The (–) value represents that no recorded concentration for that heavy metal found.
ROS- Signaling of the MAPK cascade
Over the course of time and due to their stationery nature, plants have developed various acclimatization strategies against heavy metal stresses (Majeed et al., 2023; Lamers et al., 2020). Plants have achieved these acclimatization strategies by means of various molecular systems that entails the perception and transmission of stress signals (reactive oxygen species (ROS, nitric oxide (NOS) and phytohormones) to give rise to a particular response (Majeed et al., 2023; Chen et al., 2014; Jalmi et al., 2015). These perception mechanisms involve the use cellular surface receptors/sensors to detect stress and the subsequent activation of signal transduction pathways, in order to assist the plant to adapt to the HM stress being experienced (Lamers et al., 2020; Jalmi et al., 2015). However, to exhibit a particular response, it is important for the plant to perceive the stimulus and transmit it into the nucleus of the plant cell (Jalmi et al., 2015), and one of the most important changes that occur upon perception of external stimuli is change in redox state (Jalmi et al., 2015). The change in redox state occurs due to the production and accumulation of reactive oxygen species (ROS), which accompanies HM stress (Jalmi et al., 2015). These signals undergo processing and amplification through phosphorylation and dephosphorylation as they are transmitted to plant cells (Hao et al., 2021; Mondal et al., 2022). One of the more popular studied signalling pathways include plant mitogen-activated protein kinases (pMAPKs). This is pathway is highly conserved across species (Kumar et al., 2020) and these proteins belong to the family of protein kinases which play many crucial roles in various cellular process and abiotic stress response (Kumar et al., 2020). Stresses such as heavy metals, in particular, have been shown to have a significant impact on the subsequent activation of MAPK signalling pathways (Jalmi et al., 2018), by initiating a signal perceived by the plasma membrane and subsequently cytoplasm (Majeed et al., 2023). The MAPK cascade is a three-phase system comprised of three protein kinases: MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK) and MAPK (Ma et al., 2022). These three kinases are functionally connected via activation by sequential phosphorylation (Kumar et al., 2020). In plants, the activation of MAPK usually consists of a series of reactions carried out by upstream MAP kinase kinases (MAPKKs/MKK), that phosphorylate and activate MAPKs (Sinha et al., 2011). These MAPKs are finally transported to the nucleus where it is involved in controlling the tolerance and the growth and developmental processes in plant through transcriptional programming (Kumar et al., 2020). The transcriptional factors, located downstream of MAPK, are key factors in controlling these programming (Li et al., 2022). Transcription factors possess numerous phosphorylation sites and are pivotal in regulating responses to heavy metal stress by controlling the expression of downstream genes (Jagodzik et al., 2018). They serve as central components in the regulatory networks responsible for heavy metal detoxification and tolerance (Jagodzik et al., 2018). Currently, extensive research has identified a multitude of transcription factors associated with heavy metal detoxification and tolerance in plants. Among these, transcription factors such as basic leucine zipper (bZIP), heat shock transcription factor (HSF), WRKY, myeloblastosis protein (MYB), and ethylene-responsive transcription factor (ERF) are recognized for their crucial roles in regulating heavy metal detoxification and tolerance in plants (Li et al., 2022; El Rasafi et al., 2022) (Table 2 ).
Several reports have shown that ROS acts upstream of MAPK signaling to regulate the activation and duration activity of MAPKs (Liu et al., 2019). However, as mentioned before, the relationship between particular HM-induced ROS production and MAPK signaling is still poorly understood. Therefore, in this review we focus on elucidating how 5 toxic HM’s ROS production activates the MAPK and this is done to gain further insight into early events of HM stress signaling and the involvement of the MAPK cascade in regulating a response to increase plants tolerance to these HM.
