Calcium Signaling

Ca2+ regulates a range of activities within the cell such as cell division and elongation, cytoplasmic streaming, photomorphogenesis, and plant defense against environmental stresses (Song et al. 2008; Tuteja 2009b ; Kader and Lindberg 2010). It functions as the central node in overall signaling web and has a promising role in stress tolerance (Tuteja and Sopory 2008). During abiotic stress, Ca2+ acts as a second messenger under various stress conditions (Knight 2000) . Ca2+ signatures have a leading role in numerous physiological processes such as regulation of sto-matal apertures in plants (Allen et al. 2001). Ca2+ signatures change according to the nature of stress (Kiegle et al. 2000), duration of stress (Plieth et al. 1999), earlier exposure to any stress

(Knight et al. 1997), and the type of tissue exposed to stress (Kiegle et al. 2000). For studying the role of calcium and its dynamics, various pharmacological and transgenic approaches have been utilized. The calcium reporter protein aequorin has proved to be very useful for such studies in revealing calcium fluxes within one cell and in different tissues.

Calcium-binding proteins act as stress sensors, get conformationally transformed upon binding with Ca2+, which facilitates their interaction with downstream effector molecules (Clapham 2007; Gifford et al. 2007; Tuteja 2009b). Elongation factor (EF)-hand motif, which generally occurs in pairs, is the most common motif present in these Ca2 +- binding proteins and helps in high-affinity binding of Ca2+. It has a helix-loop-helix structure and is reported in about 250 proteins of Arabidopsis (Day et al. 2002). In plants these EF-hand proteins comprise three different categories, namely, CDPKs (Ca2 +-dependent protein kinases), CaMs (calmodulins) and CMLs (CaMlike proteins), and the CBLs (calcineurin B-like proteins) (Fig. 14.1). Of these, only CDPKs act as "responders," as they are capable of directly transducing signals through their catalytic activity. CaMs/CMLs and CBLs are only sensors for regulating the downstream targets. CMLs, CDPKs, and CBLs are restricted to plants and some protists, whereas CaM is universal to all eukaryotes.

2.1 Calcium-Dependent Protein Kinases

CDPKs have been reported from several plants (Kawasaki et al. 2001; Seki et al. 2002; Ozturk et al. 2002; Asano et al. 2011) and are specific for a particular osmotic stress response. Studies on salt-tolerant and salt-sensitive rice varieties revealed specific CDPKs to be induced earlier with a sustained expression in the tolerant variety in comparison to the sensitive variety (Kawasaki et al. 2001). Arabidopsis CDPKs are also highly specific (Sheen 1996). Out of several CDPKs tested, only AtCDPK1 and AtCDPK1a were able to transcriptionally activate selected reporter

Fig. 14.1 Calcium-dependent signaling. Environmental stress responses are perceived by cell surface which in turn increases cytosolic Ca2+ concentration. Ca2+ got activated by cytosolic Ca2+ and regulates downstream targets leading to physiological responses

Fig. 14.1 Calcium-dependent signaling. Environmental stress responses are perceived by cell surface which in turn increases cytosolic Ca2+ concentration. Ca2+ got activated by cytosolic Ca2+ and regulates downstream targets leading to physiological responses

genes. CDPKs are five domain-containing proteins, and range from ~40 to 90 kDa in size. Their catalytic domain consists of a highly conserved serine/threonine kinase region, whereas, their N-terminal variable domain ranges from 21 to 185 amino acids in length (Klimecka and Muszynska 2007). Adjacent to the kinase domain is a pseudosubstrate-containing autoinhibitory junction domain that interacts with the active site and inhibits its kinase activity. Next to the autoin-hibitory domain is the CaM-like domain (CLD) which is responsible for its Ca2+-binding activity. Their C-terminal domain is relatively short and variable.

