Calcium Signaling in Plants Under Drought

Abstract

Environmental stresses are among the most important threats for food security since it adversely affects agricultural productivity. Among them, drought stress and water scarcity stand as major problems, especially in developing countries. Thus, understanding molecular mechanisms involving drought stress response is of essential importance for coping with its detrimental effects. Calcium is an essential macro-element, taking part in the regulation of many aspects of plant growth and development along with playing the role of ubiquitous secondary messenger, thus taking part in the generation of adaptive responses toward various developmental and environmental stimuli. Therefore, understanding the calcium signaling and calcium-dependent processes is crucial for improving plant productivity under environmental stresses. Formation of calcium signature through subsequent oscillations is at the onset of calcium signaling which further activates calcium-binding proteins which are acting as signal decoders, makes it possible to perceive the nature of stimuli. Further, protein kinases and transcription factors are activated in a stimuli-specific manner to create a stress-specific response. Understanding of calcium signaling processes and modulations is a promising step for unraveling the molecular mechanism of drought stress tolerance and engineering stress-tolerant crops. In this chapter, calcium-signaling components involving in drought stress signal perception and transition through calcium-signaling pathway and final formation of stress response have been discussed in detail.

1 Introduction

Calcium (Ca²⁺), an intracellular secondary messenger, known as a versatile ion takes part in modulating the numerous cellular processes (Berridge et al. 2000; White and Broadley 2003; Dodd et al. 2010) and plays a pivotal role in plant growth and development. Due to its crucial roles in various signaling pathways, it has been addressed as “signaling component of the age” (Scrase-Field and Knight 2003) and “convergence point of signaling pathway” (Tuteja and Mahajan 2007).

The presence of various intracellular Ca²⁺ stores like vacuoles, endoplasmic reticulum, mitochondria, and cell wall enables the cells to dynamic release the of the Ca²⁺ in and balance the internal Ca²⁺ concentration (Mahajan and Tuteja 2005; Mahajan et al. 2006). Interestingly, doubled-membrane organelles like mitochondria, chloroplasts, and nuclei are specialized to generate Ca²⁺ signal per se and keep a profound effect on the integrated cell function (Xiong et al. 2006). Ca²⁺ as a central component of the complicated signaling pathways modulating the cell physiology and gene expression in response to variable conditions (Dodd et al. 2010). In fact, numerous environmental stimuli such as biotic and abiotic stresses as well as extracellular signals remodel the cellular Ca²⁺ levels (reviewed by Mahajan and Tuteja 2005).

It is widely accepted that almost all kinds of stresses alter gene expression in plants (Thomashow 1999; Shinozaki and Yamaguchi-Shinozaki 2007; Hirayama and Shinozaki 2010). These gene expressions regulated by transcription factors (TFs) results in reprogramming of the transcriptome profile, which is apparently a significant consequence of stress signaling and adaptation (Brivanlou and Darnell 2002). The fact that Ca²⁺ functions as a messenger in the plants lead the researchers to introduce Ca²⁺ as a regulator of gene expression (Poovaiah et al. 1987). Because signal-induced changes in gene expression is followed by a rapid signal-specific shift in cellular Ca²⁺, it is presumed that gene expression adjustment in the cell is mediated by Ca²⁺ signaling (Hu et al. 2004; Kaplan et al. 2006; McAinsh and Pittman 2009).

During the normal development and in hypersensitive responses to pathogens, sustained high cytosolic Ca²⁺ represented an apoptosis event (Levine et al. 1996). This indicates that although an increase in cytosolic Ca²⁺ is necessary for signal transduction to trigger an appropriate response in a diverse range of biotic and abiotic stresses, a prolonged increase in cytosolic Ca²⁺ has detrimental effects. To generate an appropriate adaptive response, the cytosolic Ca²⁺ perturbations have to be either transient or of low amplitude. Once the cytosolic Ca²⁺ elevates transiently, it can be run as single (spike), double (biphasic), or multiple (oscillations) frequencies (reviewed by Evans et al. 2001; Hetherington and Brownlee 2004; Tuteja and Mahajan 2007). In addition, cellular location, extent of propagation, and amplitude during propagation are factors that vary among generated Ca²⁺ perturbation (reviewed by Tuteja and Mahajan 2007).

Spatial and temporal dynamics of stimulus-induced changes in cytosolic Ca²⁺ has been referred to as a “calcium signature” (McAinsh and Hetherington 1998; Ng and McAinsh 2003; reviewed by Lecourieux et al. 2006; Oldroyd and Downie 2008). Ca²⁺ signature is unique to a particular stimulus in regard to subcellular location and/or the kinetics of magnitude of the cytosolic Ca²⁺ perturbation which makes it possible to functions as a specific physiological response (Rudd and Franklin-Tong 2001; reviewed by White and Broadley 2003). The apoplast, across the plasma membrane, or the intracellular organelles comprise the main routes for Ca²⁺. Cellular type and localization and the abundance of Ca²⁺ permeable channels determine the spatial characteristics of cytosolic Ca²⁺ perturbations.

Calcium ion signatures have shown to be dependent on cell types (Moore et al. 2002) and the nature of the stimuli. Various stimuli can induce specific Ca²⁺ signals bearing various amplitude, duration, and frequency, in the cytoplasm or organelles of the cell (Stael et al. 2012; Nomura and Shiina 2014). These cell-stimuli-specific signatures evolved from multiple pathways to decode the natural signals and convert the stimuli message in spatiotemporal dynamics (Bothwell and Ng 2005) mediated by Ca²⁺ influx channels in the plasma membrane and endomembrane. Further, Ca²⁺ efflux transporters restate the cytosolic Ca²⁺ to stationary values by rapid removal of Ca²⁺ from the cytosol (reviewed by McAinsh and Pittman 2009).

Calcium ion-dependent protein kinase induced by drought stress in Arabidopsis (Urao et al. 1994), expression of putative Ca-ATPase under saline condition in tobacco (Perez-Prat et al. 1992) and expression of Ca²⁺-binding protein in maize and sorghum under polyethylene glycol treatment (Pestacz and Erdei 1996) have been addressed as the first evidences of Ca²⁺ calcium participation involvement in stress response. Later, many other reports confirmed the crucial role of Ca²⁺ in plant response to different stresses including biotic elicitors (Knight et al. 1991; Kosuta et al. 2008), salt, and drought stresses (Ranf et al. 2008; Seifikalhor et al. 2019), PGRs (McAinsh et al. 1990; Allen et al. 2001) ABA treatment (Knight et al. 1996, 1997), cold stress (Knight et al. 1996), light stress (Shacklock et al. 1992), osmotic stress (Knight et al. 1997), oxidative stress, and gaseous pollutants (Evans et al. 2005). It was also demonstrated that Ca²⁺ influx from the plasma membrane and intracellular Ca²⁺ oscillation is a downstream event in ABA-induced stomatal closure (Kim et al. 2010; Takahashi et al. 2007; Aliniaeifard and van Meeteren 2013).

In general, during stress conditions, a signaling network is set up in plant cell starting with stress perception at the cell membrane level and stretched to the cellular response. In this regard, Tuteja and Mahajan (2007) have described a generic signal transduction pathway defined in four biological steps:

• 1. Perception of the signal through membrane receptors.

• 2. Generation of secondary messengers.

• 3. Formation of a protein phosphorylation/dephosphorylation cascade to modulate a specific set of stress-regulated genes that may also target transcription factors.

• 4. Induction of stress tolerance and plant adaption, which may concomitant with phenotypic responses.

To create an appropriate response or signal transduction against abiotic stresses, proper spatial and temporal coordination of all signaling molecules are required. It has been widely accepted that Ca²⁺ is increased during drought stress, enhances drought tolerance of plants, and implicates in the transduction of drought and salt stress-induced signals (Yildizli et al. 2018; Seifikalhor et al. 2019). However, the question of how stimulus-specific response raises from the complex network of Ca²⁺ signaling pathways in plant cells is still open to be answered. Generally, the specificity of Ca²⁺ signaling may result from the contribution of several mechanisms to form a specific signal which is suggested to occur in the following five steps (Sanders et al. 2002):

• 1. Additional signaling events that occur accompanied by changes in cytosolic Ca^2+.

• 2. Appropriate signaling machinery takes part in the transduction of a given signal refers to as “physiological address” (McAinsh and Hetherington 1998).

• 3. A defined response must arise in cells that express different Ca²⁺ sensors (Young et al. 2006).

• 4. The compartmentalization of signaling pathways and/or the selective activation of discrete response elements takes place in response to localized elevations in Ca²⁺.

• 5. Decoding of signaling information of cytosolic Ca²⁺ oscillations and transients (Evans et al. 2001).

Plant cells have developed a complicated mechanism to sense the simple signals like alteration in the concentration of intracellular Ca²⁺, thereby, the Ca²⁺ signals fashions (amplitude and duration) are precisely regulated (Schwaller 2012). To regulate the Ca²⁺ oscillation signal and transduction pathway, calcium transporter proteins including Ca²⁺-permeable channels, divalent H⁺ antiporters, and Ca²⁺-P-type ATPases, are required (McAinsh and Pittman 2009). In addition to ascertaining intracellular Ca²⁺ signaling, the Ca²⁺-signaling components are also taking part in the regulation of their own expression (Schwaller 2012). In response to environmental stimuli, Ca²⁺ channels and transporter activities must be programmed to generate an appropriate Ca²⁺ signature to influence cytosolic Ca²⁺ concentration. The cytosolic Ca²⁺ concentration is about 100 nM at cell rest, while, it reaches the levels higher than 1 μM for signaling events (White and Broadley 2003; Dodd et al. 2010).

