Environment International 74 (2015) 221–230

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Review

Omics and biotechnology of arsenic stress and detoxification in plants:Current updates and prospective

Smita Kumar a,1, Rama Shanker Dubey b, Rudra Deo Tripathi a, Debasis Chakrabarty a, Prabodh Kumar Trivedi a,⁎a CSIR-National Botanical Research Institute (CSIR-NBRI), Rana Pratap Marg, Lucknow 226001, Indiab Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005, India

⁎ Corresponding author.E-mail addresses: prabodht@hotmail.com, prabodht@

1 Present address: Department of Biochemistry, Uni226007, India.

http://dx.doi.org/10.1016/j.envint.2014.10.0190160-4120/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 July 2014Received in revised form 21 October 2014Accepted 24 October 2014Available online 5 November 2014

Keywords:ArsenicMetabolomicProteomicRiceTranscriptomicTransgenic plants

Arsenic (As), a naturally occurring metallic element, is a dreadful health hazard to millions of people across theglobe. Arsenic is present in low amount in the environment and originates from anthropogenic impact andgeogenic sources. The presence of As in groundwater used for irrigation is a worldwide problem as it affectscrop productivity, accumulates to different tissues and contaminates food chain. The consumption of As contam-inatedwater or food products leads to several diseases and even death. Recently, studies have been carried out toexplore the biochemical andmolecularmechanismswhich contribute to As toxicity, accumulation, detoxificationand tolerance acquisition in plants. This information has led to the development of the biotechnological tools fordeveloping plants with modulated As tolerance and detoxification to safeguard cellular and genetic integrity aswell as to minimize food chain contamination. This review aims to provide current updates about the biochem-ical andmolecular networks involved in As uptake by plants and the recent developments in the area of function-al genomics in terms of developing As tolerant and low As accumulating plants.

© 2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2212. Contamination of food chain: arsenic accumulation in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

2.1. Arsenic uptake and accumulation in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2222.2. Hyperaccumulator plants for arsenic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2222.3. Source to sink mobilization of arsenic in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

2.3.1. Inorganic arsenic species uptake and transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2232.3.2. Organic species uptake and transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

3. Omics of arsenic stress response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2243.1. Transcriptome modulation during arsenic exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2243.2. Proteome modulation during arsenic exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2253.3. Arsenic induced metabolic alterations in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2253.4. Arsenic induced changes in antioxidant system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

4. Biotechnological advancements to develop arsenic tolerance in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2255. Biotechnological advances to modulate arsenic accumulation in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2276. Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

nbri.res.in (P.K. Trivedi).versity of Lucknow, Lucknow

1. Introduction

Arsenic is ubiquitous environmental contaminant, present naturallyin rocks, soil, water, air, plants and animals. Studies suggest that morethan two hundred mineral species contain As, of which arsenopyrite is

222 S. Kumar et al. / Environment International 74 (2015) 221–230

the most common (Zhao et al., 2010). About one-third of the atmo-spheric flux of As is of the natural origin which is released intothe environment through activities such as volcanic action, low-temperature volatilization, erosion of rocks and forest fires, orthrough human actions (Tripathi et al., 2007; Neumann et al.,2010). Industrial processes and use of agricultural pesticides andchemicals for timber preservation also contribute to the presenceof As in the environment (Zhao et al., 2010). Arsenic exists in differ-ent oxidation states and forms; of these inorganic forms include thetrivalent As trioxide, As trichloride, As trisulphide, sodium arseniteand pentavalent form including As pentafluoride. Organic form in-cludes arsenobetaine, arsenocholine, tetramethylarsonium salts,arsenosugars and As containing lipids. Of these, inorganic formsare more toxic, of geological origin and present in groundwaterand contaminate drinking water in several parts of the world(Lizama et al., 2011). Organic As compounds mainly arsenobetaineare found in marine organisms although some of these compoundshave also been found in terrestrial species (Newcombe et al., 2010;Zhao et al., 2013). In soil, organic form, arsenobetaine, is rapidlydemethylated to DMA and leads to toxic end product arsenateAs(V) (Huang et al., 2007). Various reports suggest the presenceof methylated forms of As including monomethylarsonic acid (MMA),dimethylarsinic acid (DMA) and trimethylarsine oxide (TMAO) in na-ture (Tripathi et al., 2012) however, capacity of plants to methylate Ashas not been reported as yet.

Arsenic, in addition to adversely affecting plant growth and pro-ductivity, also causes severe human health hazards due to its con-tamination in the food chain (Zhao et al., 2010; Banerjee et al.,2013). In humans, exposure to As has been associated with an in-creased risk of malignant arsenical skin lesions, and carcinomas(Santra et al., 2013). Chronic exposure to inorganic As affects differ-ent biological systems within the body and signs of a typical cuta-neous As exposure have been reported in different organs mainlythe liver (Banerjee et al., 2013). In addition to this, As is knownto modulate multiple pathways including those associated withgrowth factor, suppression of cell cycle check point proteins, inhibi-tion of DNA repair system and DNA methylation (Reichard andPuga, 2010; Sinha et al., 2013). Studies also suggest that As alterssignal transduction pathways, induces ROS synthesis and inhibitsDNA repair machinery which ultimately lead to the tumor develop-ment (Rodriguez-Gabriel and Russell, 2005). Animals have the ca-pacity to biotransform inorganic As to methylated forms such asmethylarsonic acid and dimethylarsenic acid which are less toxicand more readily excreted in urine, however, this process cannotsustain long term exposure (Vahter, 2002). Understanding of bio-transformation in animals and use of this information for the longterm exposure can be one of the effective strategies to combattoxic effects of As to human health.

