Arsenate reductase gene from Pityrogramma calomelanos L. enhances tolerance to arsenic in tobacco

Arsenic (As) contamination in soil, water and air is an alarming issue worldwide and has serious effects on human health and environment. Arsenic is a naturally occurring element found in rocks, soil, and water, and exposure to high levels of arsenic can lead to a range of health problems. The effects of arsenic contamination can also be felt in the environment, as it can harm plants and animals and disrupt ecological systems. The major purpose of this study was to produce transgenic plants with improved tolerance to and accumulation of arsenic via transformation of arsenate reductase gene (ArsC) into tobacco genome. Transgenic plants were screen by PCR and southern blot. Further, their tolerance and accumulation to arsenic were evaluated. In the result, we have cloned, characterized, and transformed the ArsC gene from Pityrogramma calomelanos L. (PcArsC). Its phylogenetic analysis revealed 99% homology to ArsC gene in GenBank (accession number X80057.1). Moreover, Southern blot analysis showed that ArsC gene was integrated into the tobacco genome as a single-copy. These single-copy transgenic lines showed much higher tolerance to and accumulation of As than wild type, with no other phenotypes observed. These results demonstrated that Pityrogramma calomelanos ArsC gene can improve arsenic tolerance and accumulation in transgenic tobacco lines. Thus, using Pityrogramma calomelanos L. ArsC gene for genetic engineering has potential implications in the decontamination of arsenic-containing soil.


Introduction Introduction Introduction Introduction
Contamination of toxic metals in soil, waste areas and groundwater have harmful effects on human health. It is necessary to develop effective technologies to solve this problem. Phytoremediation is a bioremediation process that utilizes plants to remove contaminants without the need to collect and discard them elsewhere (Ashraf et al., 2015). This method has gained popularity due to its numerous advantages, including large scale application, low cost, and environmental friendliness. After being proved successful at Chernobyl in 1986, this technology began to be applied more widely (Lyubenova et al., 2009). The past decade has witnessed encouraging results that opened up opportunities for developing phytoremediation technologies and for applying genetic engineering to generating heavy metal-hyperaccumulating plants (Dhankher et al., 2002;Yang et al., 2005;Li et al., 2006;Sarma, 2011).
Studying genes involved in the uptake, accumulation and transformation of heavy metals contributes to the understanding of molecular mechanisms underlying these biological processes as well as the development of genetically-modified plants that can be used as phytoremediators. Much progress has been made in the study of heavy metal tolerance in plants. Genetic engineers have already begun to successfully enhance the capacity to tolerate and accumulate heavy metals in plants. For the uptake and transport of heavy metals, critical gene families have been identified, including ion-regulated transporter (IRT) (Milner et al., 2013), ZRT, IRT-like Protein and metal transporter gene family members as natural resistance-associated macrophage protein (Ullah et al., 2018). Genes reported to play a role in the accumulation and chelation of heavy metals are phytochelatin (PC) -CAD1 (cadinene synthase gene), GmPCS1 (phytochelatin synthase) and metallothionein (MT) -MT1, MT2 genes (Liu et al., 2021). These genes are mostly universal in plants. In Arabidopsis plants, the CAX1 (Cation exchanger1), CAX2 were reported to encode the metal transporter (Hirschi et al., 1996). AtACR2 gene of Arabidopsis thaliana was transferred into the genome of tobacco (Nicotiana tabacum var. 'Sumsun'). These transgenic tobacco plants were significantly more tolerant of arsenic compared to wild type (Nahar et al., 2017). The transgenic Arabidopsis thaliana has also been used to test the function of new genes involved in arsenic tolerance. New glutaredoxin gene -PvGRX5 transferred Arabidopsis thaliana increased significantly arsenic tolerant to compared with control lines when arsenic treatment. These results suggested that PvGRX5 from As hyperaccumulating ferns can be employed as a novel biotechnological solution for decreasing As pollution in crops (Sundaram et al., 2009).
Both arsenic (As) and cadmium (Cd) are common, harmful pollutants in Vietnam and worldwide. Previous research has discovered that some plants have the capacity to accumulate As, such as rice, Holcus lanatus, watercress, ferns, duckweed, Indian mustard and Eleusine indica (Nwaichi and Dhankher, 2016). Transgenic approaches have been developed for As phytoremediation technologies. Characterization of the Ars genotype in 17 bacterial isolates (from Mandovi and Zuariestuarine water systems) revealed ArsA (ATPase), ArsB (arsenite permease) and ArsC (arsenate reductase) genes on their plasmid DNA. Phylogenetic analysis of ArsB and ArsC genes indicated their close genetic relationship with plasmid-born Ars genes of E. coli and arsenate reductase of plant origin (Sri Lakshmi Sunita et al., 2012). The arsenate reductase gene PvACR2 was reported to be isolated from Pteris vittata, an As hyper-accummulating fern (Ellis et al., 2006). Transgenic seeds could germinate in the presence of 80 μM As (III) or 1200 μM arsenate [As(V)] treatment (Chen et al., 2013). Overexpression of garlic AsPCS1 and yeast GSH1 in Arabidopsis thaliana resulted in transgenic plants with high accumulating capacity of cadmium (Cd) and As. Both single-gene transgenic lines and double transformants exhibited significantly higher tolerance to and accumulated more Cd and As than the wild-type (Guo et al.,, 2008). Transfer of Escherichia coli arsC gene encoding arsenase reductase with soybean rubisco promoter (SRS1p) into Arabidopsis plants produced transgenic plants with higher As tolerance (Dhankher et al., 2002).
As the discovery of P. vittata, P. calomelanos and other hyperaccumulators is likely to benefit arsenic removal, scientists are focusing on optimizing the ability to accumulate this toxin and detoxify contaminated soil (Chen et al., 2002). In a recent study evaluating heavy metal accumulation in three species Pityrogramma calomelanos, Cynodon dactylon and Nephrolepis biserrata, it was shown that these three species were able to take up As in the shoot and Cu in the root. Aresenate uptake in Nephrolepis biserrata was the highest, followed by Cynodon dactylon and Pityrogramma calomelanos (Hidalgo et al., 2020). In Vietnam, the first detailed investigation of soil contamination at various mine sites was published in 2018. A total of 33 different plant species samples were collected. P. vittata. and P. calomelanos. were the only two ferns identified as As hyperaccumulators, containing more than 0.1% heavy metals in their shoots. This is a potential genetic resource that could produce plants with large biomass and strong suitability for As-contaminated areas (Anh et al., 2018).
In this study, we isolated a gene belonging to the ArsC family from P. calomelanos, a local fern that can strongly accumulate As. The function of ArsC gene in tolerance and accumulation of As in transgenic tobacco lines was evaluated. We also investigated whether ArsC gene integration disturbed the overall growth of transgenic lines.

