Phytoremediation of arsenic from the contaminated soil using transgenic tobacco plants expressing ACR2 gene of Arabidopsis thaliana

Accepted Manuscript Title: Phytoremediation of arsenic from the contaminated soil using transgenic tobacco plants expressing ACR2 gene of Arabidopsis thaliana Authors: Noor Nahar, Aminur Rahman, Neelu N. Nawani, Sibdas Ghosh, Abul Mandal PII: DOI: Reference:

S0176-1617(17)30203-1 http://dx.doi.org/doi:10.1016/j.jplph.2017.08.001 JPLPH 52641

To appear in: Received date: Revised date: Accepted date:

29-3-2017 31-7-2017 1-8-2017

Please cite this article as: Nahar Noor, Rahman Aminur, Nawani Neelu N, Ghosh Sibdas, Mandal Abul.Phytoremediation of arsenic from the contaminated soil using transgenic tobacco plants expressing ACR2 gene of Arabidopsis thaliana.Journal of Plant Physiology http://dx.doi.org/10.1016/j.jplph.2017.08.001 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.

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Phytoremediation of arsenic from the contaminated soil using transgenic tobacco plants expressing ACR2 gene of Arabidopsis thaliana Noor Nahar1, Aminur Rahman1*, Neelu N. Nawani2, Sibdas Ghosh3, and Abul Mandal1

Author Affiliations 1

Systems Biology Research Center, School of Bioscience, University of Skövde, P.O. Box 408,

SE-541 28 Skövde, Sweden. 2

Microbial Diversity Research Centre, Dr. D. Y. Patil Biotechnology and Bioinformatics

Institute, Dr. D. Y. Patil Vidyapeeth, Tathawade, Pune-411033, India 3

School of Arts and Science, Iona College, New Rochelle, NY 10801, USA

*Correspondence Aminur Rahman, PhD Lecturer in Molecular Biology Systems Biology Research Center, School of Bioscience, University of Skövde, P.O. Box 408, SE-541 28 Skövde, Sweden. Telephone: +46-500 448679 Mobile: +46-7389 81928 Fax: +46-500-448499 E-mail: [email protected]

Abstract

We have cloned, characterized and transformed the AtACR2 gene (arsenic reductase 2) of Arabidopsis thaliana into the genome of tobacco (Nicotiana tabacum, var Sumsun). Our results revealed that the transgenic tobacco plants are more tolerant to arsenic than the wild type ones. These plants can grow on culture medium containing 200 µM arsenate, whereas the wild type can barely survive under this condition. Furthermore, when exposed to 100 µM arsenate for 35 days the amount of arsenic accumulated in the shoots of transgenic plants was significantly lower (28 µg/g d wt.) than that found in the shoots of non-transgenic controls (40 µg/g d wt.). 1

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However, the arsenic content in the roots of transgenic plants was significantly higher (2400 µg/g d. wt.) than that (2100 µg/g d. wt.) observed in roots of wild type plants. We have demonstrated that Arabidopsis thaliana AtACR2 gene is a potential candidate for genetic engineering of plants to develop new crop cultivars that can be grown on arsenic contaminated fields to reduce arsenic content of the soil and can become a source of food containing no arsenic or exhibiting substantially reduced amount of this metalloid.

Keywords Arabidopsis thaliana; Arsenic; AtACR2 overexpression; Phytoremediation; Heavy metal accumulation; Nicotiana tabacum.

1. Introduction

Arsenic (As) is extremely poisonous belonging to class-1 human carcinogenic substances. Long-term exposure to As may lead to several kinds of skin lesions, melanosis like hyper- and hypo- pigmentation and skin, lung and bladder cancers. (Abhyankar et al., 2012; Moon et al., 2012; Parvez et al., 2013; Seow et al., 2014; Haque, 2015). Thus, continuous release of As into ground water through natural phenomena or by anthropogenic activities contributing to environmental pollution is a severe hazard for human health globally (LeDuc and Terry, 2005; D'Ippoliti et al., 2015; Carlin et al., 2016). Contamination of ground water with high concentrations of As is reported in several countries, including Argentina, Bangladesh, Chile, China, India, Japan, Mexico, Mongolia, Nepal, Poland, Taiwan, Vietnam and some parts of the United States (Mukherjee et al., 2006; Neumann et al., 2010; Rodríguez-Lado et al., 2013; VeraAguilar et al., 2015). However, the worst arsenic contamination of the ecosystem, hence the strongest human health hazard was observed in Bangladesh and West Bengal, India due to long

