Introduction

Metals and metalloids are essential in every biological process for plant growth and development, yet in excess, it is toxic to plants. Land and water pollution by toxic heavy metals and metalloids has become a global crisis. Increased anthropogenic activities, which cause major heavy metal/metalloid (HMs) pollution, endanger plant development, the environment, and humanity. Cadmium (Cd), chromium (Cr), lead (Pb), mercury (Hg), selenium (Se), and arsenic (As) are some of the most hazardous heavy metals/metalloids (HMs), and their presence in the environment can have severe implications on all life forms (Singh et al. 2016a, b; Muthusaravanan et al. 2018). In general, the usual route of human exposure to HMs is through ingestion of contaminated food and water sources, which cause severe health issues due to their toxicity (Jadia and Fulekar 2009).

Phytoremediation is a cost-effective, efficient, environmental- and eco-friendly green technology that uses metal-accumulating plants or genetically engineered plants to remove toxic metals species from contaminated soil and water (Bernardino et al. 2019). Hyperaccumulator plant species actively absorb large amounts of one or more heavy metals from the soil without exhibiting phytotoxicity symptoms. The HMs are translocated to the shoot and accumulated in above-ground organs, especially leaves, at concentrations 100–1000 times greater than those found in non-hyperaccumulating species (Rascio and Navari-Izzo 2011; Reeves et al. 2017). They have a high bioconcentration factor, an efficient root-to-shoot transport system, alongside improved metal tolerance and detoxifying capacity. On the contrary, many slow-growing hyperaccumulators have decreased biomass yield, demanding years of decontamination from polluted sites (Rascio and Navari-Izzo 2011). There is an abundance of plants enriched with genetic resources for phytoremediation, such as Sebertia acuminata, Alyssum bertolonii, Leptospermum scoparium, Noccaea caerulescens, Pteris vittata, Brassica juncea, Arabidopsis halleri, Betula papyrifera and so on. They accumulate a wide range of HMs such as Cd, Zn, Ni, As, Cr, Se, and Pb.

Different mechanisms of phytoremediation include phytoextraction, phytostimulation, phytofiltration, phytostabilization, and phytovolatilization. The phytoextraction approach involves direct uptake of metal through absorption by plant roots, meanwhile, phytostimulation involves plant root exudates mediated microbial activity to reduce contaminants (Kaur et al. 2018). In the case of phytofiltration, plant roots, seedlings, or shoots can absorb or precipitate contaminants in surface or wastewater. The process of phytostabilization involves HMs uptake and accumulation by the rhizosphere system. In phytovolatilization, HMs such as Hg and As are converted into a volatile form through biological processes in plants, and further released into the air (Tangahu et al. 2011).

The genetic alteration of plants utilizing metabolic pathway genes or genes from microbial detoxification pathways has a possible outlook for enhanced phytoremediation potential of plants. The identification and subsequent transfer of unique genes from hyperaccumulators to rapidly flourishing species have been demonstrated to be useful as indicated by the current advancement of transgenic plants with enhanced phytoremediation potential. Currently, many biotechnological strategies are employed in the phytoremediation of HMs namely Cd, Hg, Pb, Se, and As (Mosa et al. 2016). The genetic engineering approaches improve plant life by altering features such as transport, metal uptake, accumulation, tolerance, chelation, and vacuolar sequestration, unfolding new possibilities for phytoremediation. The molecular mechanisms/strategies for the enhanced phytoremediation are signaling and regulatory proteins, genes that are involved in primary metabolism, metal transporters, metal-binding ligands, and detoxifiers (Fig. 1). In the era of high-throughput sequencing, understanding the molecular mechanism of phytoremediation has become relatively easier. The roles of miRNA, non-coding RNAs, CRISPR/Cas9, and synthetic genes in context to phytoremediation have become pertinent to get a holistic understanding of the present knowledge about phytoremediation of HMs. The effect of HMs on the triggering of signaling molecules like calcium, MAPK, and hormones besides additional regulators like miRNAs and transcription factors are certainly reported.

Fig. 1
figure 1

Molecular targets for phytoremediation. Various genes and mechanisms involved in primary metabolism, metal binding ligands, signaling and regulatory proteins, detoxifiers and metal transporters which can be utilized for enhanced phytoremediation are illustrated

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The objective here is to review the application of genetic engineering in phytoremediation for heavy metals and metalloids. Here we discussed various genes involved in signaling, phytohormones, chelation, detoxification, enhanced translocation, metal transporters, and its utilization in the amelioration of phytoremediation potential of plants. We also discussed the prospects of genome editing and gene pyramiding in phytoremediation. The limitations and challenges of genetic engineering approaches in phytoremediation were also evaluated.

Plant signaling pathways for phytoremediation

Plants have evolved specialized systems for efficient management and elimination of stress-inducing elements, both biotic and abiotic. Heavy metal stress, a type of abiotic stress, which induces metal toxicity generally results in stunted growth and leaf necrosis (Singh et al. 2016a, b). Plants detect HMs via different signaling molecules, which trigger a series of signaling events causing changes in the cell’s biochemical and molecular response. Among the several signaling cascades generated by HMs stress, MAPK signaling, hormone signaling, and calcium signaling are most important (Jalmi et al. 2018). These occur in response to critical messenger molecules, which involve a range of different signaling units that ultimately affect the cell’s transcription.

Calmodulin, calmodulin-like proteins, calcium ions dependent proteins, and calcineurin B-like proteins, activated in calcium signaling, initiate several pathways to cope with HMs stress. The hormone-triggered responses involve several plant hormones such as abscisic acid (ABA), gibberellins (GA), and auxins (Bücker-Neto R et al. 2017). Among these divergent types of responses, the most complex and imperative response involves mitogen-activated protein kinases (MAPK) (Jalmi et al. 2018). Ultimately, the transcriptional byproducts of these signaling cascades promote heavy metal chelation and transportation in plants. Below is a quick discussion of a few critical signaling pathways involved in heavy metal tolerance.

Phytohormones

Phytohormones play a critical role in plant physiology and development, growth regulation, and abiotic stress response (Bücker-Neto et al. 2017). The hormones can either function independently or in conjunction with other hormones and signals to regulate the biological processes. These chemical messengers also play a vital role in abiotic stress response. ABA, auxins, and ethylene are crucial for HMs stress tolerance in plants. Usually, the concentration of these hormones increases during heavy metal stress (Luo et al. 2016). However, studies have shown that providing an external stimulus for the synthesis of these hormones improves plant tolerance and adaptability in a heavy-metal contaminated soil (Bücker-Neto et al. 2017).

ABA is a versatile phytohormone that performs functions, including, but not limited to seed formation and dormancy (Finkelstein 2013). Its concentration increases in plant tissues during heavy metal stress, proving that it protects the plants from the stress. Auxin aids plants to build a robust root system ensuring proper growth in heavy metal contaminated sites (Hu et al. 2013; Pasternak et al. 2005). Usually, due to heavy metal stress, levels of phytohormones like auxin concentration can get disturbed, so the external application helps maintain the overall plant health, but the pathways that regulate the HMs tolerance are still unclear (Sytar et al. 2018).

