An Insight into Molecular Mechanisms of Plant Defens: Omics Approaches

Abstract

Plants face various adverse growth factors such as extreme temperatures, high salt content, heavy metals, use of pesticides, drought, and harmful pathogens. These factors result in phytotoxicity and overall retardation of growth and development, leading to yield loss. Plants produce reactive oxygen species (ROS) to counteract phytotoxicity, which results in oxidative stress. However, plants generate secondary messengers in response to threshold accumulation of ROS to activate cell signaling, leading to the activation of several transcriptional responses associated with plant defense. Heavy metal stress is a crucial constraint to plant growth, yield, and development, but plants develop several defense mechanisms to survive it. TFs gene families, phytohormones, and their signaling network play important functions in the regulation of stress using hormone-mediated pathways. OMICS approaches are needed to improve the plant's internal defense system to counteract heavy metal stress.

Keywords: Abiotic stress, Biotic stress, Crop improvement, Heavy metals, Oxidative stress

Abbreviations: ROS, Reactive Oxygen Species; HMs, Heavy Metals; TFs, Transcription Factors;

1. Introduction

Growing population, industrialization, chemicals in agriculture, and climate change expose plants to living and non-living stresses, leading to crop loss and severe diseases. Heavy metal contamination occurs through industrial water discharge and irrigation. Absorption of heavy metals by crops causes diseases in humans and animals. Controlling metal accumulation can reduce disease and enhance crop quality. Immediate measures are needed to address the situation.

Plants require essential micronutrients for their complete maturation and development. Plants absorb crucial heavy metals from soil for their normal metabolic activities (Wuana and Okieimen, 2011). Several heavy metals are present in soil and plants require some of them in minimal quantity for their growth and development. Cobalt (Co), nickel (Ni), molybdenum (Mo), manganese (Mn), vanadium (V), iron (Fe), copper (Cu) and zinc (Zn) are the most common heavy metal that are required by plants in trace amount (Singh et al., 2016). These metals affect enzyme activity, synthesis of DNA, photosynthetic pigments, proteins, and many more cellular metabolisms. Furthermore, they play an important part as a structural and functional component of plant physiology (Oves et al., 2016). Cu and Fe are components of chloroplast, which takes part in photosynthesis. Mn acts as a cofactor for many metabolic enzymes like mallic dehydrogenase and oxalosuccinic decarboxylase. Co is an essential component of cyanocobalamin (Vitamin B12) while, Fe is a crucial component of cytochrome that acts as a cofactor (Gad, 2012, Millaleo et al., 2010, Barker and Pilbeam, 2015, Thomine and Lanquar, 2011). However, any excessive amount of these heavy metals could be harmful to any organism, including plants. These metals are also known as biological essential heavy metals.

Heavy metals like lead (Pb), tin (Sn), mercury (Hg), cadmium (Cd), arsenic (As) and silver (Ag) are not so beneficial for plants growth and its development and therefore, they are called non-biological essential heavy metals. They are known to be a major threat to any organism. These metals can be tolerated by plants at lower concentrations but they become toxic as the concentration of these metal increases. Hence, plants require these metals at a low quantity to carry out its metabolic functions.

On the basis of chemical and physical properties, biologically active metals is classified as any of the two; redox-active or non-redox-active metals. Physiological and cellular homeostatis in plants are affected due to production of reactive oxygen species or oxygen free radicals, a type of oxidative stress produced by redox-active metals such as Cu, Mn, Fe and Cr. For instance, increased levels of heavy metals inside plants is responsible for the degradation of photosynthetic pigments, breakage of DNA strands, degradation of proteins and degradation of the cell wall, which subsequently activates cell death pathways (Anjum et al., 2015). In contrast, non-reactive metals namely Al, Cd, Hg, Ni, and Zn, indirectly generate oxidative stress with the help of multiple mechanisms. For instance, they induce ROS-producing enzyme NADPH oxidases which inhibit antioxidative enzyme production and glutathione depletion.

Natural processes and human activities are well-known reasons for the contamination of heavy metal in the soil. It mainly includes mining, combustion, foundries, agriculture, and smelters (Nagajyoti et al., 2010, Singh et al., 2011). Plants grown on heavy metals contaminated soil becomes the primary victim of heavy metal stress. However, plants can tolerate a certain level of heavy metal concentrations by their internal defense system. For instance, plants encode and upregulate the expression of several metal-specific transporters that bind specifically to certain heavy metals and then helps in its cellular translocation and thereby manage heavy metal toxicity (Hwang et al., 2016; Colangelo andGuerinot, 2006).

Similarly, other anatomical and physiological activities aid plants to manage heavy metal stress up to a certain concentration level. However, under excessive heavy metal concentration, the plant’s internal tolerance and defense mechanism fail to provide resistance, and hence plants become susceptive to the harmful effect of heavy metal stress. Consequently, plants show overall retardation of growth and development leading to yield loss. In this situation, specific manipulation at the genetic level could be an alternative to improve plants and make them more tolerant towards excessive heavy metal toxicity. With the advancement of science and technology, a discussion on new insight into the molecular mechanism of plant defense is highly required. Especially new approaches to understanding the mechanism of tolerance should be discussed. Hence, this review discusses several OMICS approaches to outline the plant's response towards stress like that of heavy metal, and the mechanisms of tolerance. 

