Transgenics in Phytoremediation of Metals and Metalloids

Transgenics in Phytoremediation of Metals and Metalloids

C H A P T E R 1 Transgenics in Phytoremediation of Metals and Metalloids: From Laboratory to Field Abin Sebastian1, Pawan Shukla2, Ashwini Kumar Nang...

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1 Transgenics in Phytoremediation of Metals and Metalloids: From Laboratory to Field Abin Sebastian1, Pawan Shukla2, Ashwini Kumar Nangia1 and Majeti Narasimha Vara Prasad3 1

School of Chemistry, University of Hyderabad, Hyderabad, Telangana, India, 2Central Sericultural Research and Training Institute, Central Silk Board, Pampore, Jammu and Kashmir, India, 3 Emeritus Professor, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India


plants for the removal of metals from the polluted sites. Phytoremediation is a promising technology because the method is cost-effective compared with other practices used in the remediation of metal-contaminated sites such as landfill, chemical remediation, biopiles, and bioventing (Fig. 1.1). Phytoremediation is also an in situ technology that does not require moving or excavating a large amount of contaminated soil. Successful application of phytoremediation is practiced when a low concentration of metal contamination occurs in sites. This method also helps to decrease secondary pollution occurring during physical or chemical cleanup techniques. Phytoremediation will also contribute to the stabilization of soils, biomass for biofuels, carbon sequestration, and soil fertility (Mahar et al., 2016). Thus the usage of plants for the cleanup

Phytoremediation allows eco-friendly removal of heavy metals from the environment. Mining and industrial operations resulted in the release of heavy metals in the environment (Adiansyah et al., 2015). Arable lands are contaminated with heavy metals during the application of phosphate fertilizers (Sebastian and Prasad, 2014). Most of the heavy metal polluted sites are mine tailings where vegetation is poorly developed because of harsh environmental conditions such as acidity and salinity (Adiansyah et al., 2015). But these sites feature a luxurious growth of certain plant groups known as metal hyperaccumulators. These plants are able to grow in metal polluted sites without showing any toxicity symptoms and point to the usage of

Transgenic Plant Technology for Remediation of Toxic Metals and Metalloids DOI:


© 2019 Elsevier Inc. All rights reserved.



Biotechnology-based Environmental Remediation

Biodiversity as raw material a. Cyanoremediation b. Dendroremediation c. Genoremediation d. Mycoremediation e. Nanoremediation f. Phycoremediation g. Phylloremediation h. Pteridoremediation i. Rhizoremediation

Genetic engineering and transgenic technology Search for genes a. Metal homoeostasis b. Metal chelators c. Metal transporters d. Metal uptake regulators

FIGURE 1.1 Biodiversity as raw material for promoting environmental remediation. (Cyanoremediation, blue green algae based; dendroremediation, usage of trees; genoremediation, genetic engineering based; mycoremediation; use of fungi; nanoremediation, application of nanomaterials; phycoremediation, algae based; phylloremediation; leaf and phyllotaxi dependent; pteridoremediation; usage of petridophyte; rhizoremediation, rhizosphere and PGPMO.)

Metal = metal + metalloid

of metal polluted sites can be considered as a sustainable practice. Plants uptake the heavy metal from the soil and store it in plant organs such as root, leaves, and seeds. Accumulation of metals in the root and shoot showed variation in the plant kingdom, and this difference helped to formulate different phytoremediation strategies such as phytosequestration, phytostabilization, rhizodegradation, phytohydraulics, phytoextraction, phytovolatilization, and phytodegradation (Mahar et al., 2016; Linacre et al., 2003). Phytosequestration exploited the ability of the plant roots to immobilize the metals via accumulation of metals inside the root, precipitation of metal on the root surface, and secretion of metal chelating phytochemicals to the rhizosphere. On the other hand, phytostabilization approaches helped to immobilize metals via plant uptake or precipitation in the root zone. Some plants tend to accumulate more metals in the root, and this feature helped to develop a method namely rhizofiltration where the removal of metal ions from the wastewater is in focus. Rhizodegradation dependent on the activity of bacteria thrives on roots and root exudates. These bacteria help the transformation of metal ions from one form to another that result in immobilization of metal in the soil. Phytohydraulics is a

technique that uses deep-rooted trees such as poplar for the remediation of heavy metals in the groundwater. The deep roots of the plants helps to sequester the metal in the roots or transform the metal from one form to another. Phytoextraction is the method of metal removal using metal hyperaccumulating plants. A hyperaccumulator plant accumulated heavy metal in the shoot, and harvest of the plant helps to remove the metals from the polluted sites. In the course of phytovolatilization, metal enters the plant body via root and is volatilized into the atmosphere from the aerial plant parts. Phytodegradation is the term for biotransformation of metals inside the plant body, and this process detoxifies the deleterious effect arises due to the metal ion. Plants such as Tamarix expel the excess metal accumulated in the plant body through salt glands, and this process enhances metal tolerance important in phytoremediation (Wilson et al., 2016). Plant-based cleanup of metal polluted sites has drawbacks such as relatively long duration required for the cleanup, and the periodic maintenance practices required for ensuring the luxurious growth of the plant in the metalcontaminated sites (Mukhopadhyay and Maiti, 2010). More than 5 years is required for a typical phytoremediation program prior to closure of the sites. This long duration mostly involves



