Plants cells are the depot for different ions, including calcium. Calcium (Ca⁺²) is an essential bivalent cation with varying plant utilities (Aldon et al., 2018; Yadav et al., 2022). Calcium ions (Ca²⁺) partake in several physiological parameters in plants, including cell division, cytoplasmic streaming, thigmotropism, photomorphogenesis, cell polarization, fruit development and ripening and plant microbe interaction (Gao et al., 2019; Furuichi, 2020; Eichstädt et al., 2021; Duo et al., 2022). Since the discovery of its effects on muscle contraction, the perception of calcium as a regulatory molecule in living organisms has gained wide-range recognition globally. Plant and animal cells contain a group of proteins with Ca²⁺-binding properties (Bose et al., 2011; Jia et al., 2022). These proteins alter their conformation upon stress by external stimulus, increasing the cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) concentration. Thereafter, the [Ca²⁺]_cyt couples the extracellular stimuli to their distinctive intracellular responses and synchronize a wide range of endogenous processes. Besides increasing the concentration of the [Ca²⁺]_cyt, sometimes these special Ca²⁺-binding proteins directly interact with other targets and allow them to execute their respective response (Batistič and Kudla, 2009). Several shreds of evidence have established the ubiquitous role of Ca²⁺ as an essential cellular second messenger (Marcec et al., 2019). The details on the characteristics and molecular mechanisms of calcium signalling can be found in Bickerton and Pittman, 2012 and Kudla et al., 2018.

The importance of herbicides to modern crop production is immutable. However, due to excessive application of synthetic herbicides, there has been a continuous rise in the number of cases of herbicide-resistant weeds. The evolution of herbicide resistance in weeds is an exceptional example of the adaptability of weed species to abiotic stress (Délye et al., 2013). An increase in cellular Ca²⁺ concentration is a primary signalling event when plants confront abiotic stimuli (Xu et al., 2022). Hence, assuming that calcium signalling might play a vital role in weeds toward herbicidal resistance/tolerance under herbicide stress would not be erroneous. However, to date, there have been no attempts to explore the plausible role of Ca²⁺ in this area ( Supplementary Figure 1 ). Hence, in this perspective, we will discuss the plausible roles of Ca²⁺ signalling in conferring herbicide resistance. Because of space limitations, we will cover the main aspects of calcium as a signal transducer involved in herbicide stresses.

Among the vital environmental stresses affecting agriculture, the most important are drought, salt, and temperature stresses (Hirayama and Shinozaki, 2010; Chaudhry and Sidhu, 2022; Zhang H. et al., 2022). To overcome these constraints, plants developed sophisticated mechanisms, including systematic regulation of Ca²⁺ ions within the cells, which might lead to intonations of gene expression (Reddy et al., 2011; Manishankar et al., 2018). Studies suggest Ca²⁺ plays a crucial role in regulating the physiological response to drought conditions by acting as a secondary messenger and transmitting drought signals (Hong-Bo et al., 2008; Pathak et al., 2020). It has also been elucidated that cytosolic-free calcium plays a pivotal role in stomatal movement, and changes in its concentration can regulate the opening and closing of the stomata (Wang et al., 2005; Wang et al., 2016; Thor et al., 2020). The movement of stomata regulates two of the key plant physiological processes, i.e., transpiration and stomatal conductance, which is closely related to water-use efficiency, a physiological trait of immense importance concerning drought stress. Previously, the close relationship between Ca²⁺ concentration and the degree of drought stress in wheat seedlings was examined, and it was observed that the concentration of free Ca²⁺ in the nucleus increases with increasing drought duration, indicating its potential role in maintaining nucleus structure and integrity (Hong-Bo et al., 2008; Song et al., 2008).

Ca²⁺ ions not only promote plant growth but also possess the potency to reverse the detrimental effects of salt stress (Roy et al., 2019; Borjigin et al., 2021; Lian et al., 2022). For instance, salinity causes a severe reduction of hydraulic conductivity in the primary roots of the plants. However, exogenous application of Ca²⁺ mitigated these effects in maize and other plants (Ahmad et al., 2018; Pathak et al., 2020; Lian et al., 2022). Evidence also suggests that the Ca²⁺ signalling network is closely associated with the SOS (salt overly sensitive) signal transduction pathway and regulates the homeostasis of cellular Na⁺ and K⁺ (Brini et al., 2007).