2. Adverse effects of various heavy metals on the crops

Adverse environmental stressors like those belonging to biotic and abiotic stress can affect plants in many ways. Among all of these, heavy metal is a kind of stress that can lead to multiple damaging consequences which can even be detrimental to plants. It affects plants in many ways ranging from biochemical response to crop yield (Gill, 2014). Heavy metals mainly consist of Ag, As, Cr, Co, Ni, Cd, Zn and the Pt group elements. Zinc, an important micronutrient is necessary for plants development and maturation but in trace amounts. However, when its concentration exceeds to specific permissible limit, it adversely affects the growth, metabolism and development; also leads to increased oxidative damage in numerous plant species, including Phaseolus vulgaris, Brassica juncea and tobacco (Prasad and Hagmeyer, 1999, Cakmak and Marshner, 1993, Tkalec et al., 2014); a significant increase in calcium (Ca) in leaves of Poplar was also observed (Todeschini et al., 2011). Concentrations ranging from 150 to 300 mg/kg of Zn have been reported in contaminated soils (Warne et al., 2008). Plants growing on contaminated soil with a high level of Zn showed chlorosis initially only on the younger leaves, while older leaves showed chlorosis after prolonged Zn toxicity (Ebbs and Kochian, 1997). A higher quantity of Mn and Cu, types of heavy metal due to toxicity of zinc has been reported in the shoots and roots of some plants (Nagajyoti et al., 2010). Also, it displaces the magnesium ions (Mg2 ions) and inhibits the activity of Ribulose-1,5-bis-phosphate-carboxylase/oxygenase (RuBisCO) an enzyme that is helpful is fixing carbon (Ghori et al., 2019). Moreover, phosphorous (P) deficiency results from Zn phytotoxicity which leads to the development of purplish-red colored leaf symptoms (Lee et al., 1996). It has been observed that Zn toxicity causes catalytic efficiency of several enzymes involved in photosynthesis in Phaseolus vulgaris and pea plants (Romero-Puertas et al., 2004, Somasekharaiah et al., 1992) that consequently, affects the overall production of ATP.

The permitted range of Cd concentration is approximately 100 mg per kg in agricultural soil (Salt et al., 1995). Toxicity of Cd causes chlorosis, and abnormal growth, impedes chloroplast metabolism by interfering with the biosynthesis of chlorophyll, inhibits the catalytic function of several enzymes associated with pathways related to fixation of carbon dioxide (Filippis et al., 1993). Cd toxicity manifestation initially leads to browning or blackening of the root tips and causes death with prolonged exposure (Guo et al., 2008). The overall photosynthetic quantum yield is affected severely due to inhibition of Fe (III) reductase enzyme in response to high Cd levels leading to deficiency of Fe (II)(Alcantara et al., 1994). The ATP hydrolysis function of part of the plasma membrane of wheat roots and sunflower roots are also minimized (Hegedus et al., 2001). Cd interferes with the absorption and transportation of water and numerous elements like Mg, Ca, P and potassium by the plants (Das et al., 1997). High Cd level interferes with the water balance in plants which affects plasma membrane permeability and decreases water content. (Costa and Morel., 1994). In Silene cucubalus plant, high Cd levels had been shown to inhibit nitrate absorption and transportation by inhibiting nitrate reductase activity(Hernandez et al., 1996). It was reported in a study that plants like fenugreek and Alternanthera bettzickiana treated with Cd shows a significant inhibitory effect (Alaraidh et al., 2018). In radish, Cd toxicity inhibits calmodulin-dependent phosphodiesterase enzyme activity by competitively replacing its Ca2 ions cofactor (Rivetta et al., 1997). Cd toxicity severely affects many crops such as Triticum sp. (wheat), Allium sativum (garlic), and Zea mays (maize). In case of wheat, Cd toxicity adversely gets affected by reducing the germinating efficiency of the seed; growth of shoot and root, and overall nutrient content (Ahmad et al., 2012; Yourtchi and Bayat, 2013) in comparison to the accumulation of Cd in garlic decreased shoot growth (Jiang et al., 2001). Similarly, in maize, Cd toxicity gets adversely affected by inhibiting the root and shoot growth (Wang et al., 2007).

Hg is a very toxic and unique metal as it can exist in many forms namely mercury sulfide (HgS), mercury ion (Hg2 ), mercury (Hg), and methylmercury (methyl-Hg) although, in agriculture soil, mercury is exclusively present in its ionic form that is Hg2 (Han et al., 2006). It has no beneficial function (Hameed et al., 2017), but it remains in the soil for many years as it gets incorporated into clay, organic matter, and sulfides. It is highly phytotoxic and easily accumulates in both aquatic and higher plants (Israr et al., 2006). Accumulation of mercury ions in high level induces closing of stomata in the leaf and obstructs water transport in the plants (Zhang and Tyerman, 1999). It also affects the normal mitochondrial and chloroplast function by hindering oxidative phosphorylation, elevating the oxidative stress with biomolecule oxidation, and degradation of the biological membrane (Nagajyoti et al., 2010). Together, Hg toxicity causes disruption of the lipid bilayer of the biomembrane, affected cellular metabolism and obstructs the normal physiology of the plants. (Cargnelutti et al., 2006). It has been revealed that a high-level of Hg ions can severely affect the genomic stability of plants (Malar et al., 2015).

