Prosopis juliflora

C H A P T E R

18 Prosopis juliflora: A Potential Plant for Mining of Genes for Genetic Engineering to Enhance Phytoremediation of Metals Nisha Surendran Keeran1, Usha Balasundaram2, Ganesan Govindan2 and Ajay Kumar Parida3 1

Formerly at Plant Molecular Biology Lab, MS Swaminathan Research Foundation, Chennai, Tamil Nadu, India, 2Department of Genetic Engineering, SRM Institute of Science and Technology, Kattankulathur, Tamil Nadu, India, 3 Institute of Life Sciences, Bhubaneswar, Odisha, India

18.1 INTRODUCTION Various heavy metals such as Cu21, Zn21, Mn21, Fe21, Ni21, and Co21 are released to the environment as a result of activities such as mining, smelting, manufacturing, agriculture, or waste disposal such as sewage sludges, or use of pesticides (Baye and Hymete, 2010), thus posing an ecological threat to the environment. While some of these heavy metals are essential for normal plant growth and development (known as micronutrients), at higher concentrations they can be extremely toxic to the

Transgenic Plant Technology for Remediation of Toxic Metals and Metalloids DOI: https://doi.org/10.1016/B978-0-12-814389-6.00018-3

plants causing chlorosis and necrosis, stunting, leaf discoloration, and inhibition of root growth. Some examples of micronutrients include Cu21, Zn21, Mn21, and Fe21. For example, Cu21 is involved in many electron transport reactions in photosynthesis and respiration, and Zn21 and Mn21 act as cofactors for a wide range of enzymes involved in various redox reactions. Fe21 acts as a cofactor for a number of enzymes involved in a variety of oxidation-reduction reactions such as photosynthesis, respiration, hormone synthesis, and DNA synthesis (Kobayashi and Nishizawa,

381

© 2019 Elsevier Inc. All rights reserved.

382

18. PROSOPIS JULIFLORA: A POTENTIAL PLANT FOR MINING OF GENES

2012). However, the presence of excessive amounts of metals in soils might also result in reduction of soil microbial activity and soil fertility. This in turn results in yield loss and also possible contamination of the food chain. (Varun et al., 2011) Examples of heavy metal toxicity at the cellular level include inhibition of enzyme activity by binding to sulfhydryl groups in proteins or by creating deficiency of other essential ions, disruption of cell transport processes and oxidative damage (Williams et al., 2000). They also accumulate and migrate to soils and later enter human bodies through the food chain, thus increasing the occurrences of chronic diseases such as deformity, renal dysfunction and cancer (Chang et al., 2014). A wide range of physiological and metabolic alterations are also triggered in plants exposed to heavy metals. The most widespread visual evidence of heavy metal toxicity includes leaf

chlorosis, necrosis, turgor loss, decreased rate of seed germination, and crippled photosynthetic apparatus, often correlated with progressing senescence or with plant death (Hossain et al., 2012). Heavy metals also influence homeostatic events, including water uptake, transport, transpiration, and nutrient metabolism, and interfere in the uptake of Ca, Mg, K, and P (Benavides et al., 2005) (Fig. 18.1). Despite the toxicity of these heavy metals, several plants growing in metal polluted soil are able to exclude, accumulate, or hyperaccumulate heavy metals and acquire a wide range of adaptive strategies (Sytar et al., 2012). Plants capable of growing in soils with very high metal content are known as hyperaccumulators. One of the best-known examples of a hyperaccumulator is Thlaspi caerulescens, a member of the Brassica family. Other examples include Arabidopsis halleri, Brassica napus, and Brassica juncea.

Heavy metals contamination

Effects soil

Effects plants

• Effects soil diversity • Effects soil properties • Effects actions of soil microbes

• Cellular level damages like deficiency of meta ions, oxidative damage, etc. • Physiological damages like leaf chlorosis, necrosis, decreased seed germination, and ultimately plant death

FIGURE 18.1 Effects of heavy metal contamination on soil, plants, and animals.