The deleterious effects of As to the plants and human led to ini-tiate studies to understand mechanisms related to As uptake fromsoil and transport in different plant parts. Studies were startedwith the objective to understand As homeostasis in plants and thebasis for inclusion of As in food chain and related hazards. In addi-tion, a series of events that occur at molecular, metabolic and phys-iological levels need to be explored by utilizing biotechnologicaladvancements and systems biology approach (Tuli et al., 2010). Inrecent past, various studies have been carried out which providedinformation related to biochemical, physiological and molecular re-sponses of the plant under As exposure. In addition, the biotechno-logical advancements have helped to develop transgenic plantswhich can combat As stress and have modulated mobilization ofthe metal in plant parts. Here, we have reviewed and carried outin-depth analysis of established understanding as well as identifiedkey thrust areas where further research is required to develop Asresistant and low As accumulating plant varieties to minimize foodchain contamination.

2. Contamination of food chain: arsenic accumulation in plants

2.1. Arsenic uptake and accumulation in plants

Uptake and accumulation of As in different plant parts affects thegrowth and productivity of the plants (Fig. 1). Arsenic uptake, transloca-tion and biomagnifications in various crop plants and vegetable specieshave increased the threat to humans. Accumulation of As in agriculturalplants depends on two factors: As availability in the soil and the physi-ological properties of the plant (Santra et al., 2013). Arsenic uptake byplants occurs primarily through the root system. After uptake by theroot system, As distribution is highly variable among various plantparts. Generally, roots and tubers are known to accumulate As in largeamount (Peryea, 2001), however, this varies among different plant spe-cies. Toxicity and accumulation of As in few crop plants have been stud-ied to predict and reduce the risk of As entrance into the plants as wellas to determine the effects on plant biomass and yield (Pickering et al.,2000; Takahashi et al., 2004; Zhang et al., 2009). Studies suggest that Asis not readily translocated to the shoots and edible plant parts are gen-erally low in As (b2 mg kg−1). However, some plants accumulate highlevels (5–40 mg As kg−1) of As even at soil concentrations near thebackground level (Gulz et al., 2005). In rice straw, As accumulates upto 149 mg kg−1 which is a major cause of As related health hazards(Tripathi et al., 2012). Recently,Wilson et al. (2014) investigated the ac-cumulation of antimony (Sb) and As in vegetable crops such as lettuce,spinach, raddish, carrot and silverbeet for the oral bioaccessibility ofthese toxic metalloids to humans. Analysis showed that fraction of met-alloids was soluble under typical conditions of crop production and Aswas accumulated in all the glasshouse grown vegetable species. Studieshave demonstrated that the phytotoxicity of As in crop plants is influ-enced by Fe and P which affect the phytoavailability and translocationof As in maize (Rosas-Castor et al., 2014). Among all of the As contami-nated food, seafood is reported to be containing higher As concentra-tions. However, seafood contains only a small proportion of inorganicAs forms. Themajority of As in seafood is present in the formof complexorganic compounds that are generally regarded as less toxic (Borak andHosgood, 2007).

2.2. Hyperaccumulator plants for arsenic

Naturally, several plant species are able to accumulate and detoxifyextraordinarily high levels of heavy metals. In this context, more than450 hyperaccumulator plant species from 45 families have been re-ported (Prasad et al., 2010; Sebastian and Prasad, 2014). Studies sug-gest that few members of the Pteridaceae family show tolerance aswell as hyperaccumulation of As in their fronds (Ma et al., 2001;Zhao et al., 2002). Chinese Brake fern, Pteris vittata is an efficaciousAs hyperaccumulator, and accumulates large amount of As in thefronds. P. vittata is known to tolerate maximum As concentration inthe soil (Zhang et al., 2002). Arsenic tolerance mechanism of P. vittatainvolves As uptake, and detoxification by cellular compartmentalizationinto different tissues including minor veins (Bondada and Ma, 2003).Arsenic speciation analysis of P. vittata grown in an As contaminatedsoil shows that majority of the total As in the above ground biomass ispresent in the form of As(III), which is considered to be the moretoxic form as compared to As(V) (Fayiga et al., 2005).

A number of other fern species including Pitrogram macalomelanos,Pteris criteca, Pteris longifolia and Pteris umbrosa are known to be Ashyperaccumulators (Francesconi et al., 2002; Zhao et al., 2002; Meharg,2003). Interestingly, As response, accumulation and elemental distribu-tion also identified few hyperaccumulator fern species which are sensi-tive to As (Sridokchan et al., 2005). Other plant species such as Silenevulgaris (Schmidt et al., 2004) are also known to accumulate largeamount of As and show As tolerance. Some aquatic macrophytes like,Hydrilla verticillata, Potamogeton pectinatus, Eichhornia crassipes,Egeriadensa, Ceratophyllum demersum, and watercress Lepidium sativum

Soil

RootAs(V) As(V)

As(III) As(III)

As(V) As(III)

Microbes, O 2Redox, pH

ACR

GSSG

GSHPC

PCS

PC-As(III)

MMADMA

(Anaerobic)

(Aerobic)

TMA

TMAMe2AsHVacuole

As(III)

Xylem Loading

As(V) As(III)

As(V) As(III)

Phloem Loading

MMA DMA TMA

GST

As(V)

As(III)

As(III)

Xylem parenchyma

Phloem parenchyma

Companion cells

As(III)

As(III)

Sieve elements

As(III)

As(III)

As(V) As(III)ACR

GSSG

GSH

PC

PCS

PC-As(III)GST

As(III)

GSSG

GSH

PC

PCS

PC-As(III)GSTMMA DMA As(III)

Vacuole

Vacuole

As(III) -thiol

As(III) thiol

As(III) -thiol

As(III) -thiol

As(III) -thiol

Pht1;1, Pht1;4

Aquaglyceroporins

Lsi1/ NIP2;1

Lsi2

ABCC1/2

?

NRAMP1

?

Root

Shoot

Grain

Fig. 1. Diagrammatical representation of different components involved in As uptake, transportation and detoxification in the plants.

223S. Kumar et al. / Environment International 74 (2015) 221–230

also accumulate high amount of As (Xue and Yan, 2011; Mishra et al.,2013). The rootless duckweedWolffia globosa has been known to accu-mulate and tolerate about 400mg kg−1 As because of the absence of theroot to shoot mobilization system (Zhao et al., 2009). It seems thatbetter understanding of the genetic and biochemical mechanismsunderlying response to As exposure and accumulation in identifiedhyperaccumulators may serve as a tool in designing strategies for miti-gation purposes.

