**4. Mechanisms of arsenic uptake and detoxification in aquatic weeds**

## **4.1 Mechanisms of arsenic uptake in aquatic macrophytes**

Three pathways for arsenic uptake in marine macrophytes have been described – (i) active uptake through phosphate uptake transporters, (ii) passive uptake through aquaglyceroporins, and (iii) physicochemical adsorption on root surfaces. Plants mainly uptake As(V) through phosphate uptake transporters [63, 64]. As(III), DMAA and MMAA gets into the plants by passive mechanism through the aquaglyceroporin channels [64].

### *4.1.1 Active uptake through phosphate uptake transporters*

As(V) and phosphate are chemical analogs, and compete for uptake carriers in the plasmalemma [65]. As a result, as the phosphate content rises, more As (V) is required to be desorbed in the solution. Mkandawire and Dudel. [32] and Rahman et al. [33] showed that As (V) is taken up by aquatic plants through the phosphate uptake pathway, it competes with phosphate for uptake in tissues of *L. gibba* L. and *S. polyrhiza* L.

#### *4.1.2 Passive uptake through aquaporins/aquaglyceroporins*

Physiological studies indicate that these arsenic species are transported in rice through aquaporins /aquaglyceroporins via passive uptake mechanisms [66, 67]. Molecular studies revealed that Nodulin26-like intrinsic membrane proteins (NIPs), one of the major subfamilies of aquaporins transporters that promote the transport of neutral molecules like water, glycerol, and urea, are responsible for transporting As(III) into rice roots [68]. Aquaporins and aquaglyceroporins are two of three subfamilies of water channel proteins (WCPs), the transmembrane proteins that have a specific three-dimensional structure with a pore that permeates water molecules [69], which are permeable to water, glycerol, and/or other small, neutral molecules. Glycerol and As(III) compete for uptake in rice (*Oryza sativa* L.), indicating that this arsenic species is carried via the plasma membrane by aquaporins/ aquaglyceroporins [67].

#### *4.1.3 Physicochemical adsorption on root surfaces*

Arsenic is adsorbing and accumulating on the surfaces of aquatic plants due to suspended iron oxides (Fe-plaque). Robinson et al. [70] discovered a strong association between arsenic and iron concentrations in aquatic plants, which is believed to be due to arsenic adsorption on plant surfaces' iron oxides. Rahman et al. [14] investigated arsenic species adsorption on precipitated iron oxides on *S. polyrhiza* L. roots/fronds and revealed a strong association between arsenic and iron concentrations in tissues when the plant was exposed to As (V). There was no association

*Phytoremediation of Arsenic Contaminated Water Using Aquatic, Semi-Aquatic and Submerged... DOI: http://dx.doi.org/10.5772/intechopen.98961*

between arsenic and iron in plant tissue when *S. polyrhiza* L. was exposed to As (III), DMAA, and MMAA.As (V) is primarily adsorbed on precipitated iron oxides on the roots of aquatic plants and deposited by a physicochemical adsorption process, according to the findings.

#### **4.2 Arsenic metabolism and detoxification in aquatic macrophytes**

Arsenic occurs primarily as As (V) in an oxic environment and as As (III) in a reduced environment [64]. In plants, As (V) and phosphate share the same transporter, while As(III) enters plant cells through NIPs'aquaporins [57, 64]. Because of their distinct molecular properties, these two types of arsenic elicit different biochemical responses in aquatic plants [71]. As (V) has no affinity for thiol ligands, while As(III) has a strong affinity for peptides with sulfhydryl (-SH) groups, such as glutathione (GSH) and phytochelatins (PCs) [64, 72]. Even though plants had been exposed to As, arsenic speciation in plant tissues indicates that arsenic is primarily present in the As(III) oxidation state (V).This suggests that As(V) is effectively reduced to As(III) in plant cells after uptake, and that most plants have high As(V) reduction competence [64]. The reduction of As(V) to As(III) is mediated by GSH [73] and by enzyme [74], which is thought to be a detoxification mechanism of the plants. As(V) and As(III) have been shown to generate reactive oxygen species (ROS) within cells when they are taken up [75], and plants counteract the generation of ROS by various enzymes and cellular compounds [76]. The GSH can act as an antioxidant and is required for the synthesis of Phytochelatins which are required for metalloid chelation [71].