Calcium signaling

Calcium ion (Ca2+) acts as a connection between the primary response and the physiological and developmental processes in plants. It also ensures the normal development of plants and aids in heavy metal detoxification. Its concentration inside the cell changes during the abiotic stress. Abiotic stress alters calcium ion concentration in the cells, triggering the secondary signaling cascades (Jalmi et al. 2018). Change in the cytosolic concentration of Ca2+ is detected by calcium sensor proteins, like calcineurin B-like proteins, calmodulins, calcium ion-dependent protein kinases, and calmodulin-like proteins. These bind the Ca2+ and trigger different types of responses in the cell (Mukta et al. 2019). Ca2+ has an added advantage in providing Cd tolerance to plants from other heavy metals as its radius is almost similar to that of Cd (Bridges and Zalups 2005). Several studies have shown that when Ca2+ is supplemented into the medium, it protects the plants from Cd growth inhibition. External supplementation of Ca2+ to Sedum alfredii has prominently enhanced the root growth, which was hampered by the presence of Cd in the medium (Tian et al. 2011). Studies have also suggested that it has reduced the Cd activity in the plasma membrane of root cells when added to the growth medium (Ritchie & Gilroy 2000). Due to the similar radius, Cd is also taken up by plants through voltage-gated calcium ion channels. Plants exposed to higher Cd concentrations have shown a higher cellular concentration of Ca2+, which is required to tackle the toxic effect of Cd in the system. A study on Arabidopsis shows that Ca2+ has affected the auxin concentration to deal with the harmful effects of Cd, which shows the interrelation of these signaling pathways and deal with the heavy metal stress (Hu et al. 2013). In Setaria italica, the interaction between Ca2+ and H2S signaling provides tolerance to chromium (Cr) stress (Fang et al. 2014).

Ca2+ is essential to maintain redox homeostasis in the rice plant cells as they reduce glutathione reductase and docosahexaenoic/argininosuccinic acid (Srivastava et al. 2014). The function of calcium transporters CAX1, a vacuolar cation and proton exchanger, is enhanced during Cd stress (Baliardini et al. 2015). Calcium-dependent protein kinases (CDPKs) increased Cd tolerance by enhancing hydrogen sulfide (H2S) signaling in Arabidopsis thaliana. In radish plants, Ca2+ and calmodulin proteins get activated during Cd stress (Arazi et al. 1999). During arsenic stress in rice plants, calmodulin, calmodulin-like proteins, and calmodulin kinases are activated. In Arabidopsis, CNGC1 (cyclic nucleotide-gated ion channel 1) gets triggered by lead toxicity, and in tobacco, it leads to the activation of CBP4 via calcium ion signaling (Rivetta et al. 1997). During chromium stress in foxtail millet, Ca2+ leads to the activation of TPC1 (two-pore channel 1), MRC5, and calmodulin and H2S were accumulated in the cells to cope with the stress (Chen et al. 2017a, b). Studies have also proved that calcium-dependent protein kinases work very closely with MAPK signaling in stress. A calcium-dependent protein kinase CPK18 was found to activate MAP kinase 5 in rice plants; this also proves the correlation between two signaling cascades (Xie et al. 2014). Further study needs to be conducted to find the exact role of calcium signaling in heavy metal stress and their correlation with the MAPK signaling cascade, which will open a wide range of possibilities for further research.

Mitogen-activated protein kinase (MAPK) signaling

MAPK signaling pathway is an evolutionarily conserved signal cascade that communicates between extracellular stimuli and the nucleus (Sinha et al. 2011). In tobacco, NtMPK4 gets activated due to wounding or during ozone stress, which is a part of MAPK signaling (Gomi et al. 2005). It consists of three protein kinase units activated during the signaling events: MAPK kinase kinase, MAPK kinase and MAPK (Kumar et al. 2020). Various reports have shown activation of MAPK cascade in response to HMs (Asai et al. 2002). Although the particular mode of action involved in response to individual heavy metals during the MAPK pathway is still not fully known. It was observed that cadmium chloride and copper sulfate triggered the response via MAPK3 and MAPK6 in Arabidopsis (Pitzschke et al. 2009; Kovtun et al. 2000). The involvement of the MAPKs pathway in auxin biosynthesis helped to improve the root growth in rice plants (Huang et al. 2002). ROS (reactive oxygen species) production is induced by heavy metal, which further activates MAPK cascade has also been proven in various studies (Kovtun et al. 2000).

When the rice seedlings were exposed to arsenate stress, OsMKK4 transcripts were induced in arsenite treated rice leaves and roots, whereas OsMPK3 and OsMPK4 transcript was upregulated in leaves and roots respectively (Rao et al. 2011). It has also been observed that hydrogen peroxide initiates the MAPK signaling cascade. When plants deal with the heavy metal stress, they release hydrogen peroxide, which in turn activate the MAPK genes in plants such as ANP1, OXI1, and these genes enhance the activity of MPK3 and MPK6; this was proved by the study in which the OXI1 gene was knockout, so these mutant plants were not able to activate MPK6 and MPK3 when treated with hydrogen peroxide (Kovtun et al. 2000). Arabidopsis thaliana, when exposed to Cd, shows the increased activity of MEKK1, MPK3, and MPK6. Brassica juncea, when exposed to arsenic, shows the increased activity of MAPK signaling cascade (Gupta et al. 2009). Apart from cadmium, high levels of copper can also activate MAPK pathways (Jonak et al. 2004). In Medicago sativa, under MAPKK, SIMKK gets triggered when exposed to copper stress and MAPK components, including SAMK, SIMK, MMK2, and MMK3, show an increase in activity when exposed to Cd (Opdenakker et al. 2012; Yeh et al. 2006). In Oryza sativa, OsMKK4 gets triggered in the presence of arsenic stress and OsMSRMK2, OsMSRMK3, OsWJUMK shows increased mRNA production when exposed to mercury or cadmium. OsMPK3 gets activated in arsenic stress and 40 kDa and 42 kDa MAPK shows the increase in activity during lead stress (Yeh et al. 2006). In Zea mays, components of MAPK, including ZmMPK3, show increased mRNA production, when exposed to Cd and ZmMPK5 shows increased activity during Cr stress (Wang et al. 2010). Various evidence has suggested that heavy metals like Cd and Cr affect the reactive oxygen species (ROS) and reactive nitrogen species, including nitric oxide (NO) and activate NO signaling.

Hydrogen Sulphide (H2S)

Initially considered a harmful by-product of cellular activity, H2S, at a lower concentration, is now recognized as a gaseous signaling molecule that plays a role in plant development and growth, seed formation, and other plant physiological activities. It is also vital during stress conditions such as drought, salinity, and heavy metal stress (Karle et al. 2021; Zulfiqar et al. 2020). Albeit its high water-solubility, the majority of cellular H2S exists in the form of HS− (Arif et al. 2021). It has also been reported that H2S is harmful to cells in higher concentrations and acts as a signaling molecule at lower concentrations (Paul et al. 2021). Like all other signaling molecules such as H2O2, NO, etc., H2S equilibrium is also controlled by D- cysteine desulfhydrase, cyanoalanine synthase, L- cysteine desulfhydrase, and sulfite reductase. Among heavy metal-induced stress, cadmium is most severe as it is stable, hence difficult to degrade and H2S is produced in the process. In a study on Chinese cabbage, relative expression of genes responsible for H2S metabolisms like DES1, cysteine desulfhydrase, OAS-TL homogenous family, and DCD1 were found to be increased under HM stress (Arif et al. 2021). The expression of DES1 had shown around 4.7 times increase in the expression, when the plant was exposed to Cd even in low concentrations. Another study conducted on foxtail millet seedlings in chromium stress, the genes which are responsible for H2S production, such as DES, LCD, and DCD2, were overexpressed for the first 12 h and DCD1 expression had sustained expression for 24 h (Li et al. 2016).

