Abbreviations are as follows: transcription factor (TF), basic leucine zipper (bZIP), Myeloblastosis viral oncogene homolog (MYB), and heat shock factor (HSF).

Lead, like arsenic, cadmium and coronium may enter the environment and harm plants through a variety of means, including mining (Dong et al., 2009), the burning of fossil fuels, and the use of synthetic agricultural pesticides. Out of a total of 53 naturally occurring heavy metals (HMs), 17 are biologically available and play a crucial role in the ecosystem (Alamri et al., 2018). However, for plants, animals and humans, lead (Pb) is considered a nonessential element that is categorized as the second most toxic heavy metal after Arsenic (As) (Duffus, 2002; Arias et al., 2010). When the rate of intake of heavy metals (HMs) by biological systems exceeds the rate of excretion, we refer to this as bioaccumulation of HMs in biological systems (Wang and Fisher, 1999; Ali et al., 2019). This phenomenon is especially concerning in the case of lead (Pb), which has the potential to account for approximately 10% of the total pollution caused by HMs (Collin et al., 2022). Furthermore, Pb can persist in the soil for over 2000 years, induces a wide array of toxic effects on plants, encompassed by physiological, morphological, and biochemical changes. (Kumar et al., 2020). The molecular mechanism of Pb uptake in roots has not been elucidated yet (Kumar et al., 2020). It is suggested that numerous pathways can facilitate the uptake of Pb, such as ionic channels (Kumar et al., 2020), however, Pb is a non-selective phenomenon and is independent of the H+/ATPase pump (Kumar et al., 2020). Transgenic plant studies have indicated that Pb can also penetrate into roots through other alternative non-selective pathways such as cyclic nucleotide-gated ion channels and low-affinity cation transporters. According to research on the association between lead (Pb) toxicity and an increase in ROS molecules, ROS might possibly produce phytotoxicity by causing damage to tissue ultrastructure, cellular components, and macromolecules, eventually leading to programmed cell death in plants. ROS molecules are ideal for initiating stress-signalling transduction pathways, since several processes are involved in their formation or scavenging (Hancock et al., 2001). Among these ROS processes, ionic mechanism of action believed to be sensed by calcium binding protein has been linked with affecting mineral uptake in Zea mays (Seregin et al.2004),O.sativa (Chatterjee et al.2004), Brassica oleracea (Sinha et al.2006), and Medicago sativa (Lopez et al.2007). A study by Wang et al., 2013 used transcriptomic analysis of R. sativus(radish) roots and revealed upregulation of four MAPKs (MAPKKK7, MAPK6, MAPK18 and MAPK20) in response to Pb treatments. Furthermore, looking back a study by Huang and Huang, 2008 indicated that Pb induced ROS production and activated two MAPKs (40- and 42 kDa) in O. sativaroots. By means of ROS scavenger, glutathione, and diphenylene iodonium (DPI) the authors further elucidated the relationship between Pb-induced root cell death and MAPK activation, which was that MAPK activation was dependent on Pb2⁺-cell death. The authors suggested that ROS may function in Pb²⁺-triggered cell death and MAP Kinase signaling pathway in rice roots (Huang and Huang, 2008). A study by Karanja et al., 2017 discovered 20 RsWRKY transcripts were significantly increased in Raphanus sativus L. (radish), in response to Pb treatments. RsWRKY31 and RsWRKY114 had the highest expression levels under all the abiotic stresses in their study, including Pb stress (Karanja et al., 2017). They concluded that WRKY genes may have multifunctional roles under various abiotic stresses, and could contribute to controlling signaling processes linked to transcriptional adjustments in plants under harsh conditions such as Pb stress (Karanja et al., 2017). In summary, this review gives insight on the complex effects of lead poisoning in plants, addressing environmental, molecular, and cellular issues. Understanding these consequences is critical for developing methods to reduce lead’s negative effects on plant health and the larger environment.