2.3. Source to sink mobilization of arsenic in plants

It has been observed that interconversion of As species plays an im-portant role in mobilization from one to another tissue. Analysis of thedifferent species of As suggests that among the inorganic As species,As(III) is predominant (N80%) in the xylem sap of different plant speciesincluding rice, tomato and P. vittata. Interestingly, this predominantspecies was observed in the plants grown under As(V) supplementedmedia (Su et al., 2010). It was suggested that As(V) reduction occursin the root cells before xylem loading and transportation to otherplant parts. However, in contrary to these observations, As(V) was re-ported to be present in larger proportions in comparison to As(III) inthe xylem sap of few other plant species (Pickering et al., 2000).

Most of the detailed studies on the As accumulation have been car-ried out on rice due to its efficient As assimilation characteristics fromthe paddy soil. Rice roots, shoots and seeds accumulate large amountof As compared to other crop plants as anaerobic conditions prevailin paddy soil leading to As(III) mobilization (Takahashi et al., 2004;Williams et al., 2007; Xu et al., 2008; Zhao et al., 2009). The efficient up-take and accumulation of As in rice correlate with physiological proper-ties and silicic acid uptake pathway operating in rice (Wu et al., 2011).

Studies suggest that both, influx and efflux transporters of As(III) inrice roots, mediate As(III) transport from the external medium to thexylem (Ma et al., 2008).

2.3.1. Inorganic arsenic species uptake and transportIt has been demonstrated that high-affinity as well as constitutive

low-affinity uptake systems for As are present in the plants (Mirzaet al., 2014). Translocation of As from the root to the shoot and redistri-bution between different tissues takes place through xylem (Fig. 1).Being an analogue of phosphate, As(V) competes with phosphate forits uptake by the phosphate transporter (Meharg and Macnair, 1992).After uptake, reduction of intracellular As(V) to As(III) takes place byAs reductase, ACR2, (Dhankher et al., 2006; Bleeker et al., 2006).As(III) is detoxified through complex formationwith thiol-rich peptides(Liu et al., 2010) or effluxed out of the cell (Fig. 1). As(III)-thiol-rich pep-tide complex formation and subsequent storage in vacuoles in the rootresults in lower efflux and long-distance transport to other tissues (Liuet al., 2010).

In addition to phytochelatins (PCs), detoxification of part of theAs through complex formation with glutathione (GSH) has alsobeen reported (Bleeker et al., 2006; Tripathi et al., 2007). Detoxifi-cation of GSH–As(III) complex through transport into the vacuolerequires ABC transporter in Arabidopsis thaliana (Bleeker et al.,2006). Arsenic hyperaccumulating Pteris species differs from thenon-hyperaccumulating plants because of increased uptake ofAs(V) (Poynton et al., 2004), limited As(III)–thiol complex formation(Raab et al., 2004), and efficient As(III) transportation into vacuoles byPvACR3 and through xylem (Su et al., 2008; Indriolo et al., 2010).

In addition to detoxification inside the cell, efflux of As(III) by theplant roots plays important detoxification mechanism in number of

224 S. Kumar et al. / Environment International 74 (2015) 221–230

plants (Logoteta et al., 2009) though components involved in this pro-cess have not been characterized in detail. In rice, bi-directional As(III)transporter (Lsi1, an aquaporin) has been shown to be involved in effluxof As(III), however, this process accounts for less than one fifth of thetotal As(III) efflux (Zhao et al., 2010). This observation suggests thepresence of other unknown efflux transporters in rice. Recently, Mosaet al. (2012) demonstrated that members of rice plasma membrane in-trinsic protein (OsPIP) play an important role in As(III) permeability.

Most of the studies suggest the role of thiol-rich peptides in detoxi-fication of As through transporting into vacuoles. Very limited informa-tion is available about long distance transport of As to different tissues. Ithas been suggested that the speciation of As in phloem is prerequisitebecause it determines the distribution of As in different tissues (Yeet al., 2010). Measurement of As species in the fresh rice grain using in-ductively coupled plasma mass spectroscopy and X-ray absorptionnear-edge spectroscopy suggested contribution of xylem and phloemtransport unloading of As species in the grain (Carey et al., 2010). How-ever, molecular mechanism and components responsible for this trans-portation through phloemare not available (Zhao et al., 2010). Recently,functional characterization of OsNRAMP1 suggested its involvement inxylem loading of As (Tiwari et al., 2014).

2.3.2. Organic species uptake and transportStudies suggest that uptake of themethylated As species by the roots

takes place through the aquaporin NIP2;1 (Li et al., 2009) however,withthe slower rate than the inorganic As forms. Mobility of methylated Asspecies is faster from the roots to shoots through xylem transport(Raab et al., 2007; Li et al., 2009). Studies suggest that arbuscularmycor-rhizal fungi participate in phytoremediation of soil rich inAs by differentplant species (Leung et al., 2010). Microbe-mediated As redox changesin the rhizosphere have been shown to influence As uptake in rice (Jiaet al., 2014). However, involvement of microbes in As oxidation andreduction during interaction with the root has not been studied indetail. Li et al. (2013) suggested that different degrees of radial oxygenloss affect arbuscular mycorrhizal fungi colonization and root Asaccumulation.

3. Omics of arsenic stress response

“Omics” studies are primarily aimed at the detection of biologicalcomponents such as genes, mRNA, proteins and metabolites being de-scribed by terms genomics, transcriptomics, proteomics and metabolo-mics respectively. These approaches are means for the deduction ofdifferent pathways which are altered in response to environmentalstresses. Omics investigations are increasingly being used by variousgroups to study the modulation in the transcriptome, proteome or tosome extent metabolome to explore molecular responses in the plantsdue to As stress. In recent years, mechanisms of As uptake, detoxifica-tion and accumulation have been considerably well described in fewunicellular organisms including bacterium and yeast, but limited infor-mation is available for the multicellular organisms (Tripathi et al.,2012). The updated information generated in these areas have notbeen complied together to develop meaningful hypothesis or conclu-sions. In this section, information in these areas has been summarizedto understand As stress response at cellular level and various processes.

