Transgenic Arabidopsis thaliana expressing fungal arsenic methyltransferase gene (WaarsM) showed enhanced arsenic tolerance via volatilization

Environmental and Experimental Botany 132 (2016) 113–120

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Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot

Transgenic Arabidopsis thaliana expressing fungal arsenic methyltransferase gene (WaarsM) showed enhanced arsenic tolerance via volatilization Shikha Vermaa,b , Pankaj Kumar Vermaa,b , Veena Pandeb , Rudra Deo Tripathic , Debasis Chakrabartya,* a

Genetics and Molecular Biology Division, CSIR-National Botanical Research Institute, India Department of Biotechnology, Kumaun University, India c Plant Ecology and Environmental Science Division, CSIR-National Botanical Research Institute, India b

A R T I C L E I N F O

Article history: Received 23 May 2016 Received in revised form 29 August 2016 Accepted 29 August 2016 Available online 30 August 2016 Keywords: Arsenic Arsenic methyltransferase Bioremediation Volatilization

A B S T R A C T

Arsenic contamination in agricultural soil leads to the transfer of arsenic into the food-chain and adversely affects the human health. Generation of genetically engineered plants to transform inorganic arsenic to methylated and volatile arsenic species is one of the efficient strategy to lower arsenic contamination. In the present study, we genetically engineered Arabidopsis thaliana with arsenic methyltransferase (WaarsM) gene of a fungus Westerdykella aurantiaca, isolated from arseniccontaminated sites of West Bengal. The WaarsM transgenic A. thaliana plants showed greatly enhanced tolerance to AsV and AsIII compared to wild-type (WT) plants. WaarsM expressing transgenic plants evolved 17.5 ng and 113 ng volatile arsenicals (mg 1 fresh weight) after 48 h of exposure to 250 mM AsV and 50 mM AsIII, respectively. Long-term exposure resulted in 36% and 16% less arsenic accumulation in seeds and shoots, respectively compared to WT plants. Additionally, the S. cerevisiae cells expressing WaarsM showed short lag phase in the presence of arsenic and potentially tolerate up to 5 mM AsV and 1 mM AsIII. In conclusion, WaarsM from arsenic tolerant fungus can be used in a novel biotechnological solution to decrease arsenic accumulation in food crops grows in arsenic affected areas. ã 2016 Elsevier B.V. All rights reserved.

1. Introduction Arsenic adversely affects the health of millions all over the world, particularly in Southeast Asia including India, Bangladesh, China (Martinez et al., 2011). Arsenic can find its way to the grains of cereal crops (rice, wheat), vegetables and fruit plants irrigated with arsenic-contaminated water (Roychowdhury et al., 2003; Roychowdhury et al., 2002; Tripathi et al., 2007). Rice straw also accumulates high level of arsenic and widely used as fodder presenting an alternate route for arsenic entry into the food chain (Abedin et al., 2002; Rahman et al., 2008). In the environment, arsenic exists as inorganic or organic forms. Arsenate is highly oxidized and predominant form in aerobic conditions while highly reduced arsenite (AsIII) is the predominant form in anaerobic environments, such as flooded rice paddy fields (Finnegan and Chen, 2012). Arsenate act as phosphate (Pi) analog and transported

* Corresponding author. E-mail address: [email protected] (D. Chakrabarty). http://dx.doi.org/10.1016/j.envexpbot.2016.08.012 0098-8472/ã 2016 Elsevier B.V. All rights reserved.