Arsenic may enter the environment and therefore affect plants through mining, the burning of fossil fuels and the use of synthetic agricultural chemicals. Arsenic has been noted to cause epigenetic changes in plants, such as reduced methylation at concentrations as low as 100 mg. kg^-1 (Beniwal, Yadav and Ramakrishna, 2023). Furthermore, plants exposed to As also had their nitric oxide metabolism, hormone production and nutrient acquisition reduced (Ahmadet al. , 2020; Bhat, Ahmad and Corpas, 2021; Beniwal, Yadav and Ramakrishna, 2023). It is well known that heavy metals such as arsenic have the ability to induce ROS. These ROS molecules are able to initiate stress-singling transduction pathways (Mittler et al. , 2022). Within these pathways, calcium-dependant proteins kinases (CDPKs) as well as mitogen-activated protein kinases (MAPKs) play a key role. The mechanism governing arsenic uptake and detoxification are fundamentally alike across plants, yeast, and bacteria (Navarro et al., 2022). It has been stated that arsenic can mimic natural substrates such as phosphate (Pi), glucose, and glycerol well enough to share their uptake systems, suggesting that uptake of various forms of arsenic, from the soil to the root cells, can occur via numerous kinds of transporters (Garbinski et al., 2019) such as phosphate transporters, silicon transporters and aquaporins (Khan et al., 2021). As(V) are mainly facilitated by Pi transporters (Abedi and Mojiri., 2020). Whereas shown in Oryza sativa (rice) plants, arsenite (As(III)) is transported into root cells via silicon transporters (Khan et al., 2021). It has been shown that MAPKs are activated in response to As(III), suggesting their implication in arsenic signaling (Navarro et al., 2022) and it has been documented that arsenic lead to production of ROS and NO in arsenic treatedO. sativa seedling roots (Rao et al., 2011), however, whether this MAPKs response was instigated by ROS production was to be validated. Rao et al., 2011 observed that in O. sativa roots a 42- and 44-kDa MPK was activated in response to arsenite treatment. Transcript analysis of the MAPK family revealed them to be OsMPK3and OsMPK4 (Rao et al., 2011). Gupta et al., 2009 compared two varieties of Brassica juncea with varying arsenite tolerance (Pusa bold (more tolerant) and Varuna) and observed induction of ROS under As(III) treatment in both varieties. In-gel kinetic assays revealed activation of a 46 kDa MAPK in both varieties in response to As(III) treatment (Gupta et al., 2009), with the activation more prominent in the Pusa bold variety, indicating that MAPK might be playing important role in transducing As(III) signal for an appropriate cellular response (Gupta et al., 2009). A study by (Huang et al. , 2012) performed on rice exposed to arsenic highlighted that one MAPK (OsMPK6) belonging to the GMGC group and seven MAPKKK belonging to the STE group were upregulated in response to the stress. The same study also showed that transcription factors belonging to the HsfA and HsfB were upregulated during the experiment (Huang et al. , 2012). Other transcription factors which have garnered recent attention for their potential ability to improve arsenic tolerance include the WRKY transcription factors (Mirza, Haque and Gupta, 2022). These transcription factors were found to be the substrates of MAPK, which is able to phosphorylate them. A study by (Zeeshan et al. , 2022) observed that the transcription factors under the WRKY family, GmWRKY6 (mediating arsenate transport), GmWRKY46 (ABA signalling and auxin homeostasis) (Lal et al. , 2023), GmWRKY56 and GMWRKY106 had only a slightly higher expression when grown under arsenic stress when compared to the negative control. However, the same study showed that the application of zinc oxide nanoparticles led to a significant increase in the expression of these transcription factors, improved soybean development, and lowered arsenate uptake by reducing the phosphate and arsenate transport genes (Zeeshan et al. , 2022). The findings of the aforementioned transcription factors show the importance of their activation and the need to understand their role in improving arsenic tolerance. Another transcription factor that is activated by MAPK is MYB40. In a study by (Chen, Wang and Chen, 2021) the transcriptional factor was induced by arsenate; however, when this transcriptional factor was overexpressed, it increased the plant’s resistance to arsenate by reducing the uptake of the metal. The reduction in arsenate was obtained through the expression of PCS1 (phytochelatin synthase) in Arabidopsis which was positively regulated by MYB40 (Chen, Wang and Chen, 2021). The findings of these studies show the importance between ROS stimulation of the Mitogen-activated Protein Kinases and the activation of certain transcription factors under arsenic toxicity.