The effect of five heavy metal-ROS signaling on MAPK pathways
Cadmium
Cadmium (Cd) is a toxic heavy metal, ranked 7thamongst 20 of the most toxic heavy metals (Kim et al., 2015). It is known as the most carcinogenic heavy metal, and it is introduced into the environment through natural processes such as Cd-containing rocks weathering, forest fires, volcanic eruptions, and anthropogenic systems (Haider et al., 2021; Zulfiqar et al., 2022). Cd is a high metal replacement element that has no biological function. It can replace calcium due to similar charge and ionic properties, inducing adverse effects on biological systems (Lata et al., 2019; El Rasafi et al., 2022). Cd toxicity is gaining serious research attention as it has become global hazard, with agricultural crops being the main Cd entry point into the global food chain (Aprile and De Bellis, 2020; Mikhailenko et al., 2020). Plants grown in Cd-contaminated soil can efficiently absorb Cd through their root system, and it is translocated and accumulates in the edible parts of the plant (Rabêlo et al., 2020). Cadmium occurs in various soil-bound forms; however, a substantial proportion of these forms are not readily accessible for plant uptake (Liu et al., 2018). Cadmium demonstrates a high propensity for absorption and subsequent transportation to the above ground parts of plants, as noted by Shanmugaraj et al. (2019). The extent of Cd absorption, however, exhibits variation across different plant species and genotypes due to morphological traits and physiological characteristics, as well as variations related to plant growth stages and age. (Rizwan et al., 2018). The plant root tissue plays an important role in the uptake of Cd, and it is responsible for approximately 79 – 93% soil Cd accumulation (Li et al., 2021). In general, trace elements are absorbed in their bivalent form (Fontes et al., 2014; Gupta et al., 2016). Cd2+ follows a pathway into root cells that involves the same transporters as those for Ca2+, Fe2+, Mg2+, Cu2+, and Zn2+ (Ismael et al., 2019). The passage of cadmium from the soil solution into plant roots through cell walls can occur via passive transport, primarily through diffusion (Rog Young et al., 2015). Furthermore, it has been observed that active transport mechanisms are employed by Cd to traverse the plasma membrane of root cells, facilitated by non-specific membrane transport proteins, such as zinc transporter (ZIP) and iron regulated transporter (IRT), as well as metal pumping ATPases (Wu et al., 2015; Sebastian & Prasad, 2018). Cd accumulation has been thoroughly investigated, however, the mechanism involved in Cd stress to plants is still not clearly understood. In plant systems, Cd toxicity is a major causative agent of oxidative damage, culminating in reduced plant growth induced by alterations of membrane permeability and the production of reactive oxygen species (ROS) at the organelle level. Hydrogen peroxides (H2O2), hydroxyl radical (OH) and superoxide anion (O2), are among the primary ROS responsible for membranous proteins and lipids oxidation associated with cell death (Jawad Hassan et al., 2020).
Multiple studies have been conducted attempting to elucidate the role of Cd-induced ROS production upstream of the MAPK cascade. A study by Liu et al., 2019, by means of in-gel kinase assays, identified two ERK-like MAPKs in response to cadmium treatment (CdCl2) inZea mays roots, ZmMPK3-1 (43 kDa) and ZmMPK6-1 (45 kDa). In addition, the authors also noted via CMH2CFDA fluorescence, there was Cd-induced ROS production in Z. mays roots (Liu et al., 2019). However, to validate the involvement of Cd-induced ROS production in the activation of these two ERK-like MAPKs, the authors used a two-pronged approach in order to assess 1. The influence of Cd-ROS on the activation of the MAPKs and 2. whether MAPK signaling influenced Cd-induction of ROS production. ROS inhibitors (DMTU and DPI) were used prior to the activation of the MAPKs and noted that inhibition of ROS reduced Cd-activation of both ZmMPK3-1 and ZmMPK6-1, whereas inhibition of MAPK signaling (by use of UO126) did not disturb Cd-induced ROS production (Lui et al., 2019). These two approaches indicated that Cd-treatments activated the two ERK-like MAPKs via ROS production (Lui et at., 2019). Two previous studies also validated the Cd-induced ROS activation of the MAPK cascade elucidated by Lui et al., 2019, however the two studies implemented two other approaches (using a ROS stimulus and ROS scavenger) to assess the relationship between Cd-ROS and MAPK activation. Wang et al., 2010 and Liu et al., 2010 used hydrogen peroxide stimulus (H2O2) and glutathione (ROS scavenger), respectively to elucidate the relationship between Cd-ROS and MAPK activation. Both studies identified MPK3, whereas Liu et al., 2010 also identified MPK6 in their respective plant species (Zea mays and Arabidopsis thaliana , respectively). The three studies above attempted to elucidate the relationship between Cd-ROS and MAPK activation, using different approaches (ROS inhibitor, ROS stimulus and ROS scavenger), and all concluded that the accumulation of Cd-ROS is necessary for the activation of MPK3 and MPK6 in bothZ. mays and A. thaliana , validating the role of Cd-ROS accumulation in the activation of the MAPK cascade. However, neither studies elaborated on what downstream effects these MAPK’s posed in response to Cd treatments. Zhao et al., 2021 clarified that the downstream effects of activating ZmMPK6 led to it phosphorylating ZmWRKY104 transcription factor, in Z. mays plants under drought stress. This transcription factor was further revealed via functional analysis, to be involved in abscisic acid (ABA)-induced antioxidant defence and that its activity depends on ZmMPK6 (Zhao et al., 2021). Hence, it could have been proposed that activation of the ZmMPK6 under Cd treatment could have likewise activated ZmWRKY104 in order to increase Z. mays and A. thaliana ’s tolerance to Cd-stress. Various other studies has indicated that under Cd-induced stress condition, MAPKs govern the activity of other transcription factors like bZIP, MYB, MYC, and WRKY (Ghori et al., 2019; Sharma et al., 2021) (Table 2 ). Furthermore, a crosstalk has been described between ABA, auxin, and MAPK signaling pathways, which contributes to Cd-stress tolerance in rice (Zhao et al., 2014). In soybean seedlings, the upregulation of MAPK2 has been observed in response to Cd-stress (Chmielowska-Bąk et al., 2013).
