Heavy Metal Tolerance in Plants: Role of Transcriptomics, Proteomics, Metabolomics, and Ionomics

Metallophytes under heavy metal stress

Metallophytes, also known as hyperaccumulators, have the ability to uptake large amounts of heavy metals from the soil, and this property makes them unique to be utilized in technologies such as biogeochemical and biogeobotanical prospection and phytoremediation. The absorbed heavy metals from the soil by these hyperaccumulators are not retained in the roots but are translocated to the shoots and accumulated in the aboveground organs at concentrations 100–1000-fold higher than the observed in non-hyperaccumulating species (Figures 2ia,b). However, this high concentration does not pose any toxic effect on plants (Rascio, 1997; Reeves, 2006; Prasad et al., 2010). With significant advances in our understanding of the mechanisms adopted by hyperaccumulators, there has been implication of three hallmarks that distinguish them from non-hyperaccumultors. These are (i) greater capability of heavy metal uptake, (ii) root-to-shoot translocation of heavy metal, and (iii) detoxification and sequestration of heavy metal (Figures 2iia–c). Studies on Thlaspi caerulescens and A. halleri, model plants for studying heavy metal tolerance strategies, have been done (Milner and Kochian, 2008; Singh et al., 2009; Frérot et al., 2010; Krämer, 2010). The studies have revealed that hyperaccumulation is not due to the presence of a novel gene, but it arises only from differential expression of genes that are common to hyperaccumulators and non-hyperaccumulators (Verbruggen et al., 2009). Hyperaccumulation of heavy metal includes three complex phenomena discussed below:

Plant antioxidant defense system

The term “antioxidant” refers to a class of compounds that protect cells from damage caused by exposure to certain highly reactive species like ROS. The network and coordination of antioxidants are solely responsible for removing, neutralizing, and scavenging ROS. SOD is an enzyme involved in dismutating superoxide radicals generated by oxidation of molecular oxygen into H₂O₂ and O₂ in all the cellular compartments (Fridovich, 1989).

H₂O₂ produced by the action of SOD is quite dangerous as it can diffuse through the membrane very easily and damage other cellular components, and thus, metabolites (ascorbate and glutathione) and enzymes (monodehydroascorbate reductase; MDHAR, dehydroascorbate reductase; DHAR and glutathione reductase; GR) are implicated in scavenging of H₂O₂(Foyer et al., 1997). Three types of SODs have been reported in plants on the basis of the metal containing (1) the chloroplastic or cytosolic Cu–Zn SOD; the cytosolic Cu–Zn SOD is referred to as Cu–Zn SOD I, whereas the chloroplastic one is referred to as Cu–Zn SOD II; (2) Mitochondrial Mn SOD, and (3) the chloroplastic Fe SOD. APX is regarded as a housekeeping protein in the cytosol and chloroplast, and is involved in scavenging of H₂O₂. The substrate for this enzyme is ascorbate and the product, which is a radical, is reduced to dehydroascorbate by an enzyme MDHAR in the presence of an electron donor NADPH (Asada, 1992, 1996). CAT is an important oxidoreductase enzyme that catalyzes decomposition of H₂O₂ into H₂O and O₂, and it is found in most plants and is localized in the peroxisome. CAT is a key enzyme involved in detoxifying peroxides generated during photorespiration (Morita et al., 1994; Lin and Kao, 2000). Although APX and CAT serves the same function of detoxifying, different affinities (on the basis of Km values) of APX and CAT depict the role of APX in modulating H₂O₂ for signaling and CAT in detoxifying excess H₂O₂ during stress (Mittler, 2002).

The above mentioned enzymatic components play a relevant role in mitigating heavy metal stress. Several studies have revealed that treatment of heavy metal enhances ROS formation, and thus, substantial increase in the activities of SOD, CAT, and APX was observed (Bharwana et al., 2013; Bashri and Prasad, 2015). A study by Wang et al. (2004) revealed a considerable increase in the activities of POD, APX, and SOD under Cu stress in B. juncea seedlings. Similarly, Bharwana et al. (2013) showed that under Pb treatment, there was an appreciable rise in SOD, guaiacol peroxidase, APX and CAT activities, and their activities were further enhanced with the rising concentration of Pb from 50 to 100 μM. Similar to this, Singh et al. (2013) reported increased activity of SOD and CAT under As exposure (5 and 50 μM). These results suggest that cooperative action of antioxidants is required for a detoxification mechanism under heavy metal stress.

Metabolomics

Metabolomics refers to the identification and quantification of all low-molecular weight metabolites required by the organisms during developmental stages (Arbona et al., 2013), and some metabolites have been reported to be involved under heavy metal stress tolerance strategies. In the following section, we discuss the role of metabolomics under heavy metal stress.