5 FIGURE 1.2 Transgenic approaches applicable to phytoremediation. These targets help to accumulate more metals and produce higher biomass during phytoremediation.

multiple growing seasons and affects the efficacy of phytoremediation. So climate resilient plants are required for phytoremediation. The long duration of the phytoremediation also allows downward movement of the metals, which results in metal pollution in the groundwater (Nowack et al., 2006). The chance of entry of metal pollutant in the food chain is also higher during phytoremediation until the plant material is safely disposed of. Metalcontaminated sites such as mine tailings are characterized by low nutrient content, which retards biomass productivity of metal accumulating plants. Therefore, metal removal with a single crop is often not practicable during phytoremediation. Hyperaccumulator plants are known to accumulate only specific metals. But metal-contaminated sites are often polluted with more than one metal. Therefore, phytoremediation is applicable to sites contaminated with a particular metal. Also, plants that accumulate some of the heavy metals are not yet known. This scenario limited the applicability of phytoremediation for the cleanup of

metal-contaminated sites. Space dependency is another disadvantage of phytoremediation because root growths do not occur after a certain depth. Hence phytoremediation is often limited to the cleanup of surface soil. Also, binding of heavy metals to organic matter in the soil limited plant availability of metals and decreased efficacy of phytoremediation. Drawbacks of phytoremediation can be overcome with transgenic approaches (Fig. 1.2) (Kotrba et al., 2009; Cherian and Oliveira, 2005). The gene-specific approaches help to increase metal uptake capacity of plants and thereby increase metal accumulation in plants. Transgenic approaches for the development of metal accumulator plants require knowledge about genetic loci responsible for metal accumulation, which helps to point out particular genes for developing metal hyperaccumulating plants. Transgenic approaches based on root exudates dependent on extracellular mobilization of metals as well as primary or secondary metabolite dependent intracellular metal speciation are feasible to enhance phytoremediation.




This approach enhances metal mining capacity of the plants and ability to store metal in plants respectively. Environmental stress tolerance is also an important aspect in the field establishment of the plants used for phytoremediation. Ecophysiological processes such as uptake of water and nutrients, photosynthesis, etc. are prone to metal toxicity, and result in loss of plant biomass important for successful phytoremediation (Milner and Kochian, 2008). So transgenic strategies that incorporate ecophysiological adaptations for abiotic stress tolerance are also promising to enhance metal accumulation capacity of plants. Growth requirements of crop plants are well understood, and the value-added products such as alternative fuels, construction materials, etc. derive from agroresidues. Therefore, incorporation of metal accumulating characteristics in the crop plants not only helps effective removal of metals from the sites but also provides economic gains. The following sections describe important aspects in the development of transgenic strategies for improvement of phytoremediation in a sustainable manner.

1.2 LOCALIZING GENETIC LOCI OF METAL ACCUMULATION Plants, being sessile in nature, cannot escape from the contaminated soil and so generally accumulate higher amounts of heavy metals. Several plant species have been identified that are capable of accumulating metals at a significantly high concentration in aboveground tissues. These plants are termed as hyperaccumulators and are used in phytoremediation to remediate the metal-contaminated soil (Rascio and NavariIzzo, 2010). Hyperaccumulation is reported in 450 plant species (Prasad and Freitas, 2003). Elucidation of genetics and the molecular mechanism of metal hyperaccumulation traits can be useful for their exploitation in phytoremediation. The advent of molecular techniques and

bioinformatics tools allows creating of transgenic plants with promising genes of metal hyperaccumulation (Table 1.1). The very common strategies that have been employed for understanding genes and their genetic loci associated in the shaping of metal hyperaccumulation phenotypes could be an analysis of natural variations and segregation, mapping of major genes or quantitative trait loci (QTLs), and analysis of gene expression through microarray and transcriptomics approach. Plant species have shown variation in accumulation of heavy metals. For example, Thlaspi caerulescens J. et C. Presl. accumulates Pb, Zn, Cd, and Ni, Alyssum bertolonii Desv. accumulates Ni and Co, and Arabidopsis halleri constitutively accumulates Zn and Cd, but does not accumulate Pb, while Arabidopsis thaliana is not a hyperaccumulator (Milner and Kochian, 2008; Tumi et al., 2012). Interestingly, all these species belong to Brassicaceae, and natural variations for heavy metals accumulation that exist among these species of the same family provide a good ground for comparative genome analysis as there is huge genomic, transcriptomic, proteomic, and metabolomic information available for A. thaliana. Further, interspecific crosses are usually performed for segregation analysis of metal tolerance and accumulation traits. For instance, segregation analysis of backcross progenies derived from a cross between A. halleri and Arabidopsis lyarata ssp. Patraea displayed tolerance to Cd, and the accumulation of Cd segregates as an independent trait whereas tolerance as well as accumulation of Cd and Zn cosegregate (Craciun et al., 2006). These results indicated that there are two or more genes for Cd and Zn accumulation, but only one for Cd and Zn tolerance. Quantitative trait locus (QTL) analysis is a statistical tool that links two types of information, that is, phenotypic data (trait measurement) and genotypic data (usually molecular marker), to define the genetic basis of variations in complex traits (Collard et al., 2005).