3.1. Transcriptome modulation during arsenic exposure

Through genome-wide or selective gene expression analysis variousstudies have concluded modulation in the expression of numerousdefence related genes and transporters such as heat shock proteins,metallothionines, glutathione S-transferases (GSTs), multidrug resis-tance proteins, genes of sulphate metabolizing proteins, sulphate trans-porters, multidrug and toxic compound extrusion transporter (MATE),glutathione conjugated transporters and metal transporter NRAMP1during As stress in different organisms (Abercrombie et al., 2008;

Ahsan et al., 2008; Chakrabarty et al., 2009; Gautam et al., 2012; Jinet al., 2008; Lam et al., 2006; Norton et al., 2008; Paulose et al., 2010;Rai et al., 2011). Transcriptomic and miRNA study in rice seedlingsunder As(III) stress identified several As(III) responsive genes includingtranscription factors (Liu and Zhang, 2012). Studies suggested thatmembers of miR167, miR169, and miR390 miRNA families present inmost of the plants are responsive to As(III) stress. In addition, thestudy identified few miRNA families (miR1861, miR2121, miR810, andmiR827) which have been identified only in rice as yet and are respon-sive to As(III) stress. The expression profiles of As(III)-treated rice alsoidentified genes encoding different transporters, components of phyto-hormone signalling and fatty acid metabolism (Yu et al., 2012). Apartfrom rice, microarray profiling of miRNAs in Brassica juncea identifieda total of 69 miRNAs from 18 miRNA gene families (Srivastava et al.,2013) responsive to As stress. In yeast, global transcriptome anddeletome profiles identified a set of metal responsive genes (Jin et al.,2008). Though these genome-wide studies have identified a numberof transcripts and miRNAs with modulated expression profile, there isa need to validate their involvement in As stress response and detoxifi-cation through functional genomics approaches.

Arsenic stress also modulates metabolic networks which affectmany physiological processes necessary for plant growth and develop-ment. Studies suggest alterations in the expression of a large numberof rice genes related to oxidative stress response without significantchange in the expression of PCS and ACR2 during As exposure (Raiet al., 2011). However, the detailed studyusing different species of As sug-gest modulation in metabolic networks which seems to be As species-specific. Through genome-wide expression analysis Chakrabarty et al.(2009) hypothesized differential expression of genes leading to mod-ulation in different molecular processes in response to As(III) andAs(V) stress in rice seedlings. Based on differential expression andmolecular network analysis, it was concluded that As influences thepathways involved in photosynthesis, plant defence, and signal trans-duction. The analysis also suggested that As(V) affects mainly the cellwall, and primary and secondary metabolism whereas As(III) mainlymodulates hormonal and signalling processes. Interestingly, studiessuggest that changes in expression in genes also vary with germplasmused for the study of response to As stress (Kumar et al., 2013b; Raiet al., 2011). The study utilizing different rice germplasmwith differentsensitivity towards As suggested that sulphate assimilation pathwayand antioxidant defence enzymes play an important role in differentialresponses in different genotypes (Rai et al., 2011; Tripathi et al., 2012).This was also supported by Kumar et al. (2011) with the observation ofdifferential expression in the rice sulphate transporter gene familyunder As stress in contrasting As responsive genotypes. Interestingly,splice variants of twomembers (OsSul1;1 and OsSul4;1)were observedin different rice genotypes under As stress suggesting involvement ofpost-transcription regulation of genes in response to As stress. Thestudy reported alternative splicing of OsSul1;1 during stress responsein different rice cultivars (Kumar et al., 2011). Recently, Sánchez-Bermejo et al. (2014) analysed Arabidopsis natural variation and identi-fied quantitative trait locus (QTL) for As(V) tolerance. Detailed analysisof this QTL suggested that this encodes a novel plant ACR. Identificationof many more such genes using natural variations in plants can help inidentification of other essential components related to As tolerance andaccumulation.

Global gene expression profiling of A. thaliana suggested thatAs(V) stress results in the repression of genes related to phosphatestarvation (Abercrombie et al., 2008). A comparative study of twocontrasting varieties of B. juncea exposed to As stress concluded thatAs detoxification processes operates mainly by modulating signallingcascade through different hormones and sulphate assimilation path-ways (Srivastava et al., 2009). Expression profiling of Crambea byssinicaunder As(V) stress identified genes andmolecular networks involved inAs metabolism and detoxification. Paulose et al. (2010) studied differ-ential expression pattern of transcripts encoding several genes and

225S. Kumar et al. / Environment International 74 (2015) 221–230

networks related to As metabolism and detoxification. The study sug-gested the involvement of GSTs, antioxidants, sulphur metabolism,heat-shock proteins, metal transporters, and several unknown novel pro-teins in response to physiological changes in plants under As(V) stress. Acomparative evaluation of As responsive genes inmonocots anddicots re-vealed differential expression of genes which suggest that though thereare commonmechanisms for Asmetabolism inmonocot and dicot plants,differential mechanisms may also exist (Tripathi et al., 2012).

Most of the transcriptomic studies carried out under As stress suggestmodulation in a number of genes related to defence and transporters andthose involved in plant growth and development. However, a number ofgenes from this list may be responsive to other abiotic stresses as well.Therefore, there is a need to identify genes specific to As stress so that in-formation can be utilized for better understanding of the molecular basisof As stress and detoxification. Recently, to identify common and uniqueresponses by different heavy metals, Dubey et al. (2014) studied bio-chemical and genome-wide modulation in transcriptome of rice rootafter exposure to different heavy metals such as Cd, As(V), Pb andCr(VI) in hydroponic condition. The study suggested that each heavymetal modulates specific pathways in addition to common networks.Expression of specific members of gene families under As(V) stresswas identified which includes mannitol dehydrogenase, IN2-1 pro-tein, sulphite oxidase, transporters (e.g. major facilitator superfamilyantiporter), different members of cytochrome P450 and transcrip-tion factors.