Ionome and ionomics

Ionome includes the role of mineral nutrients, namely nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), sulfur (S) and magnesium (Mg) and trace metals namely iron (Fe), copper (Cu), manganese (Mn), molybdenum (Mo), cobalt (Co), and zinc (Zn) in alleviating heavy metal toxicity. Although all the mineral nutrients and trace elements are essential for growth and development processes of plants, concentration greater than the required level becomes toxic to the plants. Apart from posing toxicity at higher concentration, nutrients under safe limit play important role in alleviating toxicity induced by heavy metals.

Nitrogen is the most essential nutrient as it is the major constituent of proteins, nucleic acids, vitamins, and hormones. It has the potentiality of alleviating heavy metal toxicity, as it enhances the photosynthetic capacity by increasing chlorophyll synthesis, often synthesizes N-containing metabolites like proline, GSH, etc. and by enhancing the activity of antioxidant enzymes (Sharma and Dietz, 2006; Lin et al., 2011). In a study by Pankovic et al. (2000), it has been shown that supplementing 7.5 mM (optimal level) of N to sunflower reduced the inhibitory effect of Cd on photosynthesis by enhancing Rubisco activity and by increasing protein content. In another study by Zhu et al. (2011), it has been shown that supplementing N fertilizer in the form of 16 mM (NH₄)₂SO₄, alleviated Cd-induced toxicity in Sedum. The alleviating potential not only depends on the supplemented level of N but also on the source of N. For instance, when N was applied in the form of ${\text{NH}}_4^{+}$-N, it reduced the Cd concentration in leaves of rice plants that was found to be below 100 mg kg⁻¹ (Jalloh et al., 2009), but when supplemented as ${\text{NO}}_3^{-}$N, it increased the Cd concentration, which suggests antagonistic behavior of ${\text{NH}}_4^{+}$- while synergistic of ${\text{NO}}_3^{-}$ toward Cd. Another mineral nutrient, phosphorus (P) is the major constituent of cell membrane and nucleic acid, and majorly required for phosphorylation reaction. It has also been reported in alleviating metal-induced toxicity either by diluting the metal or by decreasing the mobility of the metal by forming metal–phosphate complex (Sarwar et al., 2010). In addition, P can also increase GSH content and prevent membrane damage, thereby conferring tolerance to plants (Wang et al., 2009).

Potassium (K) ion is required by the plant to maintain anion–cation balance in cells and plays important regulatory role in protein synthesis and enzyme activation. By improving nutritional status of K, condition of oxidative stress in plants can be minimized (Shen et al., 2000). Supplementation of K at 60 mg kg⁻¹ alleviated the toxicity induced by Cd at 25 mg kg⁻¹ by increasing the content of AsA and GSH. Similar to nitrogen, K source may also play an important role in alleviating toxicity. A study by Zhao et al. (2004) clearly demonstrated that application of KNO₃, K₂SO₄, and KCl at the rate of 55, 110, and 166 mg.K.kg⁻¹, respectively, to the soil has differential effect on Cd (concentration 15 mg Kg⁻¹) accumulation. When KCl and K₂SO₄ were applied in increasing concentration from 0 to 55 mg kg⁻¹, there was 60–90% increase in Cd accumulation in shoots, whereas similar increasing concentrations of KNO₃ increased the Cd content very marginally, suggesting its protective action against Cd stress.

Sulfur (S), another mineral nutrient, serves as an important constituent of several coenzymes, vitamins, and ferredoxin. Wangeline et al. (2004) reported that Cd toxicity could be alleviated by the upregulation of S-assimilation pathway, thus suggesting toward alleviating role of S under heavy metal toxicity. Studies on Triticum aestivum (Khan et al., 2007), B. juncea (Wangeline et al., 2004), and Arabidopsis (Howarth et al., 2003) have shown increased ATP-sulfurylase (ATPS) and serine acetyl transferase (SAT) activities under Cd stress, and thereby conferring tolerance to these plants. As ATPS activity helps in maintaining GSH level required for regulating Ascorbate (AsA)–GSH cycle (Khan et al., 2009), it has been reported that S at 40 mg.S.Kg⁻¹ enhanced the AsA–GSH cycle, thereby reducing Cd-induced toxicity in mustard (Anjum et al., 2008). Thus, indicating toward the possibility that S supplementation to soil system might enhance the formation of S-containing defense compounds such as GSH and phytochelatins. Study by Astolfi et al. (2004) has shown that Cd (100 μM) exposure enhanced the ATPS, O-acetyl serine (OAS) thiol lyase activity, which is related to the production of phytochelatins that play the most effective detoxifying mechanism in plants (Zhang et al., 2010). Apart from enhancing the formation of phytochelatins, S also regulates ethylene signaling and thereby helping under heavy metal stress (Masood et al., 2012). Calcium (Ca) is majorly involved in activating the enzymes and also plays an important role in regulating metabolic activities. Due to chemical similarity as well as due to same channels and intracellular Ca-binding sites (Lauer Júnior et al., 2008) of Ca⁺² and Cd⁺², Cd present in external medium, replaces the Ca, and thereby affects the growth of plant. However, Ca has been shown to decrease the heavy metal-induced toxicity (Suzuki, 2005; Farzadfar et al., 2013). It has been reported that 30 mM Ca reduced the Cd content from 46.7 to 17.4 μg in Arabidopsis seedlings (Suzuki, 2005). Similar to this, Zhenyan et al. (2005) reported enhanced Cd (concentration 0.5 mM) tolerance in Lactuca sativa when supplied with 4 mM CaCl₂, which was due to enhanced expression of phytochelatin synthase. Ca reduces heavy metal-induced toxicity by reducing their uptake, influencing physiological processes, or activating expressions of other defense compounds.

