amna et al. 2015). Thereby, numerous species of plants have been utilized to remediate soil contaminated with metal (Raziuddin et al. 2011; Kamran et al. 2014; Habiba et al. 2015). However, the idea of using native plant species in phytoremediation technique has significant consequence since these plants could potentially endure their own environmental conditions in contrast to any other plant species (Kamran et al. 2014).
ACCEPTED MANUSCRIPT Thus, in the present study P. pinnata, a woody plant, has been used to test the capability of its phytoremediation potential against arsenic contamination through the morphophysiological and biochemical investigation. Arsenic treatments at higher concentration (1.5 mM) caused significant reduction in growth, chlorophylls (a and b), carotenoids and protein
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contents. Chlorophyll fluorescence parameters of P. pinnata seedlings were also affected by As
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(Kumar et al. 2014; Kumar et al. 2015). Reduction in morphological and physiological
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parameters at higher concentration of As was accompanied with enhanced level of As accumulation in root and shoot of P. pinnata which caused oxidative stress in plants (Fig. 1 and
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3). These results are in consistence with earlier findings where As stress negatively affected
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morphological and physiological traits of plants (Keller et al. 2015; Tripathi et al. 2016a; Rizwan et al. 2016). Mirza et al. (2010) have studied and documented about decline in growth of Arundo
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donax under elevated arsenic concentration. Hence, decline in biomass accumulation of P.
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pinnata might have occurred due to toxic property of arsenic on key metabolic processes such as photosynthesis and protein synthesis as reported in cork oak (Quercus suber L.), Pinus
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massoniana, Cryptomeria fortune, Cunninghamia lanceolata and Populus alba (Gogorcena et al.
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2011; Liu et al. 2011; Beritognolo et al. 2011).
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It has been well documented that characteristics of arsenic hyper accumulator plants are; arsenic content >1000 mg/kg dry weight in shoot, bioaccumulation factor >1, sometimes reaching up to 50–100 (Brooks 1998), translocation factor is >1 (Wei and Zhou 2004), and greater ability to detoxify and sequester huge amount of arsenic (Rascio and Navari-Izzo 2011). Hence, in the present study it has been well observed that P. pinnata successfully accomplished subsequent 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 (Fig. 2a and b), and
ACCEPTED MANUSCRIPT 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 P. pinnata seedlings (Fig. 1). Further, results of antioxidant enzymes showed that activities of key antioxidant enzymes like APX and CAT were significantly increased in root and shoot of P. pinnata under different
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As treatments. Similar to this, enhanced levels of antioxidant enzymes under heavy metal stress
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in plants have been reported in earlier studies which suggest that this phenomenon might be a powerful strategy for the survival of metal accumulating plants (Afshan et al.2015; Habiba et al.
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2015; Tripathi et al. 2016a). Thus, results of the present study supported one of the possible mechanisms of metal tolerance by enhancing the activities of antioxidant enzymes even under
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lower As concentration (Fig. 5) which reduced oxidative stress. It has been well reported that
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mineral elements play significant role in the accumulation of biomass and protection of plants against different stress conditions (Tripathi et al. 2014; Kumar et al. 2015; Singh et al. 2015).