across the plasmalemma by Pi transporter (PHT) proteins (Wu et al., 2011). Under low phosphate conditions, AsV may outcompete Pi for entry into the plant, amplifying Pi deprivation symptoms. Arsenite enters to the root cells through aquaporin (nodulin26-like intrinsic) proteins, such as rice OsNIP2;1/OsLsi1 facilitate AsIII uptake and transport via OsLsi2 (Ma et al., 2008). The molecular mechanism of arsenic detoxification and tolerance in plants are still elusive. Earlier, it has been shown that plants detoxified arsenic by reducing AsV to AsIII, which subsequently form complexes with g-glutamylcysteine (g-EC), glutathione (GSH) and phytochelatins (Cobbett, 2000; Dhankher et al., 2002). Recent study showed that rice glutaredoxin also modulates aquaporin to lowers arsenic accumulation in the plants (Verma et al., 2016a,b). In prokaryote, another important detoxification pathway was reported that involves removal of arsenic by converting inorganic arsenic to volatile organic compounds such as trimethyl arsine through a series of methylation reactions catalyzed by S-adenosylmethionine methyltransferases (Cullen, 2005). However, till date no functional Sadenosylmethionine methyltransferase is reported in the plants.

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Recent studies on arsenic methylation and volatilization by arsenic methyltransferase genes from bacteria, algae and fungus revealed that these genes are responsible for arsenic detoxification. Arsenic methyltransferases are the enzymes that can catalyze the biosynthesis of several methylated forms of arsenic including volatile TMAs(III) (Chen et al., 2014; Meng et al., 2011; Qin et al., 2006; Verma et al., 2016c). These studies indicated that the expression of single methyltransferase gene is sufficient to produce both volatilization and tolerance to arsenic. These results point to the possibility of engineering plants for arsenic volatilization and use for the phytoremediation of arsenic-contaminated water and soil to improve the safety of the food. However, all earlier reported studies used prokaryotic arsenic methyltransferase for arsenic detoxification. With the aim to identify an arsenic methyltransferase with higher kinetic activity, we have cloned a WaarsM gene from Wesredykella aurantica, isolated from arsenic-contaminated sites of West Bengal. We further demonstrated the enhanced arsenic resistance in vivo when expressed in arsenic-sensitive Escherichia coli strain AW3110 (arsRBC operon deleted). WaarsM catalyzes the formation of several methylated species including volatile products (TMAO) (Verma et al., 2016c). Because fungi and plants differ in their metabolism and complexity, it was important to test the role of WaarsM in planta as well. The objective of the current study was to test whether constitutive expression of WaarsM would increase arsenic tolerance and volatilization in transgenic A. thaliana. In the present study, we found that model plant A. thaliana transformed with WaarsM gene resulting in methylating inorganic arsenic including volatile arsenicals. Moreover, the expression of WaarsM increased arsenic tolerance and reduces its accumulation in transgenic A. thaliana plants. This WaarsM transgenic approach proved effective in A. thaliana and can be used for arsenic phytoremediation. 2. Material and methods 2.1. Yeast, bacterial strains and growth media The yeast Saccharomyces cerevisiae strain INVSc1 (MATa his3D1 leu2 trp1-289 ura3-52) (InvitrogenTM, USA) was used for arsenic tolerance assay (Table S1). YPD medium (1% yeast extract, 2% peptone, 2% glucose/galactose) used for yeast growth and maintenance. For selection and maintenance of yeast transformants, a semi-defined medium (SDC) (0.67% yeast nitrogen base, 0.5% casamino acids, 20 mg/l adenine, 20 mg/l L-tryptophan and 2% glucose/galactose) was used. In transformed yeast, expression of WaarsM was induced by 2% (w/v) galactose instead of 2% (w/v) glucose. The Escherichia coli strain DH5a was used for amplification of plasmid (Table S1). LB media supplemented with appropriate antibiotic (Ampicillin) utilized for the growth and maintenance of bacterial strain and transformants. 2.2. Cloning of WaarsM gene from W. aurantiaca The full-length WaarsM gene (accession KP165533.1) sequence was amplified from cDNA using WaarsM F and WaarsM R primers (Table S2). RNA was isolated from Westerdykella aurantica using the RNeasy RNA purification kit (Qiagen, USA) and the cDNA was synthesized using the RevertAid First Strand cDNA synthesis kit (Thermo ScientificTM, USA). The PCR product was cloned in the pTZ vector to derive pTZ-WaarsM and verified by sequencing.