Chromium
Recently research investigating the impact of heavy metals such as chromium (Cr) namely chromium trivalent and chromium hexavalent (Cr (VI)) in plants has gained significant attention. Studies have shown that plants grown in the presence of Cr(VI) often display a reduction in growth and development resulting in subsequent loss of quality and yield (Sharma et al., 2020). Among the various (HMs), chromium hexavalent (Cr (VI) or Cr+6), stands out due to its structural similarity to phosphate (PO43-) and sulphate (SO₄²-) ions. This similarity is notable as Cr(VI) happens to be one of the more stable forms of chromium found in soils (Liu et al., 2021). Subsequently as a result of these similarities, Cr(VI) is taken up by plant roots potentially through the use of phosphate and sulphate transporters (Cervantes et al., 2001). A study by Xu et al., 2021 reported Cr (VI) as having a high affinity for the sulphate transporter Sultr1;2. The authors confirmed that Sultr 1;2 significantly influence Cr (VI) uptake in as indicated by the increase in shoot Cr(VI) in Sultr1;2 double knockdown mutant and increased Cr(VI) uptake in Arabidopsis plants overexpressing Sultr1;2 (Xu et al., 2021). Additionally, López-Bucio et al., 2014 reported that Cr(VI) influenced the expression of low phosphorus inducible reporter genes, namely, AtPT1 and AtPT2 in Arabidopsis. Furthermore, in their study the authors also observed decreased primary root development as a symptom of Cr(VI) stress which was alleviated by the addition of phosphorus (López-Bucio et al., 2014). Their study highlighted the importance of phosphorus in Cr(VI) stress responses and to a greater degree the potential of improving plant tolerance to chromate via phosphate deficiency mediated responses.
In higher plants MAPK cascades have been shown to influence the regulation of the WRKY gene family, one of the largest families of transcription factors (TFs). Studies have demonstrated that, in response to biotic and abiotic stress, WRKY-associated TFs play crucial roles in various plant processes (Jiang et al., 2017). A study by Shen et al. (2021) observed WRKY33 as an important TF involved in the remodelling of root architecture during phosphate-deficiency (Pi deficiency) response. The authors reported that the disruption of WRKY 33 resulted in accumulation of Fe3+ at the root tips, inhibiting primary root growth and promotion of root hairs. Their study demonstrated signalling crosstalk between WRKY33 and the modulation of an aluminium mediated malate transporter 1 (ALMT1) in regulating Pi-deficiency response, through the modulation of root structural architecture (Shen et al., 2021). Furthermore, Zhang et al.(2023) showed that WRKY33 is directly involved in the direct activation of ATL31, a ubiquitin ligase, during cadmium (Cd) stress. In their study, the subsequent activation of ATL31 positively regulated Cd tolerance in Arabidopsis through controlling heavy metal uptake by regulating the activity of iron-regulated transporter 1 (IRT1) (Zhang et al., 2023). Although the above-mentioned studies highlight the importance of WRKY33 in Pi-deficiency and heavy metal stress responses. Research investigating the potential functional roles of WRKY33 during plant Cr(VI) responses remains to be limited. Given that Cr(VI) uptake utilizes mechanisms involved in phosphorus uptake, investigating the activity of WRKY33 during Cr(VI) stress responses could provide insights into the function of this TF in the regulatory mechanisms of Cr(VI) tolerance. Moreover, research has shown that in plants, WRKY33 is one of the WRKY TF that gets regulated by the MAPKs (Adachi et al., 2015). Other WRKY-TFs such as WRKY23 and WRKY47 have been shown to regulate processes such as auxin mediated root development and cell wall modification such as binding capacity respectively, all of which regulate nutrient and subsequent heavy metal up take (Grunewald et al., 2012, Trinh et al., 2014). Furthermore, a recent study by Ali and authors (2023) also reported WRKY and Ap2/ERF as important TFs in Cr(VI) signalling cascades during plant heavy metal defence responses (Ali et al., 2023). In