II. SUBJECT SPECIFIC STUDIES

Effects animals • Bioaccumulation • Production of ROS • Effects activities of enzymes • Effects various functions such as respiration, circulation, etc.

383

18.3 CANDIDATE PLANTS FOR METAL PHYTOREMEDIATION

TABLE 18.1

Some Examples of Metal Hyperaccumulating Plants

Sl. No.

Hyperaccumulator (Part Used)

Metal Accumulated

Amount (µg g21)

Reference

1

B. Napus (seed)

Cu, Mn

2.17, 22.8

¨ zcan (2006) Musa O

2

Thlaspi praecox (leaves)

Zn, Cd

4190 4530, 1221 1425

Neˇcemer et al. (2008)

3

Potamogeton crispus L. (leaves)

Cd

49.09

Sivaci et al. (2008)

4

B. Juncea (whole plant)

Cd

1450

Szczygłowska et al. (2011)

5

Amaranthus dubius (leaves)

Zn, Cu, Mn

14.45, 94.05, 96.7

Subramanian et al. (2012)

6

Hypericum perforatum (whole plant)

Mn, Zn

120 127, 425 55.9

˘ (2012) Tokalioglu

7

Helianthus annuus (seed)

Cu, Mn

18.1, 6.95

˘ (2012) Tokalioglu

8

Acacia dealbata (leaves)

Mn, Zn

74, 36

De La Calle et al. (2013)

9

P. juliflora (leaves)

Cu

775.29

Raju et al. (2013)

10

Alnus nepalensis (root)

Zn, Pb and Cd

573.9, 3550.1, 94.7

Jing et al. (2014)

18.2 HYPERACCUMULATORS The term hyperaccumulator was coined for plants that actively take up exceedingly large amounts of one or more heavy metals from the soil (Brooks et al., 1977). Hyperaccumulators accumulate 100- to 1000-fold higher heavy metals in their shoot than nonaccumulators (Mcgrath et al., 2002). Chaney et al. (1990) have reported that hyper-metal-accumulating plant species in general accumulate more than 100 µM of Cd whereas normal plants accumulate only about 0.5 5 µM of Cd. Hyperaccumulators have certain characters that distinguish them from other nonaccumulators such as enhanced rate of heavy metal uptake, a faster root-to-shoot translocation, and a greater ability to detoxify and sequester heavy metals in leaves (Rascio and Navari-Izzo, 2011). Phytoremediation is defined as the use of green plants to either remove pollutants from the environment or to render them harmless. It is noted to be one of the most cost-effective and environmentally friendly ways of cleaning up soils. The organic pollutants in the soil can be

absorbed by plants, transferred, or stored in nontoxic forms. The use of these plants for phytoremediation, however, is limited due to their low biomass and slow growth (in the case of trees). Genetic engineering of plants with high biomass and rapid growth, therefore, is an alternative solution for phytoremediation of heavy metals from the polluted soil (Table 18.1).

18.3 CANDIDATE PLANTS FOR METAL PHYTOREMEDIATION Heavy metal tolerant and hyperaccumulating plants can be found in naturally occurring metal rich sites (Baker and Brooks, 1989). However, these plants are not ideal for phytoremediation as they are usually small and have low biomass (KaE`renlampi et al., 2000). The plants used for successful phytoextraction should have substantial biomass and a significant translocation factor (TF). TF is defined as the ratio of heavy metal concentration in the shoot to that in the root of the plant (Luo et al., 2005).

II. SUBJECT SPECIFIC STUDIES

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18. PROSOPIS JULIFLORA: A POTENTIAL PLANT FOR MINING OF GENES

A plant suitable for phytoremediation should possess the following characteristics: • ability to accumulate metals intended to be extracted preferably in the aboveground parts; • tolerance to metal concentrations accumulated; • fast growth and highly effective (metal accumulating); and • easily harvestable (KaE`renlampi et al., 2000).