Materials and Methods Materials and Methods Materials and Methods Materials and Methods
Plant materials and growth conditions P. calomelanos plants were collected at Ha Thuong mine, Thai Nguyen (Vietnam), whose As level is 202-3690 ppm, 300 times higher than the national safety standard. These plants were used to isolate ArsC gene ( Figure 1). Tobacco Nicotiana tabacum K326 plants were used for the ectopic overexpression experiments and grown on a modified Murashige and Skoog (MS) medium containing 4.3 g/l MS salt mixture, supplemented with 0.2 mg/l of 2,4-dichlorophenoxyacetic acid (2,4-D), 0.18 g/l KH2PO4, 0.1 g/l myo-inositol, 1 mg/l thiamine HCl, and 30 g/l sucrose, which was designated as MST. All tobacco plants, including germinating seeds and seedlings, were grown in a controlled environment greenhouse at 26 °C, with a 16-h light/8-h darkness photoperiod, and a light intensity of 120 mmol m -2 s -1 .

Cloning of ArsC
Total RNA was isolated using TRI Reagent TM Solution (Ambion, USA). 1 μg of total RNA and an oligo dT18 primer were used for reverse transcription (Thermo Scientific, USA). ArsC full length sequence was amplified from the first strand cDNA obtained from P. calomelanos. by polymerase chain reaction (PCR) using ArsC specific primers. The primers used for the amplified genes were ArsC (forward) 5′-

Construction of pCambia 1301-ArsC vector
Isolation of the ArsC gene and construction of a binary vector for plant transformation were performed as described by Luong (2016). Briefly, the ArsC containing fragment was cut out from the pJET1.2/blunt cloning vector by restriction enzymes NcoI and Eco721. The pCambia1301 expression vector was digested with NcoI and Eco721 and ligated to the ArsC containing fragment with the T4 DNA ligase (Thermo Scientific, USA). The new plant expression vector was designated pCambia1301-ArsC. The pCambia1301 expression vector (Figure 4) contains the ArsC gene driven by the 35S promoter and terminated by nopaline synthase terminator (nos). This vector was introduced into electrocompetent Agrobacterium tumefaciens strain C58 (BTX® ECM® 600 electroporator). Transformed cells were selected using medium containing hygromycin B (50 μg/ml) and cefotaxime (500 μg/ml). Successful transformants were confirmed by PCR amplification with ArsC specific primers (Table 1) and stored at -70 °C.