Arsenic (As) is a type of metalloid; being a phosphate (P) analog it competes for the same carriers present in the plasma membrane of the plant roots (Meharg and Macnair, 1992). High-level toxicity affects various crops such as Lycopersicon esculentum (tomato), Brassica napus (canola), and Oryza sativa (rice). In canola, toxicity leads to leaf chlorosis, stunted growth chlorosis, and wilting (Cox et al., 1996). Whereas, in tomato it decreases the weight of fresh leaves and reduces the fruit yield (Barrachina et al., 1995). It adversely affects rice by reducing the germinating efficiency of the seed, dry matter weight, and by shortening the seedling height (Abedin et al.,2002). It is observed that many species of plants have tolerance for arsenic toxicity like grasses (Sharples et al., 2000) and this is due to inhibition of high affinity P/As uptake system (Meharg and Macnair, 1992).

Lead is the most ubiquitous and considered the most toxic metal which contaminates the soil. This toxic heavy metal accumulated in the soil due to natural weather processes, mining, and smelting (Ashraf et al., 2015). A high level of Pb (100-200 ppm) causes chlorosis and inhibited shoot and root growth. Additionally, it exerts a very detrimental effect on plant growth, biomass production, morphology, and photosynthetic processes membrane permeability and water imbalance (Sharma and Dubey, 2005). It triggers oxidative stress by elevating ROS synthesis in the plants (Reddy et al., 2005). It reduces carboxylating enzymes and ruins the chloroplast ultrastructure and blocks the production of chlorophyll and carotenoids, including plastoquinone which are essential pigment, thus severely affecting photosynthesis. Moreover, the electron transport chain and Calvin cycle are also responsible for closing of stomata due to which carbon dioxide reduces (Sharma and Dubey, 2005). In maize, Pinus helipensis, and Spartiana alterniflora, Pb is reported to affect seed germination (Morzck and Funicclli, 1982). A high concentration, lead toxicity also changes the plant morphology. It includes lignification of cortical parenchyma and altered thickening of roots and cell wall of the endodermal layer of pea (Paivoke, 1983). The proliferated effect on the processes related to repair in vascular type of plants is induced by Pb (Kaji et al., 1995), decrease the height of the plant, leaf numbers, and leaf size in Portia tree (Thespesia populnea) (Kabir et al., 2009), whereas in Oats plant (Avena sativa) it inhibits the function of the enzyme which ultimately badly affects the photosynthesis (Kepova et al., 2004).

Chromium is the most toxic metal to plants and is present in groundwater, soil, and sediments due to sewage and industrial waste disposal. Chromium toxicity is very detrimental to seed germination of various crops such as Echinochloa colona (Rout et al., 2000), Phaseolus vulgaris (Parr and Taylor, 1982), Lucerne (Medicago sativa) (Wang,1996), Onion (Allium cepa) (Nematshahi et al., 2012). Whereas, in Lycopersicon esculentum Cr toxicity results in the decreased acquisition of plant nutrients (Moral et al., 1995; Moral et al., 1996). Additionally, a decrease in root growth was found in wheat (Triticum sp.) (Sharma and Sharma, 1993; Panda and Patra, 2000). Usually, chromium toxicity induces the following toxic effects: (i) Changes in synthesis of necessary plant pigments, for instance, anthocyanin and chlorophyll (Boonyapookana et al., 2002) (ii) Elevated synthesis of metabolites that are detrimental to the plants, for instance, ascorbic acid and glutathione (iii) induces synthesis of newly biochemical related metabolites, such as histidine, and phytochelatins, which may show resistance/tolerance against Cr stress (Schmfger, 2001). It was observed that chromium stress decreases the function of enzymes such as amylases and thus affects the sugar transport to the axes of the embryo (Zeid, 2001). Chromium toxicity is also reported in allium plants (Nematshahi et al., 2012). The effect of chromium toxicity on electron transport chain, Co2 fixation, and photophosphorylation is well documented (Clijsters et al., 1985).

Copper is counted as one of the most essential source of micronutrient for a plants development and maturation (Gang et al., 2013). Copper has been accumulated by mining and industrial activities. It plays a vital role in ATP synthesis and carbon dioxide assimilation (Pichhode and Nikhil, 2015). Furthermore, it is a fundamental component of several metalloproteins such as plastocyanins and cytochrome oxidase (Demirevska-kepova et al., 2004). The cytotoxic effect of copper in the soil causes stress, leaf chlorosis, ROS, and retarded plant growth (Lewis et al., 2001). High-level of Co buildup in the soil affects the growth of Alyssum montanum plants and reduces the root growth in Brassica juncea and Chloris gayana (Rhodes grass) and Cucumis sativus (cucumber) (Singh and Tewari, 2003, Moreno-Caselles et al., 2000, Ouzounidou, 1994, Pichhode and Nikhil, 2015,). In Solanum melongena, Co in combination with Cd, severely affects the seed of germination, seed size and formation of secondary roots (Neelima and Reddy, 2002). Additionally, the plant mortality, biomass, and seed production are drastically reduced in black bindweed (Polygonum convolvulus) due to copper toxicity (Pichhode and Nikhil, 2015), whereas in bean (Phaseolus vulgaris) Cu toxicity is responsible for reduced plant roots and root malformation (Katare et al., 2015).