3.2. Proteome modulation during arsenic exposure

Though several studies have been performed on the physiologicaland biochemical responses of As stressed plants but not much informa-tion is available for As induced changes in proteome. Initial studies byRequejo and Tena (2005, 2006) on As exposed maize root and leaf sug-gested differential regulation of proteome on the basis of detectableproteins. In this study, the identified proteins were reported to havean important role in cellular homeostasis for redox perturbation. Fur-ther, a comparative proteomic analyses of rice roots suggested putativemechanisms involved in detoxification of As stress (Ahsan et al., 2008).Out of detectable proteins, study identified 23 differentially accumulat-ed proteins in the rice roots. On the basis of differential level of proteins,the role of GSH in protecting the plants against As stress due to synchro-nous function of S-adenosylmethionine synthetase, GSTs, cysteine syn-thase and glutathione reductase was suggested. In extension to thisstudy, Ahsan et al. (2010) also reported the first proteome map of riceleaves in response to As stress and concluded the downregulation ofchloroplast proteins and correlated this modulation with decreasedphotosynthetic efficiency.

Recently, a comparative proteomic analysis of rice shoots exposed toAs(V) was carried out which showed differential expression of 38 pro-teins mainly involved in protein metabolism and redox homeostasis. Ithas been suggested that these proteins might help in providing toler-ance towards As toxicity in rice (Liu et al., 2013). In Agrostis tenuis leaf,differential expression of enzymes of Calvin and Krebs cycle has beenreported during As stress (Duquesnoy et al., 2009). The role of glycolyticenzymes in Asmetabolismhas also been suggested due to enhanced ac-tivities of glyceraldehyde-3-phosphate dehydrogenase, phosphoglycer-ate kinase and enolase in fronds of P. vittata plants (Bona et al., 2010).However, proteins identified in above studies are known to protectplants from various environmental stresses including other heavymetals. Therefore, there is a need to carryout a detailed study takingother environmental stresses into consideration to identify proteinsspecifically involved in As stress and detoxification.

3.3. Arsenic induced metabolic alterations in plants

Many important morphological, physiological and developmentalplant processes are known to be affected by heavy metal toxicity

(Dubey et al., 2010). The study conducted by Mishra and Dubey (2006)reported that As causes alterations in the level of various molecules andmodulates the activities of RNase, protease and leucine aminopeptidasein rice seedlings. To understand the coordination between metabolicpathways of thiols and amino acids, As tolerant and sensitive cultivarswere used. Studies suggested a requirement of thiol containing ligandsand stress-responsive amino acids to achieve the effective complexationand detoxification of As by PCs (Dwivedi et al., 2010a, 2012; Tripathiet al., 2012). In the As treated rice seedlings, enhanced level of solubleproteins was observed which may be due to the decreased proteoly-sis and the synthesis of As induced proteins. Recently, Gupta andAhmad (2014) studied the response of As(V) in two rice genotypes(PB1 and IR-64) and concluded that PB1 variety is capable to detoxifyAs(V) through induction of antioxidant defence system and otherstress related parameters (cysteine, proline content) in comparisonto IR-64. However, the response of PB1 variety towards other environ-mental stresses will provide additional information regarding whetherobserved higher detoxification capacity is unique to As exposure orthis variety is in general stress tolerant. In plants, As exposure leads tochanges in the levels of starch and sugars and modulates activity ofkey enzymes of related metabolic pathways (Choudhury et al., 2010).Soluble sugars, especially sucrose and hexoses are the end products ofthe photosynthesis and their levels in plants are related to plant produc-tivity (Mishra and Dubey, 2013). Expression of growth and stress relat-ed genes in plants is regulated by sugars and plants that respond to thedifferent environmental stresses through sugar sensing mechanisms.The toxic level of As in the soil causes marked perturbations in the car-bohydrate metabolism in growing plants that may lead to the accumu-lation of soluble sugars in the tissues. Yu et al. (2012) carried out acomparative transcriptome analysis and suggested modulation in thelipid metabolism and phytohormone signalling under As(III) stress.The cumulative information related to the metabolic alterations inplants under As stress is not appropriate and needs detailed explorationusing systems biology tools.

3.4. Arsenic induced changes in antioxidant system

Several studies in different plants have suggested modulation inamino acids, mineral nutrient status and antioxidant system of plantunder As stress (Dwivedi et al., 2010a, 2010b; Tripathi et al., 2012).However, differential modulation of antioxidant system has beenreported for different species of As as well as to other heavy metals. InHydrilla verticillata, As(V) stress enhanced the antioxidant systemwhilst As(III) stress increased production of PCs suggesting differentialresponse to different As species (Srivastava et al., 2007). Similarly, inC. demersum As(V) activated antioxidant system and enhanced PCproduction suggesting that specific proteins are responsive to As stress(Mishra et al., 2008). The enhanced activities of isozymes of superoxidedismutase, ascorbate peroxidase, peroxidase, and glutathione reductaseindicated that As stress generates oxidative stress (Shri et al., 2009). Inanother study, it was suggested that thiolic metabolism, especially in-duction of PCs, plays important role in As response in different cultivarswith contrasting accumulation of As in grain (Dave et al., 2013a). In ad-dition to As response, As accumulation also showed a direct correlationwith antioxidant system. In the presence of As(V), various antioxidantenzymes guiacol peroxidase, ascorbate peroxidase and superoxide dis-mutase were stimulated in high As accumulating cultivars in compari-son to low As accumulating cultivars (Dave et al., 2013b). However,modulations in antioxidant system are not specific to As stress as suchchanges have also been reported for the other heavy metals.