INVSc1 strain using lithium acetate/heat shock method (Gietz and Schiestl, 2007) and grown on synthetic medium lacking uracil to select positive transformants. 2.4. Assessment of arsenic tolerance and growth curve analysis in engineered S. cerevisiae S. cerevisiae (INVSc1) cells transformed with pYES2-WaarsM and pYES2 were inoculated into 5 ml of SC medium with 2% glucose at 30  C overnight at 220 rpm. The cells of late exponential phase were diluted in the medium at concentration of 1 107 cells/ml and culture was further inoculated in 5 ml SC medium with 2% galactose as a carbon source and the indicated concentrations of AsV and AsIII. After 48 h incubation at 30  C for at 220 rpm optical density at 600 nm was measured. In addition to empty vector arsenic-free medium with galactose as the carbon source was used as negative control. Growth curve analysis was performed using S. cerevisiae (INVSc1) cells of late exponential phase at a concentration of 1 107 cells/ml. Cells transformed with pYES2-WaarsM and pYES2 inoculated into 100 ml of SC medium with 2% galactose as carbon source containing 500 mM AsV and 50 mM AsIII and incubated at 30  C, 220 rpm. The growth of cells was measured at 600 nm after every 4 h up to 60 h. The growth curve was prepared based on absorbance data. 2.5. Generation and selection of transgenic A. thaliana The native fungal gene, WaarsM (0.876 kb) cloned into the 35S promoter cassette of binary vector pBI121 (Clonetech) at XbaI and SacI sites (Fig. S1A). The resultant pBI121-WaarsM transformed into Agrobacterium tumefaciens (GV3101 strain) using freeze-thaw method (Jyothishwaran et al., 2007; Weigel and Glazebrook, 2006) and positive colonies were selected using colony PCR (Fig. S1B). A single colony was further used to transform A. thaliana ecotype Col-0 (wild type, WT) by floral dip method (Zhang et al., 2006). Transgenic lines were screened on 1/2 MS medium containing 50 mg L 1 kanamycin (Clough and Bent, 1998) and further tested by PCR analysis (Fig. S1C). WaarsM gene expression was analyzed by qRT-PCR (Fig. 2A). For further study, three independent transgenic lines L1, L2 and L3 of T3 generation were selected. 2.6. Plant growth and arsenic tolerance Wild type (WT, accession Columbia 0) and transgenic plants expressing WaarsM were grown at 22  C under 16-h light/8-h dark conditions. For arsenic tolerance analysis in A. thaliana, seeds were surface sterilized and sown on medium containing 1/2 MS with 2% sucrose, pH 5.8, and 0.8% agar and kept in dark at 4  C for 2 days to synchronize germination. The plates were then transferred to a growth chamber at 22  C under16-h light/8-h dark regime to facilitate germination. After germination, 3 days old A. thaliana seedlings were transferred onto plates containing the 1/2 MS agar medium without or with AsV and AsIII and grown vertically at 22  C under 16-h light/8-h dark regime for 10 days. For soil cultivation, A. thaliana seeds was germinated and cultivated in the soil-rite spiked with 250 mm AsV and 25 mm AsIII. The pots then transferred at 22  C under16-h light/8-h dark regime. After 2 months the rosette leaves, stalks and seeds were harvested for arsenic estimation.

2.3. Expression of WaarsM gene in S. cerevisiae 2.7. Estimation of volatile arsenic in A. thaliana The WaarsM gene was cloned in pYES2 yeast expression vector as described in our earlier study (Verma et al., 2016c). The pYES2 vector containing WaarsM was transformed into S. cerevisiae

For the determination of volatile arsenic, WT and all transgenic lines were grown hydroponically by the method outlined below.