18.4 PROSOPIS JULIFLORA AS A CANDIDATE PLANT FOR MINING GENES FOR GENETIC ENGINEERING Prosopis juliflora is one of the most widespread hyperaccumulating plants. It is a phreatophytic, perennial tree/shrub species, a member of the Fabaceae family and a representative species of the Sonoran Desert ecosystem. It is a thorny, deciduous, large crowned, and deep-rooted bush or tree that grows up to a height of about 10 m, depending on the variety and climatic conditions, and has been proved to be the only exotic species capable of growing in a wide variety of soils and climatic conditions (Usha et al., 2009). It has a thick, rough, gray-green bark that becomes scaly with age. The plants are often multistemmed with many sharp thorns that measure up to 5 cm. The stems of P. juliflora are often “mild zigzag” in shape, with one or two thorns present at each turn. The regional adaptation of the species and the easy dispersion as well as the lack of proper management have led P. juliflora to be considered as an invasive species (de Souza Nascimento et al., 2014). They reported that the introduced tree P. juliflora has become a serious threat to native species of the Brazilian Caatinga vegetation threatening native biodiversity and rural sustainability due to its

superior ability to adapt and establish itself in the given environment. P. juliflora is seen widely in arid and semiarid regions of Rajasthan, India, which is highly endemic to fluoride (Yadav et al., 2009). It is a highly esteemed fuel wood source in several places, a valued tree for shade, and is also used as timber and forage (Saini et al., 2013). In addition to the above mentioned uses, P. juliflora is also used to control soil erosion. It has been shown to absorb Cr(VI), lead, and other metals (Aldrich et al., 2004). In addition, X-ray absorption spectroscopy studies have shown that P. juliflora is able to bioreduce Cr(VI) to the less toxic Cr(III) (Aldrich et al., 2003). These authors have also proposed to classify P. juliflora as hyperaccumulator, as the plant can remove Cr from the environment via active transport to the aerial portion of the plant. Aldrich et al. (2007) also reported that P. juliflora has the ability to reduce As(V) to As (III) inside the plant. Rai et al. (2004) have demonstrated that P. juliflora growing in fly ash contaminated soils amended with blue-green algae or rhizobia show higher bioaccumulation of iron, manganese, copper, zinc, and chromium in its tissues. It is also a suggested bioindicator for industrial smelter pollution (Gabriel and Patten, 1994). Due to its property of heavy metal accumulation, it has been suggested as a “green” solution for soils contaminated with cadmium, chromium, and copper (Senthilkumar et al., 2005). Sharmila et al. (2013) have reported the extracts of P. juliflora to be good reducing agents of harmful factors like chloride, sulfate, hexavalent chromium, nitrate, etc. present in leather industry effluent. P. juliflora is also shown to possess high metal accumulation ratios under natural conditions. These properties coupled with its ready availability all over India, make it an ideal source plant for mining genes for phytoremediation. It could also be used for treating the effluents in an economic way.

II. SUBJECT SPECIFIC STUDIES

18.6 GENETIC ENGINEERING FOR ENHANCED PHYTOREMEDIATION

The process of phytoremediation offers a feasible and economic alternative for remediation of contaminated soils. This plant propagates through seeds even in adverse conditions and grows luxuriously throughout the year. The exceptional tolerance of P. juliflora to drought, saline soil, and waterlogging throws light on the importance of this tree. It is also shown to survive on soils with acid to alkaline reactions. Since it is not consumed by humans or livestock, it can be considered as a safe and effective choice to be used for phytoremediation (Varun et al., 2011)

