Emerging Trends in Transgenic Technology for Phytoremediation of Toxic Metals and Metalloids

C H A P T E R

3 Emerging Trends in Transgenic Technology for Phytoremediation of Toxic Metals and Metalloids Israr Ahmed1, Abin Sebastain2, Majeti Narasimha Vara Prasad3 and Pulugurtha Bharadwaja Kirti1 1

Department of Plant Sciences, University of Hyderabad, Hyderabad, Telangana, India, 2School of Chemistry, University of Hyderabad, Hyderabad, Telangana, India, 3Emeritus Professor, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India

3.1 INTRODUCTION

arsenic (As) and selenium (Se) have important roles in plant cell biology when present in the cells in an appropriate concentration. Most of these heavy metals have roles in the important enzyme activities that control several metabolic functions in the plant that include electron transport, photosynthesis, respiration, and cellular lignification. Some of the transitional metals like molybdenum and nickel are also important to the plant cells as essential micronutrients. However, they would be highly toxic beyond some threshold limits and start getting accumulated in plants posing a major threat to the natural ecosystem and human health (Hall et al., 2003). Beyond the threshold levels, they exert oxidative stress on the plant cells by producing reactive oxygen species (ROS) thereby hampering cellular metabolism, and damaging the biomolecules and membranes (Schu¨tzendu¨bel and Polle, 2002; Shahid et al., 2014). This heavy

Rapid industrialization coupled with anthropogenic activities like mining, smelting, and application of various chemical fertilizers has led to excessive toxicity in the environment with the release of toxic materials including heavy metals into soil, water, and the atmosphere. While organic contaminants can be relatively easily detoxified, these toxic metals cannot be mineralized by biodegradation, which eventually leads to their accumulation in soil and water bodies over the course of time. Plants have developed an elaborate mechanism to sustain and protect themselves in an environment that is rich in heavy metals by intake, transport across cellular membranes, and accumulation in an appropriate location in the cell. Most of the heavy metals, like cadmium (Cd), lead (Pb), mercury (Hg), and metalloids Transgenic Plant Technology for Remediation of Toxic Metals and Metalloids DOI: https://doi.org/10.1016/B978-0-12-814389-6.00003-1

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© 2019 Elsevier Inc. All rights reserved.

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3. EMERGING TRENDS IN TRANSGENIC TECHNOLOGY FOR PHYTOREMEDIATION OF TOXIC METALS AND METALLOIDS

metal induced ROS generation may not be due to their direct involvement through Haber Weiss/Fenton reactions, but through indirect routes (Shahid et al., 2014). Plants have the capacity to adapt to environmental changes by readjusting their internal cellular machinery to cope up with the stress, which might even be caused by enhanced heavy metal concentration in the microclimate of the plant. This adjustment even can sometimes be modulated by the plant associated mycorrhizal organisms (Gonzale´z-Guerrero et al., 2016). The symptoms of heavy metal accumulation are manifold and may include impairment in cellular metabolism, stunted growth, and reduction in chlorophyll content (Viehweger, 2015). Traditionally, several physical and chemical methods such as excavation, transport, soil washing, and addition of chemical metal chelators are being used to remediate the soils that were contaminated with toxic heavy metals. However, the cost of soil remediation by these methods is exorbitant and labor intensive, and might cause secondary pollution (Ali et al., 2013; Luo et al., 2016). An alternative and sustainable method to clean up and restore the contaminated soils by deploying suitable resurrection plants that are powered by solar energy is popularly known as phytoremediation. The scientific concept of phytoremediation emerged in the early 1980s after it was identified that some plants exhibit the capacity to accumulate toxic metals after “extracting” them from the soils in which they were grown. In addition to the natural environmental cleanup process, phytoremediation provides additional byproducts such as wood, pulp, and bioenergy. It also provides shelter for animals and birds, and makes available nutrients to the rhizosphere microbes involved in phytoremediation (Doty, 2008). Plants in association with their rhizosphere microorganisms restore the metal contaminated soils by different mechanisms such as (1) phytoextraction, or

removal of the metal by its uptake by plants and translocation to their aerial parts from which they can be easily harvested; (2) phytostablization, or restricting the mobility of the toxic metal by binding it in the rhizosphere of the resurrection plants; (3) phytovolatilization, or conversion of the toxic heavy metals into less toxic volatile compounds within the plant and release into the atmosphere; and (4) rhizofiltration, or adsorption and precipitation of the toxic metals by plant roots in hydroponic conditions.