Magnesium (Mg), an important constituent for chlorophyll biosynthesis, plays an essential role under heavy metal toxicity. Abul Kashem and Kawai (2007) reported that Cd (0.25 μM) -induced toxicity in Japanese mustard spinach was alleviated by Mg at 10 mM, and Cd accumulation was reduced by 40%. Mg-induced alleviation is not due to inhibition in uptake but due to enhanced antioxidant enzymes (Chou et al., 2011). Moreover, Mg-induced alleviation has been also correlated with expression of some genes OsIRT1, OsZIP1, and OsZIP3 of rice.

Trace elements are required in lesser amount for the biological system, which include iron (Fe), copper (Cu), manganese (Mn), molybdenum (Mo), cobalt (Co), and zinc (Zn), and their high levels could be toxic. The essentialities of these trace metals are due to their active participation in the redox reactions as well as because of their roles as enzyme cofactors (Sanita di Toppi and Gabbrielli, 1999). However, apart from their roles in biological system, they have been reported to play a crucial role in alleviating metal toxicity. Several trace elements have direct as well as indirect effects on heavy metal availability and toxicity (Sarwar et al., 2010). Direct effects include lowered solubility of heavy metals in the soil (Hart et al., 2005; Shi et al., 2005; Matusik et al., 2008), competition between heavy metals and trace elements for the same membrane transporters (Baszynski et al., 1980; Qiu et al., 2005), and heavy metal sequestration in the vacuoles (Salt and Rauser, 1995; Zaccheo et al., 2006). Indirect effects include dilution of heavy metal concentration by increasing plant biomass (dilution effect) and alleviation of heavy metal stress by increasing antioxidant defense system (Hassan et al., 2005; Suzuki, 2005; Jalloh et al., 2009). Zn, being an important group of metal transporter family, has been suggested to prevent damage caused by Cd toxicity. As reported in the case of Thalpsi violacea, plants supplied with 2 mgL⁻¹ Cd showed 48.5 mg Kg⁻¹ Zn accumulation than that of control (16.8 mgKg⁻¹), whereas when the plant was supplemented with 5 mgL⁻¹ Cd, Zn accumulation decreased upto 12.8 mg Kg⁻¹, suggesting Cd/Zn antagonism (Street et al., 2010). Furthermore, Zn also enhances the activities of antioxidant enzymes and competes with Cd to bind with the membrane protein in order to protect plant (Wu and Zhang, 2002). Other trace metal Fe, under Cd stress, showed reduced Cd uptake and translocation, thus increasing plant growth. Study by Qureshi et al. (2010) revealed that exogenous application of 40 μM Fe reduces the condition of oxidative stress by stabilizing the thylakoid complex under Cd stress. It was also reported that at Fe concentrations of 1.89 mg L⁻¹ (moderate) and 16.8 mg L⁻¹ (high), under low level of Cd (0.1 mM), plant height showed increment (Nada et al., 2007).

