Analysis of the rice and Arabidopsis genomes revealed presence of at least 13 and 9 members of the phosphate transporter (Pht1) family in these species, respectively, (Okumura et al., 1998; Mudge et al., 2002; Paszkowski et al., 2002). Shin et al. (2004) indicated that Pht1;1 and Pht1;4 mediate a significant proportion of the As(V) uptake in Arabidopsis. Arsenite is the dominant As species in reducing environments, such as flooded paddy soils (Meharg and Jardine, 2003). Bienert et al. (2008) showed that the Nodulin26-like Intrinsic Proteins (NIPs) OsNIP2;1 and OsNIP3;2 from rice are bi-directional As(III) channels. Direct transport assays using yeast cells confirmed their ability to facilitate As(III) transport across cell membranes. Ma et al. (2008) reported that two different types of transporters mediate transport of As(III), from the external medium to the xylem. Transporters belonging to the NIP subfamily of aquaporins in rice are permeable to As(III) but not to As(V). Mutation in OsNIP2;1 (Lsi1, a silicon influx transporter) significantly decreased As(III) uptake. Furthermore, in the rice mutants defective in the silicon efflux transporter, Lsi2, As(III) transport to the xylem and accumulation in shoots and grain decreased substantially. Mutation in Lsi2 had a much greater impact on As accumulation in shoots and grains in field-grown rice than Lsi1. The efficient Si uptake pathway in rice also allows inadvertent passage of As(III), thus explaining why rice is efficient in accumulation of As (Ma et al., 2008). However, knockout of these transporters may not provide solution to As accumulation, as NIPs facilitate the uptake of vital nutrients such as boron and silicon (Ma et al., 2008).

An As tolerance gene has been identified and mapped to chromosome 6 in rice. The collocation of As tolerance gene with a phosphate uptake QTL in another population of rice provided circumstantial evidence for a mechanism involving the behavior of As(V) as a phosphate analog, in agreement with the behavior of an As resistance gene found in populations of Holocus lanatus (Dasgupta et al., 2004). Recently, the genetic mapping of the tolerance of root growth to As(V) using the Bala X Azucena population suggested involvement of epistatic interaction involving three major genes, two on chromosome 6 and one on chromosome 10. The study provided physiological evidence that genes related to phosphate transport is unlikely to be behind the genetic loci conferring tolerance (Norton et al., 2008b). However, Zhang et al. (2007) measured As accumulation in roots and shoots at the seedling stage and brown rice at maturity of the parental cultivars (CJ06/TN1) and their double haploid lines after exposure to 210 mg As/kg As in a pot experiment. Four QTLs for As concentrations were detected in the genetic map. At the seedling stage, one QTL was mapped on chromosome 2 for As accumulation in shoots which explained 24% of the observed phenotypic variance and another QTL for As accumulation in roots was detected on chromosome 3. At maturity, two QTLs for As accumulation in grains were found on chromosomes 6 and 8, which explained 26 and 35% of the phenotypic variance, respectively. This study also showed that the QTL on chromosome 8 was identified for As concentrations in grain at maturity and shoot phosphorus (P) concentrations at seedling stage. A study targeting genetic mapping of the rice ionome in leaves and grains identified QTLs for 17 elements including As, Se, Fe, and Cd. In general, there were no QTL clusters suggesting independent regulation of each element. An epistatic interaction for grain As appears promising to decrease the concentration of this carcinogenic metalloid in the rice grain (Norton et al., 2010). Further, these workers while identifying QTL for rice grain element composition on an As impacted soil from China, found a correlation between flowering time and number of element concentrations in grains, which revealed co-localization between flowering time QTLs and grain element QTLs. Therefore, this study concluded that flowering time is one of the major factor that controls As and other element accumulation in the rice grain. Besides, an epistatic interaction for grain As also looks promising to decrease the concentration of this carcinogen (Norton et al., 2012). Recently, As concentrations in different tissues of maize was analyzed using a RIL population (Ding et al., 2011). In this study, three QTLs for As concentration in leaves were mapped on chromosomes 1, 5, and 8, respectively. For As concentration in the bracts, two QTLs were identified, which accounted for around 10% of the observed phenotypic variance. For As concentration in the stems, three QTLs were detected with around 8–15% phenotypic variance. Three QTLs were identified for kernels on chromosomes 3, 5, and 7, respectively, with around 9–11% phenotypic variance. Only one common chromosomal region between SSR marker bnlg1811 and umc1243 was detected for QTLs qLAV1 and qSAC1. These results demonstrated that the As accumulation in different tissues in maize was controlled by different molecular mechanisms.