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Germination was initiated by first placing seeds on 1/2 MS media for 2 weeks then seedlings were transfer to the vessel containing 1/2 strength nutrient solution, pH 5.6 (Somerville and Ogren, 1982). To trap TMA(III), vessel mouth was covered with nitrocellulose membrane impregnated with 2 ml of 6% H2O2, which oxidizes TMA (III) to TMAO. After the indicated time intervals, membrane, media and plant tissues were harvested. All samples for arsenic determination were dried at 60  C for overnight and digested with a concentrated nitric acid (HNO3). The ICP-MS analysis was determined arsenic concentration in the different samples.

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WaarsM tolerate up to 5 mM AsV and 1 mM AsIII. Thus, the expression of WaarsM mediates a dramatic increase in arsenic tolerance of S. cerevisiae (INVSc1) cells. WaarsM expressing cells in the presence of 500 mM AsV and 50 mM AsIII, showed short lag phase and the first significant change in the growth were observed after 16–20 h whereas, for the cells harbouring empty vector it was between 30 and 50 h. These results showed that the growth of WaarsM expressing cells were higher compared to the cells containing empty vector during arsenic stress (Fig. 1C, D). 3.2. Generation and identification of transgenic A. thaliana plants

2.8. Statistical analysis Each experiment was performed with three replicates and repeated at least, three to five times. Data are presented as the average of the mean  SE. One-way ANOVA and two-way ANOVA were used to determine significance using Graph Pad Prism (Graph Pad Software, Version 1.0, San Diego, Calif., USA) and the treatment mean values were compared at P  0.05. 3. Results 3.1. Assessment of arsenic tolerance and growth curve analysis in engineered S. cerevisiae WaarsM transformed S. cerevisiae (INVSc1) cells were grown in SC medium with 2% galactose and indicated concentrations of AsV and AsIII (Fig. 1A, B). Dose-response analysis showed that yeast cells harboring WaarsM displayed higher tolerance compared to S. cerevisiae cells bearing empty vector. The growth of S. cerevisiae cells carrying empty vector diminished in the presence of 2 mM AsV and 0.6 mM AsIII while S. cerevisiae cells expressing

To study the role of WaarsM in planta, we expressed WaarsM in A. thaliana plants under the control of CaMV 35S promoter (Fig. S1A). Eleven individual plants were determined to be NptIIpositive after PCR screening using shoot tissue DNA (Fig. S1C). qRTPCR was performed to validate the transgenic expression; three homozygous lines of T3 were selected for further analysis (Fig. 2A). 3.3. Heterologous expression of WaarsM enhances arsenic tolerance in A. thaliana To study the influence of WaarsM on plant growth in arsenic stress, transgenic and WT plants were grown on plates containing indicated concentrations of AsV and AsIII for 10 days. In the absence of arsenic, these plants appeared similar to WT plants indicating that insertion of WaarsM was not deleterious (Fig. 2B). In the presence of 250 mM of AsV, transgenic lines had uninhibited roots growth while it was stunted in WT plants (Fig. 2C, E). Transgenic lines treated with 500 mM AsV had better root growth with well-developed root systems as compared to WT plants (Fig. 2D, E). Also, transgenic plants accumulated more fresh weight

Fig. 1. Heterologous expression of WaarsM enhances tolerance in S. cerevisiae strain INVSc1 to AsV and AsIII. (A, B) Yeast cells transformed with pYES2-WaarsM and pYES2 were grown in YPD medium with indicated concentration of A) Arsenate and B) Arsenite at 30  C, growth was monitored by OD600. (C, D) Growth curve analysis of S. cerevisiae (INVSc1) cells transformed with pYES2-WaarsM and pYES2 in SC medium containing C) 500 mM AsV and D) 50 mM AsIII and incubated at 30  C at 220 rpm. Absorbance was measured at 600 nm after every 4 h up to 60 h. Error bars represent mean  SE values of three replicates.