Arsenic tolerance assay in E. coli
Tolerance assay in E. coli was evaluated via colony formation assay. E. coli containing pCambi1301 or pCambia1301-ArsC were grown in LB liquid medium to OD 0.8 -1, followed by a dilution to OD 0.1 and 0.01. Five microliters of each dilution were placed on the LB plates containing different concentrations of arsenic (0, 25 and 50 µM). The plates were incubated overnight. The surface areas of colonies were measured using a Nikon microscope (Nikon Instech, Co., Ltd.) and iSolution FL Autosoftware (IMT i-solution Inc.). Three replicates were performed.

Transformation and screening
Transformation was performed using Agrobacterium tumefaciens-mediated method (Krugel, 2002). Briefly, 2 mm in diameter of K326 leaves were mixed with 100 μl of a fresh overnight culture of A. tumefaciens C58. After 48-hour cocultivation at 27 °C in the dark, bacterial cells were washed in sterile liquid MST, and pieces of leaves were plated on MST medium (0.8% agar) containing hygromycin B (50 μg/ml) and cefotaxime (500 μg/ml). Hygromycin B-resistant transformants were visible within 3 weeks and transferred to fresh MST agar containing selective antibiotics. Viable putative transformants were transferred to soil in the greenhouse.

Molecular analysis of transgenic plants
To select tobacco lines transformed with ArsC, putative transgenic plants were primarily screened by PCR. PCR-positive transgenic tobacco lines were further analyzed by southern blot hybridization. Southern blotting was performed using Biotin DecaLabel DNA Labeling kit and Biotin Chromogenic Detection kit (Thermo Scientific, USA). Briefly, genomic DNA of transgenic lines and WT were digested into small fragments using NcoI. Digested genomic DNA was separated by gel electrophoresis, and then transferred to nitrocellulose membranes. DNA fragments were fixed onto nitrocellulose via UV light. Target DNA was detected by a labeled probe complementary to the target DNA.

Estimation of arsenic content in transgenic plants
To evaluate the tolerance and accumulation of As in tobacco, T2 seeds from transgenic T1 lines showing strong expression of ArsC gene and containing only one copy of the integrated T-DNA, were germinated on MS medium supplemented with hygromycin B (50 μg/ml). Seeds of wild type plants were germinated on hygromycin B -free MS medium. Seedlings expressing ArsC gene and harboring a single insertion of integrated transgene as well as wild type seedlings of the same size and age (21 days old) were transferred to pots containing agar-solidi ed MS medium or soil in the greenhouse supplemented with different concentrations of AsV (0, 50, 100 and 200 μM) (Nahar et al., 2012;2017). Growth conditions were the same as described above. Each treatment was done in triplicate. Plants were collected at day 40 and washed thoroughly with distilled water to remove any residual As adhering to the root surface. All ground materials were stored at 4 °C until further use. To quantify As content, dried samples were mixed with 2 mL of HNO3 (65%, Merck, Darmstadt, Germany) and 6 mL of HCl (37%, Merck). Mixture was heated to 70 °C for 1 h, and then diluted with 10 mL of deionized water. The acid digestion was ltered to remove residual particulates. Arsenic concentration was determined by inductively coupled plasma mass spectroscopy (ICP-MS) method.

Statistical analysis
All experiments were performed in triplicate. Data were expressed as means ± standard error of results. Significance between two groups determined in this study was test by the Student's t-test, and analysis of variance was utilized between three or more groups. P-values less than 0.001 are considered statistically significant. All statistical analyses were performed by Sigma Plot software.