Similarly, studies have also revealed the protective roles of trace elements in ameliorating toxic effects of heavy metals by protecting photosynthetic tissue and increasing antioxidant capacity (Zornoza et al., 2010; Tkalec et al., 2014). Pa1'ove-Balang et al. (2006) have shown that Mn-mediated amelioration of Cd toxicity was associated with a decreased Cd uptake. Apart from the beneficial role played by trace metals, there are some beneficial nutrients like selenium (Se), and silicon (Si) that also play a major role under heavy metal stress. Earlier, selenium (Se) was considered as toxic element but later on Schwarz and Foltz (1957) confirmed it to be an essential one. Studies on lettuce grown under Pb and Cd toxicity supplemented with Se showed a decrease in heavy metal accumulation as well as enhanced uptake of essential nutrients (He et al., 2004). Belokobylsky et al. (2004) and Feng and Wei (2012) have found that Se level up to 5 mg L⁻¹ has beneficial effects on Spirulina platensis and P. vittata, respectively. Filek et al. (2008) have shown that exogenous application of Se alleviates toxic effects of Cd by enhancing the activities of antioxidant enzymes such as SOD, CAT, GPX, and APX. Several reports have revealed that appropriate dose of Se can protect plants against damage by heavy metals such as Hg, Pb, Cd, Cr, and Sb (Khattak et al., 1991; Shanker et al., 1996; Belokobylsky et al., 2004; He et al., 2004; Feng et al., 2011). Role of Si under heavy metal stress is also well established (Singh et al., 2011b; Dragišić Maksimović et al., 2012; Tripathi et al., 2012). Study by Song et al. (2009) has shown that supplementation of Si under Cd stress decreased an uptake and root to shoot translocation of Cd as well as enhanced the activities of enzymes of the defense machinery in B. chinensis. Similarly, study by Bharwana et al. (2013) revealed that Si application reduces Pb uptake and enhances the activities of antioxidants viz., SOD, GPX, APX, and CAT.

The measurement of elemental composition and their changes as a response to some stimuli in living organisms comes under the study of ionomics. Alteration in ionome could be direct or indirect. Direct one includes the changes in nutrient level in soil or due to impairment of ion transporter, whereas indirect changes might be due to changes in cell wall structure (Salt et al., 2008). Heavy metals due to their interaction with nutrient elements affect the uptake and distribution of these elements and may result in deficiency of minerals thus affecting the growth. Sarwar et al. (2010) suggested that Cd affects the permeability of plasma membrane and thus interferes with the nutrient uptake. However, there exists both antagonistic as well as synergistic interaction between heavy metals and micronutrient uptake, which could be due to differences in plant species and nutrient concentration. Likewise, a study by Cataldo et al. (1983) reported antagonistic interaction between Cd and Fe, Zn, Cu, and Mn in soybean plants, whereas Nan et al. (2002) reported synergistic interaction between Cd and Zn in wheat and corn. In a study by Yang et al. (1998), decreased accumulation of Fe, Mn, and Cu in ryegrass, maize, cabbage, and white clover was observed after Cd exposure, whereas there was increased P accumulation. Similarly, Cui et al. (2008) reported decrease in Fe and Zn uptake in rice after Cd treatment in hydroponic system. A study by Safarzadeh et al. (2013) determined the effect of different doses (0, 45, and 90 mg kg soil) of Cd on uptake of Fe, Zn, Cu, and Mn in seven rice cultivar and reported decrease in Zn, Fe, Mn, and Cu uptake. Not only the uptake decreased but also there was decrease in the translocation of these minerals as Cu and Fe contents found to be greater in roots than in shoots that indicate toward impairment of ions transporters.

Similar to Cd, As has also been reported to influence nutrient uptake and their distribution in plants. Meharg and Hartley-Whitaker (2002) reported As-induced decrease in P uptake is due to chemical similarity between P and arsenate and due to which arsenate enters the plant via the phosphate transport systems. However, the concentration of As also plays an important role in P uptake. Burló et al. (1999) reported higher uptake of P at lower level of As in tomato plants. Similarly, Carbonell et al. (1998) reported increased P uptake in tomato plant when exposed to low level of As. As not only influences P uptake but also affects the uptake of other nutrients like N, P, K, Ca, etc. A study by Carbonell-Barrachina et al. (1997) observed increased concentration of N, P, K, Ca, and Mg in P. vulgaris L. plants when exposed to arsenite. Similarly, Carbonell-Barrachina et al. (1994) reported decreased uptake of K, Ca, and Mg (macronutrinets), B, Cu, Mn, and Zn (micronutrinets) in Lycopersicum esculentum Mill. The effect of As concentration on nutrient level of hyperaccumulator P. vittata L. had also been studied by Tu and Ma (2005), and the authors reported that both micro- and macronutrients were in the range of normal concentration as in non-hyperaccumulators. However, there was enhancement in P and K contents in the fronds of P. vittata L. at lower level of As. They reported molar ratio of P/As to be 1.0 in fronds of P. vittata L., which is the threshold value for normal growth of plants.

Heavy metal ions such as Cu⁺², Zn⁺², Mn⁺², and Fe⁺² are essential for plant metabolism but when they are present in excess amount become highly toxic. For instance, Zn and Mn when present in excess impairs growth and compete with Fe. Excess Fe in the plant system participates in the fenton reaction, thereby creating a condition of oxidative stress (Williams and Pittman, 2010; Shanmugam et al., 2011). In order to avoid toxicity induced by mineral elements and trace elements, these are chelated by low molecular weight compounds and sequestrated in vacuoles or excluded to extracellular spaces by transporters situated in the tonoplast or plasma membrane, which plays central role in maintaining metal homeostasis under safe limit. These transporters belong to (1) P_1B-ATPase or CPx-type ATPase, (2) Cation Diffusion Facilitator (CDF) also known as Metal Tolerance Proteins (MTPs), (3) Natural Resistance-Associated Macrophage Proteins (NRAMPs), and (4) ZRT–IRT-like Protein (ZIP) transporters.