The effect of As exposure on genome-wide expression was also examined in rice by different groups (Norton et al., 2008a; Chakrabarty et al., 2009; Yu et al., 2012). A group of defense and stress-responsive transporters including; sulphate transporters, heat-shock proteins, metallothioneins, multidrug and toxic compound extrusion (MATE) transporters, glutathione-S-transferase, multidrug resistance proteins, glutathione conjugated transportes, metal transporter viz. NRAMP1, and genes of sulfate-metabolizing proteins were commonly upregulated in rice during As(V) stress in both the studies (Norton et al., 2008a; Chakrabarty et al., 2009). In contrast, phosphate transporter, zinc transporter, aquaporin gene, amino acid transporters, and peroxidases (PODs) were commonly downregulated, however, some gene like cytochrome P450, oxidoreductase were differentially regulated in rice seedling challenged with As(V). Yu et al. (2012) first used high-throughput sequencing technology to study transcriptomes of plant response to As stress. Overall, by genome-wide transcriptome and miRNA analyses in rice seedlings treated with As(III), they found a large number of potentially interesting genes in relation to As(III) stress, especially As(III)-responsive transporters and TFs (transcription factors). The change in the expression of genes related to lipid metabolism and phytohormone pathways after As(III) exposure was striking, indicating that rice invests more energy and resources into immediate defense needs than into normal growth requirements.

Arsenate and As(III) stresses modulated metabolic pathways networks affecting various physiological processes necessary for plant growth and development (Chakrabarty et al., 2009). Arsenate stress led to upregulation or downregulation of additional genes in comparison to As(III), but one glutaredoxin (Os01g26912) is expressed specifically in the AsIII-treated shoots. In one of our studies, four rice genotypes responded differentially under As(V) and As(III) stress in terms of gene expression and antioxidant defenses (Rai et al., 2011). Arsenate challenge leads to altered gene expression in a large number of genes involved in classical oxidative stress response, however, phytochelatin synthase (PCS) and arsenic reductase (AR) levels were not altered significantly. This study suggested that the rice cultivar, IET-4786, is very sensitive to As stress due to reduction of both sulphate assimilation pathway and antioxidant defense enzymes in As detoxification in contrast to the tolerant cultivar, Triguna. The other two varieties (PNR-546 and IR-36) exhibited intermediary responses involving regulation of these genes during As stress (Rai et al., 2011). The differential expression pattern of sulphate transporters were observed for the varieties after As(V) exposure (Kumar et al., 2011). Most of the sulphate transporters studied were down-regulated in the sensitive variety IET-4786 after exposure to As(V). Interestingly, differential alternative splicing for OsSultr1;1 was also observed for all the lines after As exposure. A decrease in accumulation was observed for the largest splice variant containing both the introns at higher concentrations of As(V) and As(III). In the case of sensitive rice variety, accumulation of the entire splice variant decreased with increasing concentration of As(V). These results clearly suggest that the expression of sulphate transporter gene family with respect to heavy metal stress is regulated differentially in different cultivars.

However, several common processes were affected by As(V) and As(III). Differential expression of several genes that showed the highest contrast in a microarray analysis was validated by following the quantitative changes in the levels of individual transcripts following challenge with As(V)and As(III). Abercrombie et al. (2008) first studied the transcriptional response of a dicot plants, Arabidopsis thaliana, to As(V) stress using oligonucleotide microarrays. The study suggested that As(V) stress strongly induces Cu/Zn superoxide dismutase activity (SOD), but represses the production of Fe SOD. The study also suggested involvement of several other genes related to antioxidant systems, various transcription factors, tonoplast proteins, and proteins associated with cell wall growth. A comparative biochemical and transcriptional profiling of two contrasting varieties (tolerant and sensitive to As) of Brassica juncea indicated upregulation of sulfate transporters and auxin and jasmonate biosynthesis pathway genes, whereas, there was downregulation of ethylene biosynthesis and cytokinin responsive genes in As tolerant plant in contrast to sensitive plant (Srivastava et al., 2009). In another experiment, Paulose et al. (2010) studied expression profiling of dicot Crambe abyssinica under As(V) stress using PCR-Select Suppression Subtraction Hybridization (SSH) approach. Their study revealed novel insights into the plant defense mechanisms, and the regulation of genes and gene networks in response to arsenate toxicity. The differential expression of transcripts encoding glutathione-S-transferases (GSTs), antioxidant As_i genes, sulfur metabolism genes, heat-shock proteins, metal transporters, and enzymes in the ubiquitination pathway of protein degradation, as well as several unknown novel proteins serve as molecular evidence for the physiological responses to As(V) stress in plants.