3.2 CHARACTERISTIC FEATURES OF PLANTS FOR PHYTOREMEDIATION Plants obtain nutrients including mineral nutrients from the rhizosphere, and they will accumulate excess of what is needed for their biological processes, if they are grown in soil with high levels of metals. Depending on the species, this can be detrimental to growth or might become lethal, which can greatly limit the growth and productivity of plants (Grennan, 2009). A plant species suitable for phytoremediation should have several desirable features such as high capacity uptake and translocation of toxic metals to the aerial parts of the plant, it should be a hyperaccumulator of toxic metals, it should be fast growing with high biomass production, and it should have an extensive root architecture with versatile capability of wide climatic adaption (Ali et al., 2013; Bell et al., 2014). Hyperaccumulators are plants that possess the capacity to accumulate extremely large quantities of heavy metals in their aerial parts without exhibiting any symptoms of physiological stress that are associated with heavy metal accumulation. There are nearly 500 listed species of potential hyperaccumulators, representing approximately 0.2% of all

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3.2 CHARACTERISTIC FEATURES OF PLANTS FOR PHYTOREMEDIATION

angiosperms, that have been identified to date (Kra¨mer, 2010). Although these hyperaccumulators can accumulate large quantities of toxic heavy metals, they are often not suitable for phytoremediation due to their slow growth and reduced biomass production. With the elucidation of the physiological and molecular mechanisms of hyperaccumulation in the model hyperaccumulators such as Noccaea caerulescens, Arabidopsis halleri, Populus trichocarpa, and several other species, biotechnology has made it is possible to engineer the fast-growing plants with high biomass production to exhibit high metal accumulation capacity as genetic engineering allows the transfer of genes across the sexually incompatible species and thus helps in bringing together the desired traits in a single transgenic plant. Among the hyperaccumulators of toxic metals, Brassicaceae is the largest family representing about 25% of the total hyperaccumulator species reported to date (Kra¨mer, 2010; Sarma, 2011). This family has been extensively

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studied for its role in phytoremediation. Within this family, Arabidopsis, Noccaea, and Alyssum are considered as model plants to study the mechanism of heavy metal accumulation and detoxification (Anjum et al., 2012). Members of Brassica genus are high accumulators of Cd, Cu, Ni, Pb, and Zn (Mudasir Irfan Dar et al). It has been reported that B. juncea and B. napus can accumulate 1450 and 555 μg Cd/g dry wt respectively (Nouairi et al., 2006). In addition to high metal accumulation and detoxification properties, these species exhibit rapid growth rate and high biomass production. These features make them suitable for phytoremediation strategies (Fig. 3.1). Some of the Brassica species such as B. juncea and B. napus have well-established transformation protocols that have made the transfer of desired genes possible to further improve their phytoremediation capacity. For example transformation of Indian mustard with yeast cadmium factor1 (YCF1) has enhanced the accumulation of Cd and Pb in the transgenic plant shoots (Bhuiyan et al., 2011).

FIGURE 3.1 Brassicaceae are the best candidates amenable to genoremediation.

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3.3 STRATEGIES FOR GENETIC MANIPULATION TO ENGINEER PHYTOREMEDIATION CAPACITY IN PLANTS There are different biotechnological approaches used for the phytoremediation of toxic metals (Fig. 3.2). Genetic engineering is an efficient process in which genes across plant or prokaryotic cells can be deployed to enhance the phytoremediation capacity of the already known hyperaccumulators or species that have essential features to be converted into hyperaccumulators. In this process genes coding for proteins that have identified roles in phytoremediation such as transcription factors or functional proteins are deployed in the corresponding transgenic plants improving their soil and water reclamation capabilities (Fasani et al., 2018). The biotechnological approach used to stimulate the expression of a gene for enhanced phytoremediation is termed as genoremediation. The various genetic engineering approaches used for genoremediation are discussed in the following sections.

efficiency were made by expressing the target genes driven by constitutively expressing CaMV35S or actin promoters. More recent attempts were aimed at coexpressing two or more genes simultaneously in a single transgenic plant to augment the uptake and accumulation of toxic metals in their tissues.

3.3.2 Tissue Specific Expression of Genes Constitutive expression of genes in plants could be associated with some undesirable effects such as the accumulation of heavy metals in the edible parts or decrease in the biomass production of the plants, if these engineered phytoremediators are placed in the human food chain. In such a scenario, tissue specific expression of the target genes driven by tissue specific promoters to target the expression of the genes in tissues that are not involved in human consumption is desirable. The use of tissue specific promoters such as leaf specific promoter could lead to the accumulation of heavy metals in leaf tissues and facilitate phytoextraction and phytomining (Fig. 3.3).

3.3.1 Constitutive Overexpression of Single or Multiple Target Genes

3.3.3 Organelle Specific Expression

Initial attempts aimed at developing transgenic plants for enhancing the phytoremediation

Some genes involved in the sequestration of heavy metals in the specific subcellular FIGURE

3.2 Biotechnological approaches for genoremediation.

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3.4 ADVANTAGES OF WOODY TRANSGENIC PLANTS OVER HERBACEOUS TRANSGENIC PLANTS

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FIGURE 3.3 Genoremediation is possible by modification of plants via Agrobacterium-mediated transformation. The plasmid DNA and the gene of interest are digested with the same pair of restriction enzymes separately, and then ligated together to form the recombinant plasmid carrying the gene of interest. The recombinant plasmid is then introduced into an Agrobacterium strain. The culture of Agrobacterium strain harboring the recombinant plasmid is used infect the leaf explants. Transformed shoots regenerated from the explants are selected on selection medium and transferred to the rooting medium for root development. The plantlets with well-developed roots and shoots are transferred to soil.