Ni is a transition element; it is found everywhere in the environment. In natural soils, except in ultramafic or serpentine soils, it is present at trace concentrations. However, industrialization and human activities have increased Ni2 concentration and its toxicity in soil. These activities include mining works, smelting, burning of fuel, sewage disposal, phosphate fertilizers, and pesticides (Aziz et al., 2015). In agricultural soil, the concentration of Ni is around 10 to 1000 mg/kg, while in the polluted soil, its concentration ranges 20-30 fold higher range (200 to 26,000 mg/kg) (Izosimova, 2005). Hence, it causes various toxic effects such as chlorosis, necrosis, nutrient deficiency, and disrupted cell membrane function in different plants including rice (Das et al., 1997, Pandey and Sharma, 2002; Rahman et al., 2005). Additionally, it causes reduced water content in many plant species that belong to either dicot or monocot plants. Thus, decreased water uptake is a sign of Ni metal toxicity (Yadav 2010). In Oryza sativa plants, Ni toxicity was observed to affect the H-ATPase function and alter plasma membrane composition (Ros et al., 1992). Therefore, Ni toxicity was also found to be detrimental to the growth, development, and photosynthetic ability of plants.

3. Omics approaches for heavy metal stress tolerance

Identifying gene products like proteins, metabolites and transcripts present in a specific biological sample is important to deal with molecular-level identification, discovery and introduction of new strategies to improve certain limitation. ‘OMICS’ (transcriptomics, metabolomics, and proteomics) has opened many new possibilities for this and among the many other advances in systems biology can play a crucial role in understanding and producing crops that would be able to tolerate heavy metals (Singh.S et al., 2016). By using the techniques like transcriptomics, metabolomics and proteomics, characterization of different metabolites, TFs and proteins responsible for the stress can be carried out to understand them and build a process to overcome heavy metal tolerance in plants. Omics approaches for heavy metal stress tolerance are summarized in figure 2.

3.1. Transcriptomics study

Transcriptomics profiling is helpful to understand the TFs involved in gene expression falling under certain situation (Bräutigam. A et al., 2016). Aluminium stress in plants was found to be regulated using miRNA by using RT-qPCR approach to study the response to such metal stress and 16 different aluminium stress responses of miRNA was determined (Lima et al., 2011). Similarly, other metals which have adverse effects on plants can be studied by understanding the transcriptional factors responsible for the regulation and expression of certain genes. TFs are known to regulate many genes that are responsible for a response towards stress. These genes can be studied in detail and hormones that activate or enhance the effect of such genes can be supplied to the plant to make it tolerant towards stress. Hormones that can lead to overexpression of genes that ensure tolerance towards heavy metals can also be used (Singh.S et al., 2016).

The investigation of the basic mechanism behind the adaptation and tolerance of heavy metals is of great scientific importance. Stress tolerance in plants is induced due to signaling pathways linked to expression of protein-coding genes which are triggered by different stressors (Tran et al., 2010; Valliyodan and Nguyen,2006; Nakashima et al., 2009; Umezawa et al., 2006; Manavalan et al., 2009;). These genes encode various TFs (Table 1) which are found to be stimulating plants response towards stress (Singh et al., 2002; Shiu et al., 2005; Shameer t al., 2009; LeDuc et al., 2006), and regulate corresponding transcriptional processes. Arabidopsis thaliana (Arabidopsis) and B. juncea plants when exposed to Cd stress, upregulate the expression of transcription factors such as bZIP and Zinc-finger motif-containing transcription factors (Ramos et al., 2007). Also, Arabidopsis exposed to metal stress like cadmium, the expression of ERF1 and ERF5 belonging to AP2/ERF superfamily was found to be upregulated. Moreover, a study showed that the expression of transporters like FIT with AtbHLH38 or AtbHLH39 also triggers the expression of many other TFs like HMA3, Iron Regulated Transporter2 (IRT2) and MTP3 that serve a governing role in sustaining Fe content during Cd toxicity. Various studies have confirmed the fact that single TF controls the expression of various targeted genes (Wray et al., 2003; Nakashima et al., 2009). Dehydration-responsive element-binding protein (DREB) transcription factor helps the plants to deal with heavy metal toxicity by normalizing potential, thus flow of water contaminated with heavy metal could be slowed down in Arabidopsis roots (Nakashima et al, 2006). When the plants like Medicago sativa exposed to heavy metal stress, MAPK cascade are stimulated in its response and this cascade has its importance as transduction of the signal downstream regulated by it (Jonak et al., 2004). Therefore, the genes required for the biosynthesis of chelating compounds, activation of metal transporters, and other defensive compounds have been activated by all the transcription factors.