Mercury (Hg) is greatly distributed in nature (Bhattacharya, 2018), owing to its application in fertilizers, lime, sludges, and manures (Azevedo and Rodriguez, 2012). Mercury’s lengthy half-life and extremely detrimental effects under low concentrations, has granted it as the heavy metal of significant toxicity (Chen et al, 2014; Shao et al, 2022). Entry of mercury into plants is mainly theorized to occur through ionic channels, where this heavy metal competes with other metals such as zinc, copper, iron, and cadmium (Azevedo and Rodriguez, 2012). Once in plants, this HM has no nutritional function (Shao et al, 2022) and tends to accumulate within the roots of several plant species (Chen et al, 2014), and here its accumulation is heavily phytotoxic causing disruptions to numerous cellular-level functions and inhibits plant growth and development (Chen et al, 2014). Mercury has been shown to be connected to an excess production of reactive oxygen species (ROS), which may cause lipid peroxidation, enzyme inactivation, inhibition of photosynthesis, transpiration and nutrient transport in plants as well as causing DNA and membrane damage (Chen et al, 2014; Shao et al, 2022). A study by (Chen et al., 2014) validated this as the authors noted that mercury stress led to an induction of ROS production (superoxide and hydrogen peroxide), in Oryza sativa (rice) roots. Through whole-genome array on rice roots, Chen et al., 2014 examined the transcriptome responses to short- and long-term mercury stress (Chen et al., 2014). Through in-gel kinase activity assays the authors discovered two MAPKs of roughly 40- and 42-kDa that was dose-dependently activated in rice roots exposed to mercury (Chen et al., 2014). The authors though did not elucidate on 1. The identity of the two MAPKs and 2. The relationship between mercury-induced ROS production and these two MAPKs activation.

However, a study by Wang et al., 2012 elucidated these via comparative proteomic approach, using wild-type O. sativa and mercury (Hg²⁺)-tolerant mutant roots. Using Mass spectrometry (MS) in the signal transduction category, a MAP kinase 2 (MAPK2) was upregulated in the mutant and wild-type rice roots during Hg treatment (Wang et al., 2012), while it was weakly expressed in the untreated rice roots. The authors previous study indicated that the mercury induced ROS accumulation, and that OsMPK3 is a ROS-induced MAPK (Yeh et al., 2003; Wang et al., 2009). Therefore, in view of Hg²⁺ induced up-regulation of MAP kinase 2 and protein kinase domain containing protein, we suggest that the MAPK signaling pathways are required during plant response to Hg²⁺ stress (Wang et al, 2012). Yeh et al., 2003 indicated that H₂O₂ was involved in the induction of a 42-kDa MAPK-like kinase by CuCl₂ in rice roots, by using ROS scavenger, glutathione. The author indicated that pre-treatment of rice roots with glutathione, greatly decreased the level of copper-induced MAPK-like activation (Yeh et al., 2003). Suggesting that CuCl₂treatments result in ROS production and activation of MAPK-like kinase, at least in part (Yeh et al., 2003). The authors discovered that the 42-kDa MBP was in good accordance with those of OsMAPK2 (Yeh et al., 2003). In plants, higher amount of copper, cadmium and mercury ions lead to activation of a novel MAPK gene OsMSRMK2 from japonica-type rice (cv. Nipponbare). (Sinha et al, 2011). OsMSRMK2 was shown to be a multiple stress responsive MAP kinase gene. It was indicated that this gene accumulated rapidly suggesting the role of OsMRMK2 in transmitting signals produced in response to environmental cues (Agrawal et al, 2002). Chen et al., 2014 noted that of their multiple differently expressed genes in response to Hg-treatment, WRKY and heat shock factor (HSF) transcription factors were highly upregulated in rice roots. Numerous studies have previously reported that WRKY and HSF serves essential roles in plant resistance to biotic and abiotic stresses (Chen et al., 2014). In the authors array data, they discovered 5 group I WRKY transcription factors (OsWRKY24, 30, 38, 53 and 70 ) that were upregulated under mercury stress, hence, leading to the authors to assume that group I OsWRKY genes may serve particular roles in the mercury-stress response by activating MAPKs (Chen et al., 2014).

Regarding all the heavy metals discussed above it can be noted that among the HM-induced ROS production and MAPK activation that MPK3 and MPK6 were two of the MAPK’s that were in response to most of the heavy metals indicating their roles in HM-ROS-MAPK response pathway in various plants. Hence, these MPK should be further investigated to elucidate further how they confer a degree of tolerance to various metals and further to assess the downstream transcriptional programming that confers a degree of tolerance to HM-stress.