Arsenic treatment in pea plant seedlings resulted in increased accumulation of ROS (Singh et al. 2015). Cysteine levels were higher in plants suffering from arsenic stress. All these effects were reduced when the seedlings were treated with NaHS. NaHS is a chemical compound that induces H2S signaling molecule production in plants (Wang 2012; Lisjak et al. 2013). Further experiments noted that the activity of the ascorbic acid-glutathione cycle was reduced during H2S induced Pb stress (Kaya 2020). The experiments performed have shown that H2S could reverse the damage done to macromolecules by ROS mediation and reduce the arsenic accumulation; these results indicate that H2S plays an essential role in tackling the arsenic stress in pea seedlings (Hoque et al. 2016). Under chromium stress, the application of NaHS has improved wheat seed germination and can enhance the activity of esterase, amylases, and antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase, and ascorbate peroxidase (Shan et al. 2011).

Nitric Oxide (NO)

Nitric oxide is a gaseous signaling molecule responsible for critical regulatory processes in plants. This molecule is usually released, when the plant is under any stress, including drought, salt, or heavy metal stress. It plays a role in root formation, seed germination, closing of the stomata, and programmed cell death (Karle et al. 2021). Various studies have reported that NO also protects the plant from ROS, which may cause the oxidation of biomolecules when the plant is under Cd stress (Choudhury et al. 2016). Exogenous application of NO has been shown to increase the plant capacity to survive under HMs stress (Nabaei & Amooaghaie 2019). Exogenous application of NO donor, sodium nitroprusside (SNP), to plants increases the hemicellulose and pectin content in the cell walls of roots. NO also increases the activity of the activated reaction center, thus protecting photosystem II from Cd toxicity. It also improves the electron transport in the oxygen-evolving complex to D1 proteins (Chen et al. 2013a, b).

Some studies have also suggested that NO plays a role in the formation of root cell walls which can be helpful in heavy metal stress (Xiong et al. 2009). A study on B. juncea seedling treated with Cd and SNP showed enhanced growth compared to control plants. Photosynthetic pigments were inhibited and reduced due to Cd toxicity in control plants. In contrast, when the seedlings were treated with SNP, the photosynthetic pigments including chlorophyll a, chlorophyll b, and total chlorophyll content increased compared to control (Ahmad et al. 2016; Khator et al. 2020). Cd decreases the plants’ protein content, but the soluble protein content was increased when treated with SNP compared to non-treated plants (Khator et al. 2020). Cd stress also enhances the proline accumulation in plants. Another study conducted on Oryza sativa showed an accumulation of hydrogen peroxide during arsenic stress in the root cells, which in turn causes lipid peroxidation (Singh et al. 2016a, b). Superoxide dismutase (SOD) activity also increases during the arsenate stress, which converts superoxide anions into hydrogen peroxide and oxygen, thus increasing the hydrogen peroxide content in the cells. NO treatment regresses the activity of SOD and NO supplementation also reduces catalases and glutathione peroxidases in the root cells. Glutathione: glutathione reductase ratio increases in plants undergoing arsenate stress, when NO is supplemented to them. In contrast, NO application reduces the activity of glutathione-S transferase in both shoots and root cells (Singh et al. 2016a, b). Arsenic stress tends to increase the accumulation of iron in the roots of the plants (Singh et al. 2016a, b). NO supplementation allowed the transportation of iron to shoot instead of only root cells. It also increases OsYSL2 expression; it is a rice metal nicotinamide transporter regulated by Fe and expressed in the plant’s phloem tissues. This is used for the long-distance transport of manganese and Fe in the rice plant tissues (Ishimaru et al. 2010).

Manipulation of genes involved in chelation and detoxification

Sequestration and vacuolar compartmentalization provide effective protection against the detrimental effects of HMs by removing toxic heavy metals from cytosolic sensitive sites, where cell division and respiration take place, thereby reducing the interactions between heavy metal ions and cellular metabolic processes and avoiding damages to cell functions. This section elaborates on the genes involved in chelation and detoxification.

Metal-binding ligands as molecular targets for phytoremediation

Metal-binding ligands can be classified as phytochelatins (PCs), metallothioneins (MTs), and low molecular weight organic acids (LMWOA). The genes that encode enzymes for the biosynthesis of metal-binding ligands could be manipulated by genetic engineering, which can increase the detoxification of HMs. The thiol peptide GSH and its variant homoglutathione are observed to affect the form and toxicity of HMs such as Hg, As, Zn, Cd, and Cu, in several ways.

Metallothioneins (MTs)

MTs are cysteine-rich proteins with greater binding to cations (Cu, Cd, and Zn) and found to impart accumulation of HMs in yeast. Overexpression of metal chelators biosynthetic genes has been attempted in plants with the aim of enhancement of HMs uptake, translocation, and sequestration. For example, MT genes overexpressed in cauliflower, tobacco, and rapeseed, led to increased Cd tolerance (Hasegawa et al. 1997). Many studies have been performed where MT genes were overexpressed to enhance the phytoremediation capabilities (Koźmińska et al. 2018). MTs contain Cys-motifs that chelate HM ions, hence play a vital role in metal homeostasis. Many studies also show that MT genes are overexpressed to enhance phytoremediation capabilities. Overexpression of MT in chloroplast increased the tolerance and accumulation of Hg in transgenic plants (Ruiz et al. 2011).

Phytochelatins (PCs)

Phytochelatins are small metal-binding peptides with an n-Gly structure, where the value of n varies from 2 to 11 and several toxic metals, especially Cd, induce their synthesis (Grill et al. 1985). The thiol group (-SH) in PCs can form a coordination bond with several toxic metals, thus defending their functional significance (Cobbett 2000; Hall 2002). Even though the internal detoxification of HM in plants can be due to the production of PCs, it is unlikely that this is the only mechanism involved in HM tolerance.

Reports have shown that during PC biosynthesis in transgenic plants, the enzyme phytochelatin synthase (PCS) plays a crucial role in HMs tolerance. AtPCS1 expression in tobacco plants subjected to As and Cd increased phytochelatin levels and root As/Cd accumulation. Similarly, AtPCS1 showed increased detoxification and accumulation of As and Cd in Indian mustard, Arabidopsis, and transgenic tobacco (Zanella et al. 2015; Pomponi et al. 2005; Gasic and Korban 2007; Li et al. 2004; Brunetti et al. 2011). Arabidopsis mutants lacking PC-synthase cannot synthesize PCs and are also hypersensitive to Hg and Cd (Memon et al. 2001; Memon and Schröder 2008). In Salvinia minima (aquatic fern), the accumulation of Pb2+caused changes in the expression of the SmPCS gene. Hence, in vivo PCS activity and production of PC increased in roots and leaves, but to a lower extent (Memon and Schröder 2008).