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term geochemical changes leading to release of arsenic from its core compound, arsenopyrites (Tripathi et al., 2007). Moreover, consumption of crops grown on As polluted soils and irrigated with As contaminated water leads to potential health risk (Huang et al., 2006; Sundaram et al., 2009; Carlin et al., 2016). Due to the acute toxicity of As in humans, there is an urgent need for developing low-cost, effective and sustainable methods for remediation of As from soil. For examples, eco-friendly green technologies, such as phytoremediation (Dhankher et al., 2002, 2012; Ali et al., 2013; Hussain et al., 2017; Wang et al., 2017) or generation of “safe” crop varieties (Lund et al., 2010) can be used as alternatives to cope with excessive As toxicity in human foods. To date, many metallophytes or metal hyperaccumulator plants that grow preferably or exclusively in heavy metal contaminated soils have been described (Kapungwe, 2013; Karimi et al., 2013; Laghlimi et al., 2015; Fayiga and Shaha, 2016) and mechanisms of uptake and detoxification of As in plants have recently been reviewed in depth by many researchers (Dixit et al., 2015, Farooq et al., 2016). Several studies demonstrated that plants can protect themselves from As poisoning by reducing AsV to AsIII, which can be subsequently detoxified by forming complexes with thiol-reactive peptides such as γ-glutamylcysteine (γEC), glutathione (GSH), and phytochelatins (PCs) (Pickering et al., 2000; Dhankher et al., 2002; Vatamaniuk et al., 2004; Mishra et al., 2017). The AsIII-thiol complexes are then sequestered into vacuoles of the plant cells by glutathione-conjugating pumps (GCPs) (Dhankher et al., 2002; Mishra et al., 2017). The Arabidopsis ACR2 gene complemented the function of arsenate reductase in E. coli strain deficient in arsenate reductase, ArsC. Previously, it was confirmed that Arabidopsis lines silenced for ACR2 expression by RNAi showed a clear arsenate-dependent phenotype, which translocated 6- to 15-fold higher levels of As from roots to above-ground parts of the plant (Dhankher et al., 2006; Nahar et al., 2012). The fundamental goal of this study was to develop genetically engineered tobacco plants that can be used for phytoremediation of arsenics from the contaminated lands thus contributing to

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protect human health and the environment from severe contamination. Here we report on generation of transgenic tobacco plants expressing the A. thaliana AtACR2 gene as well as evaluation of the function of AtACR2 with respect to accumulation of arsenics in shoots and roots of these plants.

2. Materials and methods

2.1 Construction of plant transformation vector The 1356 bp open reading frame (ORF) of the AtACR2 (Dhankher et al., 2006; Nahar et al., 2012) full length gene sequence was amplified from the genomic DNA of A. thaliana by polymerase chain reaction (PCR) using forward and reverse primers as described by Nahar (2014). The amplified product was purified and cloned into the vector pSC152 (Karim et al., 2007) by replacing ScTP1 gene. Ligation or cloning of the amplified AtACR2 gene into pSC152 downstream of the Arabidopsis ribulose-1,5-bisphosphate carboxylase promoter was performed by following homologous recombination as described in Infusion® Advantage PCR cloning kit (CLONTECH 2010). The cloning vector was then renamed as pNAM19. Isolation of the AtACR2 gene and construction of a binary vector for plant transformation were as described by Nahar (2014). The resulting vector (Fig. 1) containing the AtACR2 gene driven by the pRbcS1a promoter and terminated by nopaline synthase terminator (nos) was renamed as pNAM09. The vector was then introduced into electrocompetent cells of Agrobacterium tumefaciens strain LBA4404 (Invitrogen Cat. No. 18313–015) following the electroporation transformation method (BTX® ECM® 600 electroporator). The transformed cells were selected by using the selective medium (Nahar, 2014) and stored at –70 °C until use.

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2.2 Plant transformation Transformation of tobacco leaf disks with A. tumefaciens strain LBA4404 harbouring pNAM09, culture and selection of transformed tissues as well as regeneration of transgenic plants were performed as described by (Mandal et al., 1993) with some modifications. Transformation of tobacco leaf disks as well as selection, regeneration and micropropagation of transgenic shoots were identical to those presented by Nahar (2014). To maintain several copies of the transgenic clone/line, shoots were micropropagated by culturing the nodal buds. From each line some rooted shoots were initially transferred to soil pots for hardening and later to greenhouse for further growth till maturity. T1 seeds from the kanamycin resistant transgenic lines were collected and air dried for several weeks and stored in the refrigerator (4 oC) until further use. Analyses of transgenic plants were performed on both T1 and T2 progenies.

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2.3 Molecular analysis of transgenic plants To confirm the presence of AtACR2 gene in the transgenic lines genomic DNA of the kanamycin resistant T2 plants was isolated and PCR reaction was carried out following instructions provided with the Phire Plant Direct PCR Kit (Finnzymes Cat. No. F-130). Details of the reaction mix and PCR running conditions were presented in Nahar (2014). To analyze the expression of AtACR2 gene in T2 progeny RT-PCR was performed. Isolation and purification of total RNA from the 2-week old tobacco plants were carried out following RNeasy Plant Mini Kit protocol (Qiagen, Valencia, CA; Cat. No. 74904). RT-PCR was performed using a Robust–TI RT-PCR Kit (Finnzymes, Espoo, Finland; Cat. No. F-580L). Reverse transcription reactions were performed as described by Nahar et al. (2012). To determine the number of T-DNA copies integrated in the genomic DNA of the transgenic plants Inverse-PCR (I-PCR) was carried out as described by Bragg et al. (2012) and Huang et al. (2003). For amplification of T-DNA flanking plant DNA sequences in the transgenic plants, primers designing, selection of unique restriction site on the T-DNA and self-ligation of the digested plant DNA fragments were performed as described by Nahar (2014). Genomic DNA isolated from the wild type (WT) and vector transformed control (VC) plants was treated similarly and used as controls.