Since the Ca²⁺ comprises the low diffusion through the cytoplasm while its buffering is high (0.1–1 mM), Ca²⁺ channels open to produce a local increase in cytosolic Ca²⁺ followed by a rapid dissipation in consequence of channel closure. Accordingly, the subcellular localization of Ca²⁺ channels is determinative for the operation of different cellular processes (Trewavas 1999; Tuteja and Mahajan 2007). Trewavas (1999) indicated that for coordination of cellular responses, cytosolic Ca²⁺ “waves” are produced within the cytoplasm when Ca²⁺ passes through particular Ca²⁺ channels recurrently. Suggesting that, increase in Ca²⁺ concentration might result in generation of soluble secondary messengers, such as IP3 (inositol triphosphate) or cADPR (cyclic adenosine diphosphate ribose), that diffuse through the cytoplasm by which a relay of spatially separated Ca²⁺ channels will be activated; channels close to the plasma membrane and channels that are adjacent to the vacuole, respectively (Allen et al. 1999). Ca²⁺ waves are responsible for the tandem opening of Ca²⁺ channels at the plasma membrane that activated by hyperpolarization and Ca²⁺-activated channels in the tonoplast (Schroeder et al. 2001).

In stress conditions, different waveforms are generated in cells with elevated Ca²⁺ levels. This kind of “high calcium cellular waves” has been detected in cotyledons exposed to cold stress (Knight et al. 1993) and in root tissue stimulated by mechanical injury or salinity (Fasano et al. 2002; Moore et al. 2002; Seifikalhor et al. 2019). These waves are triggered by electrical potentials, osmotic perturbations, or chemical signals as well.

2 Ca²⁺ Roles in Plant

Ca²⁺-as a bivalent cation is an essential element that plays a substantial role in all organisms. In the context of Calcium importance Trewavas (1999) stated: “Every reader knows that he or she started life as a single cell. Less familiar is the debt we owe to calcium in our earliest seconds. Penetration of the egg by the paternal sperm initiated an epigenetic calcium wave that moved quickly as a hollow band across the cytoplasm. In the wake of this calcium wave, processes were activated that led to cell division, differentiation, growth, and our eventual appearance as mature adults. As it is said in France, “Le calcium, c’est la vie.” A calcium wave marked the onset of our existence, and will quite probably mark our demise: Irreversible failure of calcium-wave generation in the heart is the most common cause of death. Therefore, calcium waves are a life-and-death issue.”

Calcium is an essential element that plays crucial structural and signaling functions in vascular plants. It is the counter-cation for inorganic and organic anions in the vacuole, as well as a secondary messenger in multiple intracellular signaling events that alleviate the adverse effect of stress by triggering stress responses. As calcium pectate, it is required for structural roles in cell wall and membranes where it accumulates and binds the cells together. One of the most well-known roles of Ca²⁺ in the plant is in membrane stability and cell integrity. Ca²⁺ takes part in cell membrane structure by stabilizing the lipid bilayers of cell membranes via bridging phosphate and carboxylate groups of phospholipids and proteins at the external site of the plasma membrane (Epstein 1972; Hepler and Winship 2010). The growth and elongation of pollen tube also require Ca²⁺. Ca²⁺ ion helps the formation of microtubules, the prerequisite for the anaphasic movement of chromosomes during cell division via mitosis. Therefore, Ca²⁺ functions in the growth and development of plant, especially in the root and shoot tips (Marschner 1986; Sanders et al. 2002).

Plant root uptakes Ca²⁺ from the rhizosphere and delivers it to the shoot via xylem. Both symplastic and apoplastic pathways are appropriate passages for Ca²⁺ through the root system. Ca²⁺ shows a high affinity to the array of Ca²⁺-binding proteins, this makes the concentration of cytosolic Ca²⁺ far higher than the concentration of free Ca²⁺ which is generally maintained at 200 nM (Bush 1995). The relative contribution of the apoplastic and symplastic pathways must be balanced in a way that root cells permitted to use the cytosolic Ca²⁺ concentration for signaling, controlling the rate of Ca²⁺ delivery to the xylem, and preventing the accumulation of toxic cations in the shoot. However, the balance between these pathways is unknown (White 2001). In this regard, Tuteja and Mahajan, 2007 stated the fact that the concentration of free Ca²⁺ within the cell is low and it is prone to rapid chelation makes the apoplastic pathway predominant rather than symplastic pathway (Tuteja and Mahajan 2007).

The ability of plants to uptake Ca²⁺ from the soil and their requirement for Ca²⁺ varies in different plant species and plant organs. It is widely accepted that for optimal growth monocots require less Ca²⁺ than the dicots. Generally, Ca²⁺ content varies between 0.1 and >5.0% dry weight indicating the contrasting Ca²⁺ requirement of various plant species in various growth stages and physiological conditions (Loneragan and Snowball 1969; Marschner 1986; Fageria 2016). Ca²⁺ competes with other cations both for entrance sites and for the uptake from the soil and enters plant cells through Ca²⁺-permeable ion channels (I_Ca) located in their plasma membranes (White 2001). The presence of other cations profoundly influence Ca²⁺ uptake as high levels of K and Mg reduces Ca²⁺ uptake, while the presence of high levels of Ca²⁺ is known to ameliorate uptake of toxic cations (Al³⁺ and Na⁺) from the soil (Burstrom 1968). In normal conditions, a high level of cytosolic Ca²⁺ is cytotoxic, therefore, Ca²⁺-ATPases and H/Ca²⁺-antiports direct the ion traffic to maintain the concentration of Ca²⁺ at sub-micromolar concentrations in unstimulated cells (Sze et al. 2000; Hirschi 2001).

3 Ca²⁺ Uptake and Transport in Plant Under Drought Stress

Ca²⁺ is taken up from the rhizosphere solution through the root system especially root tip and the regions initiating lateral roots and transported to the above-ground parts via the xylem (White 2001). The distribution of Ca²⁺ within the plant occurs mainly in the xylem vessels (Clarkson 1984). The Ca²⁺ transport to the xylem of growing plants is about 40 nmol Ca²⁺ h⁻¹ g⁻¹ root fresh weight (White 1998). The Casparian strip is the main barrier for Ca²⁺ loading into the root (White 2001). Therefore, Ca²⁺ loading into the xylem is mainly restricted to the root tip and to regions initiating lateral roots. Lack or interrupted Casparian strip in those regions permit the Ca²⁺ fluxes to the apoplast and thereafter to the xylem. Movement of Ca²⁺ toward leaves and fruits can be performed in both apoplastic and symplastic pathways (White and Broadley 2003). Ca²⁺ moves in xylem vessel in chelate form and its movement is considerably dependent on the transpiration rate. In fact, low Ca²⁺ content in plant tissues is a consequence of low transpiration rate in those tissues. Therefore, environmental cues that influence the transpiration rate would also alter Ca²⁺ uptake and movement in the plant. For instance, reducing transpiration as a result of drought exposure will decrease Ca²⁺ uptake and movement to the shoot (del Carmen Martínez-Ballesta et al. 2011). Confirming this, it has been shown that the genotype of one species having more stomatal opening and more transpiration rate is capable of higher accumulation of Ca²⁺ in their tissues and as a result, is more drought tolerant (de Freitas and Mitcham 2012; Kaczmarek et al. 2017). Lower transpiration rate of fruits in comparison with leaves is the main reason for lower Ca²⁺ concentration and the occurrence of Ca²⁺-related disorders in fruit tissue (de Freitas and Mitcham 2012; de Freitas et al. 2011).

4 Calcium Channels

Based on voltage dependence, Ca²⁺ channels are classified into three categories including hyperpolarization-activated (HACC), depolarization-activated (DACC), and voltage-independent (VICC) cation channels (Sanders et al. 2002; White and Broadley 2003; Marschner 2012). HACC opening is stimulated by ABA that play a central role in stomatal closure during water deficit conditions. This channel promotes the entrance of Ca²⁺ that depolarizes the plasma membrane and contributes to Ca²⁺-dependent events (Murata et al. 2001; Kohler and Blatt 2002). DACCs are located in the plasma membrane and are permeable to both mono and bivalent cations and participate in calcium and other cations uptake. This channel has been shown to trigger plant adaption to low temperature (White and Broadley 2003; White 2009). VICCs are encoded by genes from the cyclic nucleotide-gated channel (CNGC) and glutamate-like receptor families (GLR). Studying the Arabidopsis protein channels revealed that CNGC and GLR gene families are more involved in processes associated with Ca²⁺ signaling than with Ca²⁺ storage (Conn and Gilliham 2010), therefore, CNGC and GLR are considered as the main channel in Ca²⁺ signature.

VICCs involve in maintaining of the cytosolic Ca²⁺ hemostasis in resting cells by retaining the permanent Ca²⁺ efflux balance through Ca²⁺ ATPase and H⁺/Ca²⁺ antiporters (Marchner 2012). They are permeable to both mono- and bivalent cations. In the plasma membrane, many different VICCs are presented and characterized by their differences in cation selectivity (White and Broadley 2003; Demidchik et al. 2002). Furthermore, several membrane channels are involved in generating a Ca²⁺ influx from Ca²⁺ reservoir compartment (apoplast and organelles) to cytosol in order to create Ca²⁺ signatures (Dodd et al. 2010).

Guard cell plasma membrane I_Ca channels which play a role in Ca²⁺ influx from extracellular space (Hamilton et al. 2000), release Ca²⁺ from intracellular stores (Blatt 2000) are also involved in Ca²⁺ trafficking in guard cells of stomata.