Micro RNAs (miRNAs) has been known to work as a modulator which regulates different types toxicity due to metals in different types of plants and helps in providing tolerance toward stress.(Noman et al., 2017a; Noman and Aqeel 2017). Studies involving Arabidopsis, Medicago Sp, and P. vulgaris plants had shown miRNA regulates various types of the cellular and metabolic process throughout abiotic stress response (Budak et al., 2015; Trindade et al., 2010). It has been identified that many miRNA transcripts are involved in making plant tolerant against heavy metal stress. For example, 13 different miRNAs were found to be linked to metal stress regulation in P. vulgaris (Li et al., 2015a, b). In Brassica napus, Cd toxicity causes downregulation of miR156, miR171, and miR396a transcripts (Zhou et al., 2012a, b). Similarly, in Medicago, it was found that the miR166 transcript was downregulated. Whereas, in M. truncatula, miR171, miR319, miR393, and miR529 were found upregulated due to Hg stress (Zhou et al., 2008).

In Chinese rice, 18 different miRNA were observed to be differentially expressed in As toxicity/stress (Liu and Zhang 2012). Similarly, in B. juncea, 69 novel miRNAs were observed to be expressed in a different way due to Astoxicity/stress (Srivastava et al.,2012). Additionally, under As stress, application of phytohormones such as jasmonic acid (JA) and Indole-3-acetic acid (IAA) exogenously resulted in altered expression of various miRNAs such as miR167, miR319, and miR854. This altered expression of miRNAs helped in stimulating plant growth under As stress (Gupta et al., 2014). Likewise, in rice, oxidative stress generated by heavy metal stress results in differential expression of around seven miRNAs genes. These genes encode components of many critical physiological processes such as cell proliferation, gene regulation, phytohormone homeostasis, and nutrients transport (Li et al., 2010).

3.2. Proteomics study

Proteomics is known to be a very important technique discovered in the post-genomic era (Liu et al., 2013). It is one of the major advances in OMICS technologies as it helps in understanding the complex proteins and its characterization of interaction and response of plants with heavy metals at the molecular level. It also helps in understanding variation in protein profiles that can be compared at different developmental stages and under conditions including stresses like heavy metal stress (Ahsan et al., 2009). While genomics, transcriptomics, metabolomics have increased the basic knowledge related to the plants response towards HM stress, but these alterations are not often exhibited at the protein level (Gygi et al., 1999; Hossain and Komatsu, 2013). For example, MHX which is Mg and Zn transporter protein was found to be increased in Arabidopsis. However, there were no changes in its transcript level (Elbaz et al., 2006). This shows that there is no guarantee that functional protein can be achieved from a gene after translation. Factors like protein folding, translational modifications, various protein-protein interactions, post-transcriptional modification, stability and altered localization are determinants of protein function (Dalcorso et al., 2013b). Therefore, this tool offers a new platform to identify target proteins associated with detoxification of heavy metal and analyzing the intricate biological processes and interactions (Ahsan et al., 2009).

It was observed by using proteomics studies that proteins plays a critical role in the process of detoxification in plants. This happens when a large number of metal ions are present in soil and they activate different class of proteins that helps in detoxification for the plant as a reaction to HM stress (Ahsan. N et al., 2009). By using this study, phytohormones can be used exogenously to induce such protein detoxification processes, which in turn can increase tolerance towards HM in the plants. Additionally, proteins are caught up in plant stress response against heavy metal toxicity by changing or modulating various metabolic pathways involved in photosynthesis and mitochondrial respiration. They meet the increased energy demand of metal stressed plants by producing more high-energy compounds such as ATP, NADH, NADPH, and FADH2 (Hossain and Komatsu, 2013). For instance, to overcome the heavy metal stress, plants accumulate an increased level of the large subunit of RUBISCO proteins, NAD(P)H-dependent oxidoreductases, oxygen-evolving complex protein 1 and 2, and photosystem I and II-related proteins (Semane et al., 2010). Furthermore, protein profiling of the plants under heavy metal stress consequently activates signaling cascades and defense-related genes (Hossain et al., 2012c). Therefore, this technique helps to control the redox homeostasis in the plants (Zhao et al., 2011; Sharmin et al., 2012; Wang et al., 2012). It has been studied that the activity of proteasome present in Arabidopsis plant affected because of metal toxicity which in turn increase 20S core unit results in protein degradation of oxidized proteins and so increases the tolerance towards oxidative stress or the As treated cells (Kurepa et al., 2009).

3.3. Metabolomic profiling

Metabolomics is a very useful approach to study low molecular weight metabolites by identifying and quantifying them in an organism. Identification and quantification of different metabolites of the plants grown under certain defined environmental conditions are termed as metabolomics. This tool can be used to understand which metabolic pathway or which metabolite is getting affected by heavy metal stresses (Le Lay P et al., 2006). Few of the metabolites present in plants have shown their association with the regulation of HM stress. Some amino acids like proline and histidine tend to build up under heavy metal stress. This accumulation helps in chelation of the metal ions and supporting tolerance towards heavy metals in plants (Sharma S et al., 2006). Metal chelation, antioxidation, and signaling are some of the major techniques used by the metabolites like amino acids, organic acids, phenols, and others to act on heavy metals and make plants tolerant (Singh.S et al., 2016).