TABLE 1.1 Some Important Bioinformatics Tools Useful for Gene Discovery, Sequence Analysis, and QTL Mapping Web Address of Online Tools


Resources for plant genomics

Phytozome, the Plant Comparative Genomics portal

A. thaliana information resource (TAIR)

Brassica database (BRAD)

BLAST finds regions of similarity between biological sequences

Clustal Omega: multiple sequence alignment of nucleic acid and protein sequences

MUSCLE (Multiple Sequence Alignment Server)

MEGA (Molecular Evolutionary Genetics Analysis) is an integrated tool for conducting automatic and manual sequence alignment, inferring phylogenetic trees, mining web-based databases, estimating rates of molecular evolution

Pfam is a database of protein families that includes their annotations and multiple sequence alignments generated using hidden Markov models

UniProt is a comprehensive, high quality, and freely accessible resource of protein sequence and functional information

SMART (Simple Modular Architecture Research Tool) for protein domains and domain architectures lexington/lexington

CDART (Conserved Domain Architecture Retrieval Tool) performs similarity searches of protein database based on domain architecture

GENSCAN is a program to identify complete gene structures in genomic DNA

TargetP performs prediction of subcellular location predicts the subcellular location of eukaryotic proteins plantcare/html/

Plant care database to determine the promoter

PROSITE scan consists of documentation entries describing protein domains, families, and functional sites as well as associated patterns and profiles to identify them MapQTL software is used for the mapping of quantitative trait loci in experimental populations of diploid species WQTLCart

Windows QTL Cartographer maps quantitative trait loci (QTL) in cross populations from inbred lines

MapChart is software for the graphical presentation of linkage maps and QTLs




QTL analysis allows researchers to link certain complex phenotypes such as heavy metal accumulation traits to a specific region of chromosome with an aim to identify the action, interaction, number, and precise location of these regions. The identification of QTL regions conferring metal hyperaccumulation or tolerance holds great promise for the identification of the genes and gene networks mainly responsible for the phenomenon. The member of Brassicaceae family, A. halleri and A. thaliana, exhibited high synteny. This synteny allowed the mapping of specific genes associated with the QTL or within the QTL regions and made it possible to correlate candidate genes for metal transport to the quantitative loci. For instance, the genetic mapping of AhHMA4, a member of P-type ATPase family transporter involved in the transport of transition metals, which is colocalized with the peak of the QTL involved in both Zn and Cd tolerance (Courbot et al., 2007). The first QTL analysis involved the phenotyping of an F2 population generated from a cross between a high Zn-accumulating T. caerulescens ecotype, isolated in a nonmetalliferous soil at Lellingen, and a relatively lower Znaccumulating ecotype, from a calamine soil in La Calamine (Belgium). The crossing helped to identify two major QTLs for Zn accumulation in the root (Assunca˜o et al., 2006). A QTL mapping performed in A. halleri led to the identification of genes involved in Zn or Cd tolerance. By segregation analysis of one F2 progeny, derived from interspecific crosses between A. halleri and Arabidopsis lyrata, it was evidenced that a single major gene determined Zn tolerance (Macnair et al., 1999). Three major Zn tolerance QTLs were found in the backcross progeny of the interspecific cross A. halleri 3 A. lyrata (Willems et al., 2007). In each one of these QTLs, the allele from A. halleri increased the level of tolerance. As a whole, these QTLs explain 42.0% of the total phenotypic variance for this trait. The QTLs have been located on

chromosomes 3, 4, and 6. In the F2 of a similar interspecific cross, three QTLs for Zn accumulation were mapped on chromosomes 4, 6, and 7 instead (Filatov et al., 2007). Further, QTL analysis in A. halleri 3 A. lyrata backcross population 1 leads to the identification of the metal-pump gene Heavy Metal ATPase 4 (HMA4), Ca21/H1 antiporter, and cation/ hydrogen exchanger 1 (CAX1), as a major genetic determinant for Cd tolerance in A. halleri (Baliardini et al., 2015). QTL analyses were also reported on Cd accumulation in rice to identify Cd tolerance loci. Major QTLs associated with Cd accumulation reported presenting on all 12 chromosomes (Ueno et al., 2009; Xue et al., 2009; Ishikawa et al., 2010; Norton et al., 2010). For instance, three QTLs for shoot Cd accumulation located on chromosomes 2, 5, and 11 using an F2 population derived from Badari Dhan (a high-Cd accumulation accession) and Shwe War (a low-Cd accumulation accession) (Ueno et al., 2009). Of note, 22 QTLs associated with shoot height, root length, shoot dry weight, root dry weight, total dry weight, and chlorophyll content were identified on chromosomes 1, 2, 3, 5, 6, 7, 8, 9, and 10 using Cd stress and control conditions (Xue et al., 2009). Eight QTLs for low Cd content were detected on chromosomes 2, 3, 5, 6, 7, 10, and 12 using the LAC23/Koshihikari chromosome segment substitution lines (Abe et al., 2013). Recently, QTL analysis for two toxicity-linked traits such as leaf rolling (LR) and the green leaf ratio (GLR) in rice seedlings under Cd stress resulted in detection of two QTLs for LR (qLR-1 and qLR-9) and one QTL for GLR (qGLR-3) using 127 rice lines of doubled haploid (DH) population derived from a cross between a japonica JX17 and indicia ZYQ8 (Wang et al., 2018). Genome-wide transcriptomic analysis is a powerful tool for identification of transcripts or gene involved during certain developmental stages and different stresses including heavy metal stress (Chen et al., 2017). Heavy metal