5 Calcium Signal Transduction

Prototypical animal Ca²⁺ channels have been well characterized in the genomes of the evolutionary basal lineage of Chlorophyta algae; whereas, it was missed in land plants (Wheeler and Brownlee 2008). This can imply that the plant-specific Ca²⁺ signaling mechanism developed in compensation for the loss of these Ca²⁺ channels. Tuning of the “Ca²⁺ toolkit” on the level of the Ca²⁺ binding proteins, which decode the information presented by Ca²⁺ signatures and modulate the Ca²⁺ signal transduction to downstream responses are also involved in this evolutionary trajectory (Batistič and Kudla 2012). A more recent notion in this regard specifically considered the Ca²⁺-sensing protein CBL/CIPKs states that during land plant evolution CBL/CIPKs diversified gradually and eventually formed a complicated network involved in various aspects of stress signaling events (Beckmann et al. 2016; Kudla et al. 2018).

Despite the presence of many signaling molecules acting as a secondary messenger in plant cells, only Ca²⁺ has been shown to be involved in the regulation of gene expression of regulatory proteins that result in stress tolerance responses by altering the metabolism followed by phenotypic responses. Based on the number of genes and the type of gene and the regulation mode, the response to elevated Ca²⁺ levels varies from growth inhibition to cell death (Tuteja and Mahajan 2007). Large numbers of Ca²⁺-responsive genes (230) are identified by analysis of transcriptome changes containing known early stress-responsive genes as well as genes with unknown function. Among Ca²⁺-responsive genes, 162 genes are up-regulated and 68 are down-regulated under stress conditions (Kaplan et al. 2006).

During evolution adaption emerges upon plant response to stresses; this indicates that plant functions as a dynamic and endogenously integrated organism in response to changes in its surrounding environment. Under drought condition a wide variety of plant responses such as alterations in gene expression, accumulation of metabolites, e.g., osmotically active compounds and PGRs (especially ABA), and the synthesis of specific proteins (Ramachandra-Reddy et al. 2004) are promoted in plants; all are regulated through certain signaling pathways. Signal perception is at the onset of the signal transduction pathway. Once the signal perception takes place, the generation of second messengers occurs. Second messengers can alter intracellular Ca²⁺ levels; consequently, a protein phosphorylation cascade initiated which targets proteins that are directly involved in cellular protection or TFs controlling stress-regulated genes. Therefore, in response to stress conditions, large numbers of genes are expressed to produce proteins involved in stress tolerance pathways (Shinozaki and Yamaguchi-Shinozaki 1997). These genes may influence regulatory molecules, e.g., PGRs especially ABA. Further, these regulatory molecules take part in another signaling event with different components that may cross-talk with the above generic pathway. The proper spatial and temporal coordination of all signaling molecules should be provided for signal transduction. Therefore, coordination of certain molecules (protein modifier, scaffolds, or adapters that are known as signaling partners) in modification, delivery, or assembly of signaling components is necessary despite they do not directly relay the signal (Xiong and Zhu 2001).

In plants, studying of Ca²⁺ signaling in stomatal guard cells in legumes provides compelling evidence that signaling information can be encoded in the spatiotemporal dynamics of plant Ca²⁺ signatures. Therefore, an overview of the background of Ca²⁺ signaling in stomatal aperture facilitates addressing its role in drought stress.

Changes in guard cell cytosolic Ca²⁺ in response to a wide range of stimuli that modulate stomatal aperture led the researchers to postulate that cytosolic Ca²⁺ is at the center of the guard cell signaling network (Hetherington and Woodward 2003; Israelsson et al. 2006; Li et al. 2006; Aliniaeifard and van Meeteren 2013). The mechanism of encryption of signaling information in guard cells in response to Ca²⁺ oscillation attracted the attention of several researchers (Allen et al. 2000, 2001; Mori et al. 2006; Young et al. 2006). Reverse genetic studies on the model plant Arabidopsis provided ample evidence for the contribution of Ca²⁺-dependent signaling pathway in ABA-induced stomatal closure (Roelfsema and Hedrich 2010; Steinhorst and Kudla 2013; Murata et al. 2015; Edel and Kudla 2016). Ca²⁺-Dependent Protein Kinases (CDPKs) are activated in response to ABA-induced intracellular Ca²⁺ transients (Mori et al. 2006). In Arabidopsis thaliana 8 of the 34 detected CDPKs contribute to ABA signaling and reactive oxygen species (ROS) homeostasis in guard cells (Boudsocq and Sheen 2013; Zou et al. 2015; Simeunovic et al. 2016). CDPK3, 21 and 23 that belong to subgroup II of CDPKs are of particular importance in the regulation of SLAC1 (Slow Anion Channel-Associated 1) and KAT (potassium channel protein) channel activities in guard cell signaling (Cheng et al. 2002; Brandt et al. 2015). Mori et al. (2006) have shown that the Arabidopsis CDPK double mutant, cpk3-1cpk6-1 are characterized by impaired ABA- and Ca²⁺-induced stomatal closure. However, long-term Ca²⁺-programmed stomatal closure was not affected by mutation; suggesting that, these are functionally separated responses (Mori et al. 2006). Studying of the Vicia faba guard cells showed that cytosolic ABA application maximizes the activation of anion channel within 1.5–2.5 min (Levchenko et al. 2005). Previously the activation and autophosphorylation of a Ca²⁺-independent kinase in the same time span have been reported (Li and Assmann 1996; Li et al. 2000; Takahashi et al. 2007). However, electrophysiological studies of V. faba guard cells have shown that ABA-induced stomatal closure does not require Ca²⁺ elevation (Levchenko et al. 2005; Hubbard et al. 2011). Moreover, applying live-cell Ca²⁺ imaging technique in Arabidopsis revealed that only less than 50% of guard cells were characterized with elevated cytosolic calcium which is affected by the degree of humidity (Hubbard et al. 2011).

Localized increase and oscillations of cytosolic Ca²⁺ ([Ca²⁺]_cyt) in guard cells follow a distinct pattern (McAinsh et al. 1995). The pattern of external Ca²⁺- or ABA-induced oscillations in guard cells and the strength of the external Ca²⁺-have been shown to induce stomatal closure (McAinsh et al. 1995; Staxén et al. 1999) which provides the possibility of encoding information about the nature of stimuli (McAinsh and Hetherington 1998; Ng and McAinsh 2003; McAinsh et al. 1995). Patch-clamp studies have revealed that both rapid- and slow-type of plasma membrane anion channels are activated in a Ca²⁺-dependent manner (Hedrich et al. 1990). In Arabidopsis, steady-state stomatal closure that was programmed by Ca²⁺ oscillation have been observed in response to stimuli including external Ca²⁺, hydrogen peroxide, cold stress, and ABA (Allen et al. 2000, 2001). However, reduced vacuolar H⁺-ATPase activity in the det3 mutant (Arabidopsis de-etiolated 3) was characterized with oscillations in guard cell cytosolic Ca²⁺ and steady-state stomatal closure only in response to cold and ABA (Allen et al. 2000). The growth controlled by ABA mutant (gca2) of Arabidopsis (cv. Landsberg erecta), which causes a strong high CO₂ insensitivity have impaired stomatal closure and cytosolic Ca²⁺ transient rate in response to CO₂ treatment and elevated CO₂ levels, respectively, led the researcher to propose a new model of cellular Ca²⁺-signaling specificity in eukaryotes. Based on the proposed model, responses to cytosolic Ca²⁺-elevations in a single cell may be mediated by priming and de-priming of Ca²⁺-sensors by physiological stimuli (Young et al. 2006).

Inconformity between recent findings indicates additional complexities in the guard cell signaling network regarding the existence and importance of a Ca²⁺-independent mechanism for stomatal closure. To deal with these complexities a recent study presented a more general principle to ensure specificity and robustness of stomatal Ca²⁺-signaling on a cellular, genetic, and biochemical level. It was demonstrated that calcium-dependent protein kinase quadruple mutant of Arabidopsis has impaired ABA signal transduction in stomatal guard cells. According to the fact that protein phosphatase 2Cs prevent nonspecific Ca²⁺-signaling, it was unexpectedly revealed that Ca²⁺-dependent and Ca²⁺-independent ABA-signaling branches are interdependent; as the triple snrk2.2/snrk2.3/ost1 mutant showed impaired Ca²⁺ activation of S-type anion channels of guard cells. In addition, the in vivo simultaneous phosphorylation at S59 and S120 phosphorylation sites in SLAC1 are required intact ABA-induced stomatal closure (Brandt et al. 2015). SLAC1 channel activity is controlled by cooperative action of OST1 and CDPK3, 21 and 23 in counterbalance with competitive dephosphorylation by PP2Cs (Brandt et al. 2015), because the three CDPKs preferentially phosphorylate a residue different from that targeted by OST1 with respect to SLAC1 (Geiger et al. 2010; Brandt et al. 2012).

Notably, Ca²⁺-activation of CDPKs and ROS production by NADPH oxidases are interdependent processes. These all represent a mechanism of guard cell regulation that remained to be investigated. Moreover, in experimental studies, epidermal peels of unstressed plants or heterologous systems have been utilized thus far which hinders the researchers to investigate the shared features of SnRK2-independent ABA/Ca²⁺ signaling in stomatal closure under drought stress in the scale of whole plants (Geiger et al. 2010; Brandt et al. 2012, 2015).