The metabolite amount is proportionate to the physiological age and environmental condition of the plants (Bundy et al., 2005). It was reported that the physiological state of the plants also changes with toxicity caused by heavy metal. The effect of stress due to metals tend to increase due to production of specific metabolite and increase in stress responsive type of genes induced by receptors due signals released by plants (Nakabayashi and Saito 2015).Various bioanalytical techniques like matrix-assisted laser desorption ionization (MALDI), liquid chromatography-mass spectroscopy (LC-MS), gas chromatography-mass spectroscopy (GC-MS),nuclear magnetic resonance spectroscopy (NMR), and inductively coupled mass spectrometry has been developed in plants to identify and analyze different metabolites in the plants.

3.3.1. Free amino acids

Plants have numerous suitable solutes which are required under distinct environmental cues such as heavy metal and other abiotic stress (Serraj and Sinclair 2002; Akula and Ravishankar 2011). These solutes comprise of alanine betaine, betaine, glycine, histidine, pipecolate betaine, polyols, proline, sucrose, and trehalose and (Sharma and Dietz 2006; Ashraf and Harris 2004;). These metabolites protect the plants to with stand the negative impacts of different environmental stresses. These metabolites fortify plants by detoxification of ROS, adjusting cellular osmosis adjustment, stabilizing protein/enzymes, and maintaining the integrity of the biological membrane (Ashraf and Foolad 2007; Bohnert and Jensen 1996). Additionally, these solutes help plants to overcome heavy metal stress; proline act as a chelator for metals, signaling molecule, antioxidant protection molecule and also increases the metal metal-quenching capacity of other antioxidant enzymes (Hayat et al., 2012; Emamverdian et al., 2015). Notably, during toxicity of heavy metal, proline is elevated by many folds in both lower plants, such as algae and lichens as well as in higher plants (Nedjimi and Daoud 2009; Siripornadulsil et al., 2002; Belhaj et al., 2016; Bačkor and Loppi 2009). Additionally, another amino acid such as putrescine; aprecursor of spermine, and spermidine is known to play a significant role in response to abiotic stress of the plants. Therefore, it significantly increased in plants like rice, bean, and oat (Lin and Kao 1999; Weinstein et al., 1986).In Alyssum lesbiacum plants, high level of histidine has been reported in response to stress due to Ni (Singh et al., 2016).

3.3.2. α-Tocopherol

α-Tocopherols are mostly produced in plastids of plant cell and are known as an dynamic form of vitamin E. It plays a crucial function in combating various metal and environmental stresses by scavenging ROS and deactivate up to 220 molecules of O2. Additionally, they are found to be responsible for the termination of lipid peroxidation chain reaction (Maeda and DellaPenna 2007; Munne-Bosch 2005;). They are found to protect cell membrane integrity. In response to Cd and Cu stress, a considerable raise in tocopherols concentration was observed in many plants, including Arabidopsis plants (Lushchak and Semchuk 2012; Collin et al., 2008). Studies with two mutant Arabidopsis corroborate the link between increased α-tocopherol accumulation and alleviation of heavy metal stress. It was observed that the quantity of tocopherols increases in response to a variety of environmental stresses.

3.3.3. Glutathione

Glutathione (GSH) is chemically γ-glutamyl-cysteinyl-glycine; a non-protein metabolite with decreased molecular weight that mimics like an antioxidant and is a free radical scavenger. It is present in most of all cell organelles such as vacuoles, chloroplasts, endoplasmic reticulum, mitochondria and cytosol (Foyer and Noctor, 2003). It has a major role in various cellular and physiological functions such as growth of cell, nucleic acids and protein synthesis, signal transduction, phytochelatins synthesis, detoxification of xenobiotics, enzymes regulation, conjugation of metabolites, sulfate transport regulation and expression of the genes that respond to stress (Foyer et al., 1997). Additionally, the elevation of GR activity was studied in the Luffa seedlings contaminated with metal toxicity (Singh et al., 2015). Similar activity has been found in Helianthus annuus when exposed to multimetal toxicity (Belhaj et al., 2016).

3.3.4. Ascorbate

Ascorbate acts as a powerful antioxidant. It is known to play several important physiological activities of plants, such as cellular growth, metabolism, and differentiation and it also acts against ROS activity (Noman et al., 2014).

3.3.5. Phenols and carotenoids

Phenols such as tannins, hydroxycinnamate esters, lignin, and flavonoids are known to be one of the most abundant secondary metabolites of the plants (Grace and Logan 2000). They are capable of chelating metal ions because of the existence of carboxyl groups and hydroxy groups under heavy metal stress, specifically copper and iron (Jun et al., 2003). Therefore, they are involved in the scavenging of ROS and inhibition of lipid peroxidation. In leaves of the maize and P. vulgaris plant under aluminium and Co stress, an increased level of phenolic compounds was observed (Winkel-Shirley, 2002; Diáz et al., 2001). Similarly, phenolic compound levels were found to be elevated in lentil roots that were exposed to stress due to copper (Janas et al., 2009). High phenolic and flavonoid compounds have been reported in Trigonella foenum (Zayneb et al., 2015). Additionally, increased flavonoid accumulation was reported in wheat under heavy metal stress (Copaciu et al., 2016). It was also found in another study that induction of flavonoids can improve the antioxidant property of transgenic potato plants (Lukaszewicz et al., 2004). It has also been revealed that tea plants that are rich in tannins are found to chelate the Mn and thus reduce its toxicity (Lavid et al., 2001).