stress triggers an array of genes and several proteins of different signaling pathways (DalCorso et al., 2010). These genes are classified as regulatory genes and functional genes. The genes of the regulatory group encode various transcription factors (TFs), which contain DNA binding elements that interact with cisregulatory elements of the promoter region, which in turn regulate various stressresponsive genes cooperatively and/or separately and thus constitute a gene network. However, the genes of the functional group encode various metabolites and functional proteins, which lead to heavy metal stress tolerance. Single TF can control the expression of many genes, therefore, TFs are considered as a master switch. The induction of basic region leucine zipper (bZIP) and zinc finger TFs was observed during the transcriptome analysis in A. thaliana and B. juncea in response to Cd stress (Ramos et al., 2007). Similarly, transcriptome profiling of A. thaliana exposed to Cd showed the induction of genes related to sulfur assimilation pathways and glutathione metabolism (Herbette et al., 2006; Weber et al., 2006). Proteomics and metabolomics also helped to locate genes responsible for heavy metal accumulation. The major advantage of proteomics over transcriptomics is that it deals with the actually expressed protein rather than transcript, which may not always be translated into functional protein due to posttranscriptional and translational modifications, protein folding, stability, localization, and protein protein interactions (Chandrasekhar et al., 2014). Therefore, proteomics techniques provide a platform for identification of an array of proteins and thereby genes critical for heavy metal detoxification in plants. For example, an increased abundance of RUBISCO large subunit-binding proteins, oxygen-evolving enhancer protein 1, NAD(P)H-dependent oxidoreductase, and photosystem I or II related proteins observed under heavy metal stress (Semane et al., 2010). It was reported that 67%


of all differentially regulated proteins involved in antioxidant defense upregulated during exposure to heavy metals (Sharma and Dietz, 2009). So information about these proteins helps to focus promising genes that help to improve phytoremediation. Metabolomics approaches also help to point out changes in metabolites under metal stress. For example, synthesis of metabolites such as organic acids, α-tocopherol, glutathione, etc. increased during heavy metal stress, and hence genes responsible for the synthesis of these compounds can be targeted for transgenic approaches focusing on enhancement of phytoremediation (Antoniadis et al., 2017).

1.3 ENHANCED METAL SPECIATION WITH TRANSGENICS Mobilization of heavy metals from the soil, plant uptake of metals, and storage of metals in plant tissues depended on chemical speciation of heavy metals (Viehweger, 2014). Therefore, enhancement in plant-mediated speciation of metals helps to improve phytoremediation of heavy metals (Table 1.2). Root exudates mobilize nutrients in the rhizosphere for plant uptake. Root exudation increased during nutrient deficiency as well as metal stress (Antoniadis et al., 2017). Secretion of organic acids from plant roots is a wellstudied plant defense against Al, Fe, and Mn toxicity (Puschenreiter et al., 2003). These organic acids were intermediates of the Krebs cycle such as citrate, malate, and oxalate. Secretion of organic acid occurs through anion transporters in the plasma membrane namely aluminum activated malate efflux transporter (ALMT1) (Yamaguchi et al., 2005). Organic acids secreted into rhizosphere chelate metal ions via carboxylic group, and protect the plants from metal toxicity due to metal ions. So it is clear that overexpression of respiratory cycle enzymes will enhance metal mobilization




TABLE 1.2 Transgenic Approaches With Metal Chelators for Enhanced Metal Tolerance Plant name




Nicotiana tabacum



Pan et al. (1994)

Arabidopsis thaliana



Evans et al. (1992)

Brassica juncea



Zhu et al. (1999)

Nicotiana tabacum



Wang et al. (2010)

Arabidopsis thaliana



Ingle et al. (2005)

Arabidopsis thaliana


Cd, Cu, Ni, Zn

Kim et al. (2005)

Nicotiana tabacum



Kishor et al. (1995)

Cassia tora

Citric acid


Yang et al. (2003)

Populus tremula


Pb, As

Couselo et al. (2010)

Oryza sativa

Deoxymugineic acid


Takahashi et al. (2001)

Oryza sativa



Hong-xia et al. (2008)

Arabidopsis thaliana



Chen et al. (2015)

Arabidopsis thaliana



Li et al. (2004)

Nicotiana tabacum



Singla-Pareek et al. (2006)

Nicotiana tabacum



Ruiz et al. (2011)

efficiency of plants, and improve phytoremediation. Transformation of Nicotiana tabacum and Carica papaya with citrate synthase from Pseudomonas aeruginosa caused fourfold increases in the secretion of citrate and enabled metal tolerance (de la Fuente et al., 1997). Similarly, overexpression of malate dehydrogenase increased secretion of malate from the roots of alfalfa plants resulting metal tolerance (Tesfaye et al., 2003). These results indicate that targeting organic acid secretion pathways will be promising to increase heavy metal tolerance in plants. Plant roots also secrete mugineic acid containing compounds called phytosiderophores in response to Fe deficiency (Meda et al., 2007). Secretion of these acids helped to mobilize Fe from the soil. Phytosiderophores were also found to increase Cd tolerance in maize plants (Meda et al.,