Apart from salt and drought stresses, there has been a significant increase in research correlating Ca²⁺ ions and temperature stress tolerance. Ca²⁺ has been found to play a vital role in plants’ adaptation to cold and heat stress conditions (Hong-Bo et al., 2008; Pathak et al., 2020). For instance, in an attempt to acclimatize in the alpine subnivean conditions, Chorispora bungeana accumulates Ca²⁺ among its various tissues and organs (Fu et al., 2006). Ca²⁺-responsive protein kinases such as soybean calmodulin (GmCaM4) and Ca‐dependent protein kinase 6 (CDPK6) in Arabidopsis were found to regulate the metabolism of plant cells positively and confer cold tolerance (Pathak et al., 2020). In addition, exogenous treatment of Ca²⁺ during cold stress enhances plant growth, development, morphology, and physiology by modulating the plant’s photosynthetic capacity, ROS metabolism, and nitrogen assimilation (Yuan et al., 2018; Singh et al., 2022). In acclamatory response to heat stress, Ca²⁺ signalling causes increased expression levels of the gene coding for an enzyme called desaturase, an essential enzyme responsible for maintaining membrane fluidity under temperature stress (Huda et al., 2013). Ca-mediated heat shock proteins (HSPs) are also induced during heat stress and provide necessary cellular homeostasis (Pathak et al., 2020). In a previous study on tobacco, the heat stress caused a decrease in various photosynthetic parameters such as net photosynthetic rate, apparent quantum yield and stomatal conductance. However, the exogenous application of CaCl₂ mitigated these effects (Tan et al., 2011). In the same study, activities of ascorbate peroxidase, superoxide dismutase, catalase, and peroxidase also decreased under heat stress, but with CaCl₂ pretreatment, the levels of these enzymes showed significant alterations (Tan et al., 2011). Hydrogen peroxide (H₂O₂) tends to accumulate in plants as a response to stress like high temperatures and has been extensively used as a physiological indicator to assess the intensity of stress felt by plants (Asaeda et al., 2022). Interestingly, the levels of H₂O₂ have also been found to decrease upon exogenous calcium chloride treatment indicating enfeebling effects of Ca²⁺ during stresses (Tan et al., 2011; Pathak et al., 2020). Table 1 lists some critical Ca²⁺ signalling in abiotic stress-specific research works identified in major plant species in the last few years.

The most basic molecular mechanisms of herbicide resistance include target-site resistance (TSR) and non-target-site resistance (NTSR). Among the TSR, genetic mutations and gene overexpression are the most prevalent mechanisms. NTSR mechanisms include enhanced metabolism, reduced absorption and translocation etc. (Gaines et al., 2020; Torra and Alcántara-de la Cruz, 2022). NTSR mechanisms are more complex than TSR mechanisms and involve transcriptional reprogramming of stress-related genes such as cytochrome P450s (CYP450s) and glutathione-S-transferases (GSTs). Hence, the development of herbicide resistance in grass weeds can be considered almost similar to other abiotic stresses such as temperature and drought. Weedy plants have developed several exciting mechanisms to adapt against herbicide stress. Although numerous mechanisms have been discovered, several such mechanisms, especially different signalling events, are yet to be discovered.

Ca²⁺ signalling events amend the expression patterns of several essential genes (mainly NTSR genes) in response to several biotic and abiotic stresses (Moscatiello et al., 2018; Yadav et al., 2022). Depending upon the activation mechanisms, Ca²⁺ influx transporter channels might translate a wide range of signals into diverse Ca²⁺ signatures. To date, five different families of Ca²⁺ channels have been recognized in higher plants. These include two-pore channels (TPCs), glutamate receptor-like channels (GLRs), Cyclic nucleotide-gated channels (CNGCs), mechanosensitive-like channels (MSLs) and the reduced hyperosmolality-induced [Ca²⁺]_cyt increase (OSCAs) channels. Within the plant cells, these channels are maintained precisely in an extremely synergistic manner (Ghosh et al., 2022). Upon being stressed by herbicides, it could be assumed that these Ca²⁺ influx transporters might get triggered, and hence there might be a sudden influx of Ca²⁺, thereby creating herbicide stress-specific Ca²⁺ signatures. Thereafter, the diverse and extensive set of Ca²⁺ sensors (known as Ca²⁺ binding proteins) decodes and relays the signals for further processing (usually phosphorylation responses). These Ca²⁺ binding proteins can be classified into sensor relay proteins and sensor responder proteins. The sensor responder proteins include calcium-dependent protein kinases (CDPKs), which mainly bind to these Ca²⁺ ions and induce conformational changes (Hashimoto and Kudla, 2011). After that, the herbicide-stress-specific CDPKs decode and translate the message of elevated calcium concentration into enhanced protein kinase activity and subsequent downstream signalling events. Contrary to the responder proteins, the relay proteins lack effector domains and are not direct target proteins. The relay proteins include calmodulins (CaMs), and their primary function is to bind to these Ca²⁺ ions and induce conformational changes in the responder proteins (Hashimoto and Kudla, 2011).