organelles such as vacuole or Golgi apparatus can be expressed specifically to target the heavy metals to these regions (Antosiewicz et al., 2014; Jagtap and Bapat, 2015). Chloroplast transformation has proved to be one of the alternatives for efficient mercury accumulation and phytoremediation (Ruiz and Daniell, 2009; Ruiz et al., 2011; Hussein et al., 2007). Chloroplast or plastid transformation offers several advantages over nuclear transformation such as transgene containment ensuring biosafety, multiple transgene integration in a single transformation event, lack of position effect on transgene expression in plastid, high level of transgene expression due to multiple insertions per cell (as a cell carries several chloroplasts and each chloroplast carries up to 100

plastomes), and transgene integration by homologous recombination that aids in targeted integration in the plastome (Tabatabaei et al., 2017). Multiple genes can be engineered and expressed at a time due to prokaryotic type polycistronic mRNA transcription making pathway engineering possible through chloroplast transformation (Lu et al., 2013; Bock, 2013).

3.4 ADVANTAGES OF WOODY TRANSGENIC PLANTS OVER HERBACEOUS TRANSGENIC PLANTS In recent years, woody plants are preferred over the herbaceous plants for phytoremediation

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3. EMERGING TRENDS IN TRANSGENIC TECHNOLOGY FOR PHYTOREMEDIATION OF TOXIC METALS AND METALLOIDS

due to several advantages (Luo et al., 2016; Capuana, 2011). Woody plants have an extensive deep root system and higher biomass yield. These plants also have the advantage of being grown as short rotation coppice culture for producing energy and site restoration. Although poplar and Salix are fast growing and high biomass yielding woody plants, they are not hyperaccumulators of toxic metals (Capuana, 2011). However, they can accumulate more toxic metal compared with the herbaceous hyperaccumulators due to their massive biomass production (Luo et al., 2016). Among the various woody plants, poplar and Salix yielded encouraging results in phytoextraction of toxic metals (Wieshammer et al., 2007; Jensen et al., 2009; Kaca´lkova´ et al., 2015). Understanding the physiological and molecular basis of metal uptake, transport, and sequestration of toxic metals in the woody plants would help in devising approaches for enhancing the phytoremediation capacity of these plants. Over the years, tremendous advancement has been made in the tissue culture of poplar and Salix species, which has facilitated their regeneration and micropropagation (Yadav et al., 2009; Lyyra et al., 2006; Aggarwal et al., 2015). High-frequency genetic transformation protocols have also been established for the genetic manipulation of these plants (Yang et al., 2013; Maheshwari and Kovalchuk, 2016). Several reports are available on the genetic transformation of poplar and Salix to enhance their metal uptake, transport, and sequestration, and their detoxification capabilities (Adams et al., 2011; Turchi et al., 2012; Shim et al., 2013).

3.5 GENERAL MECHANISM OF TOXIC METAL UPTAKE AND ACCUMULATION IN PLANTS Metal solubility in soil solution is one of the critical factors that influence its uptake by the

plant roots. To enhance the metal solubility and availability for uptake, hyperaccumulators release metal chelating compounds into the rhizosphere to form complexes with metals and this process makes the complexes bioavailable for ready uptake by the plants. Some hyperaccumulators release organic acids such as citric, malic, malonic, and oxalic acids that change the rhizosphere pH, which helps in the solubilization of the metals. Upon uptake by the plants, these toxic metals subsequently enter the plant roots through both apoplastic and symplastic movements. Some of the toxic metals that come in contact with the cell wall remain bound to pectin and cellulose (Chen et al., 2013). Though the toxic metals are presumed to enter the plant roots from the soil solution through the transporters involved in the uptake of essential nutrients (Clemens, 2006; Gallego et al., 2012), no toxic metal specific transporters are known in plants to date. After crossing the plasma membrane, a part of the toxic metals are sequestered to the root cell vacuole and the rest of them reach the xylem vessel through symplastic movement (Eapen and D’souza, 2005). Translocation of metals from root symplast to xylem apoplast is mediated by several metal transporters such as cationic ATPases, ion channels, and pumps (Oveˇcka and Taka´cˇ , 2014). The excess toxic metals that reach the aerial parts of the plant are sequestered into the vacuole, cell wall, and Golgi apparatus to prevent the direct contact of toxic metals with the cellular enzymatic machinery (Luo et al., 2016). Vacuolar sequestration is mediated by metallothioneins, phytochelatins, and vacuole localized transporters (Cobbett and Goldsbrough, 2002; Sharma et al., 2016). Signaling cascades also play an important role in conferring metal tolerance in plants (Maksymiec, 2007). The signaling responses start with plasma membrane receptors and sensory proteins activated during heavy metal induced cellular damage. Heavy metal