2.4 Effect of arsenic on germination and growth of transgenic plants Seeds (T2) of transgenic plants harboring one copy of the integrated T-DNA (verified by IPCR) and expressing the AtACR2 gene (strong band in RT-PCR) as well as wild type seeds were germinated on Petri dishes containing MS medium (Murashige and Skoog, 1962) supplemented with various concentrations of AsV (0, 50, 100 and 200 μM sodium arsenate dibasic heptahydrate/ Na2HAsO4 7H2O from Sigma-Aldrich, Cat. No A6756). Fifteen days

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later, germination rates were recorded, and the seedlings were allowed to grow for additional 30 days. After the total of 45 days, plants were pulled out of the agar medium, rinsed thoroughly with distilled water and the fresh weight was estimated.

2.5 Estimation of arsenic content in transgenic plants Seeds (T2) from transgenic lines showing strong expression of AtACR2 and containing only one copy of the integrated T-DNA were germinated on MS medium supplemented with 200 mgl-1 kanamycin. Seeds of wild type plants were germinated on kanamycin free MS medium. After 21 days of germination kanamycin-resistant seedlings were again subjected to PCR, RTPCR and I-PCR analyses to further confirm the presence, expression and number of transgenes integrated into the genome, respectively as described above. Seedlings expressing AtACR2 gene and harboring a single insertion of integrated transgene as well as the wild type seedlings of the same size and age (21 days) were transferred onto baby food jars containing agar-solidified MS medium supplemented with several concentrations of AsV (0, 50 and 100 μM). After 35 days of growth on this medium the plants were collected and washed thoroughly in distilled water to remove the culture-originating As adhering to the root surface. Plant material were then separated into two groups, roots and shoots, and oven dried at 55 °C until constant weight was achieved. Dry materials from six transgenic plants were pooled and ground using a mortar and a pestle. Randomly selected six wild type plants were also pooled and prepared similarly and used as controls. All ground materials were stored at 4 °C until further use. To determine the arsenic content the dried samples were mixed with 2 mL of HNO3 (65%, Merck, Darmstadt, Germany) and 6 mL of HCl (37%, Merck). The mixture was heated to 70ºC for 1 h, and then diluted with 10 mL of deionized water. The acid-digested solution was then filtered to remove residual particulates. Arsenic concentration was determined by inductively coupled plasma

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mass spectroscopy (ICP-MS) method. All analyses were performed at Eurofins Environment Testing Sweden AB (Lidköping, Sweden).

2.6 Statistical analysis Significance of treatment effects on various parameters determined in this work was tested by one way ANOVA employing PRISM 6.0 version. Mean values obtained upon treatment were compared based on the least significant difference. All experiments were carried out with three repetitions and eight replications.

3. Results

3.1 Vector construction, plant transformation and analysis of transgenic plants The arsenate reductase2 (AtACR2) gene was cloned into a binary vector for plant transformation (Fig. 1). By employing A. tumefaciens T-DNA mediated leaf-disk transformation a total of 96 independent transgenic tobacco lines (T1) were established. Presence of AtACR2 gene in the genome of the primary transgenic T1 lines was verified by PCR using AtACR2 sequences as primers (Fig. 2A). All nine randomly selected kanamycin resistant T1 lines (Fig. 2A, lanes 2-10) contained at least one copy of the AtACR2 gene. Furthermore, transgene AtACR2 was expressed at the transcriptional level in all eight transgenic lines investigated (Fig. 2B, lanes 4-11). However, the level of expression of AtACR2 gene varied from line to line and the highest expression was observed in line no. 8 (Fig. 2B, lane 8). Out of eight investigated transgenic lines only two (Fig 2C, lanes 4 and 8) lines exhibited one copy of the integrated T-DNA. On the other hand, several copies of T-DNA insertion were found in lines no. 1, 2, 5 and 7. Based on results obtained in RT-PCR and I-PCR, transgenic

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line no. 8 exhibiting strong expression of the AtACR2 mRNA and a single copy of the integrated T-DNA was selected for further investigations.