Although the lack of higher-resolution genomic data and tools in weeds have limited our detailed knowledge of the CDPKs in these species, these proteins are well-characterized for the model plants such as A. thaliana (Shi et al., 2018). Additionally, molecular cloning and functional analysis studies have confirmed the essential roles of CPKs in abiotic stress tolerance in a wide range of plants (Atif et al., 2019). CDPKs contain a serine/threonine protein kinase domain, an autoinhibitory domain, and a CaM-like domain. The autoinhibitory domain plays a vital role in maintaining the activated state of the kinase domain. Once the cellular Ca²⁺ levels get elevated, the binding of Ca²⁺ to the CaM-like domain leads to a conformational change of the autoinhibitory domain and activates the kinase. In addition to the conformational changes of the CaM-like domain, auto-phosphorylation of these proteins further helps in the kinase activation process. Along with the CDPKs, plants contain two other sensor responder proteins: Ca²⁺/calmodulin-dependent protein kinases (CCaMKs) and CBL-interacting protein kinases (CIPKs). These kinases and their specific Ca²⁺ binding proteins form a complex cellular network in various cellular processes (Batistič and Kudla, 2009; Weinl and Kudla, 2009). Sometimes these complex cellular networks are stress-specific, and to the best of our knowledge, there are no reports or efforts to discover herbicide-stress-specific cellular signalling systems.

The final step in calcium signalling under herbicide stress will be converting these complex signals into their respective transcriptional responses. The activation of the Ca²⁺ sensor kinases further induces phosphorylation events, thus leading to the transcriptional reprogramming of essential genes (Khare et al., 2020). Usually, these transcriptional reprogramming events are mediated by activating stress-specific transcription factors (Himanen and Sistonen, 2019). Ca²⁺/CaM-mediated transcription factors relay Ca²⁺ transients generated by herbicide stress to transcriptional reprogramming of CYP450s and GSTs. RNA-seq transcriptome profiling studies have provided shreds of evidence that upregulation of CYP450 isoforms is involved in the metabolism of different herbicides in several weed species, including Echinochloa sp. (Fang et al., 2019; Pan et al., 2022), Alopecurus sp. (Zhao et al., 2017; Franco-Ortega et al., 2021). However, in most of the cases, the mechanisms of the CYP450 upregulation were not investigated. Transcription factors are potentially involved in regulating the expression of the NTSR genes, such as CYP450s, GSTs and ABC transporters. A recent study by Zhang Y. et al. (2022) on Echinochloa spp. has shown that upregulation of the bZIP88 transcription factor confers resistance to three different herbicides (Zhang Y. et al., 2022). However, the molecular mechanisms behind the upregulation were not further investigated. bZIP transcription factors are known to regulate abiotic stress responses in many plants and are expected to play a regulatory role in NTSR (Century et al., 2008; Zhang H. et al., 2022). Investigations of the calcium signalling events during the herbicide treatment might provide new understandings to such unexplained questions. A schematic diagram showing the putative role of calcium signalling in weeds in herbicide resistance can be found in Figure 1 .

Besides playing a role in conferring resistance against the herbicides, Ca²⁺ signalling might also play an essential role during stressful “herbicide-exposure memories” in weeds. Higher plants, including weeds, have developed different mechanisms (established over a long period of evolution) to respond and adapt to recurring stresses (Bhar et al., 2022). Among the two distinct mechanisms of herbicide resistance, the NTSR mechanisms play critical roles in integrating and coordinating whole plant herbicide stress responses. There is considerable evidence that [Ca²⁺]_cyt signatures get modified by previous experience with an environmental challenge (Bruce et al., 2007). There is also evidence that prior exposure to contrasting stress can alter the Ca²⁺ signatures provoked by a particular stress (Tuteja and Mahajan, 2007). These indicate a cross-talk between the signalling cascades. Frequent exposure to herbicides might lead to an attenuated response of Ca²⁺ signalling pathways, and the cells might retain the previous information. Since maintaining a cellular [Ca²⁺] homeostasis is vital for any plant, this Ca²⁺ memory is significant for herbicide-resistant weeds. These weeds can thus quickly retort better to the herbicide stress without upsetting the delicate and sensitive balance of Ca²⁺.