In the forthcoming years, heavy metals will harshly threaten crops due to climate change and continuous heavy industrialization. Therefore, there is an urgent need to validate the functional roles of MAPK genes and downstream transcriptional factor activation, in order to develop heavy metal resistant crops. This review highlights a pipeline that can be implemented across a variety of plant species to determine and elucidate the relationship between HM-induced ROS production and the activation of MAPKs, under various HM-treatments (Figure). In which the first step would be to conduct the analysis on fully sequenced genomes (Arabidopsis and rice) or on emerging sequence information (Populus, Medicago, lotus, tomato, maize, and chickpea). This is an essential aspect for validating or discovering new players within this signaling pathway and will assist with efficiently determining the function of multiple genes simultaneously (Sinha et al., 2011). The via three approaches (ROS stimulus, ROS inhibitors, or ROS scavengers) one can assess the relationship between HM-ROS production and MAPK activation. Thirdly, to identify MAPKs that are differentially regulated under HM-treatments, in-gel kinase assays can be conducted, and MAPK can be identified based on their molecular weight. In order to confirm the activity of MBP phosphorylating kinase is due to a MAP kinase, immunokinase assays can be performed (Figure). These steps above all assist in validating the MAPKs activation via HM-ROS production, however, various other approaches can be incorporated or explored to further elucidate more players within the MAPK pathway. A pipeline elucidating downstream MAPK transcription factors has not yet been properly developed. In order to identify more MAPK gene families in plants related to HM-stress and to identify their functional analysis multiple approaches such as CRISPR/Cas technology, DNA/RNA sequencing, transcriptomics, proteomics, and metabolomics can be explored to allow the analysis and clarification of a regulation network that controls HM-stress response.

To gain more insight into the cellular responses of plants to heavy metals, we could perform a large-scale analysis of the plant’s transcriptome during these heavy metal stresses (Chen et al, 2014). Identifying and further characterization of the acquired heavy metal- responsive TF and genes may be helpful for better understanding the mechanisms of heavy metals in plants (Chen et al, 2014). Once these key TF and genes have been identified, mutational studies (gene knock-ins and knock-outs) can be conducted to observe functional roles of these key players in heavy metal responses. Proteomics is another approach which could be used to address the biological function of proteins in response to the various HM-stresses, in order to assist with understanding the injury mechanisms induced under the HM-stresses as well as understanding the adaptation processes of the plants to these conditions (Wang et al., 2012). The study of plant stress responses at the proteome level has provided deeper insights into the functions of protein/protein-networks during plant adaptative stress responses. Proteins play important functional roles in the maintenance of cellular homeostasis and the regulation of adaptive stress response mechanisms such as regulating phenotypic plasticity (Zhu et al., 2023, Mackenzie and Kundariya, 2020).

In the post-genomics era, the use of mass spectrometry (MS) based proteomics has significantly improved the ability of researchers to understand how plants respond to varying conditions or stresses (Liu et al., 2019). By coupling enrichment techniques with MS-based proteomics, researchers gained crucial information about the about the function and subcellular localization of proteins as well as their post translational modification (PTM) (Komatsu and Hashiguchi, 2018, Zhao and Jensen, 2009). Of the numerous PTMS, protein phosphorylation continues to be one of most extensively researched. During this process kinases mediate the addition/removal phosphate groups present on serine, threonine and tyrosine residues which alter the function of a protein (Ramazi and Zahiri, 2021). Hence, the use of phosphoproteomics has become a powerful in understanding mechanism phosphorylation-dependant signalling response, such as MAPK in plants (Wang et al., 2020)

In the context of plant stress responses, MAPKs have been shown to be involved protein regulation via phosphorylation mediated PTMs. In a study by Forzani et al .,(2011) study and authors reported that serine/threonine protein kinase Pto-interacting 1-4 (PTI1-4) as one of the targets of MAPK (MPK6) for signalling during oxidative stress responses in Arabidopsis(Forzani et al., 2011). Additionally in separate study several MAPK’s, namely SIMK, MMK2, MMK3, and SAMK, were shown to be induced during plant heavy metal stress response in alfalfa roots (Jonak et al., 2004). In a study by Zhong et al., (2017), using phosphoproteomics the authors observed a significant increase the phosphorylation of proteins in Cd-stressed rice. In their study many of the proteins were involved in ABA mediated abiotic stress responses, some of which included the MAPK signalling protein DSM1, that has been shown to be involved in the regulation of ROS scavenging (Zhong et al., 2017).

In this review we discussed the various processes MAPKs are involved in, such as plant development and stress responses. The activation of proteins by MAPK-phosphorylation plays an essential role in plant heavy metal stress responses, affecting processes such as metal mobility, managing ion toxicity, and minimizing oxidative damage. The use of modification-specific proteomics tools such as phosphoproteomics could assist with deepening our understanding of the contribution of how phosphorylation-dependant, MAPK-mediated changes influences plant HM responses. This holds great potential for the future progression of plant heavy metal stress tolerance, benefiting both agricultural and environmental conservation.