Isolation of ArsC gene from Pityrogramma calomelanos
Total RNA isolation from selected plants was performed according to the manufacturer's protocol. Total RNA with high quality was used as template RNA for the cDNA synthesis reaction ( Figure. 2A). PCR results showed that DNA fragment with the expected size (426 bp) was amplified from the first strand cDNA of P. calomelanos ( Figure 2B). These PCR products were purified and cloned into the pBT cloning vector. The ligation mixture was used directly to transform E. coli DH5α. Colonies containing the expected size (426 bp) was confirmed by PCR amplification with ArsC specific primers (Figure 2 C). These results suggested that ArsC gene from P. calomelanos was successfully cloned into pBT cloning vector.

Protein and nucleotide sequence of the ArsC genes
The nucleotide sequence of the ArsC gene was shown in Figure. Figure 3B). Sequencing analysis revealed that the ArsC clone from P. calomelanos and ArsC gene in GenBank with accession number X80057.1 shared high similarity (99%) with each other. Using BioEdit 6 software, we compared nucleotide sequences and translated into protein sequence. Compared to the ArsC gene in GenBank (accession number X80057.1), ArsC gene sequence from P. calomelanos differed by one position (32), Thymine instead of Cytosine ( Figure 3A). The deduced amino acid at position 11 was Alanine instead of Valine ( Figure 3B).

Construction of pCambia1301-ArsC
To express target gene in transgenic tobacco lines, we used pCambia1301 vector that contains a 35S promoter. The 35S promoter is a strong constitutive promoter and has been reported to upregulate gene expression in dicots. To design pCambia1301-ArsC vector, pCambia1301 vector and pBT vector containing ArsC gene were both digested by NcoI and Eco72I. pCambia1301 vector without GUS gene (9823 bp in length) and ArsC gene were purified, ligated by T4 ligase and transformed into competent E. coli DH5α cells. pCambia1301 vector containing ArsC gene was confirmed by PCR amplification with ArsC-specific primers and restriction enzymes (Figure 4).

Molecular analysis of transgenic tobacco lines
Tobacco cultivar 'K326' (Wilt type -WT) was selected as the starting material due to its excellent regeneration ability. Transgenic tobacco lines were screened by PCR amplification. PCR results showed that 6 independent transgenic lines with the expected size of DNA fragment were observed. There was no DNA amplicon of interest in the non-transgenic lines or the blank control (Figure 5 A). Sequencing analysis confirmed that this amplified band was precisely the 426 bp partial sequence of ArsC. These results suggested that the ArsC gene was successfully inserted into tobacco genome. To verify the number of gene copies in transgenic lines, we performed southern blot analysis with a specific probe designed to complement target DNA. All 6 transgenic lines (T1) were selected by hygromycin resistance and confirmed by PCR ( Figure 5B). There were 3 transgenic lines (K1, K9 and K18) containing a single-copy of integrated T-DNA. No hybridization signal was observed in the three remain lines or the nontransgenic control. In addition, plants in the T2 generation containing a single copy of the transgene showed similar results. These data demonstrated that three transgenic lines were stably inherited. Three transgenic lines with a single copy of integrated transgene were selected for further investigation

Arsenic inhibited plant growth
To evaluate the effect of As exposure on plant growth, plant height, leaf area, and fresh weight in transgenic lines and wild type plants were measured. There was no significant difference in plant height, leaf area, and fresh weight between transgenic lines and wild type plants in the absence of As (control treatment) and at a low concentration (50 µg/ml). However, plant growth was significantly restricted at high As concentrations (100-200 µg/ml) both in vitro and in greenhouse experiment; no wild type plants survived (Figure 6 A-B). In particular, As inhibited plant growth in a dose dependent manner. Interestingly, all transgenic lines showed significantly less stunted growth than wild type when plants were treated with 100-200 µg/ml of As ( Figure 6C to 6E) and data not shown for in vitro experiment). Taken together, these results suggested that As had inhibitory activity against plant growth. However, ArsC gene in transgenic lines conferred higher resistance to As.  Quantitative data were obtained from three independent experiments, n = 3. Data represent the mean±S.E.M. (standard error of the mean) **p<0.001 compared to treated wild type plants.

Assessment of As accumulation in transgenic tobacco lines
To investigate the role of AsrC gene in As accumulation capacity in tobacco, an ICP-MS analysis was conducted on roots of plants grown in the presence or absence of 100, 200 µg/ml As for 3 weeks (Figure 7). When plants were treated with 50 μM arsenate, the amount of arsenate accumulated in the roots of the transgenic lines were approximately three-fold higher (7.133 μg/g dry wt) than that found in the wild type (2.276 μg/g dry wt). In addition, in exposure to 100 or 200 μM arsenate, transgenic lines were able to accumulate up to 17.30 μg/g dry wt of arsenate in their roots, whereas the wild type could not survive. It is important to note that no significant difference in the ability to take up arsenite was observed among three transgenic lines. These results indicated that AsrC gene improved As accumulation capacity in tobacco.