3.2 Effect of arsenic exposure on germination and growth of transgenic plants As depicted in Fig. 3A and Fig. 3C, no significant difference was observed in germination rates between transgenic (line 8) and the wild type plants when exposed to 0 and 50 μM of AsV for 15 days. However, when exposed to 100 and 200 μM AsV the rate of seed germination in the transgenic plants was significantly higher (87% and 58%, respectively) than that observed in WT controls (69% and 24%, respectively). Plants that could survive and grow even after 30 days of exposure to medium containing 50 or 100 μM AsV were collected for biomass analyses. Under these conditions no significant difference in biomass production was detected between transgenic and wild type seedlings (data not shown). 3.3 Determination of biomass and arsenic content in transgenic plants Kanamycin resistant seedlings of line 8 were subjected to PCR and RT-PCR analyses (data not shown) to reconfirm their transgenic status (presence of AtACR2 gene and its mRNA). Although the 100 μM concentration significantly inhibited the growth of both transgenic and wild type plants (compared to MS medium without AsV), the leaves of transgenic plants were greener and exhibited less chlorosis than those of the wild type plants (Fig. 4A). After 35 days of exposure to 100 μM AsV the leaves and roots of the wild type plants were damaged severely and started to decay, whereas the AtACR2-transgenic plants looked less damaged, survived arsenic exposure and can could grow further until seed maturation (Fig. 4A). For estimation of biomass production, dry weights of both wild type and AtACR2-expressing plants of line 8 grown for 35 days on MS medium containing several concentrations of AsV (0 and 50 and 100 μM) were compared. No significant difference in dry weight of shoots was observed between the transgenic and WT plants when grown on MS medium containing 0, 50

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and 100 μM AsV (Fig. 4B). However, significant differences in dry weight of roots between the transgenic and wild type plants were confirmed when grown on MS medium containing 50 and 100 μM AsV (Fig. 4C). Roots of transgenic plants exhibited approximately 6.5 and 4.6 fold higher dry weight when exposed to 50 μM and100 μM AsV, respectively. Figures 5A and 5B represent the results obtained by ICP-MS for determination of the amount of arsenic in shoots and roots. These results indicate that accumulation of total arsenic in shoots of transgenic plants was approximately 1.5 fold lower (28 µg/g d. wt.) than that (40 µg/g d. wt.) observed in shoots of wild type plants. However, accumulation of total arsenic in roots of transgenic plants was approximately 1.2 fold higher (2400 µg/g d. wt.) than that (2100 µg/g d. wt.) observed in roots of wild type plants.

4. Discussion We have generated transgenic tobacco plants expressing A. thaliana AtACR2 gene (Fig. 2) by T-DNA mediated transformation. The effect of AtACR2 gene on tolerance of plants to arsenic toxicity and accumulation of arsenic in different parts of plants were evaluated. Based on the highest level of expression of the transgene and a single copy number of the integrated T-DNA, the transgenic line no. 8 was selected for further investigations. When exposed to 50 and 100 μM concentrations of AsV, plants of this line were found to be more tolerant to As, than the wild types and could survive and grow further to a complete maturity. In addition, when exposed to arsenic stress the transgenic seeds exhibited higher rate of germination and the roots of these plants also showed an increased production of biomass in comparison with those observed in the wild types (Figure 4). Previously, it has been reported that seed germination together with root elongation are crucial stages in plant development and largely affected by abiotic stresses including environmental contaminants (Srivastava et al., 2013; Upadhyaya et al., 2014). We report that the rate of germination of the transgenic seeds on medium containing

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arsenic was higher than that of the wild type seeds validating the transgenic seeds have better capacity to overcome the sensitive and crucial stage of growth (Fig. 3A and 3B) during arsenic exposure. The most pronounced and most often measured effect of As on plants growth is the dry weight. Although As negatively affects both fresh and dry weight of diverse plants, dry weight is affected more severely. The detrimental effect of As on dry weight may be due to formation and rapid auto hydrolysis of AsV-ADP sets in place of a futile cycle that uncouples photophosphorylation and oxidative phosphorylation, decreasing the ability of cells to produce ATP and carry out normal metabolism (Finnegan and Chen, 2012). In plants, As interferes with a number of physiological processes, directly or indirectly. When exposed to As causing electrolyte leakage the cellular membranes of plants become damaged (Singh et al., 2006; Talukdar, 2013; Talukdar and Talukdar, 2013) which is often accompanied by an increase in malondialdehyde, a product of lipid peroxidation, pointing to the role of oxidative stress in As toxicity. Arsenic exposure also induces antioxidant defense mechanisms. Due to this toxic property of As the rate of seed germination (Fig. 3C) reduced significantly when exposed to 100 μM and 200 μM concentrations of AsV, compared to untreated plants. Meanwhile, treatment of plants with lower concentration (50 μM) of As had no significant effect on either seed germination or dry weight production. Biomass of both transgenic and non-transgenic plants were determined based on the relative values of dry and fresh weights in order to avoid various experimental errors that may occur due to different experimental conditions. Arsenic tolerance at the cellular level was extensively studied due to its importance to phytoremediation (Hussain et al., 2017). From these studies it was concluded that mechanisms for sequestration of As in plants could play an important role in tolerance of plants to higher level of arsenic toxicity (Zhao, et al. 2008; 2010). There are two main types of sequestration