The mechanism of arsenic accumulation and detoxification was studied by many others in aquatic plant *H. verticillata* [57, 71]. In the presence of As (III) or As(V), *H. verticillata* enhanced the biosynthesis of thiols such as PCs, and increased antioxidant enzyme activity. Although the levels of thiolic compounds such as NP-SH, cysteine, GSH, and oxidized glutathione (GSSG) were significantly enhanced in *H. verticillata* upon exposure to both As(III) and As(V), As(III) was found to enhance the activities of cysteine synthase and c-glutamylcysteine synthetase and the amount of cysteine and GSH to higher levels than As(V) . The analysis of PCs indicates that the accumulation of PC1 and PC2 in *H. verticillata* was enhanced with the increase of both As(III) and As(V) concentrations [71]. Thus, during As (III) and As(V) stress, phytochelatins and antioxidant systems in *H. verticillata* react differently, which is considered to be the plant's detoxification mechanism.

#### **5. Biotechnological interventions for phytoremediation**

Plants have been utilized for phytoremediation of toxic metals and metalloids, however due to heavy metal phytotoxicity to plants; this process has been slow and largely rendered ineffective [77]. Natural heavy metal hyperaccumulators are also available, however, they are limited to specific geo-climatic conditions and also lack the crucial biomass required for efficient phytoremediation. Phytoremediation has a lot of potential using genetic engineering technologies to improve plant tolerance and heavy metal accumulation. Furthermore, various new studies using omics technologies such as genomics, transcriptomics, proteomics, and metabolomics to elucidate the genetic determinants and pathways involved in heavy metal and metalloid tolerance in plants have been identified. Presently there are three main biotechnological approaches for the phytoremediation of heavy metals and metalloids are currently being used to engineer plants for phytoremediation of heavy metals and metalloids: (1) manipulating metal/metalloid

#### **Figure 3.**

*Potential biotechnological strategies for phytoremediation. Heavy/toxic metals can be mobilized and transported (influx) into roots through plasma membrane transporters. They can then be transported (efflux) out of the roots into the xylem and translocated into the shoots. At this stage, plant tolerance to toxic elements may be enhanced through manipulation of influx/efflux transporters or by increasing the levels of ligands/ chelators. Volatilization of the toxic elements can be achieved through enzymes that modify these toxic elements. Chelators or efflux transporters can also be used to export the toxic elements out of the cytosol and into vacuoles or the cell wall. Adapted from Dhankher et al. (2011).*

transporter genes and uptake systems; (2) enhancing metal and metalloid ligand production; (3) conversion of metals and metalloids to less toxic and volatile forms [78] (**Figure 3**).

#### **5.1 Manipulating metal/metalloid transporter genes and uptake system**

Enhanced heavy metal tolerance and bioaccumulation has been attained in different plant species by genetic manipulation of metal transporter genes. For example, the overexpression of full length *NtCBP4* (plasma membrane channel protein) in *Nicotiana tabacum* showed Pb2+ hypersensitivity and enhanced accumulation of Pb2+ in the genetically manipulated plants. However, the overexpression of a truncated version of *NtCBP4* generated by deletion of its C-terminal, calmodulin- binding domain and part of the putative cyclic nucleotide- binding domain showed improved tolerance to Pb2+ and less accumulation of Pb2+ [79]. *Nicotiana tabacum* plants expressing *CAX2* (calcium exchanger 2) gene accumulated more Ca2+, Mn2+ and Cd2+ and also showed enhanced tolerant to elevated Mn2+. It was also observed that overexpression of *CAX2* gene in *Nicotiana tabacum* increased Mn2+ and Cd2+ transport in the root tonoplast vesicles in the transgenic plants [80]. Moreover, T-DNA mutants of the *Arabidopsis CNGC1* (cyclic nucleotide- gated ion channel 1) gene, that encodes a homologous protein to *NtCBP4*, also showed Pb2+ hypersensitivity and enhanced accumulation of Pb2+ in the genetically manipulated plants. These findings suggest that *NtCBP4* and *AtCNGC1* play an important role in the transport pathway of Pb2+ [79, 81]. The overexpression of yeast *YCF1* (Yeast Cadmium Factor 1) gene in *Arabidopsis thaliana* resulted in enhanced accumulated higher amounts and tolerance to Pb2+ and Cd2+ metals in plants [82].