Discussion Discussion Discussion
In the present study, we cloned and characterized ArsC gene from P. calomelanos. Moreover, we have generated transgenic tobacco lines expressing P. calomelanos ArsC gene by using Agrobacterium tumefaciensmediated method. Transgenic tobacco lines significantly enhanced As accumulation capacity in roots. The effect of gene integration into tobacco genome on plant growth was evaluated.
Many efforts have been made to improve the phytoremediation efficiency of plants. Transgenic plants contain potential candidate genes involved in arsenic accumulation, such as PCS1, ACR, MRP1 and MRP2. Of all ACR is one of the key genes. It was first isolated from bacteria and yeast (Mukhopadhyay et al., 2000). ACR gene encodes arsenate reductase, an enzyme converting arsenate (AsV) to arsenite (As III) and the arsenite form can be sequestered in the vacuoles of aplant cell (Xu et al., 2007;Zhao et al., 2012). The reduction of AsV to AsIII also occurs nonenzymatically via glutathione (Siddiqui et al., 2015). In the presence of glutathione, AsV can be hydrolyzed to AsIII and this process depends on intracellular availability of substrates and effectors (Németi et al., 2011). Reduction of AsV to AsIII has been known as the major mechanism of As resistance in plants that are able to hyperaccumulate As Schmöger et al., 2000). In addition, hyperaccumulators tend to transfer As immediately to other organs like leaves or shoots instead of retaining As in their roots. This translocation, as seen in P.vittata, further enhances As uptake (Singh and Ma 2006). In this study, we isolated ArsC gene from P. calomelanos. Sequencing results revealed that our ArsC gene sequence was highly similar to previously reported ACR genes. Several homologous proteins from Arabidopsis (AtAsr/AtACR2), P. vittata (PvACR2), and rice (OsACR2.1 and OsACR2.2) have been known to possess ACR activity (Dhankher et al., 2002;Ellis et al. 2006).
Southern blotting is a powerful tool to analyze copy number and locus complexity in transgenic plants. In our study, we used southern blot to determine the number of integrated T-DNA copies in transgenic lines. The number of integrated T-DNA copies also affects to the growth of transgenic lines. In this study, the transgenic lines harboring single copy of the integrated transgene were selected.
In this study, transgenic lines and wild-type plants grew well under normal growth condition and at low As concentrations. No significant difference in growth parameters, including plant height, leaf area, and fresh weight, was observed between the transgenic lines and wild-type plants. However, these growth readouts for both plant types, especially wild-type plants, were significantly lower in the presence of high As. Interestingly, transgenic lines containing a single copy gene showed strong resistance to As and higher As accumulation capacity compared to wild type. Molecular mechanisms underlying As resistance in these transgenic lines remain to be investigated.
A better understanding of As tolerance is conducive to generating As-resistant plants and vital to phytoremediation and safe cropping. For soil remediation and contaminated site rehabilitation, a diverse group of resistant plants suitable for growth in a wide range of environments and able to (hyper) accumulate As in harvestable biomass are needed. In contrast, for safe cropping, As-resistant plants that can detoxify the surrounding groundwater and land and prevent As accumulation in plant products of interest are required.

Conclusions Conclusions Conclusions Conclusions
In this study, we successfully generated three transgenic tobacco lines containing ArsC gene from P. calomelanos. These transgenic accumulated a much higher amount of arsenic in the roots compared to wildtype plants. These results suggested that P. calomelanos. ArsC gene has a significant potential in genetic engineering of plants to produce transgenic plants that reduce arsenic content of soil.

Authors' Contributions Authors' Contributions Authors' Contributions Authors' Contributions
TVN and TTBL conceived and designed the experiments; HTH, TTD, NTN, LTTN and LTT performed the experiments; TVN TTBL analyzed the data, TVN, TLTB wrote the paper. All authors read and approved the final manuscript.
Ethical approval Ethical approval Ethical approval Ethical approval (for researches involving animals or humans) Not applicable.