18.5 CANDIDATE GENES FOR PHYTOREMEDIATION Enhancing the number of uptake sites, alteration of specificity of uptake system to reduce competition by unwanted cations, and increasing intracellular binding sites are a few of the approaches to increase metal uptake (Clemens et al., 2002). Vacuolar compartmentalization and ligand complexation are the two major detoxification mechanism(s) found in plants which are very important for efficient internal metal tolerance (Neerja Srivastava, 2016). Enhancing the number of uptake sites, alteration of specificity of uptake system to reduce competition by unwanted cations, and increasing intracellular binding sites are a few of the approaches to increase metal uptake (Clemens et al., 2002). Manipulation of metal transporters and vacuolar targeting of metal will have successful applications in genetic engineering of plants for enhanced phytoremediation. Molecules with selective chelation ability that are secreted into the rhizosphere are the potent scopes of research. For example, free histidine in xylem exudates was found as a metal (Ni) chelator in a Ni hyperaccumulating plant (Eapen and D’Souza, 2005). Other potential mediators of metal transport and accumulation

385

include the cation diffusion facilitation family. Hyperaccumulators are also known to tolerate high amounts of heavy metals by sequestering them into vacuoles (large storage cells), a process strongly mediated by elevated expression of specific transport proteins in various tissues (Leitenmaier and Ku¨pper, 2013). Another important strategy is cellular targeting, especially in the vacuoles, since the heavy metals can be kept in a safe compartment without affecting other cellular functions. Hence, engineering vacuolar transporters in specific cell types can be considered as a second generation approach for phytoremediation (Eapen and D’Souza, 2005). Keeran et al. (2017) have identified a novel heavy metal transporter from P. juliflora that is involved in heavy metal (Cd and Zn) uptake in yeast and tobacco.

18.6 GENETIC ENGINEERING FOR ENHANCED PHYTOREMEDIATION The phytoremediation potential of natural hyperaccumulators is limited by a number of biological and environmental factors (Berken et al., 2002) and also by the lower availability of metals to the plant (Baker et al., 2000). These limitations can be addressed by various methods like use of chelating agents, metalimmobilizing agents, plant growth promoting rhizobacteria and arbuscular mycorrhizal fungi, and genetic engineering for enhancing phytoremediation. Gene mining from metal hyperaccumulating plants or microbes and their transfer to plants that have a high growth rate is found to be a good strategy to develop plants with enhanced phytoremediation potential (Eapen and D’Souza, 2005). Maintenance of metal homeostasis, especially of transition metals, is important for almost all normal functions of the plant because many of these are redox active, generate reactive oxygen

II. SUBJECT SPECIFIC STUDIES

386

18. PROSOPIS JULIFLORA: A POTENTIAL PLANT FOR MINING OF GENES

species (ROS), may destroy DNA and cell walls, etc. (Galanis et al., 2009). A number of genes are involved in metal uptake, translocation, and sequestration. Transfer of any of these genes into candidate plants is a feasible strategy for genetic engineering of plants for improved phytoremediation traits. Such genetically modified plants can either be used in phytoremediation. A primary understanding of these processes would help us to develop plants for phytoremediation (Fulekar et al., 2009). The superfamily of ATP-binding cassette (ABC) transporters is one of the largest to be found in nature (Henikoff et al., 1997), and fulfills many essential functions in microorganisms, animals, and plants. Heavy metal transporters are ABC transporters that help in homeostasis of a variety of different essential and nonessential metals. This protein family mediates the energy driven transport across membranes of a multitude of substances ¨ ner Kolukisaoglu, 2010), and (Wanke and U plays an important role in organ growth, plant nutrition, plant development, response to abiotic stress, and the interaction of the plant with its environment (Kang et al., 2011) (Table 18.2). TABLE 18.2

2 3 4 5 6 7 8

18.7 METALLOTHIONEINS Metallothioneins (MTs) are a low molecular weight, cysteine rich (25% 33%) proteins with antiapoptotic properties. They have been demonstrated to scavenge free radicals under heavy metal stress. In a physiological context, MTs bind up to 7 zinc ions or 10 copper ions in thiolate clusters (Nielson and Winge, 1983). The first MT identified in plants was the wheat EcMT (early cysteine labeled) protein in 1987 (Lane et al., 1987). The cysteine rich domains in MTs bind the heavy metals and give them a dumbbell conformation. A variety of metals bind to the cysteine residues present in the mercaptide bonds. MTs can effectively sequester several metal ions, most notably Cu21 and Zn21 under normal physiological conditions, and more toxic metals like Cd21 or Hg1 following exposure. It has been suggested that MTs may play a role in the homeostasis of essential metal

Some Examples of Genetically Engineered Plants Developed for Phytoremediation

Sl. No. Gene Name 1

Few examples of common genes used for the development of genetically modified plants for enhanced phytoremediation are discussed in the following sections.