A comparative evaluation of As responsive genes in monocots and dicots offered interesting observations (Table 1). Various PODs genes always showed a down regulation (2–24 fold) in monocots, however, upregulation of two PODs was recorded in dicots. MT like proteins was upregulated in monocots. Five ferrodoxins were down regulated in rice but not in A. thaliana. Peptidyl prolyl cis-trans isomerase was highly upregulated in rice in contrast to A. thaliana. Interestingly, 9 GSTs were upregulated in rice, whereas only one was observed to be upregulated in Arabidopsis. At the same time, in both the plants 2 GSTs were downregulated. While most of the cytochrome P450 were upregulated in rice, none of these were upregulated in Arabidopsis. In contrast to A. thaliana, the germin like protein was upregulated in rice. Many genes encoding ferritins, ZFP (Zinc finger protein), patatin, acid phosphates, serine/threonine proteine kinases, integral membrane proteins, and hydrolases always were down regulated in both monocot and dicots (Abercrombie et al., 2008; Chakrabarty et al., 2009). The data suggests that though there are common mechanisms for As metabolisms in dicot and monocot plants, differential mechanisms may also exists.

Gasic and Korban (2007) reported that overexpression of the A. thaliana phytochelatin synthase AtPCS1 gene in Indian mustard enhanced tolerance to As and Cd. Similarly, Song et al. (2010) showed that in the absence of two ABCC-type transporters, AtABCC1 and AtABCC2, A. thaliana is extremely sensitive to As and As-based herbicides. Heterologous expression of these ABCC transporters in phytochelatin (PC)-producing Saccharomyces cerevisiae enhanced As tolerance and accumulation. Furthermore, membrane vesicles isolated from yeasts exhibited a pronounced As(III)–PC₂ transport activity. Recently, Indriolo et al. (2010) characterized two genes from P. vittata (ACR3 and ACR3;1), which encode proteins similar to the ACR3 As(III) effluxer of yeast. It was also showed that ACR3 is localized to the vacuolar membrane in gametophytes, indicating that it likely effuxes As(III) into vacuoles for sequestration. A single copy of ACR3 genes is present in moss, other ferns and gymnosperms but absent in flowering plants. The absence of ACR3 genes in both monocots and eudicots may preclude their ability to hyperaccumulate As, which is consistent with the observation that no As-hyperaccumulating angiosperm has ever been identified. Why the highly conserved ACR3 gene was lost in the angiosperm lineage may never be known, but its loss coincides with their reliance on insects and other animals for pollination and fruit dispersal. On the other hand, the selection of As hyperaccumulation in P. vittata, may have evolved as a deterrent and in response to herbivores, as it has recently been shown that insects avoid eating P. vittata grown in the presence of As, preferring those not grown in As (Rathinasabapathi et al., 2007). In another study, examining heterologous expression of the yeast As(III) efflux system, ACR3 was cloned from yeast and transformed into wild-type and nip7;1 Arabidopsis (Ali et al., 2012). At the cellular level, all transgenic lines showed increased tolerance to As(III) and As(V) and a greater capacity for As(V) efflux. With intact plants, three of four stably transformed lines showed improved growth, whereas only transgenic lines in the wild-type background showed increased efflux of As(III) into the external medium. The presence of ACR3 hardly affected tissue As levels, but increased As translocation to the shoot. Expressing Saccharomyces cerevisiae ScACR3 in rice enhanced As(III) Efflux and also reduced As accumulation in rice grains (Duan et al., 2012). In the transgenic lines, As concentrations in shoots and roots were about 30% lower than in the wild type, while the As translocation factors were similar between transgenic lines and the wild type. The roots of transgenic plants exhibited significantly higher As efflux activities than those of the wild type. Importantly, ScACR3 expression significantly reduced As accumulation in rice straws and grains. When grown in flooded soil irrigated with As(III)-containing water, the As concentration in husk and brown rice of the transgenic lines was reduced by 30 and 20%, respectively, compared with the wild type. This study reports a potential strategy to reduce As accumulation in the food chain by expressing heterologous genes in crops. Recently, Sundaram et al. (2009) overexpressed the P. vittata glutaredoxin PvGRX5 gene in Arabidopsis. It was observed that two lines of A. thaliana constitutively expressing PvGrx5 cDNA were significantly more tolerant than vector control and wild-type lines on the basis of germination, root growth and whole plant growth under As stress. A study was conducted by Grispen et al. (2009) by overexpressing AtMT2b in Nicotiana tabacum. It was observed that the highest AtMT2b expressing line exhibited a significant decrease in As accumulation in roots, but an increased accumulation in shoots, while the total amount of As taken up remained unchanged. This clearly suggested that AtMT2b expression may enhance As root to shoot transport. Rice transgenics overexpressing As(III)-S-adenosyl methyl transferase (arsM) has been found to methylate As, and gave 10 fold higher volatile arsenical, maintaining low As levels in rice seed along with MMA(V) and DMA(V) in the roots and shoots of transgenic rice (Meng et al., 2011). The upregulation of Met synthase and AdoMet synthetase could correlate with As(III) methylation. Although the amounts of methylated As were small, this may offer a new stratagem for As phytoremediation. Shukla et al. (2012) demonstrated that transgenic tobacco plants expressing Ceratophyllum demersum phytochelatin synthase CdPCS1 had a several fold increase in PC content, precursor non protein thiols, and enhanced accumulation of As without significant decrease in plant growth.