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3.6 GENETIC ENGINEERING OF PLANTS FOR ENHANCED METAL UPTAKE

triggered signal transduction operates via signals of Ca21 ions, pH change, ROS, mitogenactivated protein kinases (MAPKs) and plant hormones (Luo et al., 2016; DalCorso et al., 2010). These cellular responses also include phosphoprotein cascades, induction of transcription factors and stress responsive genes, increase in secondary metabolism, and enhancement of the antioxidant pathway. ROS generated during heavy metal stress activate the MAPK based signal transduction pathway (Jonak et al., 2004; Islam et al., 2015; Sme´kalova´ et al., 2014). This pathway results in the accumulation of repair proteins such as heat shock protein (HSP) and chaperones that ultimately assure metal tolerance (Hossain and Komatsu, 2013). Heavy metals induce redox signaling because many of these metal ions are redox active. Secondly, these metals alter redox signaling by replacing metal ions from metalloproteins such as Rubisco, oxygen evolving complex, endonucleases, and ATP sulfurylase (Viehweger, 2014). Changes in soluble redox regulators such as glutathione and ascorbic acid also cause activation of redoxdependent signaling cascades upon heavy metal stress that result in expression of antioxidant enzymes and phytochelatins or metallothioneins, thereby conferring metal tolerance in plants (Anjum et al., 2012; Sharma and Dietz, 2009).

3.6 GENETIC ENGINEERING OF PLANTS FOR ENHANCED METAL UPTAKE Higher metal uptake depends on its bioavailability, soil pH, root exudates, and microorganisms present in the rhizosphere. Recent advances in the various “omics” technologies have helped in deciphering the molecular mechanisms behind the root uptake, root to shoot translocation, sequestration, and detoxification of toxic metals in the hyperaccumulators.

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Comparative study at the transcriptomic and proteomic levels between the hyperaccumulators and nonaccumulators identified the differential expression of several genes and proteins (Weber et al., 2006; Ahsan et al., 2009). Functional characterization of these candidate genes in the model plant systems identified their potential role in metal uptake and accumulation. To combine the high biomass production with enhanced metal extraction, biotechnological tools were used to generate genetically modified plants of nonmetal accumulators with high biomass production and rapid growth. These GM plants produced interesting and promising results for efficient uptake and translocation of toxic metals in laboratory and greenhouse conditions. In the following sections, some of the key classes of genes that were selected for transformation by the various research groups are discussed in some detail (Table 3.1).

3.6.1 Genes Encoding Metallothioneins, Phytochelatins, and Other Metal Chelators Plants produce several metal binding ligands such as metallothioneins, phytochelatins, and organic acids. Manipulation of the production of these natural chelators in plants by genetic engineering not only improved the uptake of toxic metals significantly, but also facilitated their translocation through xylem. Metallothioneins are cysteine-rich polypeptides encoded by a gene family. The organic sulfur (R-OH) present in the cysteine residues binds with metals such as Ag (I), Cd (II), Co (II), Cu (II), Hg (II), and Ni (II) and forms metal complexes. These proteins play a crucial role in ROS detoxification due to the presence of cysteine residues (Akashi et al., 2004; Hassinen et al., 2011). Overexpression of the genes responsible for the synthesis of these cysteine-rich proteins resulted in enhanced

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TABLE 3.1 Transgenic Plants With Enhanced Phytoremediation of Toxic Metals Gene

Gene Product

Transformed Species

Phytoremediation Effect

Reference

METAL TRANSPORTERS OsABCC1

ABC transporter

Rice

Knockout rice plants decreased As accumulation

Song et al. (2014)

PtABCC1

ABC transporter

Arabidopsis & poplar

Mercury tolerance and accumulation

Sun et al. (2018)

AtABCC1

ABC transporter

Arabidopsis

Enhanced Cd accumulation

Park et al. (2012)

YCF1

Cadmium factor 1 in Saccharomyces cerevisiae

Arabidopsis

Enhanced accumulation of Pb and Cd

Song et al. (2003), Bhuiyan et al. (2011b), Shim et al. (2013)

Mustard Poplar

CAX2

Cation antiporter

Tobacco

Mn tolerance

Hirschi et al. (2000)

CAX4

Cation antiporter

Tobacco

Cd, Zn, Mn tolerance

Korenkov et al. (2007)

CAX2

Cation antiporter

Tobacco

Cd, Mn/accumulation, tolerance

Hirschi et al. (2000)

OsZIP4

Zinc/iron regulated protein

Rice

Zn accumulation

Ishimaru et al. (2007)

HMA4

P1B-ATPase

Arabidopsis, tobacco, & tomato

Enhanced xylem Zn loading

Hanikenne et al. (2008), Barabasz et al. (2010, 2012)

AtHMA4

P1B-ATPase

Arabidopsis

Enhances root to shoot translocation of Zn and Cd

Verret et al. (2004)

HMA3

P1B-ATPase

Tobacco

Increased Cd tolerance and accumulation

Zhang et al. (2016)

OsNRAMP1

Natural resistance-associated Arabidopsis, macrophage protein rice

As and Cd tolerance; Fe and Mn accumulation in shoot Cd accumulation in roots and leaves

Tiwari et al., (2014), Takahashi et al. (2011)

TpNRAMP5

Natural resistance-associated Arabidopsis macrophage protein

Enhances the accumulation of Cd, Co, and Mn

Peng et al. (2018)

AtATM3

ABC transporter

Mustard

Cd(II) and Pb(II) accumulation in shoots

Bhuiyan et al. (2011a)