A transgenic system for removal of As from soil was tested in A. thaliana by inserting 2 genes namely γ-ECS (γ -glutamylcysteine synthase) and ArsC (Arsenate Reductase) which are encoded by Escherichia coli and catalyze the glutathione (GSH)-coupled electrochemical reduction of arsenate to the more toxic arsenite. The transgenic plants showed 4–17 times increase in As accumulation than wild types (WT) (Dhankher et al. 2002). Likewise, overexpression of ArsC in Arabidopsis and tobacco reported a significantly increased Cd tolerance in both transgenic plants compared to the WT (Dhankher et al. 2003). Upon overexpression of γ-ECS, adenosine triphosphate sulfurylase (APS), and GS increased the rate of phytoremediation capacity (Chen et al. 2017a, b). Transgenics of GS and γ -ECS showed enhanced accumulation of Zn and Cd in shoots compared to WT. The γ-ECS transgenic showed increased levels of Cu, Pb, and Cr accumulation in contrast to WT (He et al. 2014). Studies suggested transgenic APS plants resulted in 2.5 times higher shoot accumulation of certain HMs at the seedling stage and different HMs at the mature stage (Ivanova et al. 2011). APS overexpression may be a promising approach for improved phytoextraction capacity from mixtures of metals. Genome-wide analysis of the Morus genome database resulted in the identification of two Morus alba PCS genes, MaPCS1 and MaPCS2. Under Cd2+/Zn2+ stress, the relative expression of the MaPCS gene was induced in stem, root, and leaf tissues. The overexpression of Morus notabilis MnPCS1 and MnPCS2 in transgenic Arabidopsis suggested that MnPCS1 has a major role in Cd detoxification. Transgenic Arabidopsis seedlings showed higher accumulation of Cd2+/Zn2+ concentrations in both roots and shoots indicating MnPCS1 and PCS2 genes have vital roles in HMs accumulation and tolerance (Fan et al. 2018). Studies have reported an increased accumulation of Cd when AtPCs1 was overexpressed (Lee et al. 1995). When TaPCS1 was overexpressed in Nicotiana glauca, transformed seedlings showed increased accumulation of Cd and more rooting compared to the WT (Gisbert et al. 2003).

Low molecular weight organic and amino acid (LMWOA)

LMWOAs are organic compounds that contain at the minimum one acid functional group (-COOH) and a chain of few carbon atoms. The increase in metal bioavailability in phytoremediation can be due to the potentiality of LMWOAs to form soluble compounds along with the metal cations, which may in turn improve phytoextraction rates. The efficiency in the plant metal uptake is higher when the metals are in their soluble form to increase the contact with the root cells so that they can be dissolved in the transpiration stream and can be carried out into the plants (Clemens et al. 2002). The LMWOA content is higher in plant organs because of their role as photosynthetic intermediates as well as their capable role as a metabolically active solution for osmotic adjustment. Organic acids also take part as a lead component in the mechanism that few plants use to manage the metal tolerance, as natural chelators buffering cytosolic excesses of trace elements (Clemens 2001; Martins et al. 2013; Goliński et al. 2015). Studies conducted on using tartaric, oxalic, and citric acid (OAs) as substitutes to EDTA (synthetic chelate) yielded positive outcomes for citric acid which performed better in increasing the Cu uptake by tobacco (Evangelou et al. 2006).

Manipulation of metal transporters

Genes encoding proteins involved in the transport of metal or metalloids are potential targets for increased accumulation of metal/metalloids for phytoremediation (Raskin et al. 1997). This section elaborates on the genes involved in metal transporters and its involvement in improving phytoremediation potential.

Calmodulin-binding protein

A calmodulin-binding protein NtCBP4 from tobacco, when overexpressed, exhibited enhanced tolerance to Ni2+ and hypersensitivity to Pb2+. The transgenic plant reduced Ni2+ and enhanced Pb2+ accumulation, indicating that NtCBP4 is involved in metal uptake across the plant plasma membrane and transport Pb2+ into plants for phytoremediation purposes (Arazi et al. 1999). In contrast, the calmodulin-binding domain and part of the putative cyclic nucleotide-binding domain from NtCBP4, the transgenic plants exhibited enhanced tolerance to Pb2+ and accumulated less Pb2+. Additionally, an ortholog of NtCBP4 in Arabidopsis cyclic nucleotide-gated ion channel (AtCNGC1) mutant lines demonstrated improved tolerance to Pb2+ and decreased Pb2+ accumulation (Sunkar et al. 2000). The result suggested that NtCBP4 and AtCNGC1 are components of a transport pathway responsible for Pb2+ entry into plant cells. In contrast, increased Pb2+ accumulation phenotype is dependent on the C-terminal part of NtCBP4, which harbors calmodulin-binding and putative cyclic nucleotide-binding domains (Kotrba et al. 2009; Zeng et al. 2015). Therefore, NtCBP4 and AtCNGC1 are the potential plasma membrane transporter genes, which can be downregulated for increased transportation of Pb2+ for phytoremediation purposes.

Low-Affinity Cation Transporter (LCT1)

Reports have shown that at higher calcium levels, heavy metal inhibition of growth and development of whole plants is weaker. The involvement of the gene Low-Affinity Cation Transporter1 (LCT1), which encodes putative plasma membrane low-affinity transporter for Na+ and K+ was postulated for this effect. At lower Ca2+ concentrations, the transgenic plants showed an altered phenotype in response to Pb and Cd, which could lead to the entry of Pb/Cd into the root cells (Wojas et al. 2007; Gupta et al. 2013). Salvinia minima is an aquatic fern with bioconcentration factor (BCF) values higher than 1000 and translocation factor (TF) values higher than 0.15 in exposure to 40 µM Pb (NO3)2.

Na+/H+ antiporters

The Na+/H+ antiporter gene (SmNhaD), which is not directly involved in Pb detoxification, but involved in maintaining homeostasis was highly expressed (60 times) within 3 h of Pb stress (Leal-Alvarado et al. 2018). It needs further investigation by overexpression or knockdown strategies to understand the role of SmNhaD in Pb tolerance and enhanced phytoremediation.