2007). Therefore, transgenic approaches with enzymes involved in the synthesis of phytosiderophores will help to boost phytoremediation. The enzymes of interest could be S-adenosylmethionine synthetase and nicotianamine synthase respectively. The latter enzyme was reported to enhance heavy metal tolerance as well as phytohormone responses in maize plants (Zhou et al., 2013). Plant roots also secrete compounds containing metal binding OH and COOH groups such as secondary metabolites, sugars, amino acids, and mucilage into the rhizosphere (Antoniadis et al., 2017). These compounds play an important role in the perception of soil environmental conditions, nutrient acquisition, heavy metal stress tolerance, root-to-root communication, and microbial colonization. Secretion of phenolics such as coumaric acid



and caffeic acid are well known to mobilize metals in the rhizosphere (Badri and Vivanco, 2009). Enhancing production of phenolics via transgenic strategies for heavy metal stress tolerance not only helps in the speciation of metal ions but also helps to uphold antioxidant activity against heavy metal inducible oxidative stress. Transgenic approaches targeting biosynthetic pathways of these compounds are also feasible because the increase in production of these metabolites will not affect primary metabolism dependent alterations in plant growth. Root exudation affected bacterial colonization in the rhizosphere. The colonization of siderophore-producing bacteria in the course of root exudation is well studied (Rajkumar et al., 2010). These bacteria helped mobilization of the metals in the rhizosphere. Plant growth promoting bacteria was also found to increase biomass productivity critical for successful phytoremediation (Rajkumar et al., 2010). So genetic transformation of plant genes associated with root exudation will have positive effects on the phytoremediation of heavy metals. Heavy metal entering into plant roots undergoes speciation (Hall, 2002). The metal chelators present in the plant form complexes with metal ions, and the metal complex is trafficked into various plant parts for storage. Heavy metal chelators in the plant include organic acids, amino acids, glutathione, phytochelatin, metallothionein, heat shock proteins, phenolics, and amines. The diversity of metal chelators present in the plant tissue offers a vast opportunity for transgenic technology to improve metal tolerance and metal accumulation in plants. Overexpression of malate dehydrogenase, citrate synthase, and phosphoenolpyruvate carboxylase was reported to increase organic acid content in plants (Wang et al., 2010, 2012). Therefore transgenic approaches on organic acid synthesis pathways help to increase heavy metal detoxification mediated by organic acids. Amino acids such as proline and histidine have


been implicated in heavy metal complexation (Hall, 2002). Treatment of Cd and Zn increased proline content in Lemna minor and Lactuca sativa (Bassi and Sharma, 1993; Costa and Morel, 1994). Similarly, histidine content was found to be higher in Ni hyperaccumulating plants such as Alyssum, and the treatment of nickel increased the content of histidine in plants (Kra¨mer et al., 1996). Cysteine is required for the synthesis of glutathione and phytochelatins that chelate heavy metals (Yadav, 2010). Therefore, transgenic approaches focusing on genes involved in the synthesis of histidine, proline, and cysteine are promising to enhance phytoremediation of heavy metals. Glutathione and phytochelatin help to traffic metal from the cytoplasm to the vacuole (Yadav, 2010). These macromolecules bind with heavy metals through SH groups. Brassica juncea overexpressing glutathione synthetase (GS) gene from Escherichia coli had more Cd tolerance compared with wild-type (Liang Zhu et al., 1999). Similarly, overexpression of genes involved in phytochelatin synthesis namely AtPCS conferred Cd resistance in rice plants (Li et al., 2004). Metallothionein, heat shock proteins, and heavy metal binding chaperones were produced as a result of gene expression under metal stress (Hall, 2002). Genetic transformation of Arabidopsis with BjMT2 conferred Cd and Cu tolerance (Zhigang et al., 2006). Expression of metallothionein genes from Prosopis juliflora were also found to increase Cd accumulation and metal tolerance in tobacco plants (Balasundaram et al., 2014). A heat shock protein, namely, HSP70, was found to accumulate in plants cell during metal stress (Hasan et al., 2017). This protein acts as a chaperone that prevents the proteotoxic effect of metal ions as well as metal trafficking machinery assisting sequestration of metal ions. Therefore, HSP70 could be a potential transgenic target for the improvement of metal accumulation capacity in plants.




The cellular level of cinnamic acid derivatives such as epicatechin and rutin was found to increase in Erica andevalensis during Cd stress, and this change resulted in Cd tolerance (Ma´rquez-Garcı´a et al., 2012). Phenolic acids had metal binding OH and COOH groups that decrease the occurrence of unbound metal ions in the cell, hence focusing on transgenic strategies that improve phenolics synthesis in plants is promising to enhance metal tolerance. Amines such as nicotianamine are produced under nutrient deprivation in plants (Takahashi et al., 2003). Nicotianamine is also important for the intercellular trafficking of metal ions in plants. This compound acts in metal accumulation via affecting the production of metal chelators such as mugineic acid. Studies indicated that ectopic expression of nicotianamine synthase gene of Arabidopsis thaliana in tobacco enables Ni tolerance (Douchkov et al., 2005). These plants also had a higher content of Zn and Mn. Thus it can be concluded that transgenic strategies focusing on metal speciation in plants are highly

promising to enhance phytoremediation. It is noteworthy that the vast availability of data on transition metal transporters in the plants is also very useful for enhancing metal accumulation and trafficking in the plants during the course of a transgenic approach that targets metal speciation (Hall and Williams, 2003).