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Fig. 2. Tolerance of transgenic A. thaliana plants expressing WaarsM to AsV. A) qRT-PCR analysis of relative WaarsM mRNA transcripts in transgenic lines L1, L2 and L3. (B–D) Comparative growth of 3 day old A. thaliana seedlings grown vertically on plates with B) no arsenic, C) 250 mM AsV, D) 500 mM AsV for 10 days. (E, F) Statistical analyses of E) root elongation F) fresh weight and of 10 day old seedlings of WT, L1, L2 and L3 after cultivation under different AsV concentrations (n = 5 plants per treatment per line. Plants shown are representative of three independent experiments. Scale bars, 15 mm. Asterisks indicate significant differences from the WT detected by one-way analysis of variance (ANOVA) (**P < 0.01; ***P < 0.001). Error bars, mean  SEM.

Fig. 3. Tolerance of transgenic A. thaliana plants expressing WaarsM to AsIII. (A–C) Comparative growth of 3 day old A. thaliana seedlings grown vertically on plates with A) no arsenic, B) 25 mM AsIII and C) 50 mM AsIII for 10 days. (D, E) Statistical analyses of D) root elongation E) fresh weight and of 10 day old seedlings of WT, L1, L2 and L3 after cultivation under different AsIII concentrations (n = 5 plants per treatment per line). Plants shown are representative of three independent experiments. Scale bars, 15 mm. Asterisks indicate significant differences from the WT detected by one-way analysis of variance (ANOVA) (***P < 0.001). Error bars, mean  SEM.

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Fig. 4. Arsenic volatilization in transgenic A. thaliana on exposure of arsenicfor 24 h and 48 h. All transgenic and WT plants were grown in hydroponics in the presence of A) 250 mM AsV and B) 50 mM AsIII. The experiment was performed in triplicate. Asterisks indicate significant differences from the WT determined by one-way ANOVA (***P < 0.001). Error bars, mean  SEM.

than WT plants after AsV treatment (Fig. 2F). Similar results were observed when transgenic plants were treated with 25 and 50 mM of AsIII as compared to WT plants (Fig. 3). 3.4. A. thaliana expressing WaarsM showed arsenic biovolatilization The estimated quantity of volatile arsenic in all transgenic lines was significantly higher on exposure to 250 mM AsV and 50 mM AsIII than WT plants. The volatile arsenicals evolved during 24 h were less as compared to 48 h, additionally the detected amount of volatile arsenicals was much higher during AsIII than AsV stress (Fig. 4A, B and Fig. S2). These results clearly demonstrated that the transgenic A. thaliana volatilizes arsenic by expression of the fungal WaarsM gene. The trapped arsenicals might be TMAs(III) as it was the final product of arsenic methylation pathway (Qin et al., 2009; Meng et al., 2011; Yuan et al., 2008).

3.5. WaarsM transformed A. thaliana also displayed enhanced growth and reduced arsenic accumulation in seeds and shoot tissues Soil cultivation provides optimal conditions for long-term cultivation and may optimize the bioaccumulation of arsenic in WaarsM transgenic plants whereas sterile agar plates are suitable only for short-term growth. We performed long-term soil cultivation experiments in soilrite that was spiked with AsV and AsIII. Differences were observed between the transgenic A. thaliana and WT plants in appearance and growth. The above-ground tissues of transgenic plants were better developed with higher biomass and increased seed production as compared to WT plants (Fig. 5). Arsenic accumulation in seeds and shoot tissues were also measured by ICP-MS after long-term exposure of AsIII and AsV in soilrite (described above) and revealed that ca. 24% and 26% less

Fig. 5. Transgenic A. thaliana plants accumulated more biomass in above ground tissues after long-term cultivation in soil. (A, B) Seeds of WT, L1, L2 and L3 were sown on soil spiked with A) 250 mM AsV B) 25 mM AsIII for 6 weeks. C) Dry weights of rosette leaves + stalks and D) Dry weights of seeds when plants grown in soil spiked with 250 mM AsV for 6 weeks and E) Dry weights of rosette leaves + stalks and F) Dry weights of seeds of plants grown in soil spiked with 25 mM AsIII for 6 weeks (n = 8 plants per treatment per line). The experiment was performed in triplicate. Scale bar, 5 cm. Asterisks indicate significant differences from the WT determined by one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001). Error bars, mean  SEM.