PjMT1 OsMTP1 SaNramp6 PjHMT AtACR2 PpCzcA and PpCzcB CuHR and CuHR-V

Isolated From P. juliflora

Host Plant

Sedum alfredii Prosopis juliflora A. thaliana

Cd

Balasundaram et al. (2014)

Cd

21

Das et al. (2016)

Arabidopsis thaliana Cd

21

Chen et al. (2017)

Tobacco

21

Keeran et al. (2017)

31

Nahar et al. (2017)

21

Nesler et al. (2017)

Tobacco

Tobacco

Pseudomonas putida

Tobacco

Pseudomonas fluorescens Tobacco

MnPCS1 and MnPCS2 Morus notabilis

Reference

21

Tobacco

Oryza sativa

Metal

Arabidopsis

II. SUBJECT SPECIFIC STUDIES

Cd As

Cd

21

Pe´rez-Palacios et al. (2017)

Cu

21

Zn

and Cd

21

Fan et al. (2018)

18.10 EXAMPLES OF GENES ISOLATED FROM PROSOPIS JULIFLORA FOR PHYTOREMEDIATION

ions and the detoxification of heavy metals, such as Cd21 or Hg1 (Abdullah et al., 2002).

18.8 HEAVY METAL TRANSPORTERS Heavy metal ATPases (HMAs) (P-type ATPases or CPx-ATPases) uses ATP to pump various cations across the membrane against their electrochemical gradient. Based on their substrate specificity P-type ATPases are divided into five main classes: P1 (heavy metal pumps), P2 (Ca21, Na1, K1, and H1/K1 pumps), P3 (H1 and Mg21 pumps), P4 (phospholipid pumps), and P5 (a group having no assigned substrate specificity). P1 ATPases are divided into two additional classes, P1A and P1B (Axelsen and Palmgren, 1998; Bublitz et al., 2011). In many organisms, P1B ATPases are known to exhibit two substrate specificities: either Cu21/Ag1 Or Zn21/Co21/Cd21/Pb21 (Solioz and Odermatt, 1995). They are characterized by the presence of 6 8 transmembrane helices, a soluble actuator domain and ATP-binding domain (ATPBD) and the metal binding SPC/ CPx (CPS, CPT, CPA, CPG, CPD) domain. The ATPBD includes the invariant DKTGT motif that contains the asparagine residue, which is phosphorylated during the catalytic cycle. These ATPases are known to function in the transport of metals into the subcellular compartments and target proteins, nutrition, and metal detoxification or metal hyperaccumulation. Arabidopsis genome is known to code for eight P1B ATPases (HMA 1 8) of which HMA 2, 3, and 4 encode pumps belonging to Zn21/Co21/Cd21/Pb21 transporting category and HMAs 5, 6, 7, and 8 are Cu21 ATPases. AtHMA2 and AtHMA4 are shown to play a significant role in xylem loading of Zn for long distance transport to the shoot, a process crucial to plant nutrition (Hussain et al., 2004). The role of HMA 5 in Cu compartmentalization and detoxification was shown by Andre´sCola´s et al. (2006). Similarly, AtHMA7 (RAN1) is

387

responsible for delivering Cu to ethylene receptors (Hirayama et al., 1999). Of the remaining P1B ATPase, AtHMA1 has different conserved amino acids in the transmembrane (Eren and Arguello, 2004) and has a broad substrate specificity. It is shown to be present on the inner envelope of the chloroplast and contributes to Zn(II) detoxification by reducing the Zn content of plastids (Kim et al., 2009). Moreno et al. (2008) have shown the role of AtHMA1 in cadmium detoxification and also proved that it functions as a Ca21 heavy metal pump.