Plants have a large family of Ca2 +-dependent protein kinase (CDPKs) and have an important role in signaling during abiotic stresses like drought, wounding, and cold (Fig. 14.2). Induction of CDPK by osmotic stress has been shown by many workers in various plants (Kawasaki et al. 2001; Seki et al. 2002; Ozturk et al. 2002; Witte et al. 2010; Franza et al. 2011; Wurzinger et al. 2011). Induction of CDPK exists in longer duration in tolerant varieties of rice than in the salt-sensitive variety (Kawasaki et al. 2001). Martin and Busconi (2001) have reported that cold stress activates the membrane-associated CDPK in rice plants. Saijo et al. (2001) have also

Fig. 14.2 Signaling through CDPK and protein phosphorylation by Ca2+ signatures

Fig. 14.2 Signaling through CDPK and protein phosphorylation by Ca2+ signatures

reported that overexpression of OsCDPK7 in rice provides cold and osmotic stress tolerance in these plants. Sheen (1996) has demonstrated that CDPK is involved in signal transduction. In his experiment, he demonstrated that in protoplast of maize leaf the expression of stress-responsive HVA1 was induced by active AtCDPKl. Sheen (1996) has also reported that AtCDPKl activation is blocked by a protein phosphatase type 2C (AtPP2CA). Enhanced induction of CBF1, RAB18, RC12A, and LT178 (i.e., RD29A) gene expression has been observed in AtPP2CA-silenced Arabidopsis plants during cold and ABA treatment (Tahtiharju and Palva 2001). Romeis et al. (2001) also reported that pathogen infection also activated CDPK in plants.

2.2 Calmodulin and Other Calcium-Binding Proteins

Elevated levels of calcium within a cell activate various calcium-binding proteins which in turn induce specific kinases. On the basis of function, calcium-binding proteins are classified into two groups: (1) trigger proteins and (2) buffer proteins. The trigger proteins are activated when they bind with Ca2+ and after that they interact with other proteins and alter their activity. The trigger-type CaBPs are calmodulin (CaM), CaM-binding proteins, Ca2+-dependent protein kinase, and phosphatase (Reddy 2001).

Stress causes increase in inositol 1,4,5-triphos-phate (IP3). Activation of phospholipase C (PLC) results in hydrolysis of PIP2 to IP3. IP3 is regarded as the activator of vacuolar calcium channels in plants. During stress, the increase in cytosolic Ca2+ is due to the activation of IP3-dependent calcium channels (Braam 2005; Boudsocq and Lauriere 2005). Furthermore, calcium-binding proteins (CaBP) or calcium sensors recognize and translate the information provided in calcium signatures (Tuteja and Mahajan 2007) and pass the information downstream for regulation of gene expression.

These CaBPs are partially responsible in modulating the intracellular calcium levels. The calcium-binding proteins can be regulated either in a cell- or in a tissue-specific manner. NaCl-inducible Ca2+/calmodulin-dependent protein kinase in pea (PsCCaMK) was reportedly specific to roots (Pandey et al. 2002) . A calmodulin-binding transcription activator family was shown to be specific only to the multicellular organisms (Bouche et al. 2002). Other examples of osmotic stress-activated calcium-binding proteins include

Arabidopsis protein AtCP1, the membrane-associated rice protein OsEFA27, and the Arabidopsis counterpart RD20 (Frandsen et al. 1996; Jang et al. 1998). Some CaBPs can be negative regulators of osmotic stress also; one example is the calmodulin-binding protein in Arabidopsis, AtCaMBP25. Although it is upreg-ulated by osmotic stress, its overexpression renders plants sensitive to osmotic or salt stresses and its antisense transgenics show improved tolerance (Perruc et al. 2004).

Calmodulin, an important CaBP, is a small acidic protein and is responsible for the regulation of intracellular Ca2+ levels. Increased Ca2+ concentration activates calmodulin which then induces specific kinases. Calmodulin is a very important calcium-binding protein in Ca2+ signaling and has been found to be involved in biotic and abiotic stresses (Fig. 14.3) (Reddy 2001; Tuteja and Mahajan 2007; Tuteja and Sopory 2008). Plants have been found to possess unique Ca2+ sensors like calmodulin-like proteins (CMLs), and Arabidopsis contains 50 such proteins (Tuteja and Sopory 2008). The calmodulin-like proteins differ from calmodulin in having more than 148 amino acids and also have one to six EF-hand motifs. These CMLs have been found to play a role as Ca2+ sensor during stress in plants (Vanderbeld and Snedden 2007). Certain

Ca2+-binding proteins do not contain EF-hand motifs, such as calreticulin, annexins, calnexin, phospholipase D (PLD), and pistil-expressed Ca2+-binding proteins.