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Hence, we have investigated consequences of arsenic treatments on nutrient elements
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distribution in roots and shoots of P. pinnata. Similar to the results of antioxidants, levels of mineral elements (Ca, Mg, Fe, Zn, K, Na, S and Mn) were also increased significantly under
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each treatment of As. The results further suggested that mineral elements like Ca, Fe, Zn, Mn
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and Na contents in root and shoot were significantly increased as compared to control (except K in root). In Pteris vittata and Zea mays, Ca contents enhanced significantly under arsenic stress (Li et al. 2006; Mallick et al. 2011). In agreement with our results Marschner (1995) had also demonstrated enhanced level of K contents under As exposure while in other studies Na, Zn, and Mn contents have also been reported to increase under As exposure in different plants (Tu and Ma 2005; Sakai et al. 2010; Mallick et al. 2011). Therefore, significant increase of nutrient elements under As treatments clearly supported the mechanisms of nutrient regulation under
ACCEPTED MANUSCRIPT metal stress condition in plants (Tu and Ma 2005; Sakai et al. 2010; Mallick et al. 2011). Similar results have also been observed by Kumar et al. (2015) in Wrightia arborea seedlings treated with As. Furthermore, Sakai et al. (2010) have proposed that sulfur (S) is a major component of
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glutathione, non protein thiols (metabolites antioxidants) and phytochelatins. NP-SH form
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complex with excess metals inside the cell and thus protect cell form metal toxicity (Raj et al. 2011). The outcome of present study suggests that similar to sulfur content NP-SH content was
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significantly greater in root and shoot of P. pinnata which was exposed to 0.2-1.0 mM As (Fig.4C). However, at 1.5 mM As, NP-SH content was declined significantly. These results
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clearly demonstrate the positive role of NP-SH in conferring the arsenic tolerance in P. pinnata
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at the range of 0.2–1.5 mM As concentration as supported by data of growth, pigments etc.
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5. Conclusion
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This study highlights greater arsenic accumulating potential of P. pinnata when grown in hydroponic culture. The results also suggest that status of antioxidant enzymes and mineral
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elements are actively regulated under arsenic stress. Under tested concentrations of arsenic, S and NP-SH contents have significantly increased, thereby suggesting their essential role in
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upholding tolerance against arsenic stress. Translocation factor and bioaccumulation factors also suggested that P. pinnata fulfills the criteria of arsenic hyper accumulator plants and 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 P. pinnata. Acknowledgments
ACCEPTED MANUSCRIPT Authors are thankful to the Head, Department of Botany, University of Allahabad for providing laboratory facilities. Authors are also thankful to Dr. Dhanvinder Singh, Sr. Soil Scientist, Department of Soil, Punjab Agriculture University, Ludhiana, for providing ICAPAES facility to determine As and mineral elements. Authors extend their thanks to the UGC,
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New Delhi for financial support.
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References
Acharyya, S. K., Lahiri, S., Raymahashay, B. C., Bhowmik, A., 2000. Arsenic toxicity of ground
US
water in parts of the Bengal basin in India and Bangladesh: the role of Quaternary
AN
stratigraphy and Holocene sea-level fluctuation. Environ Geol 39, 1127-1137. Adrees, M., Ali, S., Rizwan, M., Ibrahim, M., Abbas, F., Farid, M., Rehman, M. Z., Irshad, M.
M
K., Bharwana, S. A., 2015b. The effect of excess copper on growth and physiology of
ED
important food crops: a review. Environ. Sci. Pollut. Res. 22, 8148–8162.
PT
Adrees, M., Ali, S., Rizwan, M., Rehman, M. Z., Ibrahim, M., Abbas, F., Farid, M., Qayyum, M. K., Irshad, M. K., 2015a. Mechanisms of silicon-mediated alleviation of heavy metal
CE
toxicity in plants: a review. Ecotoxicol. Environ. Saf. 119, 186–197.
AC
Aebi, I.I,, 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Afshan, S., Ali, S., Bharwana, S. A., Rizwan, M., Farid, M., Abbas, F., Ibrahim, M., Mehmood, M. A., Abbasi, G. H., 2015. Citric acid enhances the phytoextraction of chromium, plant growth, and photosynthesis by alleviating the oxidative damages in Brassica napus L. Environmental Science and Pollution Research 22(15), 11679-11689.
ACCEPTED MANUSCRIPT Ahamed, S., Sengupta, M. K., Mukherjee, A., Hossain, M. A., Das, B., Nayak, B., Pal, A., Chakraborti, D., 2006. Arsenic groundwater contamination and its health effects in the state of Uttar Pradesh (UP) in upper and middle Ganga plain, India: a severe danger. Sci Total Environ 370, 310-322.
