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.