5.1 Calcium Signal Transduction Under Drought Stress

Under drought conditions, stomata closure is the main cause of limited gas exchange between the plant and the surrounding environment. Importance of stomata (aspects related to fine-tuned functioning and morphology) and natural variations among populations has been highlighted as important keys to improve plant acclimation to drought stress (Aliniaeifard and van Meeteren 2014; Aliniaeifard and Van Meeteren 2018; Daszkowska-Golec and Szarejko 2013). Abscisic acid is the main phytohormone involved in stomatal closure responses to water deficit conditions (low humidity in both above- and below-ground environments) (Aliniaeifard 2014; Aliniaeifard et al. 2014; Aliniaeifard and van Meeteren 2013; Endo et al. 2008). Drought (in both above- and below-ground environments) induces production of ABA and inhibits its catabolism in vascular tissues (Endo et al. 2008; Okamoto et al. 2009) as well as in guard cells (Aliniaeifard and van Meeteren 2013; Bauer et al. 2012; Okamoto et al. 2009). Stomatal response to ABA depends on the level of [Ca²⁺]_cyt and its oscillation (Allen et al. 2000; Kim et al. 2010; Neill et al. 2008; Wang et al. 2011). As indicated before, induction of SLAC1 and inactivation of potassium channel (e.g., inward-rectifying K⁺ channel KAT1) can be Ca²⁺-dependent and/or Ca²⁺-independent (Geiger et al. 2009, 2010; Joshi-Saha et al. 2011; Levchenko et al. 2005; Li and Assmann 1996; Marten et al. 2007; Siegel et al. 2009; Sutter et al. 2007). Among different components of ABA signaling pathway, type 2C protein phosphatases (PP2Cs) ABI1 (ABA-insensitive 1) and the SNF1-related protein kinase (SnRK2) open stomata 1 (OST1) are proposed to be Ca²⁺-independent proteins. OST1 activates anion channels in an ABA-sensitive, while Ca²⁺-independent manner (Li and Assmann 1996). Ca²⁺-sensing receptors (CAS) are localized in chloroplasts its function is crucial for the regulation of stomatal responses (Weinl et al. 2008). At the onset of water deficit condition, increase in the extracellular Ca²⁺ concentration ([Ca²⁺]_o) due to high transpiration rate will result in an increase in [Ca²⁺]_cyt with the mediation of CAS (Han et al. 2003).

CDPKs (Calcium-dependent protein kinase) are the main components in Ca²⁺-dependent ABA responses (Zhu et al. 2007), which activate the SLAC1 channels (Geiger et al. 2010; Mori et al. 2006), while inhibiting KAT1 (Li et al. 1998). Under drought and high VPD conditions, guard cell plasma membrane ICa channels will be activated by ABA, which lead to Ca²⁺ influx from extracellular space (Hamilton et al. 2000), and also Ca²⁺ from intracellular stores (Blatt 2000). As a result of [Ca²⁺]_cyt elevation, CDPK activates SLAC1 leading to stomatal closure. CAS induces reactive oxygen (H₂O₂) and nitrogen [nitric oxide (NO)] species in the guard cells in response to an increase in [Ca²⁺]_o (Wang et al. 2011). Both H₂O₂ and NO elevates [Ca²⁺]_cyt, and activates ICa and SLAC1 channels (Pei et al. 2000), and then stomatal closure (Chen et al. 2004; Li et al. 2009). A simplified model for signaling pathway involved in stomatal closure due to water deficit conditions is presented in Fig. 1.

Fig. 1

Simplified model of calcium signaling under water deficit conditions. Increase in the level of extracellular calcium as well as apoplastic ABA results in elevation of [Ca²⁺]_cyt which leads to activation of calcium and anion and inhibition of potassium channels through calcium-dependent protein kinases (CDPK)

6 Calcium-Sensing Proteins

There are many abiotic stress signals that require multiple sensors. Therefore, the presence of only one sensor for perceiving stress and controlling all subsequent signaling is unlikely, however, a single sensor might only initiate the onset of signaling cascade that is initiated by stimuli like stress signal. Therefore, various sensors are expected for stress signal transduction (Xiong et al. 2002). Studying Ca²⁺ signaling in Arabidopsis thaliana exposed to drought and salinity demonstrated that Ca²⁺ influx occurred in response to both drought and salinity. Notably, similar magnitude and duration of Ca²⁺ efflux observed for both salinity and drought stresses, leading the researchers to postulate that rather than Ca²⁺ different factor is involved in the discrimination between drought and salinity signals in Arabidopsis. Various signaling factors for drought tolerance identified including CDPK, GSK3/Shaggy MAPKKK, and SnRK2 which belong to protein kinases and other signaling factors are CBL1, 14-3-3 Protein, CC-NBS-LRR, Farnesyltransferase (reviewed by Umezawa et al. 2006). It has been demonstrated that there is a common event referred to as “calcium signature” for various types of stress transient Ca²⁺ influx into the cell cytoplasm (reviewed by Sanders et al. 1999; Knight et al. 1993; Wang and Nick 2001; Sangwan et al. 2001; Rudd and Franklin-Tong 2001). Furthermore, [Ca²⁺]_cyt elevation is required for the expression of certain stress-induced genes in plants (Knight et al. 1996). Then, activation of stress gene promoters leading to the [Ca²⁺]_cyt elevation is sufficient for their activation (Sheen 1996). It is generally accepted that Ca²⁺ is a transmitter of stress signal downstream in the pathway by interacting with protein sensors. In other words, Ca²⁺ sensor proteins synchronize alteration in metabolism, gene expression, and turnover of proteins by affecting the activity of downstream effectors (La Verde et al. 2018).

There are two groups of Ca²⁺-binding proteins including membrane-intrinsic Ca²⁺ binding protein and Ca²⁺-modulated proteins. Ca²⁺ binding proteins could be subdivided into two categories: The former modulates Ca²⁺ concentration in the environment by transporting it across cell membranes and the latter includes proteins that both contribute to the control of Ca²⁺ concentration and Ca²⁺ signals decoding, acting as Ca²⁺ sensors (Carafoli et al. 2001).

The spatial and temporal changes in cytosolic Ca²⁺ concentration form signals that are perceived and transduce through proteins termed as “Ca²⁺ sensors”, thereby, the conformation or catalytic activity of Ca²⁺ sensors changes once Ca²⁺ is bound. Among various identified signaling factors potential Ca²⁺ sensors are CDPKs, calcineurin B-like protein (CBL), calmodulin (CAM), and calmodulin-like protein (CML) and the Ca²⁺ and calmodulin-dependent protein kinase (CCaMK) (Harmon et al. 2000; Luan et al. 2002; Sanders et al. 2002; Cheng et al. 2002; Wang et al. 2015; Zhou et al. 2015a, b). Except for CDPKs, that contain a kinase domain, the other Ca²⁺ sensors had no apparent enzymatic domain, conveys their interacting role in transmitting cellular signals to their downstream targets. Having no enzymatic activity (except CDPK) these small Ca²⁺ sensors interact with their target proteins and regulate the function of their target proteins (Zielinski 1998; Luan et al. 2002; Hashimoto and Kudla 2011). The high affinity of these proteins with Ca²⁺ together with their subcellular location within the cell determines their prominent function. Binding of Ca²⁺ to related sensors results in a conformational change that renders it to associate with downstream target proteins or directly stimulate the kinase activity (Harmon et al. 2000). Both the diversity of Ca²⁺ sensors and their downstream targets bring forward the induction of the specific responses (Hashimoto and Kudla 2011).

In a general conceptual characterization, Ca²⁺-sensing proteins have been classified into two functional groups: (1) sensor relays and (2) sensor responders (Sanders et al. 2002). Among Ca²⁺ sensors, CDPKs classified as sensor responders due to their Ca²⁺ sensing (EF-hand motifs) and responding (protein kinase activity) functions. CDPKs translate the information encoded in the Ca²⁺ signatures into phosphorylation events of specific target proteins. In comparison with CDPK, CaM/CML family members have no enzymatic function thus, functions as sensor relay proteins. These Ca²⁺ sensors take part in signaling events by altering downstream target activities via protein–protein interactions in a Ca²⁺-dependent manner. CBL proteins also categorized as sensor relay proteins with representing no enzymatic activity. However, it is considered as bimolecular sensor responders when interacting with a family of protein kinases designated as CBL-interacting protein kinases (CIPKs) (Hashimoto and Kudla 2011).

6.1 CBL

CBL are structurally very similar to calcineurin B (CNB) and neuronal calcium sensors (NCS) of animals (Liu and Zhu 1998; Kudla et al. 1999). Up to now, the CIPK gene family has been identified in Arabidopsis (Kolukisaoglu et al. 2004; Yu et al. 2007), poplar (Populus trichocarpa) (Yu et al. 2007), maize (Zea mays) (Chen et al. 2011), rice (Oryza sativa) (Kanwar et al. 2014), canola (Brassica napus) (Zhang et al. 2014), and Cassava (Hu et al. 2015). To date, at least 10 CBLs and 25 CIPKs in Arabidopsis and 10 CBLs and 31 CIPKs in rice have been identified. These proteins integrate to form a network-like signaling system for coupling of specific and synergistic stimulus–response (Luan et al. 2002; Batistic and Kudla 2004; Kolukisaoglu et al. 2004; Piao et al. 2010). In the signaling pathways, they act as a threshold factor in linking the Ca²⁺ signature to the downstream components (Sanders et al. 2002). Binding to Ca²⁺ ions enables CBLs to interact with CBL-interacting protein kinase (CIPK) and form Ca²⁺-CBL-CIPK network that enables it to regulate related physiological processes (Sanyal et al. 2015).

CIPKs have a serine/threonine-protein kinase domain in the N-terminus and a self-inhibitory NAF domain in C-terminus (Albrecht et al. 2001). The activation of CIPKs is mediated when the NAF domain interacts with CBLs (Kleist et al. 2014). Therefore, CBLs modulate the activity of CBL-interacting protein kinases (CIPKs), known as sucrose non-fermenting 1 related kinase 3 (SnRK3) (Shi et al. 1999; Luan et al. 2002). The CBL-CIPK modules function downstream of Ca²⁺ flow in guard cells (Cheong et al. 2007). The CBL and CIPK proteins form Ca²⁺ decoding signaling network and play an important role in plant responses to abiotic stresses (Zhu et al. 2016).