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arsenic accumulate in seeds under AsV (Fig. 6A) and AsIII stress (Fig. 6B), respectively compared to WT plants. In shoot tissues (Shoot + rosette leaves) ca. 7% and 13% less arsenic accumulated after exposure to AsV (Fig. 6C) and AsIII (Fig. 6D), respectively compared to WT plants. Thus, after 6 weeks of arsenic treatment, transgenic lines accumulated significantly low level of arsenic in seeds and shoots in comparison to WT plants (Fig. 6A–D and Table S3). A decrease in arsenic content in seeds and shoot tissues of WaarsM expressing transgenic lines confirms the potential of WaarsM in enhancing arsenic tolerance by biomethylation and volatilization of inorganic arsenicals. Thus, the present study might provide a possible strategy for arsenic removal by transgenic crops. 4. Discussion In recent years, increased arsenic contamination in agricultural soil emerges as a grave concern for crop productivity and arsenicinduced human diseases in several countries of the world. The toxicity of arsenic depends on its binding to cellular proteins/ enzymes, uptake and translocation in plants. Toxicity of inorganic arsenic was much higher as AsIII binds with cysteine residues whereas AsV interface energy metabolism and replaces phosphate group in contrast to methylated arsenicals which are more mobile and less toxic (Finnegan and Chen, 2012). Thus, arsenic methylation is considered as an important detoxification mechanism known in members of many prokaryotes. Moreover, the end product of arsenic methylation, volatile TMAO provide an aid to arsenic removal from the soil. In the present study, we describe the capability of a fungal arsenic methyltransferase gene in phytovolatilization and bioremediation. Our previous study describes that the fungal arsenic methyltransferase (WaarsM) gene has a higher catalytic activity for arsenic methylation and volatilization (Verma et al., 2016c). Overexpression of WaarsM in S. cerevisiae displayed enhanced tolerance to both AsIII and AsV stresses due to the transformation

of inorganic into organic arsenicals and thus lowered arsenic concentration in the medium by arsenic volatilization. Furthermore, the growth curve in liquid medium also showed short lag phase in engineered yeast cells (Fig. 1). We also generated transgenic A. thaliana in which fungal WaarsM was stably integrated into the plant genome and expressed a functional WaarsM enzyme capable of methylating inorganic arsenicals. Also, the overexpression of WaarsM in A. thaliana provide higher tolerance and better growth regarding fresh weight and root length of plants during AsV and AsIII stress (Figs. 2 and 3). Arsenic volatilization is the final step in the detoxification pathway of inorganic arsenicals. Therefore, volatile arsenicals released by plants were measured and revealed that WaarsM expressing A. thaliana produces a high amount of volatile arsenicals approximately 17.5 ng and 113 ng (mg 1 fresh weight) of the total arsenic after a short-term (48 h) exposure to AsV and AsIII stresses, respectively (Fig. 4). These results clearly demonstrated that the expression of WaarsM affect the speciation of arsenic in A. thaliana plants and leads to arsenic volatilization. An important and crucial step in validating the potential of our strategy was to determine whether this transgenic approach can work on soil (glasshouse condition) that contain microorganisms and arsenic bound to the soil. To check these limitations, A. thaliana plants subjected to long-term exposure (6 weeks) to arsenic stress and found that the total arsenic accumulation in the plant parts such as seeds and shoot of the transgenic A. thaliana was significantly lower than the WT plants (Fig. 6). Earlier attempts showed that the expression of arsM produces very less amount of volatile arsenicals in comparison to the formation of methylated arsenicals (MMA, DMA) (Meng et al., 2011; Tang et al., 2016). These methylated arsenicals are more mobile and might be accumulated in plant parts to create phytotoxicity (Tang et al., 2016) in contrast, the higher production of volatile arsenic enhances arsenic tolerance in plants. Previous studies also showed that the expression of the arsM gene from R.