Heavy metal ATPase

Heavy Metal ATPase 4 (HMA4) is a member of the P-type ATPase superfamily and is more highly expressed in Arabidopsis halleri than in A. thaliana. The downregulation of HMA4 by RNA interference showed hyper-tolerance to Cd and Zn in A. halleri (Hanikenne et al. 2008). Heavy metal ATPase 3 (HMA3), a P1B2-ATPase, is a key tonoplast transporter involved in mediating the vacuolar sequestration of Cd, Zn, Co, Pb by plants. FlHMA3 isolated from Festulolium loliaceum encodes a vacuolar P1B2-ATPase that may play an important role in Cd2+ sequestration into root cell vacuoles, thereby limiting the entry of Cd2+ into the cytoplasm and reducing Cd2+ toxicity (Guo et al. 2017; Miyadate et al. 2010). Reports suggested the significance of vacuolar sequestration in rice for Cd, using transgenic strategies and positional cloning. ATPase 3 (OsHMA3) controls translocation rates from root-to-shoot. The allele of OsHMA3 that confers high root-to-shoot Cd translocation rates (OsHMA3mc) encodes a defective P(1B) -ATPase transporter. Cd over-accumulating rice cultivar Cho-Ko-Koku reported increased root to shoot translocation of Cd, controlled by a single recessive allele, qCdT7 (Miyadate et al. 2010). In rice, OsHMA3 plays a vital role in restricting Cd translocation from roots to shoots. A non-functional allele of OsHMA3 was reported in japonica rice cultivars leading to high Cd accumulation in shoots and grains (Yan et al. 2016). High Cd accumulation in grains and shoots of Japonica rice cultivars was reported by a new loss-of-function allele of OsHMA3 (Yan et al. 2016).

Natural Resistance Associated Macrophage Protein (NRAMPs)

Metal transporters are essential for the maintenance of appropriate metal ion concentrations. One of the identified metal transporters, natural resistance-associated macrophage protein genes (NRAMPs) can mediate metal ion homeostasis (Takahashi et al. 2012). Sedum alfredii (Nramp6) was cloned and functionally analyzed in transgenic A. thaliana. Transgenic Arabidopsis showed enhanced Cd accumulation, suggesting a critical HMs-responsive gene that could be useful for phytoremediation (Chen et al. 2017a, b). NRAMP1 transports metals from the apoplast into the cytosol (Seregin & Kozhevnikova 2020). In rice, OsNRAMP1 is reported to be involved in Cd accumulation (Takahashi et al. 2012). Cd tolerance and accumulation of Arabidopsis plants was affected by the knockdown of BTS, a gene that negatively regulates Fe deficiency-induced BTS ubiquitin ligase, which binds to Fe and Zn and negatively regulates various genes induced by Fe deficiency, including IRT1, by ubiquitination of bHLH transcription factors of the IVc subgroup (Vert et al. 2002). The BTS-knockdown mutant, bts-1 demonstrated enhanced Cd tolerance and accumulation in roots and shoots in comparison to WT (Zhu et al. 2020).

Arsenite antiporters

Arsenic is a toxic carcinogen, so it is crucial to decrease As accumulation in crops to reduce its risk to human health. Transgenic technology is developed for As phytoremediation. PvACR3, key arsenite [As (III)] antiporter isolated from As hyperaccumulator fern Pteris vittata, was expressed in Arabidopsis with constitutive CaMV 35 S promoter (Chen et al. 2013a, b). Transgenic Arabidopsis grown under soil containing As(V) with 10 ppm As concentration, accumulated about 7.5-times higher As in above-ground tissues. This study gives a major perception about the behavior of PvACR3 and the physiology of As metabolism in plants. ACR3 is critical for As metabolism, but ACR3 is not present in flowering plants. PvACR3;1, a novel ACR3 gene from Pteris vittata was cloned in Arabidopsis, Saccharomyces cerevisiae (yeast), and Nicotiana tobacum (tobacco) (Fu et al. 2017). Experiments conducted on yeast revealed that PvACR3;1 moderated AsIII efflux to an external medium by functioning as AsIII antiporter. Hence, the study provided a possible strategy to curb As accumulation in plant shoots, which appears as the first report to reduce translocation of arsenic by AsIII sequestering into vacuoles, dispersing light on engineering low-As crops to enhance food safety (Chen et al. 2017a, b).

Membrane Intrinsic Proteins (MIPs)

The understanding of mechanisms of arsenic uptake and translocation by the model arsenic hyperaccumulator viz. P. vittata provides promising tools to generate transgenic plants for phytoremediation purposes. The As (III) uptake mechanism and phytoremediation potential of P. vittata were extensively reviewed (Danh et al. 2013). P. vittata showed increased As(V) uptake due to increased expression of PvPHT1;3 (a phosphate transporter) and higher affinity for As(V) over phosphate (Cao et al. 2019). As(III) is transported in plants via members of membrane intrinsic proteins (MIPs) mainly by the NIP subfamily (Lindsay & Maathuis 2016; Kumar et al. 2018). Arabidopsis NIP subfamily members viz. NIP3;1 and NIP7;1 are involved in the xylem loading of arsenic and imparted shoot translocation of As (Lindsay & Maathuis 2016). In rice, the silicic acid efflux transporter Lsi2 is involved in the xylem loading of As(III) (Ma et al. 2008), which explains the reason for the higher translocation of As(III) in rice. The bidirectional arsenite permeability of rice PIPs was shown by the heterologous expression of OsPIP2;4, OsPIP2;6, and OsPIP2;7 in Arabidopsis, which resulted in increased biomass and enhanced As(III) tolerance (Mosa et al. 2016). Apart from NIPs and PIPs, a TIP subfamily PvTIP4;1, mediated As(III) uptake in P. vittata. Thus, for designing phytoremediation strategy, the high biomass crop can be genetically engineered by overexpression of the candidate MIP genes particularly NIP3;1, NIP7;1, PIPs, Lsi2, and PvTIP4;1, which could increase more As uptake and translocation and leads to enhanced As accumulation in genetically engineered plants.

Manipulation of genes involved in detoxification

HMs, namely Se and Hg can be detoxified by gene products obtained from animals, plants, and bacteria that may confer tolerance to these metals (Dhankher et al. 2012). Few bacteria have a natural capacity for metabolic conversion and express genes that are organized in the mer operon namely, transport (merP, merF, merC, merT), mercury regulation (merR), and electrochemical reduction (merB, merA) (Hsieh et al. 2009). An operon encodes mercury resistance genes in Gram-negative bacteria that comprise nearly 5 to 6 genes. MerA is a soluble NADPH-dependent, FAD-containing disulfide oxidoreductase (Kärenlampi et al. 2000). Several reports from the past suggested that the merA gene when expressed in plants confer metal tolerance. This enzyme transforms a more toxic form of Hg2+ to the least toxic metallic mercury (Hg0). Escherichia coli expressing merA in addition to reducing Hg2+ possessed a weak reduction activity toward Au3+ and Ag+ (Summers and Sugarman 1974). Hg detoxification and volatilization studies showing ectopic expression of merA in transgenic poplar, rice, tobacco, and hybrid sweetgum were reported (Haque 2010; Rugh et al. 1998; Che et al. 2003; Dai et al. 2009; Heaton et al. 2003, 2005; He et al. 2001). Studies showed that a toxic form of Hg2+ is converted to the least toxic form of metallic mercury (Hg0) by mercuric ion reductase (MerA). A mutagenic merA, merApe9, was constructed by modification in the flanking region, 9% of the coding region. The mutagenic merA transgenic seedlings grew well, flowered and set seeds on a medium containing HgCl2. Transgenic MerApe9 seedlings resulted in considerable amounts of increase in Hg0 relative to control plants (Rugh et al. 1996). A study showed a new function of metallothionein-like gene OsMT1e from rice plants. OsMT1e was expressed in roots at all developmental stages and was not much expressed in leaves at seed filling and vegetative stages. OsMT1e is targeted to the nucleus and induced by the exposure of Cd (Rono et al. 2021). A new study proposed that Jasmonic acid (JA) transporters in Subgroup 4 of ATP-Binding Cassette G (ABCG) transporter might also play a crucial role in heavy metal detoxification (Chen et al. 2012). Some studies also suggest that many proteins which are important for the accumulation and detoxification of heavy metals and metalloids are also traced to the ancestral green algae (Song et al. 2017; Verma et al. 2018).