Alternative splicing (AS), in general, generates multiple mRNA isoforms from the same pre-mRNA and is alleged to contribute to increasing transcriptomic and proteomic diversity (Chaudhary et al., 2019; Mandadi et al., 2022; Tognacca et al., 2022). The putative roles of AS in a wide range of physiological processes (such as plant metabolism, plant immunity, plant disease resistance etc.) have been well explored in many plants. However, despite the evidence of multiple copies of important herbicide target genes (such as ALS, ACCase and EPSPS), the area of alternative splicing has remained unexplored in weedy plants (Xu et al., 2014; Iwakami et al., 2017; Yakimowski et al., 2021). Although no experiments have shown AS in weeds, NGS and omics approaches in plants have revealed that most of the plant multi-exon genes undergo AS (Chaudhary et al., 2019). We hypothesize that Ca²⁺-signalling might influence the alternative splicing of these herbicide target genes by controlling the relevant splicing factors. Earlier, we discussed that elevation of the intracellular Ca²⁺ level activates a variety of signalling kinases; after that, the Ca²⁺-binding sensor proteins trigger phosphorylation responses during abiotic stresses (Xu et al., 2022). So, it is possible that when exposed to herbicides, Ca²⁺-binding sensor proteins might stimulate the splicing factors via phosphorylation. Furthermore, Ca²⁺-signalling regulates the subcellular redistribution of splicing regulatory proteins (Razanau and Xie, 2013). For example, in rats, thapsigargin treatment triggered an increase in the intracellular calcium concentration, leading to hyperphosphorylation and further accretion of a splicing factor in the cytoplasm (Daoud et al., 2002). Moreover, polarization and depolarization might also significantly impact the chromatin accessibility of the variable exon by regulating the histone modifications (Razanau and Xie, 2013). As a result, Ca²⁺-signalling is thought to play an important role in mRNA isoform expression by controlling chromatin status. In summary, comprehensive experiments are needed to elucidate possible regulatory roles of Ca²⁺-signalling in alternative splicing in herbicide-resistant weeds.

Even though many indicators are available for plants, none of those have been used in weedy plants to understand the role of Ca²⁺ signalling in alleviating herbicide stress. Since changes in the intracellular and cytosolic Ca²⁺ concentration are stimulus-specific, Jaffe posited three critical criteria to ensure that a particular Ca²⁺ signal pattern is associated with a particular stress response. These criteria include: (a) inhibition of the levels of Ca²⁺ must also impede the physiological response; (b) an artificial increase in the Ca²⁺ levels must provoke the physiological response, even in the absence of the stimulus; and (c) an increase in the cytosolic Ca²⁺ concentration must either precede or accompany the response (Jaffe, 1980). The first two criteria can be accomplished by various chelators and ionophores, whereas the last criterion requires techniques that can measure intracellular resting and stimulated Ca²⁺ levels in weeds. Hence, monitoring the Ca²⁺ signal patterns associated with the herbicide stress response is crucial.