18.9 PHYTOCHELATINS Synthesis of phytochelatins (PCs) by plants is yet another general mechanism that has been developed by plants to achieve metal detoxification. The process involves the chelation of metal ions by specific high affinity ligands that reduce the concentration of free metal ions, and consequently decrease their phytotoxicity. Several studies have reported an increase in the PC concentration in plants upon exposure to heavy metals. For example Heiss et al. (2003) have reported a significant increase in PC synthase in B. juncea leaves after prolonged exposure to cadmium. Mendozaco´zatl et al. (2008) have reported high levels of PCs in the phloem sap of B. napus using combined mass spectrometry and fluorescence high-performance liquid chromatography analysis within 24 hours of Cd exposure.

18.10 EXAMPLES OF GENES ISOLATED FROM PROSOPIS JULIFLORA FOR PHYTOREMEDIATION 18.10.1 PjMTs MTs constitute 4.13% of the ESTs examined in P. juliflora (George et al., 2007). Usha et al. (2009)

II. SUBJECT SPECIFIC STUDIES

388

18. PROSOPIS JULIFLORA: A POTENTIAL PLANT FOR MINING OF GENES

Day 0

Leaf (µg/g)

Stem (µg/g)

Root (µg/g)

% Cd in MS mediu m

0.008±0.0 0.3±0.17 3.04±0.57 04

79

PjMT 0.019±0.0 0.4±0.08 7.133±0.7 1 07 8 5

70

Ctrl

C

Day 3

Day 3

T

C

Day 8

T

C

Day 6

T

Day 30

Day 8

Phytoextraction of metal by PjMT1 plants from medium containing 200 µ Cd

C

T

C

T

Survival of transgenic plants at 0.3 mM Cadmium

C

T

Necrotic and chlorotic symptoms of cadmium toxicity. Control on the left and transgenic on the right side.

FIGURE 18.2 Effect of PjMT1 overexpression on tobacco plants under cadmium stress. C, control; T, transgenic.

isolated type I, II, and III MTs from P. juliflora. Out of the three MTs isolated, PjMT1 has been shown to possess a typical CXCTNCXC motif at the N-terminal region, which is different from the proposed CXCGSXCXC motif (Roosens et al., 2005). However, it also shows a conservation of CxC motifs at the C-terminus. PjMT1 was also found to have the maximum ability to bind to Cd, Zn, and Cu possibly due to the presence of six CxC motifs (MT2 and MT3 have five and four CxC motifs respectively). The CxC motifs at the N-terminal region are crucial to cellular metabolism and gene regulation (Robinson et al., 1996). Plant MTs are also considered potent scavengers of ROS (Cobbett and Goldsbrough, 2002). All three MTs from P. juliflora were shown to be induced under H2O2 treatment, thus suggesting a role in maintaining the local redox balance either by sequestering ions and/ or preventing potentially deleterious Fenton chemistry reactions or by directly scavenging deleterious oxygen radicals (Usha et al., 2009). The increase in the expression of all three PjMTs under abscisic acid and oxidative stress treatment signifies a cytoprotective role of PjMTs. PjMT1 and PjMT2 overexpressed Nicotiana tabacum plants demonstrated better survival and their atomic absorption spectroscopy

revealed nine-fold and five-fold increase of Cd than wild-type plants under 0.3 mM CdSO4. The phytoextraction capability of PjMT1 transgenic plants was also compared with the control plants. It was found that PjMT1 transgenic plants could extract 30% Cd while transgenic plants could extract only 21% Cd from the medium containing 200 µM Cd. These results identified PjMT1 as a better candidate gene for phytoremediation of cadmium (Fig. 18.2).