Carotenoids are the lipophilic compounds; it absorbs energy from sunlight and transfers it to chlorophyll pigment. They act as antioxidants and inhibit ROS (Young 1991). Besides this, they also act as a signaling molecule that perceives environmental cues and communicates them to the plants (Li et al. 2008). The plants that are exposed to heavy metal stress accumulate a high level of carotenoids (Piotrowska-Niczyporuk et al. 2015; Soares et al. 2016; Wang et al. 2014).

4. Strategies for metal tolerance

Heavy metals have shown a destructive effect on the growth of plant, its development and yield of the plants. However, plants evolve various strategies to combat metal toxicity like chelation, selective uptake, compartmentalization, and toxic metals secretion (Pourrut et al., 2013).A few of them are described here in brief.

4.1. Phytochelatins and metallothioneins

It is very vital to develop resistance towards heavy metal stress when plants uptake a huge quantity of these toxic metals. Phytochelatins and metallothioneins are cysteine-rich protein molecules that bind to heavy metals. They play an important function in detoxification of the heavy metal in plants by formation of mercapeptide bonds with several heavy metals (Maestri et al. 2010; Jiang and Liu 2010). They act as natural metal-binding chelators and transport toxic metals from the cytosol to vacuoles of plants by metal/H antiporters or ABC transporters (Israr et al., 2011; Xu et al., 2011). Cd is the strongest inducer of phytochelatins as all the metals do not tend to form a complex with phytochelatins. PC-metal complexes are highly resistant towards proteolytic degradation (Bertrand and Guary, 2002). Metallothioneins also contribute to antioxidant protection and plasma membrane repair mechanisms (Goldsborough, 2000). Heavy metal sequestration to the vacuoles via different mechanisms is shown in figure 3.

4.2. Antioxidant defense system and ROS

In plants, heavy metals accumulation leads to the synthesis of ROS, namely, hydroxyl radicals (OH.), hydrogen peroxide (H2O2) and superoxide radicals (O2.-). Among them, hydrogen peroxide plays a crucial function as an intermediate signaling molecule to control the defense mechanism (Rastgoo and Alemzadeh, 2011; Peleg and Blumwald, 2011). Accumulation of heavy metal causes elevation of ROS production, which results in the peroxidation of some crucial constituents of the cell. They contributed by developing an antioxidant response and induce resistance to heavy metals. They work best at moderate temperatures but damages cells at greater concentrations. It was reported that Ni stress in wheat, Cd stress in pea plant, Pb stress in Viciafaba, Cd and Cu stress in Arabidopsis can results in elevated level NADPH oxidase-dependent ROS (Hao et al., 2006, Rodr ́ıguez-Serrano et al., 2006, Pourrut et al., 2008, Remans et al., 2010). Various enzymatic antioxidants such as superoxide dismutase, peroxidase, catalase, and glutathione-S-transferase assist to convert superoxide radicals into hydrogen peroxide and finally to water and oxygen. Additionally, Fenton reaction can form • OH from H2O2 or H2O2 and O2- using the Haber-Weiss reaction in response to metabolic ROS resources with redox-active HMs (Fe, Cu, Cr, V, and Co) (Halliwell et al., 2006). Moreover, previous studies had shown that proline helps in the regulation of plant growth and maintains the osmotic balance as it acts as an osmolyte as well. Under the heavy metal stress, it also defends plants from the accumulation of ROS in regards to Cd toxicity (Islam et al., 2009).

5. Genotoxicity in response to heavy metal stress

Heavy metals can impose a harmful effect on the plants by entering and binding to the nucleus and results in promutagenic damage that comprises DNA base modifications, rearrangements, intra- and inter-molecular cross-linkage of DNA and proteins, DNA strand breakage, and depurination. These destructive effects are caused by the induction of oxidative species that are generated in response to metal toxicity. It results in the production of 7,8-dihydro-8-oxoguanine, which is promutagenic adduct that base pairs with adenine leading to transversion of C to T (Cunning- ham 1997; Kasprzak 1995). It has been observed that there is a reduction in the mitotic activity of meristem in the roots of maize due to the accumulation of nickel. Additionally, it has been reported that Helianthus annuus shows clastogenic effects in response to time and concentration-dependent copper, cadmium, and nickel (Chakravarty and Srivastava, 1992).

6. Plant hormones and heavy metal stress

When some plants are exposed to a variety of abiotic or biotic stresses, it uses its own defense mechanism against these stresses to become tolerant. But many times, due to the increase in stress, these defense mechanisms are not sufficient. This defense mechanism can be stimulated by phytohormones presents in plants. Several strategies have been introduced to supply these hormones to plant and stimulate the defense mechanisms. These strategies are; to supply phytochemicals like auxins, gibberellins, and cytokinins exogenously or by manipulating their content endogenously by using genetic engineering methodologies. This process can not only help plants tolerate the abiotic stresses but also help in promoting crop yield and productivity (Egamberdieva et al., 2017). It was observed that application of Brassinosteroids, ethylene, and salicylic acid exogenously in plants containing heavy metals show enhanced growth and development as the heavy metals tolerance rate is increased (Sytar et al., 2018).