1.4 TRANSGENICS FOR ECOPHYSIOLOGICAL ADAPTATIONS OF METAL ACCUMULATION Heavy metal stress affects ecophysiological processes in plants (Emamverdian et al., 2015). Therefore, the usage of transgenics for upholding ecophysiological functions under metal stress helps to increase the efficacy of phytoremediation. Plant growth under varying soil conditions is a prerequisite in developing transgenic plants for use in phytoremediation (Fig. 1.3). Overexpression of genes involved in the organic acid secretion pathway is well FIGURE 1.3 (A,B) Transgenics for ecophysiological adaptation of phytoremediation. Mine tailings with poorly developed soil diminish plant growth, and therefore it is important to incorporate nutrient mining traits to boost plant growth in metal-contaminated sites such as mine tailings for rehabilitation. (C,D) Crowded plant growth is inevitable in phytoremediation, and hence the incorporation of adaptation such as more perception of light and nutrients with the help of transgenic techniques helps to overcome limitations of phytoremediation such as low biomass productivity.



suited to incorporate adaptations for plant growth under varying soil pH and nutrient status. For example, overexpression of malate dehydrogenase gene, which helps to secrete more malate into the rhizosphere, not only helps to survive plants under low pH but also helps to mobilize nutrients such as Fe in calcareous soils (Xue et al., 2016). Transgenic strategies for genes involved in secretion of coumarins and caffeic acids are also promising for the growth of plants in a calcareous environment contaminated with heavy metals. The incorporation of genes such as deeper rooting 1 (DRO1) helps to develop deeper root systems for growth under water and the nutrient deficit that prevails in mine tailings (Uga et al., 2011). The deeper root system also will have the advantage that it helps to apply phytoremediation for groundwater cleanup. Genes such as heme oxygenase 1 (HO1), phosphorus-starvation tolerance 1 (PSTOL1), ADP-ribosylation factorlike (ARL1), abscisic acid responsive elementsbinding factor 2 (ABF2), transport inhibitor response 1(TIR1), and cytochrome P450 family 2 (CYP2) are highly promising to initiate more lateral roots in plants, and transgenics using these genes are promising to accumulate more metals in the plant root (Sebastian and Prasad, 2015b). Light availability varies in different habitats. Therefore, plants that can survive under the varying light are very important for phytoremediation of metal-contaminated sites. Perception of light in plants is associated with plant pigments and photosystems. An increase in the production of accessory pigments such as chlorophyll b is often found in low light environments (Havaux et al., 1999). Plants also adapted to high light with a decrease in the production of chlorophyll a (Kouˇril et al., 2013). So the genetic control of the light-harvesting process can play an important role in the promotion of plant growth under the varying light. A gene, namely high photosynthetic efficiency1 (HPE1), helped to control the synthesis of chlorophyll a


and b with a hike in Chl a/b ratio (Jin et al., 2016). This change in the light-harvesting pigments decreased antenna size and allowed efficient light capture resulting in enhancement in photosynthetic quantum yield during photosynthesis. So plant transformation with HPE1 helps to increase biomass productivity important for phytoremediation. Transgenics with light sensor proteins such as cryptochrome circadian regulator 2 (CRY2) also allow plants to grow more vigorously during phytoremediation in a crowded and shady environment (Duan et al., 2017). This gene acted independently of phytohormones and hence heavy metal induced fluctuations in plant hormones will not affect bioproductivity of the transgenic line developed with CRY2 gene. Genetic improvement of thermal energy dissipation (qE) is a very important factor that can increase photosynthetic efficiency of plants used in phytoremediation. Transgenics for controlling the synthesis of proteins such as Lhca4 and Lhcb is feasible to control qE (Teramoto et al., 2001). Phytoremediation in high light environment must be focused with transgenic plants having a higher capacity for qE whereas those plants grow in the low light environment must be developed with low qE capacity to avoid resource wastage. Control of leaf orientation is another important aspect in enhancing the photosynthetic efficiency of plants used in phytoremediation. The new plant type (NPT) rice varieties characterized with erect leaves had more perception of light in lower leaves (Taglea et al., 2016). So transforming grass plants such as Vetiver with NPT traits allows more biomass productivity during phytoremediation. Heavy metal stress is well known to cause oxidative stress in plants (Hall, 2002). Exposure to heavy metals results in the formation of reactive oxygen species. Heavy metal inducible reactive oxygen species arise from the activity of plasma membranelocalized NADPH oxidase activity as well as peroxidases and amine oxidases present in the cell wall (Keunen et al., 2011). Alterations in




photosynthetic electron transport also resulted in the formation of reactive oxygen species under heavy metal stress (Sebastian and Prasad, 2015b). Hence transgenic approaches that focus on the enhancement of antioxidant activity will be promising to assure heavy metal tolerance. Among various antioxidants, glutathione (GSH) plays an important role in detoxification of heavy metal stress. The activity of GSH during heavy metal stress includes redox regulation, metal chelation, metal trafficking to the vacuole, and phytochelatin synthesis (Hasanuzzaman et al., 2017). Hence overexpression of GSH will be very much feasible to enhance phytoremediation of heavy metal. Expression of γ-glutamylcysteine synthetase-glutathione synthetase (StGCS-GS) from Streptococcus thermophilus in sugar beets was found to enhance accumulation of Cd, Zn, and Cu and metal tolerance (Liu et al., 2015). Transgenic strategies focusing on increase in cellular GSH for metal tolerance can also be achieved via transformation of the gene encoding glutathione synthetase (GSH2), ATP sulfurylase, cystathionine synthase, serine acetyltransferase (SAT), and glutathione reductase (Hasanuzzaman et al., 2017). Transformation using genes encoding phytochelatin synthase and glyoxalases (glyoxalase I and II) are also promising to boost metal tolerance in plants. The role of GSH based transgenic strategy for the enhancement of phytoremediation revealed during the transformation of Arabidopsis with SAT from Thlaspi goesingense (Na and Salt, 2011). Transgenic Arabidopsis overexpressing SAT had more GSH content as well as higher Ni tolerance. Transgenic plants with genes responsible for the production of antioxidants such as ascorbic acid, anthocyanin, tocopherol, polyamines, etc. are also promising to enhance metal tolerance in plants. Polyamines in plants are depicted as antioxidants. Polyamines such as putrescine, spermidine, and spermine are