OsIRT1

Ferrous transporter

Rice

Increased Fe and Zn accumulation

Lee and An (2009) (Continued)

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3.6 GENETIC ENGINEERING OF PLANTS FOR ENHANCED METAL UPTAKE

TABLE 3.1 (Continued) Gene

Gene Product

Transformed Species

PvACR3

As[III] antiporter

CsMTP9 OsMTP1

Phytoremediation Effect

Reference

Arabidopsis

Transgenic plants accumulated 7.5fold more As in the aboveground tissues compared with untransformed plants

Chen et al. (2013)

Metal transport protein 9

Arabidopsis

Enhanced translocation of Mn and Cd from roots to shoots

Migocka et al. (2015)

Metal transport protein 1

Tobacco

Cd hyperaccumulation

Das et al. (2016)

PHYTOCHELATINS AtPCS1

Phytochelatin synthase

Tobacco

Cd and As accumulation

Pomponi et al. (2006), Zanella et al. (2016)

TcPCS1

Phytochelatin synthase

Tobacco

Cd hyperaccumulation

Liu et al. (2011)

METALLOTHIONEIN ThMT3

Metallothionein

Salix matsudana

Enhanced Cu tolerance and root growth

Yang et al. (2015)

PsMTA1

Metallothionein

Poplar

Enhanced Cu and Zn tolerance

Turchi et al. (2012)

MT1

Metallothionein

Tobacco

Accumulation of Hg (transplastomic)

Ruiz et al. (2011)

BcMT1 and BcMT2

Metallothionein

Arabidopsis thaliana

Tolerance to Cd and Cu; decrease ROS Lv et al. (2013) production

EhMT1

Metallothionein

Tobacco

Cu tolerance, accumulation, decreases ROS production

Xia et al. (2012)

OsMT1a and OsMT2b

Metallothionein

Rice

Plays roles in Zn homeostasis and enhance drought tolerance transgenic in rice

Yang et al. (2009)

SaMT2

Metallothionein

Tobacco

Tolerance and accumulation in transgenic tobacco plants by chelating metals and improving antioxidant system

Zhang et al. (2014)

γ-ECS

γ-Glutamylcysteine synthetase

Poplar

2.5 3.0 times higher Cd accumulation in transgenics compared with WT

Koprivova et al. (2002)

CSase

Cysteine synthase

Tobacco

Cd, Se, and Ni tolerance

Kawashima et al. (2004)

γ-ECS

γ-Glutamylcysteine synthetase

Mustard

Transgenics accumulated 2.4- to 3-fold Bennett et al. more Cr, Cu, and Pb (2003)

ANTIOXIDANTS

(Continued)

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TABLE 3.1 (Continued) Transformed Species

Gene

Gene Product

γ-ECS 1 AsPCS1

γ-Glutamylcysteine synthetase 1 phytochelatin synthase

StGCS-GS

A novel Streptococcus Sugar beet thermophilus glutamylcysteine synthetaseglutathione synthetase

Arabidopsis

Phytoremediation Effect

Reference

10 times higher Cd accumulation than the WT plants

Guo et al. (2008)

Resisted two or three of the metal combinations (50 mM Cd-Zn, Cd-Cu, Zn-Cu, and Cd-Zn-Cu) with greater absorption in shoots

Liu et al. (2015)

TRANSCRIPTION FACTORS RsMYB1

Transcription factor

Petunia

Ai et al. (2018) Transgenic plants tolerant to heavy metals like cadmium, copper, zinc, etc.

TaHsfA4a

Heat shock protein

Rice and wheat

Cd tolerance

Shim et al. (2009)

ZAT6

Zinc-finger transcription factor

Arabidopsis

Enhanced Cd tolerance by increasing phytochelatin synthesis in transgenic plants

Chen et al. (2016)

FIT 1 AtbHLH38 or AtbHLH39

FER-like deficiency induced transcription factor basic helix loop helix transcription factor

Arabidopsis

Cadmium tolerance by increasing root sequestration and iron homeostasis in shoots

Wu et al. (2012)

metal tolerance, uptake, and accumulation. For example, expression of a pea metallothionein PsMTA1 in white poplar enhanced Zn and Cu tolerance and accumulation (Turchi et al., 2012). Phytochelatins (PCs) are small peptides of general formula (γ-Glu-Cys)nX (where n 5 2 to 11, X represents Gly, Ser, β-Ala, Glu, Gln, or no residue). These peptides are synthesized from gluthathione by the action of phytochelatin synthase (PCS) enzyme (Cobbett and Goldsbrough, 2002). The synthases of PC are induced by toxic metal and metalloids and their role in conferring tolerance to Cd, As, Hg, and Pb has been well established (Fischer et al., 2014; Gasic and Korban, 2007). These peptides can efficiently bind several metal and metalloids forming stable metal(loid)-thiolate complexes and are sequestered into vacuoles

by ABC transporters (Clemens, 2006; Park et al., 2012). Transgenic approaches to overexpress PCS from many species have shown their considerable potential in phytoremediation. Heterologous expression of AtPCS1 in Brassica juncea enhanced the tolerance of this species to Zn and Cd (Gasic and Korban, 2007). Overexpression of PCS from the Zn/Cd hyperaccumulator Thlaspi caerulescens enhanced Cd tolerance and accumulation in transgenic tobacco (Liu et al., 2011). Similarly, the overexpression of Populus tomentosa PCS increased Cd tolerance and accumulation in tobacco (Chen et al., 2015). Nicotinamide acts as a natural metal chelating agent secreted by roots. It is present in higher concentrations in the hyperaccumulators like A. halleri and N. caerulescens, when compared with the closely related nonaccumulators