By using the OMICS approach, it becomes easier to understand how a plant responds to the heavy metal, thus making it easier to develop a strategy to promote the plant’s tolerance towards heavy metal. Using phytohormones to stimulate the plants growth and development and tolerance towards heavy metals is a new approach, and OMICS technologies can be used to study the response of plants after the application of these hormones towards heavy metal stress. This can be done by using transcriptomics to study the effect of hormones on the transcriptional level, metabolomics to understand if the phytohormones elevate any metabolic pathways, and proteomics to understand if proteins play any part in the process of heavy metal tolerance after the supply of plant hormones. Brassinosteroids and gibberellin are found to be the most effective plant hormone that increases the plants ability to tolerate heavy metals stress.

ABA is an important plant defense hormone; it plays an important role to overcome different kinds of abiotic stressor including that due to heavy metals. In heavy metal stress conditions, the expression and signaling of biosynthetic genes of ABA were found to be enhanced (Bücker-Neto et al. 2017). Additionally, 10% of protein-coding genes were found to be transcriptionally controlled by ABA (Wani et al., 2016). It has been reported by using ABA deficient and insensitive mutants that decreased growth of the plants due to Cd toxicity is not because of ABA signalling. Instead, the elevated ABA level is due to triggered HM stress to regulate the water balance by opening and closing stomata (Sharma and Kumar, 2002). However, the role and mechanism of ABA under HM stress is needed to be explored. Moreover, ABA maintains a balance between the growth and survival of plants under heavy metal stress. A study found in Solanum tuberosum that the expression of 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) and ABA synthesis induced in response to exogenous supply of Cd, which in turn leads to elevated production of PCs by increasing the expression of phytochelatin synthase (PCS) gene.

Gibberellins belong to the tetracyclic diterpenoid carboxylic group of compounds made up of four isoprene units, but only one form of GAs i.e., GA1, in its active form regulates the plants growth and its development under HM stress (Sponsel and Hedden, 2004). It functions as an important growth regulator influencing seed germination, flower and trichome initiation leaf expansion, stem elongation, enzyme induction, dormancy, sex expression and development of fruits (Yamagu- chi 2008). It also helps in adapting tolerance and protecting plants from heavy metal stress. In wheat, gibberellic acid causes increased expression of the TaMYB73 gene in reaction to HM stress (He et al., 2012). Another report has assured that DELLA proteins, a repressor of GA responses, take part in helping plants in stress prevention (Wild and Achard 2013). Additionally, the concentration of GA3 enhances over small doses of Zn while decreases on its high doses (Atici et al., 2005). Furthermore, GAs play a significant function in signalling pathways, but their exact transport mechanism to different parts is still a mystery and needs to be explored (Gupta and Chakrabarty 2013). It was found that GA reduces Cd-stimulated detrimental effect on growth and seed germination of Brassica napus by regulating oxidative stress and ROS damage (Meng et al., 2009). Additionally, under the metal stress, adenosine-5’-phosphosulfate reductase (APR) expression is induced, which is required for sulfate assimilation in Arabidopsis (Koprivova et al., 2008).

Cytokinin is also a crucial hormone that regulates plant development. It was observed that its endogenous concentration changed under heavy metal stress, showing its involvement in stress tolerance (Brien and Benkova 2013). Heavy metal stress causes a decrease in CKs and hinders its transport from roots to different plant parts. It has been reported that cytokinin crosstalk and interacts with other hormones during HM stress (Ha et al., 2012). For instance, the concentration of CKs level is interdependent on other hormones like ABA under HM stress as they often act opposite to each other (Pospíšilová 2003a). It has been reported that cytokinin response 1(CRE1) gets downregulated in reaction to As stress which elevates the expression of group 1 sulphate transporters in Brassica juncea (Srivastava et al., 2009). Additionally, an alleviated level of cytokinin was reported to repond towards Cu stress in Arabidopsis (Lequeux et al., 2010). Additionally, thiol compounds like Phytochelatins (PCs) were also found to be accumulated in response to As tolerance mechanism, which is activated by increased expression of cytokinin oxidase/dehydrogenase 1 (CKX1) (Mohan et al., 2016). Also, in a transgenic approach, tobacco plants having a high expression of isopentenyl transferase (IPT) gene; thus, more accumulated cytokinin was found to have a high tolerance against Cu stress (Thomas et al., 2005). Therefore, cytokinin plays a significant function in developing strategies towards heavy metal stress.

Auxin is the first known phytohormone; it plays a significant function in plants yield under normal as well as under stressful situaion. Various naturally present auxins are known, for example, indole 3-butyric acid (IBA), indole 3-acetic acid (IAA) and phenyl acetic acid (PAA). IAA is the principal naturally occurring auxin and the multifunctional phytohormone (Kazan 2013). It was found to be taking part in adapting plants under high salinity and heavy metal stressed conditions. A significant increase in IAA level has been seen in heavy metal stress response, which results in very little or no growth and production of higher plants (Fahad et al., 2015). Exogenous supply of IAA induces hormone interaction and expression of miR167, miR319, and miR854, which in turn helps in the regulation of As stress in the Brassica juncea (Srivastava et al., 2013). Furthermore, the interaction of IAA, ROS, and ethylene plays a crucial role in signalling pathways with modification in the root of the plants (Camacho-Cristóbal et al., 2015).