Transcriptomics

Investigations on the basic mechanisms of heavy metal tolerance and adaptation are the area of great scientific interest and an intensive research. Various stressors induce an expression of a set of genes in plants (Nakashima et al., 2009).

At molecular level, the regulation of gene expression is very important for the biological processes, which determines the fate of plant development as well as tolerance to heavy metal stress. Stressors trigger large number of genes and several proteins in order to link the signaling pathways that confer stress tolerance (Umezawa et al., 2006; Valliyodan and Nguyen, 2006; Manavalan et al., 2009; Tran et al., 2010). These genes are classified into two groups: the regulatory genes and the functional genes (Tran et al., 2010). The genes of regulatory group encode various transcription factors (TFs), which can regulate various stress-responsive genes cooperatively and/or separately and thus, constitute a gene network. However, the genes of functional group encode metabolic compounds such as amines, alcohols, and sugars, which play a crucial role in heavy metal stress tolerance. The TFs, which are reported to be master regulators, control an expression of gene clusters and usually members of multigene families. Studies reveal that a single TF can control the expression of many target genes via specific binding of the TF to the cis-acting element in the promoters of its target genes (Wray et al., 2003; Nakashima et al., 2009). Most of the TFs contain a DNA-binding domain that interacts with cis-regulatory elements in the promoters of its target genes and via a protein–protein interaction domain that helps in oligomerization of TFs with other regulators (Wray et al., 2003; Shiu et al., 2005). This type of transcriptional regulatory system is referred as “regulon” (Nakashima et al., 2009). Various TFs families such as AREB/ABF, MYB, AP2/EREBP, WRKY, bHLH, bZIP, MYC, HSF, DREB1/CBF, NAC, HB, ARID, EMF1, CCAAT-HAP2, CCAAT-DR1, CCAAT-HAP3, CCAAT-HAP5, C2H2, C3H, C2C2-Dof, C2C2-YABBY, C2C2-CO-like, C2C2-Gata, E2F-DP, ABI3VP1, ARF, AtSR, CPP, E2F-DP, SBP, MADS, TUB, etc. are known to influence stress response in plants (Singh et al., 2002; Shiu et al., 2005; Shameer et al., 2009). LeDuc et al. (2006), in a transcriptome analysis on plants, reported that plants treated with heavy metals could induce transcription factors that regulate corresponding transcriptional processes.

Liang et al. (2013) reported first FER regulatory gene involved in Fe uptake in tomato, and the functional analog of FER is FER-like Deficiency Induced Transcripition Factor (FIT) that has been conferred to play an important role under Fe deficiency in Arabidopsis (Yuan et al., 2005). In addition to this, there are several other subgroups of bHLH family viz., AtbHLH38, AtbHLH39, AtbHLH100, and AtbHLH101 that have been shown to be upregulated under Fe deficiency in roots and leaves of Arabidopsis (Wang et al., 2007; Yuan et al., 2008). Later, several researchers proposed that AtbHLH38 or AtbHLH39 interacts with FIT and forms heterodimers and directly activates transcription factors for ferric chelate reductase and ferrous transporters, which are the two major genes regulating Fe uptake under deficient condition (Varotto et al., 2002; Vert et al., 2002; Yuan et al., 2008). In Arabidopsis, IRT1 has been reported to be the most essential ferrous transporter. Beside transporting Fe, it can also transport Zn, Mn, Co, Ni, and Cd, and thus, these metals get accumulated under Fe deficiency (Vert et al., 2002; Schaaf et al., 2006). A recent study by Wu et al. (2012) in Arabidopsis revealed that expression of FIT with AtbHLH38 or AtbHLH39 further activates expression of several other transporters viz., HMA3, (MTP3), Iron Regulated Transporter2 (IRT2) that play regulatory role in maintaining Fe content under Cd exposure.