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mechanisms: (1) cytosol sequestration via glutathione, glutathione-related phytochelatins and (2) vacuolar sequestration via the tonoplast Cd+/H+ antiporters, ABC-type transporters for Cdglutathione and Cd-phytochelatins along with other transporters such as CDF and NRAMP3 HMA proteins (Martinoia et al., 2007; Verbruggen et al., 2009; Wu et al., 2011). We can only speculate that the increased As tolerance exhibited by AtACR2-transgenic tobacco plants might result from enhanced AsIII sequestration into the vacuole, where AsIII can do the least harm to vital cellular processes. This enhanced sequestration was probably due to the additional reduction of AsV provided by the AtACR2 expression. In fact, many cation/H+ transporters are localized in the vacuolar membrane and are involved in the transport of AsIII and other divalent cations (Zn, Mn and Cd) into the vacuole (Korenkov et al., 2007; Wu et al., 2011). Recently, Wu et al. (2011) and Baliardini et al. (2015) reported the enhanced vacuolar sequestration of Cd following the expression of an Arabidopsis CAX1 variant (CAXcd) in petunia. In case of As, expression of Arabidopsis vacuolar transporter AtMRP1/2 in plants increased sequestration in the vacuole (Lund et al., 2010). Our investigation finds that at 100 uM AsV the roots of transgenic plants accumulated 1.2 fold higher amount of arsenic than those of wild types, whereas the shoots of transgenic plants accumulated 1.5 fold lower amount of arsenic than those of wild types. Thus, AtACR2 may contribute to limit As transport from root to shoot, leading to accumulation of more arsenic in roots which is in accordance with the observations by Wojas et al. (2009), who demonstrated that expression of the AtMRP7 in plants increased vacuolar sequestration of Cd and higher accumulation of Cd in the roots than in the shoots. Most importantly, our studies revealed that AtACR2-transgenic tobacco plants when exposed to arsenic stress accumulated lower amount of arsenic in the shoots (Fig. 5A) as compared to the wild type. Also, transgenic tobacco plants seem to transport less amount of As to their aerial parts compared with wild type plants. Taking together, these results indicate that it is possible to regulate arsenic accumulation in different parts of the plants by employing genetic

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engineering. Obviously, this is a complex mechanism and it requires optimum regulation of many other known and unknown genes and pathways. Nevertheless, our results support our suggestion that plants expressing AtACR2 gene can potentially be grown on cultivated land that has been contaminated with arsenic due to various anthropogenic activities. Uptake of arsenic by the roots and its accumulation in different parts of the plants will ultimately result in significant reduction in the arsenic content of the cultivated lands. Further, to the best of our knowledge, this is the first report ever that AtACR2 can be engineered to develop a plant variety that accumulate relatively less arsenic in the above ground parts which are usually used for production of human foods and animal fodder. One major drawback of our studies is that we have not (yet) confirmed if the above features observed in the transgenic lines are related solely to transgene activation leading to As sequestration into the vacuole and/or activation of other native genes involved in stress tolerance and As detoxification. These need to be explored further.

Conclusions In this study we have developed transgenic tobacco plants harboring the AtACR2 gene of A. thaliana. Amount of arsenic accumulated in roots and shoots of both transgenic and WT plants were measured and evaluated. The transgenic plants accumulated higher amount of arsenic in the roots compared to the wild-type control plants suggesting that these plants can be used for phytoremediation of arsenics. In addition, the amount of arsenic accumulated in the shoots of the transgenic plants was much lower than that in the control plants. This indicates that AtACR2 can be used as a potential candidate for developing new variety of cultivated crops which will

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accumulate significantly reduced amount of arsenic in the above ground edible parts such as shoots, even when grown on arsenic contaminated land.

Acknowledgments

The study was financially supported partly by the Swedish International Development Cooperation Agency (SIDA, grant no. AKT-2010-018), and partly by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS, grant no.2292007-217). Authors also acknowledge a grant from the Nilsson-Ehle Foundation (The Royal Physiographic Society in Lund) in Sweden. Authors would like to thank Dr. Kjell-Ove Holmström for providing vectors for plant transformation.

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References Abhyankar, L.N., Jones, M.R., Guallar, E., Navas-Acien, A., 2012. Arsenic exposure and hypertension: A systematic review. Environ. Health Perspect. 120(4), 494–500. Ali, H., Khan, E., Sajad, M.A., 2013. Phytoremediation of heavy metals—Concepts and applications. Chemosphere. 91(7), 869–881. Baliardini, C., Meyer, C.L., Salis, P., Saumitou-Laprade P., Verbruggen N., 2015. Cation Exchanger1 cosegregates with cadmium tolerance in the metal hyperaccumulator Arabidopsis halleri and plays a role in limiting oxidative stress in Arabidopsis Spp. Plant Physiol. 169(1), 549–559. Bragg, J.N., Wu, J., Gordon, S.P., Guttman, M.E., Thilmony, R., Lazo, R. G., Gu, Q. Y., Vogel, P. J., 2012. Generation and Characterization of the Western Regional Research Center Brachypodium T-DNA Insertional Mutant Collection. PLoS ONE. 7(9), e41916. Carlin, D.J., Naujokas, M.F., Bradham, K.D., Cowden, J., Heacock, M., Henry, H.F., Lee, J.S., Thomas, D.J., Thompson, C., Tokar, E.J., Waalkes, M.P., Birnbaum, L.S., Suk, W.A., 2016. Arsenic and environmental health: state of the science and future research opportunities. Environ. Health Perspect. 124, 890–899. Dhankher, O.P., Li Y.J., Rosen, B.P., Shi, J., Salt, D., Senecoff, J.F., Sashti, N.A., Meagher, R.B., 2002. Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and gamma-glutamylcysteine synthetase expression. Nat. Biotech. 20, 1140−1145. Dhankher, O.P., Pilon-Smits, E.A.H., Meagher, R.B., Doty, S., 2012. Biotechnological approaches for phytoremediation. In: Altman A., Hasegawa PM., eds. Plant Biotech. Agri. 309– 328.