Reportedly, CIPK gene family could be divided into an intron-rich clade and an intron-poor clade, which subgroups in Arabidopsis, maize, rice, and canola CIPK genes were induced by drought (Chen et al. 2011; Ye et al. 2013). Recently, in an attempt for increasing drought tolerance in soybean, 52 CIPK family members have been identified suggesting that intron-poor CIPK genes might be originated in seed plants. Using quantitative real-time polymerase chain reaction (qRT-PCR) confirmed that 18 genes were drought-inducible (Zhu et al. 2016).

It has been shown that overexpression of CBL1, CBL9, CIPK1, and CIPK6 increased drought tolerance in Arabidopsis (D’Angelo et al. 2006; Pandey et al. 2004; Tsou et al. 2012), conversely, the cbl1, cbl9, and cipk3 mutants are shown to be more sensitive to drought stress (Cheong et al. 2003; Pandey et al. 2008). It was demonstrated that the CBL1 gene is induced by drought, cold, and wounding (Kudla et al. 1999). Cheong et al. 2003 studied the functions of a CBL type Ca²⁺ sensor—CBL1 in stress gene expression and stress tolerance in plants. Their research revealed that CBL1 functions as a critical Ca²⁺ sensor in abiotic stress responses. Interestingly, investigating the role of CBL in both cold and drought stress showed that, the same Ca²⁺ sensor can act as both positive and/or negative regulators in different signaling pathways. Overexpression of CBL1 exhibited elevated drought and salt tolerance and up-regulated the expression of stress-responsive genes. Moreover, it was revealed that the CBL1b gene is expressed during all developmental stages of plants and in all plant organs. Overexpression of CBL1 in transgenic plants activated the expression of some late stress-responsive genes under “normal” conditions and their tolerance to salt and drought was enhanced however reduced tolerance to freezing was reported as well. Suggesting that, the quantity of CBL1 is a rate-limiting factor in Ca²⁺-mediated stress signaling with respect to the level of expression of downstream target genes (Cheong et al. 2003, 2010). In another study, the function of CBL1 was investigated by analyzing T-DNA-induced knock-out mutant and plants that overexpressed CBL1. Plants lacking CBL1 have shown impaired response to drought and salt stress though their sensitivity to ABA was not affected by the mutation. On the other hand, overexpression of CBL1 enhanced stress tolerance via the induction of stress-regulated genes. These findings suggest that CBL1 may form an integrative node in abiotic stress signaling and take part in the regulation of early stress-related TFs of CBF/DREB type (Albrecht et al. 2003). Overexpression of CBL5 has shown to result in enhanced tolerance to high salt or drought stress. Furthermore, these transgenic plants were more resistant to high salt or hyperosmotic stress during the early developmental stage; nevertheless, their response to ABA didn’t change. In addition, the expression of stress gene markers, such as RD29A, RD29B, and Kin1 was modified by overexpression of CBL5, indicating that, CBL5 may play a functional role in the regulation of salt or drought stress responses in plants (Cheong et al. 2010). In transgenic tobacco, ZmCIPK8 involved in drought stress tolerance by regulating the expression of related stress genes (Tai et al. 2015). Different studies in rice revealed that OsCIPK23 acts as a positive regulator under drought stress as oscipk23 was sensitive to drought (Yang et al. 2008). Moreover, overexpression of the BrCIPK1 in rice plant resulted in enhanced proline concentration which subsequently led to osmoregulation and further drought stress tolerance (Abdula et al. 2015). Studying the grapevine under drought stress, clarified that, a CBL Ca²⁺ sensor 1–protein kinase, CIPK23 network is expressed in grape berries and activates grapevine Shaker inward K⁺ channel which triggers in drought stress response (Cuéllar et al. 2010). Another type of CIPK2 was detected in Arabidopsis under drought and osmotic stress treatments that took part in osmotic stress tolerance when it was overexpressed (Li et al. 2012). Quantitative RT-PCR studies in Arabidopsis revealed a multifold increase in CIPK21 transcript in seedlings treated with NaCl or mannitol in comparison with control. Moreover, treatment with polyethylene glycol, abscisic acid (ABA), cold, and drought stress-induced CIPK21 transcript, which suggests the pivotal role of CIPK21 in salinity and osmotic stress tolerance (Pandey et al. 2015). CBL-CIPK complex also triggers stress response indirectly by regulating ABA signaling. In this regard, CBL9 has shown to function as a negative regulator in the ABA-signaling pathway (Nakashima and Yamaguchi-Shinozaki 2013; Pandey et al. 2004). The role of CIPK3 as a negative regulator of ABA signaling has been also revealed by studying seed germination of cipk3 mutants of Arabidopsis (Kim et al. 2003) and increased germination rate of overexpressed AtCBL9/AtCIPK3 Arabidopsis exposed to ABA (Pandey et al. 2008). CIPK6 can also involve in the regulation of the ABA-signaling pathway as it was found that cipk6 mutant can accumulate more ABA under ABA treatment (Chen et al. 2013a).

In a recent study, it was shown that TaCIPK27A confers drought tolerance and sensitivity to exogenous ABA in transgenic Arabidopsis. It was found that TaCIPK27 is up-regulated by multiple abiotic stresses. TaCIPK27 interacts with AtCBL1, AtCBL3, AtCBL4, AtCBL5, and AtCBL9. In Arabidopsis, ectopic overexpression of TaCIPK27 enhanced drought tolerance which was concomitant with the regulation of some drought-related genes and ABA concentration (Wang et al. 2018). In another study, the participation of TaCIPK23 in the regulation of several drought- and ABA-responsive genes has been demonstrated. Biochemical and reverse genetic studies confirmed the contribution of TaCIPK23 to plant ABA signaling and drought tolerance. Exogenous ABA induced the expression of TaCIPK23 and delayed seed germination and small stomatal aperture under exogenous ABA treatment observed. Accordingly, under drought stress, the lower stomatal conductance in TaCIPK23-overexpression lines resulted in the reduced water loss and consequent drought tolerance. Based on these results, researchers concluded that TaCIPK23 positively modulates plant drought tolerance through ABA-dependent and -independent pathways (Cui et al. 2018).

Interaction network and co-expression analyses of cassava CIPKs, besides transcriptome analysis of different cassava accessions in response to drought indicated that the pathways controlled by CIPK networks may contribute to responses to drought stress in different tissue or different accessions of cassava (Hu et al. 2015). Overexpression of a cotton CIPK gene, GhCIPK6, in Arabidopsis was induced by drought, and enhanced plant tolerance to drought (He et al. 2013). It was demonstrated that the expression pattern of several ZmCIPKs under drought stress involved in ABA and H₂O₂ signaling (Tai et al. 2013) and in tobacco overexpression of ZmCIPK8 enhanced drought tolerance by inducing the expression of the NAC, CBF, and Rd29A genes (Tai et al. 2016). It was also revealed that, although CBL1 and CBL9 are structurally and evolutionary homolog, they function differently. Both cbl1 and cbl9 mutants are highly sensitive to abiotic stress, but cbl1 did not respond to stresses under ABA treatment, which led the researcher to postulate that CBL1 may trigger abiotic stress via ABA-independent pathway (Albrecht et al. 2003; Cheong et al. 2003). Reportedly, the diverse CBL–CIPK Ca²⁺ signaling pathway plays its role in stress tolerance by regulation of ions transport in plant under stress conditions. The CBL–CIPK pathways have been shown to operate the transport of sodium, potassium, nitrate, and phosphorous in plant (reviewed by Thoday-Kennedy et al. 2015).

Up to now, the majority of research focused on identifying the interactions between CBLs and CIPKs and the location of their interaction in Arabidopsis and the downstream targets of CIPKs signaling have been identified poorly while it is essential to be identified for unraveling the mechanism controlling plant ontogeny under abiotic stresses and the genes expressed and protein synthesized during this process. Studies on rice and Arabidopsis revealed that in each species CBL families follow species-specific evolutionary pathways with specific gene duplication events, despite the equal number of CBL genes encoded in the rice and Arabidopsis genome. Thus, precise gene-for-gene predictions of CBL function from one genus to the other may not be reliable (Batistic and Kudla 2004).

6.2 CAM

Calmodulin (CAM), one of the best distinguished Ca²⁺ sensors is a small acidic protein consist of four EF-hands. The binding of Ca²⁺ to CaM induces a conformational change in this protein. These changes enable the protein to contribute to hydrophobic and electrostatic interactions with its target proteins by exposing hydrophobic surfaces surrounded by negative charges (Snedden and Fromm 1998; Hoeflich and Ikura 2002). Up to now, more than 50 different types of CaM-binding proteins were identified. Ca²⁺/CaM complex modulates the activities of numerous target proteins which yields in regulation of a variety of cellular responses including regulation of metabolism, cytoskeleton function, phytohormone signaling, ion transport, protein folding, protein phosphorylation and dephosphorylation, phospholipid metabolism, and transcriptional regulation (Snedden and Fromm 2001; Yang and Poovaiah 2003; Bouché et al. 2005).

The presence of CaM in the nucleus beside the identification of several nuclear molecules as CaM-binding proteins indicates the crucial role of CaM in decoding of Ca²⁺ signaling, in both of the cytosol and nucleus (Dauwalder et al. 1986; Schuurink et al. 1996). Mechanism of Ca²⁺ responses in nuclei is yet to be understood; however, the independent responses of cytosolic Ca²⁺ and nucleic Ca²⁺ have been revealed (Pauly et al. 2000). The identification of various TFs regulated directly or indirectly by CaM strongly supports the crucial role of CaM as a nuclear Ca²⁺ signaling decoder, Therefore, a role for CaM in regulation of gene expression that regulate cellular responses to developmental and environmental stimuli is highly likely (Snedden and Fromm 2001; Yang and Poovaiah 2003; Bouché et al. 2005). The possible role of nucleus-translocated CaM53 protein in the Ca²⁺-mediated sugar sensing pathway in plant cells (Rodríguez-Concepción et al. 1999) accompanied with identification of numerous nuclear CaM-binding proteins like pea nuclear apyrase that binds to CaM in a Ca²⁺-dependent manner which is activated by Ca²⁺/CaM-binding proteins (Hsieh et al. 2000) and the potato CaM-binding protein (PCBP) (Reddy et al. 2002) are among evidence indicating significant role of CaM as a nuclear Ca²⁺ signaling decoder.