In most of the studies, metabolites involved in antioxidant systems, PCs and related molecules involved in biosynthesis of PCs have been analyzed during As stress. A list of several As responsive metabolites has been mentioned in the Table 3. The raised level of some metal detoxifying thiolic ligand such as glutathione were also noticed in ferns such as P. vittata, P. ensiformis (Singh et al., 2006), aquatic plants such as H. verticilata and C. demersum (Srivastava et al., 2007; Mishra et al., 2008), and crop plants like B. juncea and O. sativa (Srivastava et al., 2009; Tripathi et al., 2012). While concomitant reduction in glutathione and S-nitrosoglutathione (GSNO) content were observed in A. thaliana suggesting the altered GR and S-nitrosoglutathione reductase activities during higher As exposure (Leterrier et al., 2012). Similarly induced level of PCs were also observed in H. verticillata, C. demersum, and O. sativa (Srivastava et al., 2007; Mishra et al., 2008; Tripathi et al., 2012) under As stress. Earlier, As-PC complexes were studied in H. lanatus and P. cretica using parallel metal(loid)-specific-ICPMS and organic-specific-ESI-MS detection systems (Raab et al., 2004). In an in vitro experiment using a mixture of GSH, PC₂, and PC₃, As preferred the formation of the As(III)-PC₃ complex over GSH-As(III)-PC₂, As(III)-(GSH)₃, As(III)-PC₂, or As(III)-(PC₂)₂. In H. lanatus, the As(III)-PC₃ complex was the dominant complex, although GSH, PC₂, and PC₃ were found in the tissue extract. P. cretica only synthesizes PC₂ and forms dominantly the GSH-As(III)-PC₂ complex. In both plant species, As is dominantly in non-bound inorganic forms, with 13% being present in PC complexes for H. lanatus and 1% in P. cretica. Phytochelatin synthesis was induced upon exposure to As(V) in P. vittata, with only PC₂ detected in the plant (Zhao et al., 2003). The As concentration correlated significantly with PC₂ concentration in roots and shoots of P. vittata, but not with GSH. Chelation of only a small proportion (1–3%) of the As with PCs in P. vittata suggests that PCs play a limited role for the hypertolerance of As in P. vittata. Similarly, Raab et al. (2007) demonstrated the As concentration-dependent formation of As–PC complex, redistribution and metabolism of As after arrested As uptake in Helianthus annuus. The amount and number of As–PC complexes increased exponentially with concentration up to 13.7 μM As and As(III)–PC₃ and GS–As(III)–PC₂ complexes were the dominant species. In another study, Liu et al. (2010) quantified As(III)-thiol complexes and free thiol compounds in A. thaliana exposed to As(V). In wild-type roots, 69% of As(III) was complexed as As(III)-PC₄, As(III)-PC₃, and As(III)-(PC₂)₂ while in roots of the GSH-deficient mutant (cad2-1) and the PC-deficient mutant (cad1-3) very little of As was complexed with As(III)-PCs and As(III)-(GS)₃, respectively. This conferred approximately 20 times more tolerance for the wild type than the mutants. These mutants showed significantly higher accumulation of As(III) in shoots and effluxed larger amount of As(III) than the wild type, suggesting that enhancing PC synthesis in roots may be an effective strategy to reduce As translocation to the edible organs of food crops. Furthermore, transgenic Arabidopsis expressing very high levels of the bacterial γ-glutamylcysteine synthetase (ECS) gene and PCS had several fold higher concentrations of γ-glutamylcysteine (EC), GSH, and PCs than the wild type, and show tolerance to As (Dhankher et al., 2002; Li et al., 2004).