15

16

Dhankher, O.P., Rosen, B.P., McKinney, E.C., Meagher, R.B., 2006. Hyperaccumulation of arsenic in the shoots of Arabidopsis silenced for arsenate reductase (ACR2). Proc. Natl. Acad. Sci. USA. 103, 5413–5418. D'Ippoliti, D., Santelli, E., De Sario, M., Scortichini, M., Davoli, M., Michelozzi, P., 2015. Arsenic in drinking water and mortality for cancer and chronic diseases in central Italy, 1990– 2010. PLoS One, 10 (9), e0138182. Dixit, G., Singh, A.P., Kumar, A., et al. 2015. Sulfur mediated reduction of arsenic toxicity involves efficient thiol metabolism and the antioxidant defense system in rice. J. Hazardous Materials. 298, 241–251. Farooq, M.A., Islam, F., Ali, B., Najeeb, U., Mao, B., Gill, R.A., Yan, G., Siddique, K.H.M., Zhou, W., 2016. Arsenic toxicity in plants: Cellular and molecular mechanisms of its transport and metabolism. Envirnt. Exp. Botany. 132, 42–52. Fayiga, A.O., Saha, U.K., 2016. Arsenic hyperaccumulating fern: Implications for remediation of arsenic contaminated soils. Geoderma. 284, 132–143. Finnegan, P.M., Chen, W., 2012. Arsenic toxicity: the effects on plant metabolism. Front Physiol. 3, 182. Haque, U.I., 2015. Arsenicosis Case Identification, Diagnosis and Management Protocol for Early Patient Medicare Administration and Treatment. Int. J. Environ. Monitor. Anal. 3(3-1), 1–9. Huang, R., Gao, S., Wang, W., Staunton, S., Wang, G., 2006. Soil arsenic availability and the transfer of soil arsenic to crops in suburban areas in Fujian Province, South East China. Sci. Tot. Environ. 368, 531–541. Huang, S., Chen, S., Jong, A., 2003. Use of inverse PCR to clone cDNA ends. Methods Mol. Bio. 221, 51–58.

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Hussain, S., Akram, M., Abbas, G., Murtaza,B., Shahid, M., Shah, N. S., Bibi, I., Niazi, N.K., 2017. Arsenic tolerance and phytoremediation potential of Conocarpus erectus L. and Populus deltoides L. Int. J. Phytoremediation. 21. Kapungwe, E.M., 2013. Heavy metal contaminated water, soils and crops in peri urban wastewater irrigation farming in Mufulira and Kafue towns in Zambia. J. Geogr. Geol. 5(2), 55–72. Karim, S., Aronsson, H., Ericson, H., Pirhonen, M., Leyman, B., Welin, B., Mäntylä, E., Palva, E.T., Van Dijck, P., Holmström, K.O., 2007. Improved drought tolerance without undesired side effects in transgenic plants producing trehalose. Plant Mol. Biol. 64(4), 371–386. Karimi, N., Ghaderian, S.M., Schat, H., 2013. Arsenic in soil and vegetation of a contaminated area. Int. J. Environ. Sci. Technol. 10(4), 743–752. Korenkov, V., Hirschi, K., Crutchfield, J.D., Wagner, G.J., 2007. Enhancing chloroplast Cd/H antiport activity increases Cd, Zn and Mn tolerance, and impacts root/shoot Cd partitioning in Nicotiana tabacum L. Planta. 226, 1379–1387. Laghlimi, M., Baghdad, B., Hadi, H., Bouabdli, A., 2015. Phytoremediation Mechanisms of Heavy Metal Contaminated Soils: A Review. Open J. Ecol. 5, 375–388. LeDuc, D.L., Terry, N., 2005. Phytoremediation of toxic trace elements in soil and water. J. Ind. Microbiol. Biotecnol. 32, 514−520. Li, W., Khan, M.A., Yamaguchi, S., Kamiya, Y., 2005. Effects of heavy metals on seed germination and early seedling growth of Arabidopsis thaliana. Plant Growth Reg. 46, 45–50. Lund, D., Larsson, D., Nahar, N., Mandal, A., 2010. Arsenic accumulation in plants – outlining strategies for developing improved variety of crops for avoiding arsenic toxicity in foods. J. Biol. Sys. 18, 223–224. Mandal, A., Lång, V., Orczyk, W., Palva, E.T., 1993. Improved efficiency for T‐DNA‐mediated transformation and plasmid rescue in Arabidopsis thaliana. Theor. Appl. Genet. 86, 621–628.