It is widely accepted that the Ca²⁺/CaM complex mediates plant response to biotic and abiotic stimuli (Snedden and Fromm 1998; Bender and Snedden 2013; Virdi et al. 2015). There are two defined pathways through which Ca²⁺/CaM complex functions either directly or indirectly to trigger a stress response. Direct pathway is binding to target proteins that are a coordinator of a variety of cellular functions and the indirect pathway is regulating the expression of genes encoding downstream effectors which result in cellular responses.

A novel CaM-binding transcription factor family that contains a CG-1 DNA-binding domain distinguished in plant proteins during the isolation of a partial cDNA clone encoding a sequence-specific DNA-binding protein from parsley (da Costa e Silva 1994). Later, homologs of CG-1 identified in various plant species including Arabidopsis, rice, tobacco, and rapeseed (Reddy et al. 2000; Yang and Poovaiah 2000; Bouché et al. 2002; Yang and Poovaiah 2002; Choi et al. 2005a, b). Several studies suggest that the CG-1 proteins act as transcriptional activators of downstream gene expression (Mitsuda et al. 2003; Choi et al. 2005a). Two different kinds of CaM-binding motifs of CG-1 proteins exist; a Ca²⁺-dependent CaM-binding domain and a Ca²⁺-independent CaM-binding domain, termed the IQ motif. The function of IQ motif is not clearly understood (Choi et al. 2005b).

Calmodulin-binding transcription activator (CAMTA) TF triggers the expression of several downstream genes involved in stress response (Janiak et al. 2015). Altered expression of CAMTA gene family in response to drought stress has been reported. Enhanced drought susceptibility has been observed in Arabidopsis thaliana camta mutant in comparison to the wild-type plants (Pandey et al. 2013). The possible role of CAMTA family in the regulation of drought stress indicated by the involvement of members of CAMTA family in regulation of different drought-responsive genes like ethylene-responsive element-binding factor 13 (ERF13), C-repeat/DRE binding factor 2 (CBF2), and WRKY33 (Pandey et al. 2013). In a recent study, the expression pattern of MuCAMTA (Musa acuminate Calmodulin-binding transcription activator) genes was identified by qRT-PCR which up-regulated (40-fold) under drought stress; suggesting that MuCAMTA1 plays an important role in drought stress tolerance in banana plants (Meer et al. 2019).

A plant-specific family of TFs that consists of CBP60 proteins bind to CaM (Bouché et al. 2005). One of the members of this family, interact with CaM in a Ca²⁺-dependent manner (Wang et al. 2009). Overexpression of CBP60g in Arabidopsis resulted in increased sensitivity to ABA as well as increased tolerance to biotic and drought stress (Wan et al. 2012). A family of trihelix TFs or GT factors, identified in Arabidopsis and rice (Riechmann et al. 2000; Wang et al. 2014). One of the members of this family, GTL1 (GT-2 LIKE 1), negatively regulates water use efficiency, inasmuch as loss of function mutation in this gene in Arabidopsis showed enhanced tolerance to water stress (Yoo et al. 2010). On the other hand, overexpression of the same gene homolog in poplar (Populus tremula × Populus alba) downgraded the drought tolerance. Interestingly, the Ca²⁺-dependent interaction of PtaGTL1 with CaM through the C-terminus amino acid residues 528–551 and 555–575 have been reported (Weng et al. 2012).

Beside direct regulation of TFs including DNA-binding or transcription activities, Ca²⁺/CaM complex also regulates gene expression indirectly by either modulating posttranslational modification of TFs (Snedden and Fromm 2001) or by its function through a CaM-binding protein kinase and a CaM-binding protein phosphatase (Liu et al. 2007, 2008). The indirect regulation of gene expression by modulating the transcription elongation reaction has been also reported (Nelissen et al. 2003).

6.3 CML

CMLs are another type of Ca²⁺ sensor protein, which abundantly present in plants. In Arabidopsis, various CMLs exist with various lengths ranging from 83 to 330 amino acids. At the primary sequence level, CLMs shows between 16 and 75% amino acid identity with CaMs. Contrary to CaMs that contain 4 EF-hands, CMLs contain one to six EF-hands and not all motifs are functional. This variation in the number of EF-hands is hypothesized to be the reason for different responses to Ca²⁺ signals (Bender and Snedden 2013). Studying biochemical and structural properties of CMLs, revealed that CMLs generally, but not always function as Ca²⁺ sensors (Song et al. 2004; Bender and Snedden 2013).

A research on expression patterns of Arabidopsis CML37, CML38, and CML39 genes under drought stress treatment demonstrated the increased expression of all three CMLs within 8 h in the shoot apex, hypocotyl and root (primarily in the vasculature) compared to the controls, suggesting that, these CMLs likely play important roles as Ca²⁺-sensors and take part in stress response pathways (Vanderbeld and Snedden 2007). Genome-wide analyses of Ca²⁺ sensors indicated the possible involvement of Ca²⁺ sensors in storage root deterioration in Cassava. Interaction network and co-expression analyses identified some gene pairs uniformly up-regulated at 48 h/0 h after harvest. Based on the transcriptomic data, some Ca²⁺ sensor genes including MeCML-4, -10, -16, -17, -20, -22, -25, -26, and MeCBL2 induced after drought treatment and during storage root deterioration which can be possibly nominated for genetic improvement of cassava resistance to drought and storage root deterioration (Hu et al. 2018). In a recent study, TaCML20 overexpressing transgenic lines in wheat have shown to accumulate higher water-soluble carbohydrate concentrations in the shoots and a significant increase in transcript level of sucrose (sucrose-1-fructosyltransferase (1-SST) in the internodes compared with the control plants. Moreover, a role for TaCML20 in drought stress suggested, as a significant increase of TaCML20 transcripts observed in the shoots of wild-type plants under water deficit condition (Kalaipandian et al. 2018). Microarray and quantitative real-time RT-PCR analyses have revealed that expression of OsMSR2, a novel calmodulin-like protein gene isolated from rice Pei’ai 64S (Oryza sativa L.) was strongly up-regulated by various ranges of stresses in different tissues at different developmental stages of rice. In Arabidopsis, the expression of OsMSR2 concomitant with altered expression of stress/ABA-responsive genes resulted in enhanced tolerance to high salt and drought stresses (Xu et al. 2011). Moreover, The ERECTA-overexpressing Arabidopsis exhibited reduced stomatal density (Masle et al. 2005). Interestingly, the transcript level of ERECTA in OsMSR2-transgenic plants is significantly higher than that in the wild type. Possibly, increased expression of ERECTA by expression of OsMSR2 may take part in the reduced stomatal density. As far as stomatal density is a determinative factor in water and CO₂ exchange, it is possibly responsible for lower respiration rate in the transgenic plant (Hetherington and Woodward 2003; Xu et al. 2011). Atcml9 null mutants showed enhanced tolerance to drought and salt stress concomitant with a hypersensitive response to ABA during early growth stages. Moreover, alterations in the expression of several stress and ABA-responsive and defense-related genes have been reported (Ranty et al. 2016).

In this regard, Scholz et al. (2015) reported antagonist involvement of AtCML37 and AtCML42 in drought stress. It was revealed that CML37 knock-out line, cml37, showed high susceptibility to drought stress, in contrast, CML42 knockout line, cml42, showed no difference in drought stress response compared to wild type (WT) plants. In accordance, a significant reduction of ABA under drought stress observed in cml37 plants, whereas in cml42 plants, an increase of ABA was detected which led the researchers to speculate that, CML37 is a positive regulator of ABA, while CML42 is a negative regulator of ABA accumulation, which is inducible by drought stress. Therefore, both CML37 and CML42 are antagonistically involved in drought stress response (Scholz et al. 2015). The antagonist regulation of CML37 and CML42 has been demonstrated in herbivory defense response as well (Vadassery et al. 2012; Scholz et al. 2014). Besides, cml9 knockout mutants also showed enhanced tolerance to drought and salt stresses in Arabidopsis suggesting that CML9 negatively regulated drought tolerance responses (Magnan et al. 2008).

6.4 CDPK

The CDPKs are encoded by a multigene family and have been identified in a wide range of vascular and nonvascular plants as well as in green algae and certain protozoa (Harmon et al. 2001). CDPKs have been characterized in various plant species, such as Arabidopsis (Cheng et al. 2002), rice (Wan et al. 2007), cotton (Huang et al. 2008), and wheat (Li et al. 2008). The expression of CDPKs occurs both ubiquitously or in a tissue-specific manner in response to environmental stimuli (Hrabak et al. 2003). CDPKs function diversely by occurring in various subcellular locations including cytosol, nucleus, the plasma membrane, endoplasmic reticulum, peroxisomes, mitochondrial outer membrane, and oil bodies (Harper et al. 2004). Containing EF-hands, CDPKs have the feature of Ca²⁺-dependence. Degeneration and loss of some EF-motifs alter their Ca²⁺ dependence or sensitivities. The ability of CDPKs in Ca²⁺ perception and loading might be affected by specific cellular circumstances and the surrounding regulators. Therefore, the interactive protein effectors and other surrounding regulators are determinative in Ca²⁺ loading of CDPKs (Shi et al. 2018).