*Phytoremediation of Arsenic Contaminated Water Using Aquatic, Semi-Aquatic and Submerged... DOI: http://dx.doi.org/10.5772/intechopen.98961*

Recent research findings have revealed arsenite is transported in plants by proteins belonging to the aquaporins [83, 84]. It is observed that in efficient arsenic hyperaccumulators such as *Pteris vittata* has highly well-organized system of arsenic translocation from root to shoot tissues [85, 86], However, most non-hyperaccumulators show low mobility rate compared to *P. vittata*, also variable Arsenic mobility rate is observed among different plant species, suggesting that it is controlled by genes. Arsenic loading to the xylem is a critical stage in arsenic translocation from root to shoot, however it is a poorly known mechanism. Ma et al. [87, 88] has identified and characterized *Lsi2* gene encoding an efflux protein, plays an important role in loading arsenite into the xylem. Mutation in *Lsi2* gene caused about 50% reduction in arsenic accumulation in the shoot. The *Lsi2* gene is a homolog of the *E. coli* ArsB gene, an As (III)/H+ exchanger that confers bacterial arsenite tolerance [89].

Genome-wide gene expression analysis in *Oryza sativa* roots treated with different heavy metals and metalloids; As(V), Cr(VI), Pb, and Cd, showed numerous differentially expressed genes as well as unique genes. Various genes belonging to different transporter families were identified [90]. Recently Wang et al. [91], has identified genes for Cu tolerance in the *Paeonia ostii* with the help of *de novo* transcriptome sequencing approach. Such genes may further be transferred to crop plants for enhancing heavy metal tolerance. Therefore, strategies of developing transgenic plants for arsenic (As) phytoremediation include enhancing plant uptake for phytoextraction, decreasing plant uptake, improving the plants' tolerance to As contamination, and increased methylation for enhanced food safety.

#### **5.2 Enhancing metals and metalloids ligand production**

Complexation of Arsenic with phytochelatins (PCs), or metallothionein (MTs) or glutathione (GSH) is an proficient way to detoxify As(III), since these complexes are sequestered in the vacuoles, this process is catalyzed by the homologs of multidrug resistance proteins (MRPs) [92, 93]. Enhancing the accumulation or synthesis of PCs and/or GSH and/or MTs may be one way to increase phytoremediation of arsenic. The overexpression of *PCS* in *Brassia juncea* enhanced its tolerance to arsenic but no significant increase arsenic accumulation was observed, this may be due to the fact that PC synthesis is also limited by the production of GSH [94]. The overexpression of *AtPCS1* and *GSH1*genes, that encode g-glutamylcysteine synthetase (g-ECS), the rate-limiting step in GSH biosynthesis, individually in *Arabidopsis thaliana* increased both arsenic tolerance and as well as accumulation [95].

Arsenic (As) tolerance in plants can also be increased by modifying GSH and PCs. Dhankher et al. [96] transferred and co-expressed two bacterial genes, *E. coli* arsenate reductase (*arsC*) and γ-glutamylcysteine synthetase (*γ-ECS*), in *Arabidopsis thaliana*, the transgenic plants grown in the presence of 125 μM sodium arsenate accumulated threefold more arsenic in the aboveground biomass and showed almost 17-fold higher biomass than wild type WT plants. The overexpression of AtPCS1 under constitutive promoter in *A. thaliana* enhanced tolerance to arsenate but failed to enhance arsenic accumulation [97]. These studies showed that manipulation of genes for increasing the production of metal chelation agents hold great potential for improving heavy metal and metalloid tolerance and accumulation in plants.