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Martinoia, E., Maeshima, M., Neuhaus, H.E., 2007. Vascular transporters and their essential role in plant metabolism. J. Exp. Bot. 58, 83–102. Mishra, S., Mattusch J., Wennrich, R. 2017. Accumulation and transformation of inorganic and organic arsenic in rice and role of thiol-complexation to restrict their translocation to shoot. Sci. Rep. 7, 40522. Moon, K., Guallar, E., Navas-Acien, A., 2012. Arsenic exposure and cardiovascular disease: an updated systematic review. Curr. Atheroscler. Rep. 14 (6), 542–555. Mukherjee, A., Sengupta M. K., Hossain M.A., 2006. Arsenic contamination in ground water: A global perspective with emphasis on the Asian scenario. J. Health Popul. Nutri. 24(2), 142– 163. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Planta. 15, 473–497. Nahar, N., 2014. Studies of genes involved in uptake and metabolism of arsenic in plants. Doctoral dissertation/PhD thesis, Skövde/Krakow., ISBN 978-91-981474-3-8. Nahar, N., Rahman, A., Moś, M., Warzecha, T., Algerin, M., Ghosh, S., Johnson-Brousseau, S., Mandal, A., 2012. In silico and in vivo studies of an Arabidopsis thaliana gene ACR2 putatively involved in arsenic accumulation in plants. J. Mol. Model. 18, 4249–4262. Neumann, R.B., Ashfaque, K.N., Badruzzaman, A.B.M., Ali, M.A., Shoemaker, J.K., Harvey, C.F., 2010. Anthropogenic influences on groundwater arsenic concentrations in Bangladesh. Nat. Geosci. 3, 46 – 52. Parvez, F., Chen, Y., Yunus, M., Olopade, C., Segers, S., Slavkovich, V, Argos, M., Hasan, R., Ahmed, A., Islam, T., Akter, M.M., Graziano, H.J., Ahsan, H., 2013. Arsenic exposure and impaired lung function. Findings from a large population-based prospective cohort study. Am. J. Respir. Crit. Care Med.188 (7), 813–819.

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19

Pickering, I.J., Prince, R.C., George, M.J., Smith, R.D., George, G.N., Salt, D.E., 2000. Reduction and coordination of arsenic in Indian mustard. Plant Physiol. 122, 1171–1177. Rodríguez-Lado L., Sun G., Berg M., Zhang Q., Xue H., Zheng Q., Johnson C.A., 2013. Groundwater arsenic contamination throughout China. Science. 341 (6148), 866–868. Seow, J.W., Kille, L.M., Baccarelli, A.A., Pan, C.W., Byun, M.H., Mostofa, G., Quamruzzaman, Q., Rahman, M., Lin, X., Christiani, C.D. 2014. Epigenome-wide DNA methylation changes with development of arsenic-induced skin lesions in Bangladesh: a casecontrol follow-up study. Environ. Mol. Mutagen. 55, 449–456. Singh, N., Ma, L.Q., Srivastava, M., Rathinasabapathi, B., 2006. Metabolic adaptations to arsenic induced oxidative stress in Pteris vittata L. and Pteris ensiformis L. Plant Sci. 170, 274– 282. Srivastava, S., Srivastava, A.K., Singh, B., Suprasanna, P. and D’souza, S.F., 2013. The effect of arsenic on pigment composition and photosynthesis in Hydrilla verticillata. Biol. Plant. 57, 385-389. Sundaram, S., Ma, L.Q., Wu, S., Rathinasabapathi, B. (2009) Expression of a Pteris vittata glutaredoxin PvGRX5 in transgenic Arabidopsis thaliana increases plant arsenic tolerance and decreases arsenic accumulation in the leaves. Plant cell Environ. 32, 851–858. Talukdar, D., 2013. Arsenic-induced oxidative stress in the common bean legume, Phaseolus vulgaris L. seedlings and its amelioration by exogenous nitric oxide. Physiol. Mol. Biol. Plants. 19(1), 69–79. Talukdar, T., Talukdar D., 2013. Response of antioxidative enzymes to arsenic-induced phytotoxicity in leaves of a medicinal daisy, Wedelia chinensis Merrill. J. Nat. Sci. Biol. Med. 4, 383-388.