IP
T
Ali, S., Bharwana, S. A., Rizwan, M., Farid, M., Kanwal, S., Ali, Q., Ibrahim, M., Gill, R. A.,
CR
Khan, M. D., 2015b. Fulvic acid mediates chromium (Cr) tolerance in wheat (Triticum aestivum L.) through lowering of Cr uptake and improved antioxidant defense system.
US
Environ. Sci. Pollut. Res. 22, 10601–10609.
AN
Ali, S., Chaudhary, A., Rizwan, M., Anwar, H. T., Adrees, M., Farid, M., Irshad, M. K., Hayat, T., Anjum, S. A., 2015a. Alleviation of chromium toxicity by glycinebetaine is related to
M
elevated antioxidant enzymes and suppressed chromium uptake and oxidative stress in
ED
wheat (Triticum aestivum L.). Environ. Sci. Pollut. Res. 22, 10669–10678.
PT
Amna, Ali, N., Masood, S., Mukhtar, T., Kamran, M. A., Rafique, M., Munis, M. F. H., Chaudhary, H. J., 2015. Differential effects of cadmium and chromium on growth,
CE
photosynthetic activity, and metal uptake of Linum usitatissimum in association with
AC
Glomus intraradices. Environ. Monit. Assess. 187, 1–11. Arditti, J., Dunn, A., 1969. Environmental Plant Physiology-Experiments in Cellular and Plant Physiology. New York: Holt, Rinehart and Winston Inc., pp. 312. Beritognolo, I., Harfouche, A., Brilli, F., Prosperini, G., Gaudet, M., Brosche´, M., Salani, F., Kuzminsky, E., Auvinen, P., Paulin, L., Kangasjarvi, J., Loreto, F., Valentini, R., Mugnozza, G. S., Sabatti, M., 2011. Comparative study of transcriptional and physiological
ACCEPTED MANUSCRIPT responses to salinity stress in two contrasting Populusalba L. genotypes. Tree Physiol 31, 1335–1355. Bhattacharyya, D., Mukherjee, P. K., Ray, A. K., Sengupta, S., 2004. Arsenic-polluted
T
groundwater in West Bengal: A cost-effective remedy. Current Science 86(9), 1206-1209.
IP
Brooks, R. R., 1998. Plants that Hyperaccumulate Heavy Metals: Their Role in
CR
Phytoremediation, Microbiology, Archaeology, Mineral Exploration and Phytomining. CAB
US
Internationsl, Oxford, UK.
Bureau of Indian Standards (BIS), 2003. Indian Standard: Drinking Water. Specification (first
AN
revision), Amendment No. 2, New Delhi, September 2003.
M
Chauhan, V. S., Nickson, R. T., Chauhan, D., Iyengar, L., Sankararamakrishnan, N., 2009. Ground water geochemistry of Ballia district, Uttar Pradesh, India and mechanism of arsenic
ED
release. Chemosphere 75, 83-91.
PT
Ehsan, S., Ali, S., Noureen, S., Mahmood, K., Farid, M., Ishaque, W., Shakoor, M.B., Rizwan,
CE
M., 2014. Citric acid assisted phytoremediation of cadmium by Brassica napus L.
AC
Ecotoxicol. Environ. Saf. 106, 164–172. Ellmann, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Elstner, E. F., Heupel, A., 1976. Inhibition of nitrite formation from hydroxyl ammonium chloride: a simple assay for superoxide dismutase. Anal Biochem 70, 616-620. Gogorcena, Y., Larbi, A., Andaluz, S., Carpena, R. O., Abadía, A., Abadía, J., 2011. Effects of cadmium on cork oak (Quercus suber L.) plants grown in hydroponics. Tree physiology 31(12), 1401-1412.