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3.6 GENETIC ENGINEERING OF PLANTS FOR ENHANCED METAL UPTAKE

(Kra¨mer, 2010). Arabidopsis plants overexpressing NAS1 gene that encodes the enzyme nicotinamide synthase exhibited enhanced the accumulation of Ni, Zn, Fe, and Mn (Douchkov et al., 2005; Pianelli et al., 2005).

3.6.2 Metal Transporters Several “omics” technologies have identified metal transporters as the candidate gene involved in the uptake of metals from the soils. Functional characterization of these genes in the model plants has enhanced the efficiency of metal uptake and tolerance. The major classes of metal transporters are shown in Fig. 3.4. Accumulating evidence suggests that toxic metals enter the plant cell through the mediation of nutrient transporters. To date, no heavy metal specific metal transporter has been identified (Luo et al., 2016). Metal transporters across the plasma and endomembranes play a pivotal role in the uptake, translocation, and sequestration of metals from the soil. Excessive

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heavy metals in the plasmatic compartments such as stroma, cytosol, nucleoplasm, and matrix interfere with the metabolic activities of the cells. Ion homeostasis in these compartments is maintained by the sequestration of the excessive metals in the vacuole or extracellular space. To increase the metal uptake by the plants for phytoremediation, transporter proteins involved at three levels, i.e., metal uptake from the soil, their translocation to the aerial parts, and sequestration to the vacuoles can be exploited. Toxic metals can enter the plant cells with the help of different types of transporters such as zinc/iron regulated protein (ZIP), iron regulated metal transporter (IRTs), zinc transporters (ZNTs), and natural resistance and macrophage proteins (NRAMPs) (Luo et al., 2016; Kotrba et al., 2009; Clemens and Ma, 2016). The ZIP family of proteins are shown to be involved in the uptake of heavy metals such as Zn, Fe, Cd, and Ni (Xu et al., 2012; Nishida et al., 2011; Li et al., 2016). Overexpression of a ZIP family transporter, FIGURE 3.4

Different classes of metal transporter involved in maintaining metal ion homeostasis in plants.

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NcZNT1, from a Zn hyperaccumulator, Noccaea caerulescens, resulted in the accumulation of more Zn in the roots of transgenic Arabidopsis plants compared with nontransformed control plants (Milner et al., 2012; Lin et al., 2016). The family of natural resistance-associated macrophage protein (NRAMP) transmembrane proteins can transport the divalent cations Mn21, Zn21, Cu21, Fe21, Cd21, Ni21, and Co21 into the cytoplasm or across the tonoplast (Nevo and Nelson, 2006; Kra¨mer et al., 2007). Cd21 and Pb21 can enter the plant root cells through the Ca21 channels (He et al., 2015; Rodriguez-Hernandez et al., 2015). Efficient phytoremediation capacity depends on the capacity of plants to translocate heavy metals through root to shoot translocation. HMAs (heavy metal transporting ATPases) constitute an important class of transporters that are involved in xylem loading of heavy metals for root to shoot translocation. Overexpression of AtHMA4 enhanced root to shoot translocation of Zn21 and Cd21in transgenic Arabidopsis plants (Verret et al., 2004). YSL family of proteins is shown to be involved in the long distance root to shoot transport of NA-complexed toxic metals (Gendre et al., 2007). Wang et al. (2013) demonstrated that the transgenic tobacco plants expressing BjYSL7 exhibited vigorous root growth that was associated with enhanced capacity of Cd21 and Ni21 translocation to shoots compared with WT plants. Another YSL family gene, SnYSL3, characterized from Cd21 hyperaccumulator Solanum nigrum enhanced the translocation ratios of Fe and Cd under Cd exposure (Feng et al., 2017). ABC transporters are a class of proteins that are involved in transporting PC-HM complexes into the vacuole (Park et al., 2012). In addition, certain other tonoplast pumps such as V-ATPase and V-PPase generate proton motive force on the tonoplast for the compartmentalization of HM within the vacuole. Overexpression of these metal transporters in