In the past years, Ca²⁺-selective microelectrodes were used to evaluate the modifications of the intracellular Ca²⁺ concentration. However, owing to their convenience, in recent years, these microelectrodes have been replaced by luminous Ca²⁺ indicators, such as Fura Red, Fluo-4, quin-2 and indo-1 (Bush and Jones, 1990; Kanchiswamy et al., 2014). Confocal laser scanning microscopy (CLSM) with these fluorescent Ca²⁺ indicators might be an excellent option for measuring Ca²⁺ in living plant cells. Besides CLSM, calcium imaging can also be done based on Förster Resonance Energy Transfer (FRET) and Selective Plane Illumination Microscopy (SPIM). Even though FRET has been used in Arabidopsis, to our knowledge, these techniques have not been used to understand calcium signalling in any weedy plants. In addition to these Ca²⁺ dyes, protein-based Ca²⁺ indicators (such as aequorin–based and GFP–based indicators) are also used to measure Ca²⁺ in living plant cells (Mithöfer and Mazars, 2002; Granatiero et al., 2014; Kanchiswamy et al., 2014). Aequorin is a calcium-activated photoprotein composed of apoaequorin (as apoprotein) and coelenterazine (a luciferin molecule, as a prosthetic group). When Ca²⁺ occupies the Ca²⁺-binding sites of the aequorin, the coelenterazine gets converted into coelenteramide and is released together with carbon dioxide. Thereafter, on returning to its ground state, blue light (λ = 469 nm) is emitted, which can be detected with a luminometer. Bioluminescent probes paired with Ca²⁺-sensitive aequorin can be used in the weeds for real-time measurement of the Ca²⁺ signal patterns linked with herbicide stress. In addition to the aequorin-based Ca²⁺ sensors, since their discovery in 1997, the GFP-based Ca²⁺ indicators have also gained the interest of plant biotechnologists (Kanchiswamy et al., 2014).

To understand the mechanisms of Ca²⁺-mediated molecular signalling processes, understanding the expression patterns and correlations with their biochemical activities is vital. Additionally, conducting these analyses in a spatiotemporal fashion will be better. Even though these Ca²⁺-induced changes are well documented in some model plants (Cui et al., 2020; Schulz et al., 2021; Wang J. et al., 2021; Gao et al., 2022; Li et al., 2022), reckonable data at high spatiotemporal resolution are still inadequate. In weeds, to date, there are no attempts to study both Ca²⁺-induced changes and high spatio-temporal resolute data. Elucidation of the molecular signalling mechanisms in model species is mainly based on high-resolution genomic analyses under controlled experimental conditions. Quite the reverse, the unattainability of high-resolution genomic data hampers the identification of the connection between various Ca²⁺ signatures with the phenotypic effects in non-model organisms such as weeds. Additionally, no practical tools for in-vivo Ca²⁺ quantification are available for the weed system. However, attempts must be made using existing tools available for Ca²⁺ quantification within the plant system. Additionally, In-silico analysis of the gene families involved in the Ca²⁺ signalling will help detect Ca²⁺ transporters in weeds (Goel et al., 2011; Mohanta et al., 2015; Yadav et al., 2022).

Despite extensive research on the mechanisms of herbicides in recent years, the mechanisms of herbicides and the herbicide stress signalling network have not been investigated and discovered. Much progress has been made in understanding several Ca²⁺-mediated signal networks in crops and plants under various environmental stresses such as cold, heat and drought (Kudla et al., 2018). Similar to plant abiotic stresses, herbicides are known to cause oxidative stress (Caverzan et al., 2019). Hence, the resistance against these chemicals involves the increased expression of several stress-responsive genes, such as CYP450s and GSTs (Délye et al., 2013). Several signal pathways might be involved during herbicide stress signalling (Alberto et al., 2016). In this perspective, we attempted to describe a possible hypothesis on how a Ca²⁺-mediated signal network might confer herbicide resistance. However, due to the lack of any solid experimental attempts evidence, we had to confine ourselves to the theoretical concept as discussed in Section 3 and Figure 1 . Currently, research efforts are increasing to obtain reference weed genomes. In addition, bioinformatics-based modelling approaches, RNA-seq transcriptome and gene editing experiments (using RNAi and/or CRISPR/Cas systems) are needed to elucidate the exact roles of Ca²⁺ signalling in weeds under herbicide stress. Implementing the multi-omics data-based approaches (generated from genomics and transcriptomics) will be very useful in identifying the candidate Ca²⁺ transducing proteins, channels, exchangers and pumps involved in weed-herbicide interactions and other abiotic stress responses in weeds. Weeds have developed resistance against all the major herbicidal modes of action. Hence, further genetic engineering of these stress-specific Ca²⁺-signalling regulators might enable the herbicide industries to discover novel targets and use them for herbicide discovery. Research on weed molecular communication during herbicide stress (particularly Ca²⁺ signalling) is still in its infancy. This area of research should be an exciting area for future research in terms of novel and applied science.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

MS and AR conceived the idea. MS, KH, RB and PK drafted the manuscript. MS and RAC designed and created the figure. AR and JS edited the manuscript MKS revised the manuscript. All authors contributed to the article and approved the submitted version.