Other important genes for enhancing heavy metal phytoremediation potential

Few genes responsible for different cellular activities are crucial members to increase phytoremediation capacity for HMs in plants. For instance, the Arabidopsis MAN3/XCD1 gene encodes an enzyme endo- β-mannanase, when overexpressed, resulting in increased Cd accumulation and tolerance, while a loss-of-function mutant gene indicates an inverse phenotype (Chen et al. 2014). The Lhcb2 gene from hyperaccumulator Sedum alfredii (SaLhcb2) encodes chlorophyll a/b-binding, when overexpressed in tobacco, showed 6–35% increased accumulation of Cd in shoots when compared to WT (Chen et al. 2014). PSE1 encoding Pb-sensitive 1 protein regulates Pb tolerance by induction of PC biosynthesis genes (GSH-dependent) expression and PDR12/ABCG40, an ABC transporter. AtPSE1 overexpression in Arabidopsis increased Pb accumulation and tolerance, whereas the loss-in-function mutant PSE1 displayed Pb sensitivity (Fan et al. 2016).

The enzyme in plants for heme biosynthesis is Ferrochelatase-1 (FC1), which might act as a positive regulator of Cd stress tolerance. The overexpression of AtFC1 guided to increased Cd tolerance and accumulation of Cd and nonprotein thiol components in Arabidopsis (Song et al. 2017). On the other hand, FC1 mutant depicted Cd sensitivity and transcriptome examination of loss-in-function mutant disclosed distinctive gene expression linked to metal transporters, GSH/PC biosynthesis, response to oxidative stress, and metal detoxification (Song et al. 2017). Fungal WaarsM gene encoding arsenic methyltransferase on ectopic expression in rice led to an increased As tolerance and a decreased As accumulation of about 50–52% in roots, shoots, and grains. Enhanced methylation of harmful As species and volatilization of As was exhibited by transgenic plants (Verma et al. 2018).

Another study showed that overexpression of the OsMTP1 gene encoding a metal tolerance protein resulted in significantly decreased Cd-induced toxic effects and increased Cd tolerance in tobacco plants. MTP1 is located at the tonoplast and is also responsible for the transport of Zn, Co, Fe, and, possibly, Ni, in many plant species, both excluders, and hyperaccumulators in the vacuoles (Das et al. 2016). Transgenic plants displayed more biomass, increased content of vacuolar thiol and vacuolar sequestration, and Cd hyperaccumulation (Deng et al. 2021). The role of adenosine 5’-phosphosulfate reductase 2 (APR2) of reductive sulfate assimilation pathway in Arabidopsis, was shown in Cd stress tolerance, and found that APR2 regulates Cd accumulation and tolerance by modulating GSH-dependent antioxidant capacity and Cd-chelation mechanism. For this experiment, the transgenic plants overexpressing APR2 and knockout mutants were raised, where the overexpression enhanced Cd tolerance, while knockout resulted in lowered Cd tolerance. Further, APR2-overexpressing plants with enhanced Cd accumulation and Cd tolerance also had higher GSH and phytochelatin levels than the WT and apr2 mutant plants, but lower H2O2 and TBARS contents upon Cd exposure (Xu et al. 2020). Higher Cd translocation from roots to shoots was observed in bts-1 mutants compared to WT plants (Zhu et al. 2020). The increased Cd accumulation in the bts-1 mutant in the roots and shoots was due to the positive regulation of Fe nutrition through the upregulation of Fe-related genes. An additional study was conducted on the accumulation and detoxification of Cd in rice shoots. The study was verified by ectopic expression of the CAL2 gene in rice and Arabidopsis that induced the Cd uptake without affecting the uptake of various other essential nutrients (Luo et al. 2020). Hemoglobin (Hb) proteins are ubiquitous in plants, and among them, Hb1 is a non-symbiotic class 1 hemoglobin is involved in various biotic and abiotic stress. The expression of Nicotiana tobacum hemoglobin gene NtHb1 in Arabidopsis showed higher Cd tolerance and lower accumulations of Cd, NO, and ROS (Bahmani et al. 2019).

Enhanced translocation and storage

Usually, HMs are not bioavailable to plants since they exist as insoluble forms in soil. Different types of root exudates increase the bioavailability of HMs, alter the pH of the rhizosphere, and enhance HMs solubility (Mishra et al. 2017). Once the metals are bioavailable, they are absorbed at the roots and transported across the cellular membrane inside the root cells. The soil pH is maintained by the release of protons, which are controlled by proton pumps located in the plasma membrane, thus lowering its pH. The two pathways namely the symplastic pathway and apoplastic pathway are responsible for the uptake of HMs into roots. HMs uptake occurs by a symplastic pathway, an energy-dependent mechanism led by complexing agents or metal ion carriers (Dalvi and Bhalerao 2013).

Once the HMs enter the root cells, HMs form complexes with various chelators, such as organic acids. Some of these complexes are carbonate, sulfate, and phosphate precipitate (Ali et al. 2013). The complexes are immobilized in the intracellular spaces (symplastic compartments, mostly vacuoles) and extracellular space (apoplastic cellular walls). The metal ions sequestered inside the vacuoles may transport into the stele and enter into the xylem stream via the root symplasm (Thakur et al. 2016) and eventually translocated to the shoots via xylem vessels (Ali et al. 2013). HMs uptake and translocation in plants is led by a diversity of molecules, that include complexing agents and metal ion transporters. H+-coupled carrier proteins are the specialized transporters situated inside the root cells plasma membrane and take up HMs from the soil. These transporters transport certain metals across cellular membranes and moderate influx-efflux of metal translocation from roots to shoots (DalCorso et al. 2019). By lowering the intensity of accumulation and metal uptake, metal tolerance can be increased, as seen in the case of Cd in tobacco plants by overexpressing endogenous NtHb1, a class 1 hemoglobin gene (Bahmani et al. 2019). Some organic acids such as LMWOA can cause an increase in HMs absorption. Adding LMWOAs increases metal movement by establishing complexes with metals that make them soluble and accessible for uptake. The LMWOAs are biodegradable, and more efficient unlike synthetic chelates such as EDTA (Renella et al. 2004). The transformed plants sometimes show negative effects on metal homeostasis due to metal accumulation. For instance, Arabidopsis metal transporter AtHMA4 when overexpressed in tobacco, resulted in decreased Cd uptake/accumulation in roots and shoots. It was shown that the down-regulation of genes that control the Cd uptake was not responsible for reduced Cd uptake/accumulation in AtHMA4 transformants. But surprisingly, upon exposure to Cd, genes involved in cell wall lignification such as peroxidase (NtPrx11a), catechol o-methyltransferase (NtOMET), and hydroxycinnamoyl transferase (NtHCT) were up-regulated in transformants (Siemianowski et al. 2014). The expression of HMA4 is up-regulated only when the plant is subjected to high levels of Zn and Cd, while it is downregulated in non-hyperaccumulator relatives. The metal ions which are sequestered inside vacuoles might be transported towards the stele and later go inside the xylem stream through the root symplasm (Kumar et al. 2018) and eventually get translocated to the shoots via xylem vessels.