different plants enhanced the metal uptake and its translocation to the foliar parts. For example, yeast cadmium factor 1 (YCF1) belongs to the ABC transporter gene family. Its encoded protein can form a complex with thiol groups and transport Pb21 and Cd21 to vacuoles. Overexpression of YCF1 in Arabidopsis and B. juncea imparted tolerance to and accumulation of Pb21 and Cd21 in the transgenic plants (Song et al., 2003; Bhuiyan et al., 2011b). Overexpression of two Arabidopsis transporters, AtABCC1 and AtABCC2 conferred Cd21 and Hg21 detoxification in transgenic Arabidopsis plants (Park et al., 2012). Rice ABC transporter, OsABCC1 reduced As21 toxicity in transgenic plants by transporting the PC-As complex to the vacuole (Song et al., 2014). Another class of transporter proteins known as cation diffusion facilitators (CDFs), which are also otherwise known as metal tolerance proteins (MTPs), play an important role in maintaining metal homeostasis and detoxification in plants (Oveˇcka and Taka´cˇ , 2014; Migeon et al., 2010). These transporters are involved in the efflux of divalent cations Zn21, Fe21, Cd21, Co21, and Mn21 from cytoplasm to the subcellular compartments like vacuole and Golgi bodies, thereby reducing the metal toxicity in cellular metabolism. CDF family proteins are subdivided into three groups based on the substrate specificity, that is, Zn-, Fe/Zn-, and Mn-CDF (Montanini et al., 2007). Overexpression of the vacuole localized rice OsMTP1 improved Zn21, Ni21, and Cd21 transport in transgenic rice and Arabidopsis plants (Yuan et al., 2012; Menguer et al., 2013).

3.6.3 Antioxidants Glutathione plays an important role in hyperaccumulators because of its metal chelation and antioxidative properties. Glutathione acts as a substrate for phytochelatin synthesis by the action of the enzyme phytochelatin

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3.6 GENETIC ENGINEERING OF PLANTS FOR ENHANCED METAL UPTAKE

synthase. In plants, GSH is synthesized by a two-step ATP dependent reaction catalyzed by the enzymes γ-glutamylcysteine synthase (γ-ECS) and glutathione synthetase (GSHS). In metal hyperaccumulators such as Thlaspi goesigense and Sedum alfredii, GSH overproduction was implicated in sustaining Cd21 and Ni21 tolerance (Freeman and Salt, 2007; Sun et al., 2007). Transgenic plants expressing either of these enzymes were developed and tested in several species for metal tolerance. Transgenic poplar plants expressing γ-ECS accumulated 2.5 3 times more cadmium than the untransformed plants in their young leaves (Koprivova et al., 2002). Expression of the bacterial γ-ECS in Indian mustard improved the phytoextraction capacity of Cd21 and Zn21 by 25% and 6%, respectively, in greenhouse experiments with metal contaminated soil (Bennett et al., 2003).

3.6.4 Chemical Transformation of Metals With Transgenic Plants Several efforts have been made to convert toxic metals and metalloids to the less toxic and volatile forms using plants. Phytovolatilization is a potential phytoremediation method that involves absorption of the metals or metalloids from the soil, their conversion to the less toxic volatile forms within plant tissues, and subsequently releasing the volatiles into the atmosphere. The main advantage of phytovolatilization is the omission of harvesting and disposal steps in the process of phytoremediation. This method of phytoremediation is often regarded as controversial as it releases the toxic Hg into the atmosphere. However, it was reported that the advantage of dilution of the volatile form of metal(loid) in the atmosphere minimizes the potential risks associated with the method (Moreno et al., 2005; Kotrba et al., 2009; Lin et al., 2000). Some of the phytovolatilization attempts using transgenic plants are shown in Table 3.2.

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Some bacterial strains have the capacity to survive on high mercury concentration due to their ability to metabolize the toxic form of inorganic mercury Hg(II) or methylmercury into less toxic elemental form Hg(0). Bacterial resistance to mercury is due to expression of merA and merB genes that encode mercuric ion reductase and organomercurial lyase, respectively (Silver and Phung, 2005). Mercuric ion reductase is involved in the conversion of Hg21 to nontoxic volatile Hg. Organomercurial lyase encoded by merB gene releases Hg21 from organic mercuric compounds (CH3Hg1). This potential of bacterial gene encoded proteins to detoxify the mercuric compounds has been transferred to plants through genetic engineering. Ectopic expression of either or combination of merA and merB in Arabidopsis, tobacco, rice, poplar, and cottonwood provided tolerance to Hg21 and other organomercurial compounds (Ruiz and Daniell, 2009). The merA expression was further modified by codon optimization. Higher level of mercury volatilization was reported when MerB was targeted to endoplasmic reticulum (Bizily et al., 2003). Targeting the MerA and MerB proteins to tobacco chloroplasts also provided moderate levels of methylmercury resistance (Ruiz et al., 2003). The phytotransformation strategy has also been used to volatilize selenium. Selenium is mainly available in the form of selenate in the soil. Because of chemical similarity with sulfur, selenium can replace sulfur in the sulfur containing amino acids such as cysteine and methionine forming selenocysteine and selenomethionine, respectively. Methylation of selenocysteine and selenomethionine renders them inactive. Methylated selenocysteine and methylated selenomethionine can be metabolized as dimethylselenide or dimethyldiselenide. In plants, selenocysteine is converted to selenothenionine. Overexpression of cystathionine gamma synthase, the first enzyme involved in the conversion of selenocysteine to selenomethionine

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3. EMERGING TRENDS IN TRANSGENIC TECHNOLOGY FOR PHYTOREMEDIATION OF TOXIC METALS AND METALLOIDS