Transcriptome analysis in A. thaliana and B. juncea exposed to Cd stress has revealed the induction of basic region leucine zipper (bZIP) and zinc finger transcription factors (Ramos et al., 2007). ERF1 and ERF5, two transcription factors belonging to AP2/ERF superfamily (characterized by AP2/ERF domain; Nakano et al., 2006), have been reported to be induced when A. thaliana was exposed to Cd (Herbette et al., 2006). Similar induction of TFs has been reported in A. halleri under Cd stress (Weber et al., 2006). Differential expression of ERF factors under Cd indicates toward their responses to various levels of Cd stress. A study by Nakashima and Yamaguchi-Shinozaki (2006) reported down-regulation of dehydration-responsive element-binding protein (DREB) transcription factor (involved in cold and osmotic stress responsive genes) in roots of A. thaliana under heavy metal treatment and suggested it could be acclimation response and DREB might have helped in normalizing osmotic potential, so that flow of heavy metal-contaminated water could be reduced, thus helping plants to avoid toxic effects of heavy metal. Therefore, acquiring a deep knowledge of the interrelated mechanisms, which regulate the expression of these genes, is a crucial issue in plant biology and necessary to generate genetically improved crop plants for extreme environments like heavy metal stress (Umezawa et al., 2006; Valliyodan and Nguyen, 2006; Nakashima et al., 2009). Summary of an involvement of TFs in conferring heavy metal and other abiotic stresses tolerance is given in Table 2.

Proteomics

Proteomics is a well-established technique in the post-genomic era (Liu et al., 2013). Proteomics deals with the study of large-scale expression of proteins in an organism encoded by its genome (Anderson and Anderson, 1998). Proteomics not only serves as a powerful tool for describing complete protein changes in any organisms but it can also be used to compare variation in protein profiles at organ, tissue, cell and organelle levels under various stress conditions including heavy metal stress (Ahsan et al., 2009). Although genomic analysis has enhanced our understanding regarding plants' response to heavy metal toxicity, transcriptomic changes in the genome are not always reflected at protein level (Gygi et al., 1999; Hossain and Komatsu, 2013). For instance, putative Zn and Mg transporter protein MHX was more abundant in Arabidopsis even though its corresponding transcript level was not different (Elbaz et al., 2006). This suggests that transcription of any gene is not a guaranty that gene would be translated into a functional protein. This may occur due to the potential impact of post-transcriptional and translational modifications, protein folding, stability and localization, protein–protein interactions, which are considered important determinants of a protein function (Dalcorso et al., 2013b). Therefore, depth analyses of proteomics offer a new platform for identifying target proteins, which take part in heavy metal detoxification, and in studying complex biological processes and interactions among the possible pathways that involve a network of proteins (Ahsan et al., 2009).

Furthermore, it is known that proteins directly take part in plant stress responses, and plant adaptations to heavy metal stress are always accompanied with deep proteomic changes. Therefore, technique of proteomics can be exploited for deciphering the possible relationships between proteins abundance and plant stress adaptation. It can contribute to better understanding of physiological mechanisms under heavy metal stress such as perception of stress and further signaling cascade that leads to changes in the expression of huge numbers of genes at transcriptional level and in metabolite profile, which could be used for an acquisition of an enhanced plant tolerance under heavy metal toxicity (Kosová et al., 2011). Studies have revealed that an abundance of defense proteins was increased for scavenging of ROS, and molecular chaperones play a role in re-establishing the conformation of a functional protein that contributes in helping heavy metal stressed plants to maintain the redox homeostasis (Zhao et al., 2011; Sharmin et al., 2012; Wang et al., 2012). Under heavy metal stress, modulations of various metabolic pathways occur such as photosynthesis, respiration, nitrogen metabolism, sulfur metabolism, etc. particularly in photosynthesis and mitochondrial respiration that help stressed plants to produce more reducing power such as NADPH, NADH, and FADH₂ and assimilatory power ATP to compensate high energy demand of heavy metal-challenged plants (Hossain and Komatsu, 2013). For example, an increased abundance of RUBISCO large sub unit (LSU)-binding proteins, oxygen-evolving enhancer protein 1 and 2, NAD(P)H-dependent oxido-reductase, and photosystem I and II-related proteins is an adaptive feature to withstand heavy metal stress (Semane et al., 2010). The cellular mechanism of stress sensing and further transduction of signals into the cell appear to be the first reactions in the plant cell against heavy metal. Furthermore, an intracellular communication of stress signals plays a fundamental role in signal transduction pathways under stress, which ultimately activate defense-related genes and thus signaling cascades (Hossain et al., 2012c). Therefore, to decipher an underlying molecular mechanism of alterations in the protein signature of a plant cell in order to withstand stress, a deep study on the cellular as well as organelle proteomics would be of great importance in developing heavy metal-tolerant crops. Alterations in protein profile under heavy metal stress, which could be utilized for developing heavy metal-tolerant plants, are given in Table 3.

Apart from inducing synthesis of amino acids (proline and histidine), amines, organic acids, and plant antioxidant α-tocopherol and glutathione, some nitrogen containing metabolites like some peptides (phytochelatins, metallothioneins, and ferritins) have been reported to play an important role under heavy metal stress. In the following section, we will discuss about the roles of peptides in heavy metal tolerance.