4. Biotechnological advancements to develop arsenic tolerancein plants

Arsenic in different forms iswidespread innature and affects croppro-ductivity. In recent past, efforts have been made to develop transgenic

226 S. Kumar et al. / Environment International 74 (2015) 221–230

plants through biotechnological approaches to combat As stress to in-crease productivity in As rich soil. The genes identified as componentsof As uptake and detoxification mechanism have been important targetsto engineer plants for the survival in the presence of As (Table 1). Themain targets being used are part of the mechanism involved in inhibitionof uptake of As by the roots (Duan et al., 2012), chelation by cysteine-richthiol-reactive peptides and intracellular compartmentalization (Duanet al., 2012; Shukla et al., 2012), efflux from the roots (Xu et al., 2007)as well as biotransformation to methylated As forms (Zhao et al., 2010).

Overexpression of PC synthase genes contributed to As tolerance inplants (Li et al., 2004; Shukla et al., 2012, 2013a;Wojas et al., 2010). Ex-pression of the A. thaliana PCS gene in Indian mustard increased toler-ance to As and Cd (Gasic and Korban, 2007). Tolerance through PCsrequires efficient transport of the PC–metal complex in vacuole. Forthis, vacuolar PC-transporters are essential for the sequestration ofmet-alloid into the vacuole. Song et al. (2010) identified two ABC trans-porters in A. thaliana, essential for the sequestration of As into thevacuoles. Arabidopsis double knockout mutants (atabcc-1 and atabcc-2) showed hypersensitivity to As and As-based herbicides. Recently,Shukla et al. (2013b) proposed that the use of synthetic phytochelatins(ECs) as potential candidates for increasing themetal-accumulation ca-pacity of plants. Using three synthetic genes of different lengths the

Table 1Transgenic plants developed for modulated As tolerance and accumulation.

S. No. Source Target Gene name

1. E. coli A. thaliana Arsenate reductase (ArsC)γ-Glutamyl cysteine synthetase

2. A. thaliana A. thaliana Phytochelatin synthase (PCS1)3. B. juncea B. juncea ATP sulphurylase4. E. coli A. thaliana γ-Glutamyl cysteine synthetase5. B. juncea B. juncea γ-Glutamyl cysteine synthetase

Glutathione synthetase (GS)6. A. thaliana B. juncea Phytochelatin synthase (PCS1)7. Garlic and baker's yeast A. thaliana Phytochelatin synthase (AsPCS1

Glutathione (GSH1)8. E. coli B. juncea γ-Glutamyl cysteine synthetase

Glutathione synthetase9. A. thaliana N. tabacum Metallothionein

(MT2b)

10. P. vittata A. thaliana Glutaredoxin (GRX5)

11. S. cerevisiae S. cerevisiae Aquaglyceroporin (FPS1;Farnasyldiphosphate synthase 1transporter (HXT7)

12. A. thaliana S. cerevisiae ABCC transporters13. A. thaliana N. tabacum Phytochelatin synthase (PCS1)

14. C. elegans N. tabacum Phytochelatin synthase (PCS1)

15. E. coli Eastern Cottonwood γ-Glutamyl cysteine synthetase

16. O. sativa O. sativa As(III)-S-adenosyl methyl trans(arsM)

17. O. sativa O. sativa Phosphate transporter (Pht1;8)Phosphate Starvation Response

18. Yeast A. thaliana Arsenate reductase (ACR3)

19. S. cerevisiae O. sativa Arsenate reductase (ACR3)

20. S. pombe A. thaliana Phytochelatin (PC)-Cd transpor(SpHMT1)

21. C. demersum N. tabacum Phytochelatin synthase (PCS1)22. C. demersum A. thaliana Phytochelatin synthase (PCS1)23. O. sativa X. laevis Plasma membrane intrinsic pro

(OsPIP2;4, OsPIP2;6 and OsPIP224. P. vittata A. thaliana Arsenate reductase (ACR3)25. O. sativa A. thaliana Glutathione S-transferase (GSTL26. O. sativa A. thaliana Natural Resistance-Associated

Macrophage Protein (OsNRAMP

study demonstrated that the expression of EC genes can rescue the sen-sitive phenotype of the PC-deficient Arabidopsismutant under As stress.

Not only PCS, but also other genes involved in PC biosynthesis havebeen shown to provide tolerance to heavy metals including As. Li et al.(2005, 2006) developed Arabidopsis transgenic plants expressing bacte-rial gamma-ECS gene. Transgenic lines, in response to As, Hg and Cdshowed significantly higher accumulation of the gamma-EC and GSHand PCs in transgenic plants in comparison to wild-type plants. Com-pared to other heavy metals, As treatment significantly enhanced thelevels of these molecules. Analysis showed higher tolerance in transgeniclines towards As andHg stress and sensitivity towards Cd stress. In anoth-er study, genetically engineered eastern cottonwood with a bacterial ECSgene showed elevated thiols and ECS activity (Le Blanc et al., 2011). ECS-expressing transgenic plants showed tolerance towards As(V) stress inwild type plants. Furthermore, the roots of ECS-expressing plants accu-mulated significantly more As than control roots whilst shoots accumu-lated significantly less As than wild type shoots.

Our study related to expression of rice Lambda class GST subfamilysuggested differential expression of this gene family in As sensitiveand tolerant genotypes under As stress (Kumar et al., 2013a, 2013b).To studywhethermembers of this gene family can provide As toleranceto the plants, one member, OsGSTL2, was expressed in heterologous

Consequences References

(γ-ECS)Increased tolerance and As accumulation Dhankher et al. (2002)

Increased tolerance and As accumulation Li et al. (2004)Increased tolerance and As accumulation Wangeline et al. (2004)

(γ-ECS) Increased As tolerance Li et al. (2005)(γ-ECS) Higher As accumulation Navaza et al. (2006)

Significantly higher tolerance to As Gasic and Korban (2007)); Increased tolerance and As accumulation Guo et al. (2008)

(γ-ECS) Increased tolerance and As accumulation Reisinger et al. (2008)

Significantly decreased As accumulationin roots and increased accumulation inshoots.Increased root to shoot transport of As.