PLD activity has been reported to be involved in ethylene and ABA responses, synthesis of a-amylase in aleurone cells, closing of stomata, responses to pathogens, leaf senescence, and drought tolerance (reviewed by Tuteja and Sopory 2008) .

Annexins have been reported to be involved in biological membrane organization and functions (Tuteja and Mahajan 2007). The exact function of annexins is not clear yet but are thought to play a role in secretary processes and they have ATPase and peroxidase activities (Tuteja and Mahajan 2007) . Annexins have been regarded to have a role in stress responses (Gorecka et al. 2007a). Annexin At1 of Arabidopsis thaliana (AnnAt1) plays a vital role in pH-mediated cellular responses to environmental stress (Gorecka et al. 2007b).

Calnexin (CNX) is an important calcium-binding protein and is an endoplasmic reticulum (ER) type 1 integral membrane protein. CNX behaves as a molecular chaperone and has a leading role in the recognition of misfolded proteins, lectin-like activity, and Ca2+ binding (Sarwat and Tuteja 2007) ; Till date, the role of calnexin in stress has not been reported but it is considered that it has a role in ER stress response in plants (reviewed by Tuteja and Sopory 2008).

In plants, another group of Ca2+ sensors is the SOS (salt overly sensitive) family and is responsible for calcium-mediated pathway for salinity stress tolerance (Mahajan et al. 2008). Zhu (2003) has isolated sos mutants (sosl, sos2, and sos3) from Arabidopsis which are hypersensitive to salt. The sos mutants have been shown to accumulate more proline under salt stress which gives protection to the salt-stressed plants (Liu and Zhu 1998). Cloning and characterization of sos genes (SOS1-SOS3) has opened new doors for ion homeostasis and plant tolerance to salt. It has been reported that sos1, sos2, and sos3 function in a common pathway leading to salt tolerance (Halfter et al. 2000; Zhu 2000). Sosl is activated by sos3-sos2 complex and has been reported by many workers. Shi and Zhu (2002) demonstrated that if sos1 alone was allowed to express in yeast cells, slight enhancement in salt tolerance was observed. However, if sos1 was expressed with sos3 and sos2 the yeast cells showed more tolerance to salt. Qiu et al. (2002) have reported very low Na+/H+ exchange activity in sos mutant plants in comparison with wild type in plasma membrane vesicles. Addition of activated sos2 protein to isolated membrane vesicles of mutant plants showed that the exchange activity increased in sos2 and sos3 but remains unaffected in sos1 mutants. The results lead to the conclusion that Na+/H+ exchange activity of sos1 was stimulated by sos3 and sos2 (reviewed by Xiong et al. 2002). sos3-sos2 also regulate Na+ transporter AtHKTl, which is a salt tolerance effector (Uozumi et al.

2000). Homologs of HKT1 in other plant species revealed that it can be either a K+ transporter or a Na+/K+ cotransporter (Horie et al. 2001; Liu et al.

2001). Rus et al. (2001) demonstrated that mutation in AtHKT1 of Arabidopsis suppressed the salt hepersensitivity phenotype of sos3, leading to the concept that activity of AtHKT1, the Na+ influx transporter, may be inhibited by SOS3 (reviewed by Xiong et al. 2002).