It is widely revealed that CDPKs take part in plant responses to various abiotic stresses, including cold, salt, drought, and wounding (Cheng et al. 2002; Ludwig et al. 2004; Klimecka and Muszynska 2007; DeFalco et al. 2010; Seifikalhor et al. 2019). It has been shown that in normal conditions activation of some CDPK members is sufficient to activate a stress gene promoter in maize protoplasts, suggesting that these CDPK members are positive regulators of stress gene expression (Sheen 1996). The expression of CPK13 in Arabidopsis guard cells and the inhibition of light-induced stomatal opening via CPK13 overexpression have been also reported (Ronzier et al. 2014). Transgenic Arabidopsis plants overexpressing PeCPK10 a CDPK gene cloned from Populus euphratica has shown stronger ABA-induced promotion of stomatal closure in response to drought stress (Chen et al. 2013b). Another study indicated that AtCPK6 functions redundantly as a positive regulator of salt/drought stress in Arabidopsis as AtCPK6 overexpressing plants showed altered regulation of several stress-regulated genes and enhanced tolerance to salt/drought stresses (Xu et al. 2010). Modulation of ABA- and Ca²⁺-regulated stomatal movements by CPK10 (At1g18890) and involvement of a heat shock protein, HSP1 (At4g14830), as a CPK10-interacting protein has been indicated in plant drought stress responses which led scientists to conclude that for plant response to drought stress, especially for regulation of stomatal movements, the interaction between CPK10 and HSP1 is of particular importance (Zou et al. 2010). CPK8 have been also found to function in ABA-mediated stomatal closure in response to drought stress via CAT3 (CATALASE 3) regulation. It has been verified that CPK8 is able to interact and phosphorylate CAT3 (catalyzes the dismutation of H₂O₂ to water and oxygen). In addition, both cpk8 and cat3 mutant plants were more sensitive to drought stress than wild-type plants, and inhibition of K⁺ flow by ABA and Ca²⁺ was reduced in guard cells of mutant plants which suggested the possible role of CPK8 functions in ABA-mediated stomatal movement in response to drought stress through the regulation of CAT3 (Zou et al. 2015). Overexpression of ginger CDPK1 gene (ZoCDPK1) in tobacco resulted in high percentage of seed germination, higher relative water content, induction of expression of stress-responsive genes, higher leaf chlorophyll content, increased photosynthetic efficiency, and other photosynthetic parameters and 50% more growth than the wild-type plants, indicating increased tolerance to salinity and drought stress. This confirms the involvement of ZoCDPK1 in positive regulation of salinity and drought stress response signaling pathways in ginger (Vivek et al. 2013). Increased tolerance to drought stress was also observed when a rice CDPK, OsCPK13, was overexpressed (Saijo et al. 2000). Phosphorylation of drought-responsive zinc-finger domain protein (Di19) by AtCPK4/1, proposing a role for CDPKs in the regulation of abiotic stress responses (Milla et al. 2006). In Arabidopsis, five of the seven AtDi19 family members are known to function in ABA-independent drought and salinity tolerance pathways, these AtDi19 members are phosphorylated by AtCPK3 and AtCPK11 (Milla et al. 2006). In other reports, both ABA-dependent and -independent drought-response pathways have been assigned to AtCPK11 and AtCPK3 (Mori et al. 2006; Zhu et al. 2007). It turns out from the evidence that AtCPK3 participates in drought stress responses in both ABA-dependent and -independent manners. At the onset of functional analysis of AtCDPKs, it was revealed that transcription of AtCPK10 and AtCPK11 was induced by drought and high salt stresses in A. thaliana (Urao et al. 1994). In Mesembryanthemum crystallinum exposure to dehydration stress and salinity increased transcription of McCPK1 was observed (Chehab et al. 2004). AtCPK32 is an ABA-signaling component that regulates the ABA-responsive gene expression via ABF4 (leucine zipper class transcription factors) (Choi et al. 2005a). Two Arabidopsis guard cell expressed CDPK genes, CPK3 and CPK6, have been investigated. Among single and double mutants, cpk3 cpk6 showed impaired ABA and Ca²⁺ activation of slow-type anion channels and ABA activation of plasma membrane Ca²⁺-permeable channels which resulted in impaired stomatal closure; therefore, CPK3 and CPK6 identified as important regulators of guard cell ion channels and ABA-regulated stomatal signaling (Mori et al. 2006). It was demonstrated that ABA, PEG, and NaCl treatments induced the transcription of a rice CDPK gene, OsCPK9. Overexpression of OsCPK9 (OsCPK9-OX) and OsCPK9 RNA interference (OsCPK9-RNAi) analyses showed that OsCPK9 induced drought stress tolerance by enhancement of stomatal closure and improvement of osmotic adjustment (Wei et al. 2014).

In contrast with mentioned evidences, it was reported that the deficiency of CPK21 and CPK23 enhanced the tolerance to hyperosmotic stress and drought/salt stresses, while the overexpressing lines showed reverse phenotypes proposing that CPK21 and CPK23 act as negative regulators in Arabidopsis responses to corresponding stresses (Franz et al. 2011; Latz et al. 2013). Based on biochemical analysis CPK21 and CPK23 are able to phosphorylate the guard cell anion channel SLAC1 in a Ca²⁺-sensitive and Ca²⁺-insensitive manner. Geiger et al. (2010) provided evidence of a new Ca²⁺-dependent ABA-signaling pathway in guard cells, composed of CPK21 (strongly dependent)/CPK23 (weakly dependent), SLAC1 and ABI1. Two distinct cpk33 mutants displayed a significantly smaller stomatal aperture in comparison to wild-type plants and showed drought tolerance and an increased activity of the slow anion channel under both drought stress and ABA treatment, whereas, CPK33 overexpressing lines showed impaired stomatal closure, increased water loss, and less tolerance to drought stress (Li et al. 2016). In this regard, it was shown that overexpression of AtCPK23 increased stomatal apertures. On the other hand, stomatal apertures reduced when AtCPK23 expression was disrupted, suggesting that, modified Arabidopsis response to drought stress caused by the alteration of stomatal apertures due to alteration in AtCPK23 expression (Ma and Wu 2007).

7 Involvement of Calcium in Other Signaling Pathways

7.1 ABA-Signaling Pathways

Ca²⁺ plays an important role in plant cell signaling and known as an important secondary messenger involved in ABA signal transduction (reviewed by Finkelstein and Rock 2002). The sensitivity of stomatal closing to increased intracellular Ca²⁺ in response to stimuli especially ABA termed as “Ca²⁺-sensitivity priming” (Hubbard et al. 2011). Three regulatory steps bring about ABA signaling including: (1) perception by receptors, (2) protein kinases and phosphatases mediators, (3) targets such as transcription factors. The Ca²⁺ signals and ABA signaling can be integrated at all three steps (Kudla et al. 2018). It was revealed that a family of small proteins harboring a Ca²⁺-binding C2 domain, namely CAR proteins (C2-domain ABA-related proteins) controls the establishment of ABA receptors in the membrane (Rodriguez et al. 2014).

Activation of onion channels occurs in both Ca²⁺-independent and a Ca²⁺-dependent manner (Levchenko et al. 2005; Stange et al. 2010). In both signaling pathways, the same ABA receptors and PP2C phosphatases such as ABI1, ABI2, and HAB1 are affected (Ma et al. 2009; Melcher et al. 2010; Hua et al. 2012; Edel and Kudla 2016). Recent experiments found that Ca²⁺-sensing proteins such as CDPKs and CBL/CIPKs potentially link the Ca²⁺-signaling with ABA responses (Edel and Kudla 2016). In addition, ABA sensitivity and consequent ABA response are affected by altered expression of CDPKs and CBLs/CIPKs (Choi et al. 2005a, b; Zhu et al. 2007; Lynch et al. 2012). Strong ABA-insensitive phenotypes in for both stomatal closure and inhibition of stomatal opening have been observed in double mutants of Arabidopsis cpk4-1 cpk11-1 and cpk4-1 cpk11-2. These mutants are characterized by more water loss in comparison to single mutants. Moreover, in vitro experiment demonstrated that immunoprecipitated natural proteins of both CPK4 and CPK11 phosphorylate the ABA-responsive ABF1 and ABF4 TFs. Notably, ABA treatment significantly stimulated the phosphorylation of these TFs. These observations revealed evidence of participation of Ca²⁺-dependent kinases in stomatal closure in an ABA-dependent manner via regulating ABA-responsive TFs (Zhu et al. 2007). In maize (Zea mays) leaf protoplasts, stress- and ABA-inducible promoter activated as a result of constitutive ectopic expression of two A. thaliana CDPKs, CPK10/CDPK1, and CPK30/CDPK1a, clarified the linkage between CDPKs and ABA signaling (Sheen 1996). Moreover, it was shown that ZmCPK4—a maize CDPK gene, positively regulated ABA signaling and enhanced drought stress tolerance in transgenic Arabidopsis. Researchers suggest that ZmCPK4 may play its role in alleviating drought stress via ABA-mediated regulation of stomatal closure (Jiang et al. 2013).

The Ca²⁺-regulated protein kinases share similarities with SnRK2s in their PP2C interaction and target regulation (Cutler et al. 2010; Raghavendra et al. 2010). ABA response regulatory SnRK2s such as OST1 directly phosphorylates and activates the ABA-responsive TFs by binding to PP2Cs (Kobayashi et al. 2005; Ng et al. 2011; Xie et al. 2012). The impairment of ABA signal transduction in stomata of CDPK quadruple mutant plants clarified the interdependence of CPKs and SnRK2s for a functional ABA-signaling pathway (Brandt et al. 2015). It was revealed that despite SnRK2s, CDPKs and CIPKs specifically address distinct targets, they have overlapping substrate spectrum (Steinhorst and Kudla 2014; Edel and Kudla 2016).