Variation in amino acid content was observed in different plant species during As exposure. Dwivedi et al. (2010) performed a simulated pot experiment, using environmentally relevant concentrations of As, analyzed the amino acid profile in grain of various rice genotypes. This study demonstrated that Specific As Uptake (SAU, μg g⁻¹dw), which indicates the ability of As uptake by rice per unit root under As exposure, was different between rice genotypes, and found in the order of As tolerant Triguna (134) > IR-36 (71.5) > PNR-519 (53) > sensitive IET-4786 (29). However, the grain As concentration (μg g⁻¹dw) order was IR-36 (1.5) > Triguna (1) > PNR-519 (0.5) > IET-4786 (0.3). They concluded that most of the essential amino acids (EAAs) metabolites such as valine, metheionine, leucine, alanine, and nonessential amino acids (NEAAs) viz. histidine, alanine, proline, glutamic acid, and cysteine increased in most of the rice genotypes during As(V) exposure. Further to validate this finding a field experiment was conducted, determining the amino acid profile of sixteen rice genotypes differing in grain As accumulation, grown at three sites with different soil As concentrations in West Bengal and India. Grain As accumulation negatively correlated with EAAs which were more prominent in high As accumulating rice genotypes (HAARGs). Conversely, NEAAs showed an increase in low As accumulating rice genotypes (LAARGs) but a decrease in HAARGs. EAAs like isoleucine, leucine, valine, phenylalanine, and tyrosine also decreased in most of the genotypes (Dwivedi et al., 2012). Some other amino acids for example proline, glutamic acid, aspartic acid, and alanine also increased during As(V) stress in Spinacia oleracea (Pavlík et al., 2010). Among stress responsive amino acids, proline is a much studied molecules and can function as an osmolyte, free radical scavenger and also protects the cell membrane against damage. The level of proline has also been observed to be elevated in O. sativa during As (III) stress (Mishra and Dubey, 2006). The S-containing amino acid, cysteine, plays a central role in As detoxification, as it is a primary metabolite for synthesis of GSH and PCs. The cysteine content increased in some aquatic plants such as H. verticillata, rootless plant C. demersum, and crop plants B. juncea, O. sativa during As stress (Srivastava et al., 2007, 2009; Mishra et al., 2008; Tripathi et al., 2012).

Some low molecular antioxidant like ascorbate (AsA) and dehydroascorbate (DAsA), which work as non-enzymatic antioxidants in the glutathione-ascorbate cycle for free radical scavenging, were also analyzed in some plants during As(V) exposure. As(V) exposure caused an increase in the ratio of AsA/DAsA in P. vitatta, P. ensiformis, H. verticillata, and O. sativa (Singh et al., 2006; Srivastava et al., 2011; Tripathi et al., 2012) indicating the significant role of ascorbate for As induced stress amelioration. Malondialdehyde (MDA), the byproduct of lipid peroxidation was also increased during As(V) expsoure in P. vittata, P. ensiformis, H. verticillata, C. demersum. Nitric oxide, a signaling molecule was also found to be induced during As(V) stress condition in H. verticillata, O. sativa, and A. thaliana (Srivastava et al., 2011; Tripathi et al., 2012; Leterrier et al., 2012). The protection provided by polyamines against oxidative stress has been proposed to involve scavenging free radicals (Drolet et al., 1986) and the reduction of lipid peroxidation (Borrell et al., 1997). Mascher et al. (2002) demonstrated that levels of polyamines viz., spermidine, spermine increased only at lower doses but diamine increased at higher doses during As(V) stress in red clover (Trifolium pretense). Another study concluded that redox state and energetic equilibrium analyzed in terms of ATP/ADP NADH/NAD, NADPH/NADP, GSH/GSSG, and AsA/DAsA ratios, were found to be altered due to As toxicity in H. verticillata (Srivastava et al., 2011). Hence, variation in metabolite profiling during As exposure in different plant species signify that plants modulate their metabolome to respond against As stress.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.