TABLE 3.2 Transgenic Plants Engineered for Phytotransformation of Toxic Metals and Metalloids to Less Toxic Forms Gene

Gene Product

merA and merB

Mercuric reductase and organomercurial reductase

merApe9 and merB

Methylmercury lyase

Transformed Species

Phytoremediation Effect

Reference

N. tabacum

Doubled biomass yield with seedlings grown on medium with 400 μM phenyl Hg1

Ruiz et al. (2003)

A. thaliana

Volatized 760 ng Hg0 [g FW]21 min21

Bizily et al. (2003)

merA 1 merB Mercuric reductase and organomercurial reductase

P. deltoides

Transgenic plants were more resistant to Lyyra et al. (2007) phenyl mercuric acetate and detoxified organic mercury 2 to 3 times more than the control

SMT

Selenocysteine methyltransferase

B. juncea

Se tolerance and accumulation

LeDuc et al. (2004)

CGS1

Cystathionine-γ-synthase

B. juncea

Enhances Se volatilization

Van Huysen et al. (2003)

APS 1 SMT

ATP sulfurylase 1 selenocysteine methyltransferase

B. juncea

4 to 9 times increase in Se accumulation

LeDuc et al. (2006)

arsM

Arsenite O. sativa S-adenosylmethyltransferase

As[III] converted into less toxic monomethylarsenate and dimethylarsenate; transgenic plants gave 10-fold higher arsenical volatiles

Meng et al. (2011)

AtACR2

Arsenic reductase 2

As content was 1.2 fold higher in the roots of transformed plants

Nahar et al. (2017)

N. tabacum

in B. juncea showed two- to threefold higher volatilization rates compared with the WT plants (Van Huysen et al., 2003).

3.7 POTENTIAL RISKS ASSOCIATED WITH THE USE OF TRANSGENIC PLANTS AND THEIR MITIGATION STRATEGIES Although the transgenic plants offer several advantages, they are associated with some potential risks that should be carefully addressed before their field testing. One of the possible risks of the transgenic plants with higher metal accumulation in the edible parts

is the entry of these heavy metals and metalloids into the human food chain, which could have a negative impact on animal and human health. To make the genetic transformation for phytoremediation biosafe, it is advisable to choose fast-growing plants with higher biomass production that do not have a place in the food chains involving humans and animals. This is important for animals too, as they have a significant presence in the human food chain. Also if such a situation exists, the deployment of transgenic plants at the field level would attract fewer biosafety concerns with respect to human and animal health. Spread of the transgene to the wild relatives of the transgenic plants through cross

I. EMERGING ISSUES

REFERENCES

pollination could destroy the natural diversity of the ecosystem. This can be controlled by harvesting the biomass before flowering or by developing male sterile plants with enhanced phytoextraction capacity. The spread of the heterologous transgenes can be controlled also by developing transplastomic transgenic plants. Chloroplasts are maternally inherited; thus the spread of transgenes through pollen can be minimized by expressing the desired genes in the chloroplasts. The effect of the transgene on the plant associated microorganisms should also be taken into consideration. The use of marker or antibiotic resistance genes for the selection of transgenic plants often raises public and regulatory concerns due to the risk of horizontal transfer of marker or herbicide resistance genes to the pathogenic microorganisms, wild relatives, or weeds. Hence, the use of marker free transgenics is often desired and publicly more acceptable (Tuteja et al., 2012; Yau and Stewart, 2013). Biotechnological advancement has made it possible to develop marker free transgenics by a number of methods such as Cre-lox autoexcision strategy, Ac/Ds transposable genetic system, or cotransformation/segregation methods among others. Each transgenic plant should pass through rigorous environmental risk assessment tests prior to its commercialization by evaluating them for any potential risks to humans, animals, and the environment.

3.8 CONCLUSION AND FUTURE PERSPECTIVES Phytoremediation is an eco-friendly method for the cleanup of toxic metal contaminated sites. Progress in the various omics technologies has helped in the identification of the genes and the corresponding proteins encoded by them that are involved in the uptake, root to shoot translocation, sequestration, and

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detoxification of toxic metals. Some naturally occurring metal hyperaccumulators can accumulate high amounts of toxic metals but are not suitable for commercial use due to their slow growth rate and low biomass yield. Transgenic technology has been successfully used in modifying the fast-growing high biomass producing plants with metal hyperaccumulation property. Genetic engineering to manipulate the expression of these genes has shown considerable success in augmenting the phytoremediation efficiency under controlled laboratory conditions. However, the results of these experiments are not extrapolated under field conditions due to complex regulatory issues associated with the GM plants. The availability of the complete genome sequence of several plant species and advanced gene editing tools such as zincfinger nuclease technology, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat (CRISPR) or CRISPR associated protein 9 system can allow precise genome editing at predetermined sites of the genome. These edited plants would be publicly more acceptable and bypass the regulatory issues associated with the GM crops (Waltz, 2018). The use of nonfood crops should be selected for genetic manipulation to avoid the entry of toxic metals and metalloids in the food chain. Additionally, phytoremediation can be integrated with bioenergy production to further improve the cost-effectiveness of this method.

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