Salicylic acid (SA)

In recent years, SA has gained much scientific attention due to its function as an endogenous signaling molecule conveying local and systemic plant–pathogen defense responses. Besides this, it has been reported that SA also plays a role in plant response against abiotic stresses such as heavy metal toxicities, chilling, drought, osmotic stress, and heat. In this sense, SA appears to be an “effective therapeutic agent” for plants as in the case of mammals (Rivas-San Vicente and Plasencia, 2011). Salicylic acid is a phenolic compound biosynthesized in all the plant kingdoms through the phenylpropanoid pathway (Métraux, 2002).

Being well characterized and studied role of SA in pathogen resistance, an exogenous application of SA could also provide protection against several types of abiotic stresses such as heavy metals, high or low temperature, salinity, radiation, etc. (Horváth et al., 2007; Hayat et al., 2010). Since under stress condition, reduced plant growth could result from an altered hormonal status, and thus, an exogenous application of plant hormones like SA has been an attractive approach to attenuate heavy metal stress. Studies carried out so far demonstrated that SA treatment to plants evoke acclimatization effect, which causes an enhanced tolerance toward heavy metal stress primarily due to the adjustment of metabolic processes such as enhanced antioxidative capacity. In one of the first works, it was demonstrated that SA may induce protective effects against Cu toxicity in tobacco and cucumber (Strobel and Kuc, 1995). Later, an increasing numbers of studies have demonstrated SA-mediated amelioration of toxicities produced by various heavy metals. Zhou et al. (2009) reported that 0.2 mM of SA ameliorates Hg toxicity in alfalfa by increasing activity of APX, POD, and NADPH oxidase, and amounts of ascorbate, glutathione, and proline, and decreased lipid peroxidation, and an increase in NADPH oxidase activity. It indicates a role of ROS signaling in such an amelioration process.

In maize plant, Cd declined the growth by inhibiting chlorophyll synthesis, ribulose 1,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase, and enhancing oxidative damage such as lipid peroxidation and electrolyte leakage, whereas SA pretreatment of seeds reversed these toxic effects (Krantev et al., 2008). In cucumber, an exogenous application of SA has also been reported to enhance Mn tolerance by modulating nutrients' statuses and antioxidant defense system (Shi and Zhu, 2008). Similarly, in pea seedlings, Cd toxicity caused decline in growth due to an inhabited photosynthetic process and enhanced oxidative damage, whereas SA pretreatment alleviated damaging consequences of Cd on growth and photosynthesis (Popova et al., 2009). Moreover, Guo et al. (2009) have demonstrated that SA pretreatment alleviated Cd toxicity in rice by enhancing antioxidant components such as SOD, CAT, POD, glutathione, and non-protein thiols, which in turn depressed oxidative damage induced by Cd. Conversely, Metwally et al. (2003) reported that SA down-regulates activities of antioxidant enzymes such as CAT and APX under Cd stress and concluded that SA alleviates Cd toxicity not at the level of antioxidant defense system but by affecting other mechanisms of Cd detoxification. Contrary to this, SA at higher concentration may also cause tissue damage and cell death by inducing oxidative stress (Horváth et al., 2007). For instance, SA has been shown to potentiate generation of ROS in photosynthetic tissue under abiotic stresses and thus causes tissue damage (Borsani et al., 2001). Therefore, it can be concluded that the concentration of SA appears to be important in regulating stress responses. The SA-mediated alterations in genes that are involved in mediating stress tolerance are listed in Table 4.

It is known that SA also involves in the regulation of oxidative stress caused by various stress factors (Yang et al., 2004). An enhanced level of SA under heavy metal stress suggests a connection between the extent of plant tolerance to heavy metal, which is mediated by the SA signal and the redox balance (Metwally et al., 2003; Sharma and Dietz, 2009). In the SA signaling under heavy metal stress, several signaling molecules such as nitric oxide (NO), H₂O₂, Ca⁺², etc. and their interactions have been reported (Rodríguez-Serrano et al., 2009; Xu et al., 2014). Moreover, Cui et al. (2012b) have reported a cross-talk of haem oxygenase-1 and SA in alleviation of Cd stress in M. sativa. In spite of considerable progress in the understanding of SA signaling, molecular events, which are involved in the SA signaling in order to alleviate heavy metal stress, are still poorly known (Figure 4).