plants, basic region/leucine zipper motif (bZIP) has also been shown to be regulated by MAPKs. These TFs play a role in many essential processes such as seed maturation, pathogen defences as well as stress responses to various abiotic stresses (Yu et al., 2020). Furthermore, studies have shown that bZIPs can be phosphorylated during plant HM stress responses to various heavy metals (i.e Cd, Zn and Pb etc.) (Li et al., 2022). Additionally, Dubey and colleagues reported that bZIP Tfs influenced shielding responses against various heavy metals like Cr(VI), As, Cd, and Pb in Oryza sativa (Dubey et al., 2014). Furthermore, in a study by Fang et al. (2017), the authors observed increased activity in the bZIP (TGA3) in response to Cr(VI) stress. This triggered hydrogen sulfide (H2S) mediated defence responses as a result the subsequent increase in the transcription of L-cysteine desulfhydrase (LCD) by TGA3. (Fang et al., 2017). A recent study by Chai and authors (2022), described GmbZIP152, a soybean bZIP as an important TF involved in multiple stress responses (i.e., abiotic and biotic) (Table 2 ). In their study, the overexpression of GmbZIP152 improved S. sclerotiorum­ -disease resistance as well as tolerance to salinity, drought and heavy metals in soybean. The overexpression of GmbZIP152 improved the expression levels of antioxidant genes, namely superoxide dismutase, peroxidases, and catalase, resulting in the subsequent improved biotic/abiotic stress resistance/tolerance (Chai et al., 2022). In a study by Fan et al., (2019) the authors observed the downregulation of RsbZIP010 in response to Cr(VI) in radish, (Fan et al., 2019). Furthermore, earlier studies have shown bZIP10 to be involved in plant oxidative stress responses and cell death though its interaction with lysine-specific demethylase 1 (LSD1) (Kaminaka et al., 2006). Myeloblastosis viral oncogene homolog (MYB) transcription factors (MYB) form one of the largest families of TFs involved in the biosynthesis of secondary metabolites in plants. Transcription factors in this family have been shown to be involved in a plethora of essential biological processes such as cellular morphogenesis, growth/development as well environmental stress responses (Cao et al., 2020) (Table 2 ). In a study by Baek et al., (2013), the authors reported AtMYB2 as an important regulator of P-deficiency response through its activation of miR399f transcript which regulated the root system architecture (Baek et al., 2013). Given that Cr (VI) uptake occurs via P and S transporters. The mechanism of regulating Cr in plants could also be influenced by the activity TFs such as MYBs. Studies investigating the functional roles of MYB in plant heavy metal stress have been done. A study by Wang et al. (2017) reported on the function of OsARM1, an arsenite-responsive MYB1 transcription factor, and its subsequent binding to key promoters involved in As transporters (Wang et al., 2017). Additionally, Hu and authors also observed increased sensitivity to Cd in OsMYB45 mutants, attributed to an increase in ROS accumulation and decreased antioxidant activity compared to the wild type (Hu et al., 2017). Although these studies highlight the importance of MYB in plant responses to HM-stress via adaptive root remodelling and redox regulation, research on the functional roles of MYB with relation to Cr (VI) stress responses and MYB2 in plant Cr(VI) sensitivity is still limited. Furthermore, many studies have alluded to the notion that MYB TFs form part of the downstream targets of MAPKs as indicated by the dual alteration in gene expression of both MAPKs and MYBs in plants during heavy metal stress (Jalmi et al., 2018). Therefore, investigating the role of MAPK-mediated MYB activity in response to Cr(VI)-stress could provide insights to the functional contribution of MYB to chromate sensitivity versus tolerance (Table 2 ).
Table 2: Summary of the MAPK activated transcription factors involved in regulating heavy metal stress in plants. The four major MAPK-activated transcription factors are shown. Furthermore, the genes activated, and their roles involved in various heavy metals are shown.