report to accumulate during heavy metal stress (Gill and Tuteja, 2010). Also, transgenic eggplants expressing a key enzyme in the polyamine synthesis pathway namely arginine decarboxylase under the control of a constitutive promoter of cauliflower mosaic virus, CaMV35S, had heavy metal tolerance (Prabhavathi and Rajam, 2007). So it is clear that enhancing antioxidant properties in plant cells will be a practical solution to improve phytoremediation of heavy metals. Plant hormones such as abscisic acid, auxin, and ethylene responded to heavy metal accumulation (Bu¨cker-Neto et al., 2017). Water balance was often affected in the course of heavy metal stress, which in turn triggered ABA accumulation in plants (Bu¨cker-Neto et al., 2017). The increase in ABA accumulation also helps to prevent a decrease in water potential of guard cells and prevent water balance related retardation of plant growth. Transgenic Arabidopsis lines overexpressing G subunit of Juglans regia (JrVHAG1) involved in ABA synthesis found to have more Cd tolerance (Xu et al., 2018). Therefore transgenic approaches targeting enhanced ABA accumulation is a promising strategy to increase metal accumulation capacity of plants used in phytoremediation. Transgenic rice plant with the cytochrome P450-like gene (Os08g01480) important for auxin biosynthesis was reported to enhance heavy metal tolerance in Arabidopsis (Rai et al., 2015). Ethylene and auxin had synergistic effects in the regulation of root elongation, root hair formation, and growth in A. thaliana (Negi et al., 2008). Hence, transgenic approaches targeting biosynthesis of these hormones help to promote plant growth during phytoremediation. Apart from plant hormones, transgenic approaches based on components involved in heavy metal signaling pathways such as mitogen activated protein kinase (MAPK) are also promising to enhance phytoremediation of heavy metals.



1.5 TRANSGENIC CROPS IN PHYTOREMEDIATION Genetic manipulation through modern biotechnological tools has gained a lot of popularity for the crop improvement (Gascuel et al., 2017). Introduction or inactivation of specific genes in plants provides a powerful tool to test some hypotheses in plant physiology or gene function in plants that has been difficult to resolve through biochemical methods. In addition to this, its utilization in heavy metal stress tolerance for improvement of important crop plants has an enormous scope. The important requirements for genetic manipulation are an efficient protocol for tissue culture regeneration system, the desired gene construct to be transformed into the plant cell, an effective genetic transformation method for transfer of a gene in a plant, and a screening procedure to select the transformed plant at a satisfactory frequency (Gascuel et al., 2017). In the last few decades, regeneration and transformation protocols have already been standardized in most of the agriculturally important crop plants. The floral dip method has become a routine procedure for in planta transformation in Arabidopsis plants (Bent, 2006). Similarly, in planta transformation has also been tried in mustard, rice, and groundnut (Jan et al., 2016). The advantage of doing in planta transformation is that it does not require a tissue culture regeneration system. Gene construct is an important requirement in plant transformation. Generally, a binary vector has a gene cassette for selection marker and target gene cassette cloned in between the left and right border of the T-DNA (Fig. 1.4). The target gene can be a metal transporter, metal chelator, metal detoxifying gene, etc. that provide heavy metal tolerance. These target genes should be driven by a suitable promoter. A constitutive promoter like CaMV35S promoter is often used, but a tissue-specific promoter can also be tried for expression of genes in a


particular tissue or at specific developmental stages of plants (Pauli et al., 2004). Selection marker gene associated with the gene of interest is an integral part of plant transformation system. Selection marker genes can be antibiotic resistant genes (neomycin phosphotransferase, nptII; aminoglycoside resistance protein, aadA) or herbicide-resistant genes (bialaphos resistance, bar; phosphinothricin N-acetyltransferase, pat) (Goodwin et al., 2005). They provide a selective advantage by conferring resistant to the transformed cells over nontransformed cells, when they are grown on medium with a selective agent such as antibiotics (kanamycin, hygromycin) and herbicide (phosphinothricin). Agrobacterium-mediated plant transformation is a quite popular method for gene transfer in crop plants (Gelvin, 2003). Agrobacterium has the ability to transfer T-DNA having the gene of interest into the host genome. However, there are also other gene transfer methods such as particle bombardment and protoplast fusion (Husaini et al., 2010). Application of transgenic techniques with crop plants is promising for phytoremediation because of well-established growth requirements of these plants. Crop plants often grow in monoculture and the maturity of these plants happens uniformly. This helps to remove the crop in a single harvest. The low nutrient status often seen in the heavy metal polluted sites evokes the need for fertilizer management for successful phytoremediation (Adiansyah et al., 2015). So the well-developed fertilizer application chart of crop plants helps to overcome poor growth of the plants in the metal-contaminated sites. The various byproducts generate from the farms are being converted into value-added products (Srinivasan et al., 2016). Hence the usage of crop plants in phytoremediation allows sustainable phytoremediation. Crop plants such as jatropha, castor, sunflower, and rice are examples of candidate crop plants that can be used for genetic transformation to increase the efficacy