18.10.2 PjHMT Keeran et al. (2017) have shown the role of PjHMT, a HMA peptide from P. juliflora, in metal tolerance especially of Cd and Zn in yeast and tobacco. Expression of PjHMT in mutant yeast resulted in uptake of Cd up to eight times compared with the vector control at 50 µM Cd and 1.9-fold at 30 and 40 µM Cd. The Zn concentration was also found to be double in the PjHMT expressing cells compared with the vector control cells at the concentrations of 100 and 125 µM Zn. PjHMT overexpressing N. tabacum was also shown to accumulate 1.8-fold more cadmium at 300 µM concentration of cadmium. These experiments suggest an influx of metals into the cell as the

II. SUBJECT SPECIFIC STUDIES

389

18.12 CONCLUSION

(A)

(B)

Cd accumulation (mg/g)

0.4

WT VC

0.3

L2 L17 L20

ns

0.2 0.1

(C)

A.

L2 0

L1 7

L2

VC

W

T

0.0

Cadmium accumulation in the leaves of two month old control and PjHMT overexpressing tobacco plants measured using Inductively Coupled Plasma (ICO) analysis.

B.

Effect of 300 µM Cd on 2 month old control and PjHMT overexpressing tobacco plants on Day 0 and Day 7.

C. Necrotic and chlorotic symptoms of Cd toxicity on leaves of control and PjHMT overexpressing tobacco plants on Day 3

FIGURE 18.3

Effect of PjHMT overexpression on tobacco plants under cadmium stress. Asterisks indicate a P value ,0.05 fromStudent’s t test. ns not significant, * significant, ** highly significant.

possible mechanism of action, which is similar to AtHMA1 (Kim et al., 2009) (Fig. 18.3). It is also interesting to note that PjHMT sequence shows high similarity to the Cterminal region of the HMA1 pump. PjHMT shows the presence of conserved regions/motifs like the ATP-binding consensus sequence MVGEGINDAPAL, the metal-binding domain HEGGTLLVCLNS, and metal binding motifs MLTGD, GEGIND (Moreno et al., 2008) and HEGG (Seigneurin-Berny et al., 2006), which play important roles in metal transport or ATP binding. However, it is devoid of the highly conserved DKTGT.

Keeran et al. (2018). The results showed that PjHMT PjMT1-transformed plants accumulated higher amounts of Cd (11.3-fold) compared with untransformed tobacco control plants. Furthermore, the heterologous expression of the same construct in yeast Saccharomyces cerevisiae (INVSc1) improved the Cd tolerance compared with wild-type yeast cells. These results indicate that coexpressing of PjHMT and PjMT1 has a positive correlation with the phytoremediation potential of plants and could be adopted for Cd removal from contaminated soils.

18.12 CONCLUSION 18.11 OVEREXPRESSION OF AND FOR ENHANCED METAL TOLERANCE The metal tolerance and accumulation of N. tabacum plants overexpressing both PjHMT and PjMT1 (double construct) were studied by

P. juliflora is an evergreen, well-adaptable tree species that can be used as animal feed and also as a source of wood and fodder. The ability of this phreatophytic tree species to grow well in extreme climatic and soil conditions, coupled with the excellent potential to

II. SUBJECT SPECIFIC STUDIES

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18. PROSOPIS JULIFLORA: A POTENTIAL PLANT FOR MINING OF GENES

uptake heavy metals from the soil, makes P. juliflora an ideal source for mining of genes for phytoremediation. Results have shown that heavy metal transporter and MT (PjHMT and PjMT1) isolated from P. juliflora show promising results on the phytoremediation potential of the genes in transgenic system. Further detailed studies on this plant would yield beneficial results in formulating future strategies for enhancing heavy metal tolerance and would be instrumental in cleaning up heavy metal contaminated soils thus making them viable for agriculture production. More studies on the phytorememdiation potential of P. juliflora in long term would also contribute to its better understanding and use thereby restoring soil fertility and thus in increased crop productivity in marginal lands and food security.

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