ACCEPTED MANUSCRIPT Habiba, U., Ali, S., Farid, M., Shakoor, M. B., Rizwan, M., Ibrahim, M., Abbasi, G. H., Hayat, T., Ali, B., 2015. EDTA enhanced plant growth, antioxidant defense system, and phytoextraction of copper by Brassica napus L. Environmental Science and Pollution Research 22(2), 1534-1544.
IP
T
Heath, R. L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts I. Kinetics and
CR
stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125, 189-198. Hu, Y., Nan, Z., Jin, C., Wang, N., Luo, H., 2014. Phytoextraction potential of poplar (Populus
AN
cadmium. Int J Phytoremed 16(5), 482-495.
US
alba L. var. pyramidalis Bunge) from calcareous agricultural soils contaminated by
Huang, H., Yu, N., Wang, L., Gupta, D. K., He, Z., Wang, K., Yang, X. E., 2011. The
M
phytoremediation potential of bioenergy crop Ricinus communis for DDTs and cadmium co-
ED
contaminated soil. Biores Technol 102(23), 11034-11038.
PT
Kacálková, L., Tlustoš, P., Száková, J., 2015. Phytoextraction of risk elements by willow and
CE
poplar trees. Int J Phytoremed, 17(5), 414-421. Kamran, M. A., Mufti, R., Mubariz, N., Syed, J. H., Bano, A., Javed, M. T., Chaudhary, H. J.,
AC
2014. The potential of the flora from different regions of Pakistan in phytoremediation: areview. Environ. Sci. Pollut. Res. 21, 801–812. Keller, C., Rizwan, M., Davidian, J. C., Pokrovsky, O. S., Bovet, N., Chaurand, P., Meunier, J. D., 2015. Effect of Silicon on wheat seedlings (Triticum turgidum L.) grown in hydroponics under Cu stress. Planta 241, 847–860.
ACCEPTED MANUSCRIPT Kumar, D., Singh, V. P., Tripathi, D. K., Prasad, S. M., Chauhan, D. K., 2015. Effect of arsenic on growth, arsenic uptake, distribution of nutrient elements and thiols in seedlings of Wrightia arborea (Dennst.) Mabb. Int J Phytoremed17, 128-134. Kumar, D., Tripathi, D. K., Chauhan, D. K., 2014. Phytoremediation potential and nutrient status
IP
T
of Barringtonia acutangula gaerth. Tree seedlings grown under different chromium (CrVI)
CR
treatments. Biological trace element research 157(2), 164-174.
Li, W., Chen, T., Huang, Z., Lei, M., Liao, X., 2006. Effect of arsenic on chloroplast
US
ultrastructure and calcium distribution in arsenic hyperaccumulator Pteris vittata L.
AN
Chemosphere 62, 803–809.
Lichtenthaler, H. K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic
ED
M
biomembranes, Methods Enzymol 148, 350-382.
Liu, T., Wu, F., Wang, W., Chen, J., Li, Z., Dong, X., Patton, J., Pei, Z., Zheng, H., 2011. Effects
PT
of calcium on seed germination, seedling growth and photosynthesis of six forest tree
CE
species under simulated acid rain. Tree Physiol 31, 402–413. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J., 1951. Protein measurement with the
AC
Folinphenol reagent. J Biol Chem 193, 265–275. 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.
ACCEPTED MANUSCRIPT Mirza, N., Mahmood, Q., Pervez, A., Ahmad, R., Farooq, R., Shah, M. M., Azim, R. M., 2010. Phytoremediation potential of Arundo donax in arsenic-contaminated synthetic wastewater. Bioresour Technol 101, 5815–5819. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate specific
IP
T
peroxidasein spinach chloroplasts. Plant Cell Physiol. 22, 867–280.
CR
Nriagu, J. O., 2002. Arsenic poisoning through the ages. Marcel Dekker, Inc.: New York,pp. 1-
US
26.