Grispen et al. (2009)

Increased As tolerance. Decreased Asaccumulation in leaves

Sundaram et al. (2009)

) hexoseIncreased As accumulation Shah et al. (2010)

Enhanced tolerance and As accumulation Song et al. (2010)Increased tolerance and As(V)accumulation

Wojas et al. (2010)

Increased tolerance and As(V)accumulation

Wojas et al. (2010)

(γ-ECS) Increased tolerance and accumulationof As

Le Blanc et al. (2011)

ferase Decreased As in rice grain. Meng et al. (2011)

2 (PHR2)Increased phosphate and As(V) uptakeand translocation

Wu et al. (2011)

Increased As tolerance and greatercapacity for As(V) efflux.

Ali et al. (2012)

Increased As(III) efflux.Decreased As accumulation in rice grain.

Duan et al. (2012)

ter Enhanced tolerance and As accumulation Huang et al. (2012)

Increased As accumulation Shukla et al. (2012)Enhanced As accumulation Shukla et al. (2013a, 2013b)

teins;7)

Enhanced As(III) tolerance and higherbiomass accumulation

Mosa et al. (2012)

Increased tolerance to As Chen et al. (2013)2) Increased tolerance to As Kumar et al. (2013b)

1)Increased tolerance and As accumulation Tiwari et al. (2014)

227S. Kumar et al. / Environment International 74 (2015) 221–230

system A. thaliana. The analysis suggested that expression of OsGSTL2provides tolerance towards different abiotic stresses including As andother heavy metals. Our study suggested that OsGSTL2 transgeniclines were resilient towards As overdose/challenge (Kumar et al.,2013b). In a study glutaredoxin gene family has been shown to partici-pate in As tolerance. Transgenic Arabidopsis lines expressing brake fernglutaredoxin, PvGRX5, showed increased tolerance towards As in com-parison to control lines based on various developmental parametersunder As stress (Sundaram et al., 2009). The study concluded the roleof PvGRX5 in As tolerance via improvingAs(V) reduction and regulatingcellular As levels.

Not only in plants but also in microbes, As detoxification mecha-nism has been explored. Studies suggest that the most effectivestrategy of As detoxification in microbes is As(III) efflux from thecells. In E. coli, ArsB ATPase, an antiporter of As(III) confers resistanceto As(III) (Rosen, 2002). Saccharomyces cerevisiae As resistancetransporters including Acr3p and Ycf1p mediate As(III) efflux andprovide resistance towards arsenicals (Maciaszczyk-Dziubinskaet al., 2011). As(V) detoxification involves reduction of As(V) toAs(III), a process catalyzed by As(V) reductase enzyme (Meng et al.,2004; Rosen, 2002; Wysocki and Tamas, 2011). Expression of ScACR3gene in the yeast increased As(III) tolerance linked with decreased Asaccumulation (Ghosh et al., 1999). Similar genes were isolated fromhyperaccumulator fern P. vittata, PvACR3 and PvACR3;1 (Indriolo et al.,2010). Heterologous expression of yeast As(III) efflux transporters ACR3in Arabidopsis conferred resistance to As(III) and As(V) and a greater ca-pacity for As(V) efflux. The presence of ACR3 increased As translocationto the shoot without affecting tissue As levels (Ali et al., 2012). In a recentstudy, PvACR3, was expressed in Arabidopsis (Chen et al., 2013). Trans-genic plants showed increased tolerance towards As stress. Transgenicseeds germinated and grew at concentrations of As species whichwas le-thal to non-transgenic seeds.

A recent study suggests that heterologous expression of membersof rice plasma membrane intrinsic protein (PIP) subfamily genes inXenopus laevis oocytes enhanced the uptake of As(III) (Mosa et al.,2012). Heterologous expression of these OsPIPs in Arabidopsis increasedtolerance towards As(III) in addition to higher biomass accumulation.The study concluded bidirectional As(III) permeability of OsPIPs andsuggested to use these genes in developing As tolerant crop plantswith enhanced productivity without increased accumulation of toxicAs. In a recent study, involvement of plasmamembrane localised trans-porter, OsNRAMP1, has been shown to provide tolerance towards Asstress though with higher As accumulation (Tiwari et al., 2014). It wasdemonstrated that this transporter assists in xylem loading for theroot to shoot mobilization of As(III).

5. Biotechnological advances to modulate arsenic accumulationin plants

The understanding developed through different studies have helpedin employing strategies for developing As resistant plants as well as todevelop low and high As accumulating varieties (Table 1). Coexpressionof two bacterial genes, As(V) reductase (arsC) and γ-glutamyl cysteinesynthetase in Arabidopsis showed enhanced As accumulation in freshshoot in the transgenic lines as compared to non-transgenic plants.Such a strategy to develop As tolerant plants with enhanced accumula-tion of As through gene pyramiding of ArsC and γ-ECS can be useful forAs remediation (Dhankher et al., 2002). Recently, phytochelatin syn-thase gene from C. demersum (CdPCS1) was expressed in transgenictobacco (Shukla et al., 2012) and Arabidopsis (Shukla et al., 2013a).Transgenic lines harbouring CdPCS1 showed enhanced PC contentwith significantly increased heavy metal accumulation without affect-ing plant growth.

Grispen et al. (2009) expressed Arabidopsis metallothionein gene,AtMT2b, inNicotiana tabacum and observed that transgenic lines exhibita significant decreased accumulation of As in the roots, but an enhanced

accumulation in shoots without any change in total amount of As takenup by the plant. Overexpression of genes encoding for gamma-glutamylcysteine synthetase and glutathione synthetase in B. juncea showedhigher accumulation of As and Cd (Navaza et al., 2006). Huang et al.(2012) reported that phytochelatin (PC)-Cd transporter, SpHMT1, offission yeast (Schizosaccharomyces pombe) in Arabidopsis enhances tol-erance and accumulation of different heavy metals including As. Trans-genic lines developed by overexpressing AsPCS1 and GSH1 (derivedfrom garlic and baker's yeast) in Arabidopsis showed increasedtolerance and accumulation of Cd and As (Guo et al., 2008). SinceAs(V) and As(III) utilize the same transport system of essential or ben-eficial elements, these transporters cannot be blocked. However, identi-fication of variants of these transporters can be more discriminativeagainst As.