The root to shoot transportation depends on xylem loading. A certain type of ATPase, specifically heavy metal transporting ATPases (HMAs), mediates this mechanism (Hanikenne et al. 2008). HMAs role is to balance the quantity of metal absorbed and help the plant live under severe HMs stress. HMA4, a gene that controls protein transporter, is engaged in translocation of Pb and hyperaccumulation of Cd and Zn. The transport of HMs occurs inside leaves and sequestered in cells either extracellularly or intracellularly. Before they are stored in the leaves, HMs are detoxified by chelate ligands such as MTs, PCs, organic acids like citrate and amino acids like histidine in different hyperaccumulators, various metals are stored in different parts of leaves (Krämer et al. 2000; Küpper et al. 1999; Cosio 2005). An interesting strategy was utilized to increase vascular storage of HMs by expression of the TaVP1 gene that encodes wheat vacuolar H+-pyrophosphatase. Mutants were efficient in tolerating and accumulating more Cd than WT as TaVP1 role is to generate an H+ gradient. TaVP1 produces the driving force to the metal H+ antiporter further helps in the translocation of Cd across the tonoplast (Khoudi et al. 2012). The mechanism involved in uptake, translocation and sequestration of HMs in plants is schematically presented (Fig. 2).

Fig. 2
figure 2

Schematic representation of mechanism involved in uptake, detoxification, and sequestration of heavy metals in plant shoot and root system. The plants usually acquire heavy metals from contaminated soil and water and absorb them via the root system. Exudates containing chelating agents such as EDTA help in stabilizing and the uptake of the metal ions. The transporter proteins such as HMA4 and NIP help in the influx of Zn, Cd, and As(III) respectively into the xylem where they get translocated to other parts of the plant with the help of apoplastic loading and root pressure. The process of bioaccumulation occurs uniformly across the different cells and tissues of both the shoot and root systems. In the root cells, the As(III) is transported into the cell via the aquaporin (NIP) transporter and later stabilized by PCs to form a stable complex. The complex As(III)-PC is imported into the vacuole by ABCC for its storage. In addition, the shoot cells methylate the As(III)-PC complex to form organic MMA and DMA which can be volatilised. In both shoot and root systems, the divalent cation transporter NRAMP1 is responsible for the influx of HMs such as Mn, Cd, Zn and Fe. Metal transporter NRAMP3/4 helps to mobilize these ions from the vacuole into the cytoplasm. Pb and Cd ions imported via LCT1 are transported to the vacuole by ATPase HMA3. Monomethyl mercury (CH3-Hg), is converted to the elemental mercury (Hg0) before volatilisating it. [Phytochelatins (PCs); NRAMPs (Natural resistance-associated macrophage protein); Cation/proton exchangers (CAXs); ATP binding cassette(ABC); nodulin 26- like intrinsic protein (NIP); heavy metal ATPases (HMAs); Arsenite AS(III); Low Affinity Cation Transporter (LCT1)]

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Prospect of genome editing and gene pyramiding for phytoremediation

Advances in genome editing technology (CRISPR-Cas9) that enables precise editing of the targeted genome along with the haploid induction technology are promising prospects in plants for phytoremediation. A proof of concept for gene pyramiding using haploid genetics has been demonstrated in A. thaliana by pyramiding 6 genes of the TRF-like family which is a huge feat in itself (Thondehaalmath et al. 2021; Fulcher and Riha 2016; Muthu et al. 2020; Shailani et al. 2020) have accurately emphasized the need for gene pyramiding to fortify the crop plants and to improve their yield in hopes to counter world hunger. Gene pyramiding has also been attempted to alleviate different biotic/ abiotic stresses (Zhang et al. 2018), similar mechanisms could be used for heavy metal accumulation in planta, i.e., phytoremediation. Techniques such as CynoGate (Vasudevan et al. 2019) are being developed to incorporate and express multiple genes in the plant system and need more refining. Several plants are already known for their phytoremediation ability and could be a good resource for exploring the key molecular elements of phytoremediation processes. For example, Arabidopsis halleri (Cd and Zn hyperaccumulator) (Küpper et al. 2000), Hirschfeldia incana (tolerance to Pb toxicity) (Auguy et al. 2013), Noccaea caerulescens (Ni, Zn, and Cd hyperaccumulator) (Callahan et al. 2016; Kozhevnikova et al. 2020; Deng et al. 2016), Pteris vittata (As hyperaccumulator) (Li et al. 2020; Xiao et al. 2021), Brassica juncea (Chaudhry et al. 2020). Common salt marsh halophytes such as Suaeda monoica Forsk. and Sesuvium portulacastrum have been reported to be excellent candidates for phytoremediation of heavy metals like Cr, Cd, Cu and Zn; studies also reported that the plants were healthier when they were continuously exposed to heavy metals when compared with the control plants. Since halophytes are capable of tolerating high salt content (storing excess salt in vacuoles and increasing the water uptake to maintain the physiological osmolarity), the mechanism is also applicable for the accumulation of heavy metals in certain tissues and cell organelles (trichomes, cell wall, and vacuole), ensuring the ions don’t hamper the cellular machinery in any manner (Ayyappan et al. 2014; Ayyappan et al. 2015). Genomes of these plants hold a key in finding out the gene/s responsible for phytoextraction/ accumulation of heavy metals without hampering the plants’ growth. Plant-bacteria duo has been observed to be more efficient in carrying out phytoremediation than the plants alone. Several plant growth-promoting bacteria (PGPB) help in the phytostabilization and accumulation of the HMs in the roots (Mesa-Marin et al. 2020; Basu et al. 2018; Agarwal et al. 2020). These symbiotic bacteria and/or the plant may be genetically modified to further enhance the uptake of heavy metals; this notion still requires more research (Bernardino et al. 2019).

Genome editing may be applied to modulate the gene expression to enhance the metal-ligand synthesis (like PCs and MTs), metal transport proteins, plant growth hormones, and root exudates (like LMWOA and siderophores). The choice of the target site, Cas9/Cpf action, design of gRNA, delivery systems, and off-target effect, needs to be considered carefully. Modification in genes of interest, expression of genes of entire pathways, and pollutant homeostasis networks that regulate hyperaccumulation, degradation, or tolerance, could be revolutionary for cleansing the environment through plants. Liu et al. (2013) have compiled all the technologies used for the genetic engineering of plants. ZFNs (Zinc-finger Nucleases) and TALENs (Transcription activator-like effector nucleases) have been used for genome editing, but had certain setbacks; the major problem being the linkage of the meganuclease domain with their catalytic domain, which makes it difficult to engineer the nucleases according to the requirements. Also, limited knowledge of the recognition loci for such nucleases creates a hindrance in utilizing the whole genome for editing. This led to the development of more easily customizable and usable CRISPR-Cas9 technology (Basharat et al. 2018). A report of OsNramp5 knock-out by CRISPR/Cas9 led to the development of low Cd accumulating indica rice without reducing yield (Tang et al. 2017). Apart from editing the genome, CRISPR technology has also been used for transcriptional activation or repression (CRISPRa or CRISPRi) respectively to micro-manipulate the expression of genes playing an important role in phytoremediation (Thakur et al. 2019).