Raj, A., Pandey, A. K., Sharma, Y. K., Khare, P. B., Srivastava, P. K., Singh, N., 2011.
M
Bioresour Technol 102, 9827–9832.
AN
Metabolic adaptation of Pteris vittata L. gametophyte to arsenic induced oxidative stress.
ED
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.
PT
Raziuddin, Farhatullah, Hassan, G., Akmal, M., Shah, S. S., Mohammad, F., Shafi, M., Bakht, J.,
CE
Zhou, W., 2011. Effects of cadmium and salinity on growth and photosynthesis parameters
AC
of brassica species. Pak. J. Bot. 43, 333–340. Rizwan, M., Meunier, J. D., Davidian, J. C., Pokrovsky, O. S., Bovet, N., Keller, C., 2016. Silicon alleviates Cd stress of wheat seedlings (Triticum turgidum L. cv. Claudio) grown in hydroponics. Environmental Science and Pollution Research 23(2), 1414-1427. 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.
ACCEPTED MANUSCRIPT Shah, B. A., 2010. Arsenic-contaminated groundwater in Holocene sediments from parts of Middle Ganga Plain, Uttar Pradesh, India. Curr Sci 98(10), 1359-1365. Sharma, P., Pandey, S., 2014. Status of phytoremediation in world scenario. International Journal
T
of Environmental Bioremediation & Biodegradation 2(4), 178-191.
IP
Singh, S., Srivastava, P. K., Kumar, D., Tripathi, D. K., Chauhan, D. K., Prasad, S. M., 2015.
CR
Morpho-anatomical and biochemical adapting strategies of maize (Zea mays L.) seedlings against lead and chromium stresses. Biocatalysis and Agricultural Biotechnology 4(3), 286-
US
295.
AN
Smith, A. H., Goycolea, M., Haque, R., Biggs, M. L., 1998. Marked increase in bladder and lung cancer mortality in a region of northern Chile due to arsenic in drinking water. Amr J
ED
M
Epidemiol 147, 660-669.
Tauqeer, H. M., Ali, S., Rizwan, M., Ali, Q., Saeed, R., Iftikhar, U., Ahmad, R., Farid, M.,
PT
Abbasi, G. H., 2016. Phytoremediation of heavy metals by Alternanthera bettzickiana:
CE
growth and physiological response. Ecotoxicology and environmental safety 126, 138-146. Tripathi, D. K., Singh, S., Singh, V. P., Prasad, S. M., Chauhan, D. K., Dubey, N. K., 2016a.
AC
Silicon Nanoparticles More Efficiently Alleviate Arsenate Toxicity than Silicon in Maize Cultiver and Hybrid Differing in Arsenate Tolerance. Frontiers in Environmental Science, 4, p.46. Tripathi, D. K., Singh, V. P., Chauhan, D. K., Prasad, S. M., Dubey, N. K., 2014. Role of macronutrients in plant growth and acclimation: recent advances and future prospective. In Improvement of Crops in the Era of Climatic Changes. Springer New York, pp. 197-216
ACCEPTED MANUSCRIPT Tripathi, D. K., Singh, V. P., Prasad, S. M., Chauhan, D. K., Dubey, N. K., Rai, A. K., 2015. Silicon-mediated alleviation of Cr(VI) toxicity in wheat seedlings as evidenced by chlorophyll fluorescence, laser induced break down spectroscopy and anatomical changes. Ecotoxicol. Environ. Saf. 113, 133–144.
IP
T
Tripathi, D. K., Singh, V. P., Prasad, S. M., Dubey, N. K., Chauhan, D. K., Rai, A. K., 2016b.
CR
LIB spectroscopic and biochemical analysis to characterize lead toxicity alleviative nature of silicon in wheat (Triticum aestivum L.) seedlings. Journal of Photochemistry and
US
Photobiology B: Biology 154, 89-98.
AN
Tu, C., Ma, L. Q., 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.