The *de novo* transcriptome sequencing analysis in *Raphanus sativus L.* roots under cadmium stress was carried out to discover differentially expressed genes and microRNAs (miRNAs) involved in Cd-responsive regulatory pathways. Various candidate genes encoding PCs, GSHs, and MTs; and other genes belonging to zinc iron permease (ZIPs) and ABC transporters were identified [98]. Likewise, in *de novo* transcriptome analysis in radish roots under chromium stress, showed that

1561 unigenes down-regulated and 1424 unigenes were up-regulated, various transcription factors such as Chromium stress-responsive genes involved in chelate compounds, signal transduction and antioxidant biosynthesis were discovered [99]. Such candidate genes can further be transferred into the crop plants to enhance heavy metal tolerance as well as accumulation.

#### **5.3 Conversion of metals and metalloids to less toxic and volatile forms**

There are several reports for developing phytoremediation strategies for heavy metals with the help of biotechnological interventions by conversion of these metals to less toxic and volatile forms. It is observed that many organisms, including bacteria, fungi, and animals, methylate arsenic. Methylated arsenic have been discovered in several plant species, including rice grain [100, 101], and suggest that this is the process is a result of endogenous methylation by the plants themselves. The final product of this pathway is the gas trimethylarsine (TMAs(III)), that can be volatilized from the plant. Qin et al. [102] have cloned a gene encoding an As(III)-S-adenosylmethionine methyltransferase (arsM) from the soil bacterium *Rhodopseudomonas palustris*. Expression of the *arsM* gene in an arsenic-sensitive strain of *E. coli* that resulted in the biosynthesis of several methylated forms of arsenic, including volatile TMAs(III) and conferred arsenic tolerance in the plants. These findings show that the expression of the single methyltransferase (*arsM*) gene is sufficient to produce both volatilization and tolerance to arsenic (As). A gene for an ArsM homolog in a primitive plant, the eukaryotic alga Cyanidioschyzon merolae has been idenfied [103]. Cells expressing *CmArsM* methylates As(III), as like the purified enzyme. In a rice microarray study, a putative gene annotated as a methyltransferase was found to be upregulated upon exposure to arsenate in the growth solution [104]. These findings indicate the possibility of engineering arsenic volatilization for the phytoremediation of arsenic-contaminated water and soil and also to improve the safety of the food supply.

## **6. Conclusions**

Contamination of soils and water by arsenic is one the serious threat for food security and human health in throughout the world. Some severe skin and other diseases occur due to continuous consumption of As contaminated foods and water. This necessitates a suitable technology to handle arsenic contaminated water carefully, so that above mentions points can be satisfied. Phytoremediation of arsenic contaminated water by aquatic and semi aquatic weeds offers low cost, economically feasible and eco-friendly technology to remove arsenic from contaminated water for long term. Some weeds have tremendous potential to accumulate higher amount of arsenic in their plant parts such as Eichhornia crassipes, Hydrilla verticillata, Spirodella polyrhiza, Arundo donax and Vetivaria spp. More specifically semi aquatic weeds like Arundo donax and Vetivaria sp. (perennial) can be used with in combination with Eicchornia, Spirodella and Hydrilla to remove arsenic more efficiently from treatment tanks or constructed wetland system. Although management of plant biomass will be another concern for disposal, but these plant materials can be used for making fiber (water hyacinth), handcraft items (Arundo and Typha stems) and biofuel purpose. Moreover, with advancement of molecular genetics in future As tolerance genes can be transferred to food crops (specially rice) which can store huge amount of As in their roots or very low transfer coefficient from root to grain so that transgenic rice crops will able to grow using As contaminated water and contribute in food security in upcoming days.

*Phytoremediation of Arsenic Contaminated Water Using Aquatic, Semi-Aquatic and Submerged... DOI: http://dx.doi.org/10.5772/intechopen.98961*