As compared to caltractin and calmodulin, sos3 binds with Ca2+ with low affinity and has three EF-hand motifs (Ishitani et al. 2000). In sos3 mutation, one of the EF-hand motifs gets mutated, thus preventing it to bind Ca2+ (Ishitani et al. 2000). sos4 and sos5 have recently been characterized (Mahajan et al. 2008). sos4 encodes a pyridoxal (PL) kinase which has a role in the biosynthetic pathway of pyridoxal-5-phosphate, which is an active form of vitamin B6. Sos5 is a putative cell surface adhesion protein and has a role in normal cell expansion. Under salt stress, sos5 has been found to have a role of maintenance of cell wall integrity (reviewed by Tuteja and Sopory 2008) .

2.3 Calcineurin B-Like Proteins

These proteins possess four Ca2+-binding EF-hand domains and have significant identity to calcineu-rin B subunit and neural calcium sensor from yeast and animals (Kudla et al. 1999). The CBL protein family and their corresponding kinases (CIPKs) together form a complex and dynamic Ca2+ decoding signaling network (Fig. 14.4). Together they show Ca2+-binding functionality and kinase activity (Mahajan et al. 2006a).

CIPKs have a conserved N-terminal kinase domain and C-terminal regulatory domain, separated from the kinase domain by a variable junction domain. A conserved NAF domain present in the divergent regulatory domain is required for interaction with CBLs. CBL binds to the NAF domain of CIPKs, releases the C-terminal (auto-inhibitory) domain from the kinase domain, in turn transforming the kinase into its active state (Guo et al. 2001; Mahajan et al. 2006a).

Bioinformatic analysis shows a number of components of both proteins in the families of CBLs and CIPKs; 10 CBLs and 26 CIPKs in Arabidopsis, 10 CBLs and 30 CIPKs in rice (Albrecht et al. 2001; Kolukisaoglu et al. 2004; Weinl and Kudla 2009). However, several species of green algae possess single CBL and CIPK genes, other plant species can have multiples, like Physcomitrella contains four CBLs and seven CIPKs and the fern Selaginella moellendorfii has five CBLs and five CIPKs (Batistic and Kudla 2009; Weinl and Kudla 2009); thus showing evolutionary complexity of these CBL and CIPK protein families from lower to higher organisms.

Fig. 14.4 Signaling through CBL/CIPK and protein phosphorylation by Ca2+ signatures

CBLs are found to be localized all through the cell. In Arabidopsis itself, four CBLs are present at the plasma membrane, four at the vacuolar membrane, and two in the cytoplasm and nucleus (Batistic et al. 2008; Cheong et al. 2007; D' Angelo et al. 2006; Kim et al. 2007; Weinl and Kudla 2009) . The pea CBL was reported to be exclusively localized in the cytosol whereas pea CIPK is localized in the cytosol and the outer membrane (Mahajan et al. 2006b). The pea CBL and CIPK were reported to be coordinately upregulated in response to high NaCl, cold, wounding and also in response to calcium and salicylic acid, whereas drought and abscisic acid had no effect on the expression of these genes (Mahajan et al. 2006b) .

Our knowledge has been greatly facilitated by the reverse genetic approaches. A CIPK3 loss-of-function (LOF) mutant showed its involvement in regulating ABA-induced gene expression and ABA responses during seed germination (Kim et al. 2003). Through these studies, CBL1 has came out to function in an ABA-independent manner in controlling responses to drought, cold, and salinity (Albrecht et al. 2003; Cheong et al. 2003), whereas its closely related Ca2+ sensor CBL9 renders plants hypersensitive to ABA (Pandey et al. 2004). Interestingly, when CIPK1 complexes with

CBL1, it mediates the ABA-dependent pathway (D' Angelo et al. 2006). CBL1 and CBL9 activate CIPK23, and the complex regulates the activity of the shaker-like K+ channel ARABIDOPSIS K+ TRANSPORTER1 (AKT1) and thus contributes in K+ homeostasis within the cell (Kudla et al. 1999). This complex also has a role in stomatal regulation under drought conditions.

CBL-CIPK network is a central and critical system functioning in response to a broad variety of stimuli in order to decode Ca2+ signals. Each CBL and each CIPK can make alternative protein interactions and are part of multifunctional signaling component, thus determining the flow of information (Mahajan et al. 2006a).

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