M
Velikova, V., Yordanov, I., Edreva, A., 2000. Oxidative stress and some antioxidant system in
ED
acid rain-treated bean plants, Plant Sci151, 59-66.
PT
Wei, S. H., Zhou, Q. X., 2004. Discussion on basic principles and strengthening measures for
CE
phytoremediation of soils contaminated by heavy metals. Chinese J Ecol 23, 65–72.
edn.
AC
WHO, 1993. Guideline for drinking water quality. Recommendations, Geneva, 1993, vol. 1, 2nd
Wuana, R. A., Okieimen, F. E., 2011. Heavy metals in contaminated soils: a review of sources, chemistry, risks and best available strategies for remediation. Isrn Ecology, 2011. Ye, N., Zhu, G., Liu, Y., Li, Y., Zhang, J., 2011. ABA Controls H2O2 Accumulation Through the Induction of OsCATB in Rice Leaves Under Water Stress. Plant Cell Physiol 52, 689-698.
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Fig. 1. Impact of different As treatments on root and shoot length (A), fresh mass (B), dry mass (C), As accumulation in root and shoot (D), correlation between As concentrations (0-1.5 mM) and As content in root and shoot (E), and bioaccumulation factor and translocation factor (F).
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Table 1: Correlation (r) between arsenic treatments (0.2-2.0 mM), and root, shoot and leaf with different parameters in seedlings of Pongamia pinnata Investigated parameter
Arsenic Shoot
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IP
T
r P r p R p Length -0.987** 0.002 -0.951* 0.013 Fresh mass -0.989** 0.001 -0.962** 0.009 Dry mass -0.993** 0.001 -0.993** 0.001 SOR 0.993** 0.001 0.985** 0.002 H2O2 0.983** 0.003 0.977** 0.004 MDA 0.996** 0.001 0.999** 0.001 Total protein -0.991** 0.001 -0.976** 0.004 CAT 0.942* 0.017 1.000** 0.000 APX 0.990** 0.001 0.991** 0.001 NP-SH -0.595 0.290 -0.582 0.303 Chlorophyll a -0.996** 0.001 Chlorophyll b -0.993** 0.001 Carotenoids -0.990** 0.001 Fv/Fm -0.986** 0.002 Fv/F0 -0.959* 0.010 Fm/F0 -0.962** 0.009 qP -0.919* 0.027 NPQ 0.992** 0.001 **correlation (r) is significant at p<0.01 significance level, and *correlation (r) is significant at p<0.05 significance level
ACCEPTED MANUSCRIPT
Table 2: Effect of different concentrations of As on chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll a/ chlorophyll b (Chl a/Chlb ratio), carotenoids (Car), chlorophyll/carotenoids ratio (Chl (a+b)/Car ratio), and total protein; root and shoot in seedlings of Pongamia pinnata
0.8 03 0.7 22 0.6 49 0.5 63 0.4 31
2.7 52 2.8 12 2.8 20 2.8 24 2.8 31
0.6 43 0.6 11 0.5 87 0.5 59 0.5 13
4.6 86 4.5 04 4.2 23 3.8 52 3.2 18
±0.02 6a ±0.02 7a ±0.02 8ab ±0.02 2b ±0.02 3c