Left border

Poly A terminator

Constitutive promoter

Plant selection marker gene

Constitutive/ tissue-specific promoter

Heavy metal tolerance gene

Poly A terminator

Right border

Gene construct in binary vector plasmid

Introducing gene construct into agrobacterium

Bacteri al se marke lection r gene

Replication origin for agrobacterium

Replication origin for E. Coli

Incubation of explants with agrobacterium suspension Transgenic plants with desired traits

Transfer the elongated shoots to rooting media

Suspension of agrobacterium having gene construct

Transfer the explants to shoot regeneration media with selective agents

Transfer the transformed plantlets into the pots

Regeneration of shoots from explants

FIGURE 1.4 Generation of transgenic crops for phytoremediation. Incorporation of metal accumulating gene in plasmids followed with genetic transformation of plants help to enhance metal accumulation capacity of crop plants.

of phytoremediation. Jatropha curcas is an industrial crop used in biofuel production. This plant was found to survive on degraded land having fragile soils. Jatropha plants had shown a higher tendency to accumulate as well as translocate the metals such as Cd, Cr, Ni, and Zn (Warra and Prasad, 2016).

Transgenic Jatropha lines with vacuolar transporter Na1/H1 Antiporter (NHX1) from Salicornia brachiata had significant Na tolerance (Jha et al., 2013). This approach is highly promising to use Jatropha plants for phytoremediation in coastal areas. The transformation of Jatropha plants with phosphoribosyl



pyrophosphate amidotransferase, nuclear TF Y subunit beta, and glycine sarcosine N-methyltransferase genes found to enhance drought tolerance (Tsuchimoto et al., 2012). Expression of these genes is important for the biosynthesis of CoA, nuclear TF Y subunit beta, and glycine betaine respectively. These compounds are well known for conferring oxidative stress tolerance in plants. Therefore, the transgenic Jatropha lines overexpressing these macromolecules will have the ability to overcome heavy metal-inducible oxidative stress. Castor plants had low growth requirement and produce higher biomass in soil with poor nutrient status. Genetic engineering of castor plants focused on improving disease resistance and oil production. But it was also reported that transgenics with SbNHX1 enhance salinity tolerance in castor plants, which will have beneficial effects on the survival of castor plants under heavy metal stress (Patel et al., 2016). Sunflower is a well-known oil-producing plant and grows well across the globe. The short life cycle of this plant enables efficient metal removal in a relatively short period. Sunflower plants have been applied for phytoextraction of Pb, Cr, Ni, and Zn from polluted soil (Dhiman et al., 2017). Transgenics with yeast metallothionein gene (CUP1) enabled heavy metal tolerance in sunflower plants (Watanabe et al., 2005). The increase of salinity tolerance observed during the transformation of sunflower with ProDH1 gene from Arabidopsis increased proline accumulation, which is a prerequisite for tolerance against heavy metal stress (Tishchenko et al., 2014). Rice plants are well known to accumulate heavy metals from polluted sites (Sebastian and Prasad, 2014). The very short duration required for maturity, and ability to survive under varying oxygen level, made this plant ideal for remediation of submerged lands polluted with heavy metals. The merA transgenic lines of rice showed Hg tolerance (Ruiz and Daniell, 2009). This plant line efficiently converts the toxic form of Hg to a less toxic volatile form.


Agrobacterium-mediated transformation of cadmium tolerance gene (YCFI) in rice was found to increase uptake of more Cd from the soil, and hence was considered as a potential gene for enhancing phytoremediation (Islam and Khalekuzzaman, 2015). Organ localized expression of many of the transition metal transporters reported in rice plants also helps to design transgenic rice plants having more metal accumulation capacity (Sebastian and Prasad, 2015a). The opportunities for transgenic approaches to improve metal accumulation in crop plants are vast; testing for metal tolerance during a transgenic approach exploring stress tolerance or increment in yield are economically feasible to look for enhanced metal accumulation capacity useful for phytoremediation.

1.5.1 Outlook Transgenic approaches are important to enhance metal removal efficiency of plants. The localization of traits associated with the metal accumulation and metal tolerance in the plant genome helps to design plants with enhanced metal removal capacity. Transgenic approaches that exploit metal speciation inside the plants are promising to enhance the metal accumulation capacity of the plants. A focus on more organic acid synthesis and GSH content in the plants is highly feasible for accumulating more metals in the plant body. Transgenic strategies that target secondary metabolite pathways involved in the secretion of phenolics such as coumarin, caffeic acids, etc. increase mobilization of metals from the soil during phytoremediation. Genetic engineering focusing on the incorporation of ecophysiological adaptations helps to establish phytoremediation in a variety of habitats. The development of the transgenic crop plants with enhanced metal accumulation capacity helps large-scale field application of phytoremediation with minimum economic input.




Acknowledgment AS gratefully acknowledges the Dr. DS Kothari postdoctoral fellowship UGC (No. BL/14-15/0162). Thanks are due to Crystalin Research Pvt. Ltd, Technology business incubator and the University of Hyderabad for the technical support.

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