Fig. 6. Transgenic A. thaliana plants accumulated low arsenic in above ground tissues and seeds after long term cultivation in soil. (A, B) Arsenic accumulation in seeds when plants grown in soil spiked with A) 250 mM AsV and with B) 25 mM AsIII for 45 days. (C, D) Arsenic accumulation in rosette leaves + stalks when plants grown in soil spiked with C) 250 mM AsV and with D) 25 mM AsIII for 45 days (n = 8 plants per treatment per line). The experiment was performed in triplicate. Asterisks indicate significant differences from the WT determined by one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001). Error bars, mean  SEM.

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palustris in rice was too little to produce abundant volatile arsenicals. These transgenic rice plants produce only about 0.2– 0.9% volatile arsenicals in shoots (Meng et al., 2011) might be due to the use of prokaryotic gene which required codon optimization to achieve an efficient expression in higher plants (Quax et al., 2015; Wang et al., 2012). Recently, Tang et al. (2016) also generated transgenic A. thaliana plants expressing CrarsM gene from algae, although CrarsM was highly expressed in transgenic plants only a moderate amount of volatile arsenicals was produced (only 0.01%). The reason for the low arsenic volatilization could be because C. reinhardtii itself produces a little amount of volatile arsenic (Myashita et al., 2011; Tang et al., 2016). Thus, CrarsM gene expressed in transgenic plants was not efficient to catalyze the final step of arsenic methylation and leads to enhanced phytotoxicity (Tang et al., 2016). In contrast, the WaarsM gene used in the present study was an eukaryotic origin and isolated from arseniccontaminated areas, highly expressed in transgenic A. thaliana plants and able to produce high amount of volatile arsenicals leading to lower arsenic accumulation in shoots and seeds during arsenic stress. Thus, the expression of WaarsM in A. thaliana provide an approach which may be suitable for engineering arsenic tolerance and achieving enhanced volatilization in plants for arsenic phytoremediation. The present study suggested that the WaarsM could be used to engineered transgenic crops with the ability to accumulate less arsenic in the shoot tissues (shoot and leaves) and other edible parts. Therefore, WaarsM may help to alleviate health risks associated with arsenic accumulation in rice and other crops. For arsenic-free drinking water many physical and chemical strategies were developed (Amrose et al., 2015). Whereas soil arsenic remediation by physical methods such as land burial or removal is much expensive, labor intensive and environmental invasive (Chen et al., 2013). In this context, bioremediation strategies including the use of plants and microorganisms for arsenic cleanup from contaminated soil are of significant advantage (Kramer and Chardonnens, 2001; Shukla et al., 2010; Valls and de Lorenzo, 2002). Thus, our study strongly suggested the WaarsM could be utilized to produce transgenic plants for bioremediation. Conflict of interest Authors have no conflict of interest. Acknowledgments The authors acknowledge the Director CSIR-NBRI for extending the facilities to carry out this work and the Council of Scientific and Industrial Research (CSIR), Govt of India, for the funding through the Project BSC-0107. S.V. and P. K.V. thankfully acknowledge Council of Scientific and Industrial Research (CSIR), India for senior research fellowship. RDT is grateful to CSIR for Emeritus Scientist scheme. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.envexpbot. 2016.08.012. References Abedin, M.J., Cresser, M.S., Meharg, A.A., Feldmann, J., Cotter-Howells, J., 2002. Arsenic accumulation and metabolism in rice (Oryza sativa L.). Environ. Sci. Technol. 36, 962–968. Amrose, S., Burt, Z., Ray, I., 2015. Safe drinking water for low-Income regions. Annu. Rev. Environ. Resour. 40 (40), 203–231.

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