±0.1 6a ±0.1 8a ±0.1 9ab ±0.1 7ab ±0.1 4c
CR
±0.08 1b ±0.08 6b ±0.09 0ab ±0.08 8a ±0.08 3a
US
±0.03 1a ±0.03 5a ±0.02 9ab ±0.02 8b ±0.02 2c
AN
±0.1 10a ±0.1 01a ±0.0 87b ±0.0 81b ±0.0 73c
T
2. 21 2. 03 1. 83 1. 59 1. 22
Total protein (mg g-1 FW)
IP
Chl (a+b)/Car ratio
M
1.5
Car (mg g-1 FW)
ED
1.0
Chl a/Chl b ratio
PT
0.5
Chl b (mg g-1 FW)
CE
0.2
Chl a (mg g-1 FW)
AC
Treatm ents As (mM) 0.0
8. 91 8. 08 7. 43 6. 55 4. 86
Root ±0.2 7a ±0.3 5a ±0.3 3a ±0.2 5ab ±0.2 0c
Shoot 16. ±0.4 74 2a 15. ±0.6 38 0a 14. ±0.6 47 9ab 13. ±0.6 81 1ab 11. ±0.4 79 6c
ACCEPTED MANUSCRIPT
Table 3: Correlation (r) matrix among nutrient contents in roots and shoots of Pongamia pinnata seedlings, under different As concentrations (0.2-2.0 mM) FeS
FeR Mn
Mn
Zn
Zn
Na
Na
Ca
Ca
S
R
S
R
S
R
S
R
.87 2 .88 3* .88 1* .96 1** .84 0 .97 5** .88 8* .94 8* .86 7
.94 3* .86 4 .72 3 .99 7** .91 6* .99 2** .81 9 .85 6
.96 9** .81 2 .92 2* .86 9 .91 2* .81 4 .97 6**
.85 6 .95 9* .95 6* .96 5** .93 4* .90 9* .85 6 .94 6* .98 5** .97 9** .94 0* .88 4*
M gS
.77 9
.91 8*
.77 9
.77 1
.92 8*
.78 8
M gR
.61 1
.76 5
.66 4
.57 4
.90 1*
SS
.99 5**
.93 3*
.96 4**
.95 0*
SR
.74 3
.79 9
.80 6
.67 1
S
K R
M gS
M gR
SS
IP
.83 6 .84 5 .80 6 .94 8* .99 3** .85 3 .76 5 .79 8 .77 9 .82 2 .99 3**
.87 0 .99 7** .96 2** .88 7* .73 2 .99 2** .90 2* .98 0** .80 7 .88 4*
CR
M nS M nR Z nS Z nR N aS N aR C aS C aR K
KR
US
R
.86 0 .82 9 .85 5 .84 6 .86 5 .99 3**
.67 6 .88 9* .73 8 .91 1* .85 2
.89 4* .98 7** .78 3 .82 1
.94 7* .96 .86 7** 5 .82 .82 3 4
.76 9
.82 6
.89 1*
.75 2
.95 5*
.84 3
.58 5
.61 7
.68 6
.94 6*
.53 8
.84 9
.63 7
.87 9
.93 1*
.99 7**
.98 3**
.81 6
.90 7*
.86 8
.90 4*
.93 5*
.66 0
.76 0
.80 7
.99 3**
.61 2
.84 2
.67 2
ED
Fe
PT
S
CE
A sR Fe
KS
T
.94 6* .93 4* .97 5** .88 4* .95 9* .99 8** .96 2** .79 9 .94 1* .88 1* .93 2* .82 2 .96 5**
AC
R
AN
As
M
AsS
.81 6 .99 7** .89 7* .83 3 .86 1
.77 0 .65 4
.90 0*
.98 4**
.79 .62 0 5
.87 0
.84 .94 0 8*
.7 6 0
ACCEPTED MANUSCRIPT *p<0.05 probability level, **p<0.01 probability level, S= Shoot, and R = Root
IP
P. pinnata successfully tolerate the arsenic concentration upto 0.2-1.0 mM without
CR
T
Research Highlights
showing As toxicity while at 1.5 mM of As shows toxicity symptoms. Mineral nutrients and antioxidant activity were increased significantly due to increased As concentration
US
Bioaccumulation and translocation factor was >1 and also had greater ability to detoxify
AN
and sequester huge amount of arsenic
Thus, P. pinnata may be useful for arsenic phytoremediation program
Further, non-protein thiols played important role in imparting arsenic tolerance
AC
CE
PT
ED
M