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20

Tripathi, R., Srivastava, S., Mishra, S., Singh, N., Tuli, R., Gupta, D.K., Maathuis, J.M., 2007. Arsenic hazards: strategies for tolerance and remediation by plants. Trends Biotech. 25, 158– 165. Upadhyaya, H., Shome, S., Roy, D., Bhattacharya, M.K., 2014. Arsenic induced changes in growth and physiological responses in Vigna radiata seedling: Effect of curcumin interaction. A. J. Plant Sci. 5, 3609-3618. Verbruggen, N., Hermans, C., Schat, H., 2009. Molecular mechanisms of metal hyperaccumulation in plants. New Phytol. 181, 759–776. Vatamaniuk, O.K., Lang S.M., Chalasani, S., Demkiv, L.O., Rea, P.A., 2004. Phytochelatin synthase, a dipeptidyltransferase that undergoes multisite acylation with γ-glutamylcysteine during catalysis: stoichiometric and site-directed mutagenic analysis of Arabidopsis thaliana pcs1-catalyzed phytochelatin synthesis. J. Biol. Chem. 279, 22449–22460. Wang, L., Ji, B., Hu, Y., Liu, R., Sun, W., 2017. A review on in situ phytoremediation of mine tailings. Chemos. 184, 594-600. Wojas, S., Hennig, J., Plaza, S., Geisler, M., Siemianowski, O., Sklodowska, A., Ruszczyńska, A., Bulska, E., Antosiewicz, D.M., 2009. Ectopic expression of Arabidopsis ABC transporter MRP7 modifies cadmium root-to-shoot transport and accumulation. Environ. Pollut. 157, 2781–2789. Vera-Aguilar, E., López-Sandoval, E., Godina-Nava, J.J., Cebrián-García, M.E., LópezRiquelme, G.O., Rodríguez-Segura, M.A., Zendejas-Leal, B.E., Vázquez-López, C., 2015. Arsenic Removal from Zimapan Contaminated Water Monitored by the Tyndall Effect. J. Environ. Protec. 6(5), 538–551. Wu, Q., Shigaki, T., Williams, K.A., Han, J.S., Kim, C.K., Hirschi, K.D., Park, S., 2011. Expression of an Arabidopsis Ca2+/H+ antiporter CAX1 variant in petunia enhances cadmium tolerance and accumulation. J. Plant Physiol. 168, 167–173.

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Zhao, F.J., Ma, J.F., Meharg, A.A., McGrath, S.P., 2008. Arsenic uptake and metabolism in plants. New Phytol. 181, 777–794. Zhao, F.J., McGrath, S.P., Meharg, A., 2010. Arsenic as a food chain contaminant Mechanisms of plant uptake and metabolism and mitigation strategies. Annu. Rev. Plant. Biol. 61, 1–25.

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Figure legends Figure. 1 A schematic diagram of the binary vector pNAM09 for plant transformation. RB and LB, right and left borders of the T-DNA, RuBisCo promoter, the ribulose-1, 5-biphosphate carboxylase small subunit of A. thaliana; pNos, A. tumefaciens nopaline synthase gene promoter; 3’nos, A. tumefaciens nopaline synthase gene terminator; AtACR2, A. thaliana arsenate reductase2 gene; NPTII, neomycin phosotransferase II gene conferring kanamycin resistance.

Figure. 2 Molecular analysis of transgenic tobacco plants harboring AtACR2. (A) PCR amplification of genomic DNA of wild type and transgenic tobacco (AtACR2) plants using AtACR2 gene specific primers. L, 2-log DNA molecular size marker; lane 1, positive control pNAM19 plasmid; lanes 2-10, amplification from individual transgenic tobacco plants; lane 11, wild type plant DNA; lane 12, DNA from vector transformed control plants. (B) RT-PCR analysis of transgenic tobacco plants using AtACR2 specific sequences as primers. M, molecular size maker; lanes 1 and 2, WT samples from wild type tobacco plants and only water, respectively; lane 3, samples from vector transformed VC control plants,; lanes 4 -11, samples from individual transgenic plant lines. (C) I-PCR amplification of genomic DNA of wild type and transgenic tobacco (AtACR2) plants using T-DNA- specific primers. L, 2-log DNA molecular size marker; lane 1-8, samples from individual transgenic tobacco plants; lanes 9-10, samples from wild type DNA

Figure. 3 AtACR2-transgenic tobacco seeds and seedlings exhibiting enhanced tolerance to arsenic stress. (A) Seeds of WT and transgenic plants were surface sterilized and germinated directly on MS medium supplemented with different concentrations of AsV. Photographs were taken after 15 days. (B) Seedlings of WT and transgenic plants were grown on MS medium

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supplemented with different concentrations of AsV. Photographs were taken after 45 days. (C) percentage of germination of transgenic and wild type seeds on various concentrations of As. Values are means ± SE of three replicates: each replicate represents a Petri dish containing 50 seeds.

Figure. 4 Analysis of arsenic tolerance of transgenic and WT tobacco plants. (A) Phenotype of transgenic and wild type WT tobacco plants after six weeks of exposure to 0, 50 and 100 μM of AsV. (B) Relative values of dry and fresh weights of shoots. Error bars denote standard error of mean ± SE (n= 6); (C) Relative values of dry and fresh weights of roots, Error bars denote standard error of mean ± SE (n= 6).

Figure. 5 Accumulation of total arsenic in transgenic and WT tobacco plants. Seeds (T2) of transgenic lines were grown on MS medium supplemented with kanamycin, whereas seeds of WT plants were grown on MS medium without kanamycin. Kanamycin-resistant 21-day old seedlings were double checked by PCR analysis and grown in jars containing various concentrations of AsV along with wild type seedlings of same size. (A) Arsenic content in shoots of transgenic and wild type plants. (B) Arsenic content in roots of transgenic and wild type plants. Figure 1.

Figure 2.

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Figure 3.

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Figure 5.

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