Brassinosteroids (BRs)

Brassinosteroids are group of hormones having ability of regulating ion uptake in plant cells and very effectively reducing the heavy metal accumulation in plants. BRs can also impart plant stress tolerance against variety of biotic and abiotic stresses such as heavy metal, salinity, drought, low and high temperatures, and pathogen attack (Bajguz and Hayat, 2009; Hao et al., 2013). An increasing numbers of studies have shown that an exogenous application of BRs is widely used in order to improve crop yield as well as stress tolerance in various plant species (Divi and Krishna, 2009; Peleg and Blumwald, 2011; Li et al., 2013). Cadmium, a heavy metal, very toxic even when present in trace amount, have been found to retard chlorophyll biosynthesis, activity of several enzymes, and inhibit light and dark reactions of photosynthesis by limiting the energy/reducing sources (Vassilev and Yordanov, 1997). However, it has been reported that Cd-induced toxicity can be lowered with BR. For instance, Janeckzo et al. (2005) reported that Cd-induced inhibition in pigments content, cotyledon growth could be minimized with exogenous epibrassinolide (EPL: another BR). Hayat et al. (2007) have verified the role of HBL under Cd stress in B. juncea. In Vigna radiata L. Wilczek, Al stress caused a reduction in length, fresh and dry mass of root and shoot; activity of carbonic anhydrase; water use efficiency; relative water content; chlorophyll content; and the rate of photosynthesis, whereas addition of BR reversed these toxic effects and protected the plants via elevated level of proline in an association with an antioxidant defense system which at least in part was responsible for the amelioration of Al stress (Ali et al., 2008). In B. juncea, BR alleviates Cd toxicity through enhanced level of antioxidants (Hayat et al., 2007). The Cr, a known toxic metal, reduced the growth performance of Raphanus sativus L., whereas BR protects plants from adverse consequences of Cr toxicity by regulating antioxidant defense system (Sharma et al., 2011). Micronutrients such as Cu and Ni are essential for growth and development, but in excess, they cause severe toxic effects. Cu, which has increasingly attained interest due to its use in fungicides, fertilizers, and pesticides, is also highly toxic to plants, but when seeds of B. juncea primed with epibrassinolide (a form of BR) were exposed to Cu stress, improvement in shoot emergence and biomass accumulation, along with reduced Cu uptake and accumulation, was noticed (Sharma and Bhardwaj, 2007). Similar protective responses of exogenous BR on B. juncea and V. radiata under Cu and Ni toxicities have been reported (Alam et al., 2007; Sharma et al., 2008; Fariduddin et al., 2009; Yusuf et al., 2012b). Besides higher plants, BR has also been found to be effective in alleviating heavy metals such as Cu, Pb, and Cd toxicities in algae, Chlorella vulgaris, through the regulation of antioxidant defense system (Bajguz, 2010). The BR-mediated alterations in the gene expressions and their roles in stress tolerance are listed in Table 2.

Gibberellic acid (GA)

The gibberellins (GAs) are a large family of tetracyclic diterpenoid plant growth hormone associated with the plant growth and developmental processes (Matsuoka, 2003). To alleviate deleterious effects of stress, different types of plant hormones have been used that might complement decreased and/or imbalanced hormone level during exposure of stress. Of these, GA has been a focus of plant scientists (Hisamatsu et al., 2000; Iqbal et al., 2011; Zhu et al., 2012).

Several studies revealed that GA alleviates various abiotic stresses including heavy metal toxicity. In A. thaliana, GA (5 μM) is reported to ameliorate Cd toxicity by reducing Cd uptake and lipid peroxidation (Zhu et al., 2012). Furthermore, authors demonstrated that GA reduces NO level which in turn down-regulates expression of IRT1 gene, a Fe transporter (might be involved in Cd absorption) as indicated by no effect of GA in reduction of Cd uptake in an IRT1 knockout mutant irt1. It is reported that an exogenous addition of GA reprograms the growth of soybean under stress conditions by enhancing the levels of daidzein and genistein contents, suggesting protective role of GA in mitigating adverse consequences of stressors (Hamayun et al., 2010). In wheat seedlings, Ni (50 mM) has been shown to decline growth, chlorophyll content, and carbonic anhydrase activity by enhancing oxidative stress, whereas an addition of GA ameliorates Ni-induced toxic effects (Siddiqui et al., 2011). Gangwar et al. (2011) have also reported that an exogenous addition of GA ameliorates toxic effects of Cr (50–250 μM) on growth and ammonium assimilation of pea seedlings by regulating oxidative stress and an antioxidant system. In B. napus L., GA (50 μM) has been shown to alleviate Cd (10–400 μM)-induced negative impact on seed germination and growth by regulating oxidative stress and damage (Meng et al., 2009). It has been observed that Pb and Zn affect seed germination in Cicer arietinum cv. Aziziye-94 by altering hormonal balance, and an exogenous application of GA reverses the toxic effect of heavy metals (Atici et al., 2005). Furthermore, Sharaf et al. (2009) have also reported that GA mitigates detrimental effects of Cd and Pb on broad bean and lupin plants by regulating activities of proteases, CAT, and POD. These studies clearly indicate that GA plays an important role in protecting plant metabolism against various stresses; however, this may occur via various routes suggesting complex GA signaling during plant acclimation against stresses. The GA-mediated alterations in genes and their relation with stress tolerance are summarized in Table 4.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.