Not only heavy metal stress tolerance but microRNAs have also been identified to be up or down-regulated during other abiotic stresses as well. In wheat roots, 108 miRNA members belonging to 82 miRNA families were differentially expressed during extended exposure to polycyclic aromatic hydrocarbons (PAHs) such as phenanthrene (Li et al. 2017). Such microRNAs can be used to manipulate the housekeeping and other genes which will eventually aid in the function of the heavy metal tolerance genes. Fu et al. 2019 have shown that on exposure of Cd to Brassica napus, there are five families of microRNAs (MIR395, MIR397, MIR398, MIR408, and MIR858), which were differentially expressed in shoot and root tissues. In addition, they were also able to identify 3 novel microRNAs. On differential gene expression analysis, the microRNA families were known to alter the expression of 389 and 399 genes in shoot and root tissues respectively. Some of these genes were known to be important in the pathways concerning photosynthesis, amino acid biosynthesis, secondary metabolism-related genes, and some genes related to Cd stress tolerance. Similar miRNA-mRNA regulatory networks on Cd stress were attempted in potato (Yang et al. 2021).

Enhancement of phytoremediation ability of plants has greater prospects due to gene stacking or pyramiding. In some cases, modulating single gene expression does not work and requires two or more genes for this purpose. For example, combined expression of γ-glutamyl cysteine synthetase and PCS in Arabidopsis, which are taken from baker’s yeast and garlic (ScGSH1 and AsPCS1) resulted in enhanced accumulation and greater tolerance for Cd and As, in contrast to single-gene transformants (Guo et al. 2008). In another gene pyramiding effort, a bacterial merB gene was integrated into ppk/merT transgenic tobacco. The double transgenic plant showed increased tolerance of organic methyl mercury and mercury accumulation (Nagata et al. 2010). Methyl mercury was transformed to ionic Hg (II) by MerB, which is simultaneously chelated by polyP resulting in the least toxic Hg-polyP complex, hence avoiding the discharge of volatile Hg (0) in the surrounding and producing reusable plant residues which are rich in mercury (Nagata et al. 2010). Even though there is evidence for engineering an entire plant-based metabolic pathway for xenobiotic deterioration via expression of multiple-gene rather than lone-gene transformation, further experiment has to be carried out in this order for the systematic elimination of toxic chemicals from polluted region utilizing gene pyramiding/stacking approaches. Several studies like Liu et al. (2020) have found genes responsible for selective uptake for Cd and Se to improve the quality of rice grains. These genes could be alternatively regulated and expressed in inedible parts of the plants to safely store the heavy metals. With the discovery of new genes and mutant alleles, new ways to improve the plant system for phytoremediation must be continued (Sharma and Yeh 2020; Zhao and Huang 2018).

Plants that accumulate metals of about various percentages of their biomass could be better sources for phytoremediation. Such hyperaccumulator plants would be best suited to understand the underlying mechanism of phytoremediation. Proteins that are overproduced and involved in intracellular metal sequestration (vacuolar transporters, phytochelatin synthase, MTs) might efficiently enhance metal tolerance. However, they might not be helpful for an accumulation of HMs. The list of genes involved in signaling, metal-binding ligands, chelators, metal transporters, detoxifiers, and associated genes related to phytoremediation are summarized in Table 1.

Table 1 List of genes involved in signaling, metal-binding ligands, chelators, metal transporters, detoxifiers and associated genes related to phytoremediation
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Limitations and challenges of genetic manipulation for phytoremediation

The major limitation of the phytoremediation method is that the process is too slow and seasonal. The mechanisms of detoxification and accumulation of heavy metals involve multiple genes and their genetic manipulation to improve the phytoremediation potential, which is time-consuming as well as expensive (Yan et al. 2020). Another limitation of the genetic engineering of plants for phytoremediation is that genetically modified plants are difficult to gain approval for field testing based on the fear of unknown risks due to the long-life cycle of plants and trees (Koźmińska et al. 2018; Yan et al. 2020). However, the risk of contamination of food with an engineered metal hyperaccumulator, for example, is low because plants used for phytoextraction would be in isolated, industrial-type areas, not in agricultural areas. Furthermore, crops used for phytoextraction would be harvested before the seed set, thus reducing the threat of crossing with other crops intended for food or entering the food supply (Doty 2008).

Even though genetic engineering has a huge potential in phytoremediation, there have been a few problems related to the testing and release of these transgenic plants for commercial utilization due to the environmental risks and contamination to wild and non-transgenic plants. One of the ways to solve this problem would be to grow these plants in contaminated sites that are isolated. Before the flowering stage, the transgenic crops are harvested or the usage of male-sterile plants would ensure an efficient strategy to lower the chance of interbreeding and uncontrolled spread of seeds or pollen. A greater problem is when phytoremediation affects the food chain via omnivores and herbivores, which may engulf metal-rich leaves. Plants are best suited for situations where the elements are present within the range of plant roots and take longer to clean the environment than other remediation strategies. Metal bioavailability is also a concern; for example, if the metal is securely linked to the organic portions of the soil, it may not be bioavailable; on the other hand, if the metal is water-soluble, it will flow through. Another issue would be the impact of introducing non-native plant species and the usage of synthetic chemical chelates on the plant extraction of lead to enhance the bioavailability and uptake in plants, indicating the direction for future investigations.

Conclusion and future perspective

Considering the severity of HMs contamination in soil, water, and plants entering the food chain, our focus should be to understand the key underlying mechanism to modulate the phytoremediation process. For this, the multi-pronged strategies based on metal transporters and cellular targeting to particular cell types (vacuoles), for secure compartmentation of HMs and keeping cellular functions intact. Chloroplast genome-based safer genetic manipulation can help achieve high gene expression and control the risk of transgene escape through pollen. Plants having high metal tolerance capabilities are to be maintained separately from the crop plants to prevent the transfer of metals into the food chain. Transgenic plants with enhanced plant-microbe interaction and those capable of secreting metal selective ligands to solubilize elements are good candidates for phytoremediation. Stacking of multiple genes into plants to remove mixed contaminants and genome editing of targeted regulatory elements is also a viable option. Intragenic and cisgenic approaches would be helpful in breeding programs that would ease biomass production and the growth of natural hyperaccumulators.

With the rise of new technologies, such as in situ editing by the CRISPR/Cas9 gene or gene silencing, it is now possible to determine and engineer the gene function to increase the phytoremediation ability. Micro-RNAs and long non-coding RNAs, which regulate plant stress, remain the subject of study in future studies. In addition to genetic engineering methods such as increasing the absorption of pollutants by a strengthened root system, stimulating synergies between transgenic plants and gene-modified microbes, and determining new genes to improve the xenobiotic-chelating ability hold good promise to create plants that are more efficient in handling the HM stress.