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