Though studies mentioned above have developed transgenic plantswith low As accumulation, there is an urgent need to develop “As-free” rice to minimize health hazards due to contamination of foodchain. There is a need to understand various processes in detail whichoperate in rice and involved in As uptake and accumulation. All acrossthe globe, researchers are trying to develop As-free rice but no suchbreakthrough has been achieved from any corner because this is a mul-tistep and multigene phenomenon. However in recent past, few suc-cesses have been achieved in minimizing As content in rice grain.Duan et al. (2012) demonstrated that expressing S. cerevisiae ACR3gene in rice increased As(III) efflux and also lowered As accumulationin rice grains. The transgenic lines showed 30% lower As concentrationin the root and shoot as compared to wild type without change in theAs translocation factor between transgenic and wild type plants. Over-expression of bacterial As(III)-S-adenosyl methyl transferase (arsM) inrice was found to methylate As leading to reduced toxicity. Trans-genic lines expressing arsM produced 10 fold higher volatile arseni-cal, maintaining low As levels in rice seed along with organic Asspecies MMA(V) and DMA(V) in the root and shoot of transgenic rice(Meng et al., 2011). Very recently, heterologous expression of CdPCS1in rice significantly decreased As accumulation in grain (Shri et al.,2014). This study demonstrated that CdPCS1 expression in rice en-hanced accumulation of As in the roots and decreased As accumulationin aerial parts including rice grain. This strategy might help in develop-ing low As accumulating rice varieties in the near future.

6. Conclusions and outlook

Arsenic toxicity poses a serious threat to the environment. Plantsexposed to elevated or toxic concentrations of As exhibit considerablyreduced growth, productivity and yield. Arsenic toxicity causes discrep-ancies in the plantmetabolism affecting various physiological pathwaysinvolved in the photosynthesis, water/nutrient balance and oxidativestress. In humans, As causes acute and chronic health issues. In thelast few years, studies have generated preliminary and scattered infor-mation associated with molecular mechanism of As uptake and accu-mulation in plants. There was a need to compile established resultsand their in depth analyses to develop correlation that may be helpfulin understanding the mechanisms and networks involved in As uptakeand transport to some extent. The detailed analysis of the existingknowledge suggests a molecular network comparing enzymes relatedto transport, detoxification, antioxidant system regulated by transcrip-tion factors and small RNA that plays important role in stress responseand detoxification of As (Fig. 2). Analysis also suggests that thesemolec-ular responses are species- and genotype-dependent and a commonhy-pothesis cannot be developed to provide general response of As to theplants.More in depth experiments using different toolsmay be requiredto develop a common hypothesis.

In order to clean up the ecosystem from As toxicity, a few variousbiotechnological interventions have been carried out in recent past.Modulation in the expression of numerous defence related genes andtransporters have helped in developing As resistant plants which can

AsmiRNA

Transcription Factors

Transporters

GSH Metabolism

Sulphur AssimilationDetoxification Mechanism

Hormone

Redox Homeostasis

Antioxidant System

Fig. 2. Involvement of variousmolecular components in As stress and detoxification. Dashed arrows represent direct effect of As on differentmolecules and processes. Bidirectional arrowsrepresent networking between different molecular entities and processes which play a role during As stress and detoxification.

228 S. Kumar et al. / Environment International 74 (2015) 221–230

grow at elevated As levels in soil, have developed low As varieties andalso have maximized a plant's As removal potential (Table 1). In mostof the cases, gene targeting mechanisms involved in reduced As uptake(Duan et al., 2012), chelation by cysteine-rich thiol-reactive peptidesand intracellular compartmentalization (Duan et al., 2012), effluxfrom the roots (Xu et al., 2007) and biotransformation into organic Asspecies (Zhao et al., 2009) have beenused. However, it has been noticed,in few cases, that expected results are not obtained due to the use of im-portant cellular components (GSH) in synthesis of metal binding pep-tides (PCs) which leads to sensitivity to plants during As stress (Wojaset al., 2010). Recently, it has been proposed that the use of syntheticPCswhich do not require GSH for the synthesis may be utilized as an al-ternative approach for phytoremediation of heavy metal(loid)s (Shuklaet al., 2012).

Importantly, manipulation in the level of expression of genes re-sponsible in As transport from the root to shoot and distribution ofAs in the grain needs to be studied in detail. Molecular level ap-proaches combining genomics and proteomics would make it possi-ble to target specific transporters of interest in the roots, which arethe organs in contact with potential harmful As. The developmentof transgenic plants with efficient xylem loading of As by trans-porters can be an important strategy to develop resistant but highAs accumulating plants. Recently such a transporter has been identi-fied and been demonstrated to help in xylem loading of As (Tiwariet al., 2014).

Studies related to differential gene expression analysis identifiednumerous differentially expressed genes, not much information isavailable about the regulatory components comprising transcriptionfactors and miRNA which can participate in As stress response inplants. In addition, studying genetic variation in As response and ac-cumulation can provide major insights into the potential of plant ad-aptation to divergent environmental contamination. Exploitingnatural variation to uncover candidate genes that are As responsiveand regulate As accumulation will not only help in gene identificationbut may also lead to evolve better technologies for developing As-freecrops through biotechnological tools andmay serve in as a tool for effec-tive bioremediation processes. Another beneficial target could be to en-hance As methylation i.e., conversion of the inorganic As form to themethylated As species, or to volatile forms of As. Identification of sucha gene from plant source and use for the biotransformation of toxic Asspecies will be an important area for phytoremediation as well as forhuman health.

Acknowledgements

Financial support from the Council of Scientific and IndustrialResearch (CSIR), New Delhi, as Network Project (BSC-0107) andDepartment of Biotechnology, Government of India, New Delhi(BT/PR13446/AGR/36/665/2009) is acknowledged. SK thankfully ac-knowledges the Department of Science and Technology (DST),Government of India, New Delhi for DST-INSPIRE Faculty Award.

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