It has been suggested that certain types of Ca²⁺-sensing proteins negatively regulate ABA signal transduction. The CBL-interacting protein kinase CIPK11/PKS5 negatively impacts the ABA responses by phosphorylation of the bZIP transcription factor ABI5 (Zhou et al. 2015a, b). CIPK15 interacts with two calcium-modulated protein phosphatases 2C, ABI1 and ABI2, known as negative regulators of ABA signaling (Gosti et al. 1999; Merlot et al. 2001; Guo et al. 2002). An AP2 transcription factor (At ERF7kinase) as a substrate of CIPK15, negatively regulates the ABA responses, therefore, CIPK15 may regulate ABA signaling directly by phosphorylating a TF and modulating gene expression (Song et al. 2005). CIPK3 and CBL9 are also characterized as negative regulators of ABA signaling (Kim et al. 2003; Pandey et al. 2004). It was also reported that disruption of CIPK3 function does not influence gene induction by drought and hyperosmotic stress. This evidence suggests that CIPK3 regulates specific pathways that lead to the stress gene expression. It was also stated that CIPK3 functions as a cross-talk “node” mediating the interaction between ABA and abiotic stress signal transduction pathways (Kim et al. 2003).

Despite the numerous reports on involvement of Ca²⁺- and ABA in stress response, evidence for the role of Ca²⁺-regulated kinases in activation of ABA-related TFs is still missing, however, an alternative scenario is CDPKs and CIPKs perceive the cues from environmental stimuli such as drought, salinity or flooding via endogenous Ca²⁺-release and function in controlling the machinery of ABA signal transduction; therefore, these protein kinases turn out to be the prime candidates for acclimation responses in stress condition (Kudla et al. 2018; Seifikalhor et al. 2019).

7.2 ROS Signaling Pathway

ROSs play an essential role as signaling molecules in the regulation of numerous biological processes such as growth, development, hormonal signaling, polar growth, autophagy, programmed cell death, and responses to biotic and abiotic stimuli in plants (reviewed by Demidchik 2015; Foyer and Noctor 2016).

In the plasma membrane of Arabidopsis guard cells, activation of hyperpolarization-dependent I_Ca channels by ROS induces an increase in [Ca²⁺]_cyt (Pei et al. 2000). Other cell types also indicated ROS-induced increase in [Ca²⁺]_cyt indicating that the activation of Ca²⁺ channels could be a key step in many ROS‑mediated processes (Mori and Schroeder 2004) (Fig. 1).

Reactive oxygen species and Ca²⁺ signals have been shown to regulate the same physiological reactions in the same time span; notably under biological conditions, H₂O₂ as relatively long-lived ROS does not exist for longer than one second. Apparently, ROS and Ca²⁺ stimulate each other in a way that ROS target the same or nearest cells that are effective autocrine/paracrine stimulators of Ca²⁺ signaling (Halliwell 2006). Increase in [Ca²⁺]_cyt results in an elevated ROS production and vice versa (Foreman et al. 2003). This self-amplification extends the duration and amplitude of weak signals; thereby, appropriate signaling causes dramatic ROS-Ca²⁺ alterations (Demidchik and Maathuis 2007; Demidchik et al. 2009).

Reactive oxygen species are generated by multiple biological systems including electron-transporting chains (ETC) of chloroplasts and mitochondria, small ETC of peroxisomes, class III cell wall peroxidases, oxalate oxidase, quinine reductase, and polyamine oxidase. These systems produce ROS as by-products of their constitutive physiological functions. NADPH oxidases contribute to extracellular ROS synthesis for mediation of signaling and regulation processes (Bose et al. 2014; Demidchik 2015; Foyer and Noctor 2016). In addition to direct ROS synthesis, primary stress stimuli induce NADPH oxidases leading to ROS production during signaling events. Superoxide generated by NADPH oxidases is at the onset of ROS generation and other signaling events and scavenging superoxide is a leading mechanism for terminating ROS and Ca²⁺ signals (Foyer and Noctor 2016). The joint function of Ca²⁺ and NADPH oxidase is due to a lack of NADPH oxidase function at the absence of Ca²⁺. This Ca²⁺-depended function is due to the presence of two Ca²⁺-binding helix–loop–helix structural domains (EF-hands) in the NADPH oxidase structure. The conformational change and intramolecular interaction of the N-terminal Ca²⁺-binding domain with the C-terminal superdomain in consequence of binding to Ca²⁺, enables electron transfer (Bànfi et al. 2004). Reportedly, binding of CaM to the NADPH binding domain or phosphorylation of serine/tryptophan residues in the FAD-binding domain by protein kinase C enhances the capability of binding to Ca²⁺ (Jagnandan et al. 2007; Kobayashi et al. 2007; Tirone et al. 2010). Biding capacity to Ca²⁺ increases after CaM attachment to the NADPH binding domain or phosphorylation of serine/tryptophan residues in the FAD-binding domain by protein kinase C (Jagnandan et al. 2007; Kobayashi et al. 2007; Tirone et al. 2010).

A parallel increase in NADPH oxidase activity and CDPK expression have been reported (Xing et al. 2001). It was also reported that an increase in NADPH oxidase activity via small G proteins (Rac/Rop GTPases) occurs in a Ca²⁺-dependent manner (Baxter-Burrell et al. 2002). It was hypothesized that the Ca²⁺-activated NADPH oxidases work cooperatively with the ROS-activated I_Ca channel to generate and amplify stress-induced Ca²⁺ and ROS signals which are known as the “ROS-Ca²⁺ hub” (Demidchik and Maathuis 2007; Demidchik 2015; Shabala et al. 2015). It was reported that overexpression of OsACA6 (Ca²⁺ATPase gene from Oryza sativa) is triggered during salinity and drought stresses. Besides several physiological aspects, overexpressing lines also showed higher leaf chlorophyll and reduced accumulation of ROS compared to the wild-type. Transcription profiles of transgenic plants declared altered expression of genes encoding many TFs, stress-related proteins and signaling components. This suggests that Ca²⁺-ATPases have diverse roles as regulators of stress signaling pathways (Huda et al. 2013).

Transcriptomic analysis of H₂O₂-regulated genes in Arabidopsis resulted in the identification of 175 H₂O₂ responsive genes; among them 113 up-regulated and 62 down-regulated by H₂O₂. Fourteen ESTs with the known function were selected from up-regulated genes for expression studies by RNA blots. Rapid dehydration of plants induced the expression of a couple of the selected genes; it was speculated that H₂O₂ is the mediator of observed effects, in a way that, these genes encoding CaM, a Ca²⁺‑binding protein, the DREB2A transcription factor, the Arabidopsis MAP kinase ATMPK3, and a zinc-finger protein, suggesting that H₂O₂ plays a pivotal role in plant drought stress response regulation by modulating the H₂O₂ signaling, MAPK cascades, and gene expression (Desikan et al. 2001).

8 Signal Transduction for Rhizobium-Induced Nodule Formation

Establishing of legume-rhizobium endosymbiosis requires an exchange of molecular signals between two partners (plant and bacterium). Bacterial signals (Nod factor) are perceived by host plants receptors and trigger a plethora of symbiotic responses, ranging from nuclear Ca²⁺ oscillation to symbiotic gene expression in host plants (Oldroyd 2013). Activation of the Nod factor receptors induces intracellular signaling, which is associated with oscillations of the Ca²⁺ concentration in the perinuclear region. These oscillations may occur through localized Ca²⁺ influx from outside of the cell or the intracellular release of Ca²⁺. Bacterial induced Ca²⁺ flux has been reported in diverse legume plants indicating the common role for Ca²⁺ in endosymbiosis establishment. The Nod factor induced Ca²⁺ oscillation signal is—most probably—decoded by a nuclear-localized calcium and CaM-dependent kinase (CCaMK). This protein triggers a transcriptional network and phosphorylates the LjCYCLOPS as a target protein and makes it an active transcription factor. LjCYCLOPS binds to the promoter of the ERN1 and NIN gene eventually leads to the root nodule initiation.

The essential role for ROS in the generation of the Ca²⁺ gradient in root-hair growing has been reported (Foreman et al. 2003). It has been revealed that ROS activates the Ca²⁺-dependent protein kinase (CPK5) phosphorylation which is required for systemic defense responses including drought stress (Dubiella et al. 2013). This will let us speculate that ROS-mediated responses in the cell can play a key role in the signal transmission of distinct stimuli. Recent evidence also represent tolerance to drought stress by rhizobium infection in legume plants. Delayed leaf senescence and induced growth rate in soybean plants inoculated with rhizobium exposed to drought stress imply that rhizobium endosymbiosis commit a function to relief drought stress (Staudinger et al. 2016) though the central question that remains is which regulatory mechanism/s occurred so that upon the activation of nodule symbiosis drought tolerance is triggered.

9 Concluding Remark

Various aspects of Ca²⁺ signaling have attracted the attention of numerous researchers so far. The growing trend of research attempts in this issue highlights the Ca²⁺ signaling as a distinct realm of research. Despite the presence of myriad numbers of reports on Ca²⁺-signal transduction under drought stress, there are not ample evidence on the cooperative mechanism of Ca²⁺-dependent and -independent signaling network and the underlying molecular principles in regulation of stress response. The circumstance of the specificity of Ca²⁺ signals within the same cell is yet to be investigated. Certain Ca²⁺-sensing proteins have shown to up-regulate drought tolerance signaling events, whereas negative regulation of drought stress is also attributed to these proteins. Unraveling the specific mechanism underlying the Ca²⁺ signaling events may simplify the complex network addressing the role of Ca²⁺ sensing proteins. And pave the way to provide drought-resistant plants in the coming future.