Introduction Calcium ions (Ca2+) are important for signaling in eukaryotic cells.

The Ca2+ concentration in cytoplasm ([Ca2+]cyt), amplitude and timing is interpreted by Ca2+ binding proteins to induce various cellular responses which are specific to stimuli (Dodd et al., 2010). To understand how this is interpreted differently by calcium binding protein, calcium signature hypothesis is developed and it is postulated that in plants, Ca2+ signatures regulate cellular signaling in response to abiotic stress, stomatal aperture, fertilization, interaction with pathogenic and symbiotic microorganisms, development of tip-growing structures, circadian responses etc. (Dodd et al., 2010). The [Ca2+]cyt dynamics is regulated by opening and closing of Ca2+ channels and by Ca2+-ATPase activities located in the plasma membrane as well as endo-membranes (e.g. endoplasmic reticulum, tonoplast, etc.). Various stimuli evoke calcium signaling, which is complex process involving spatio-temporal patterns of calcium influx (Kim, Cheong, Grant, Pandey, & Luan, 2003). This encoded calcium signature is decoded by different downstream sensor and responders (see figure 1). Despite clear experimental evidence that calcium signatures are correlated with specific cellular and physiological response, downstream events are still not well understood. An array of calcium binding proteins (CBPs) has been implicated in decoding the calcium signatures (DeFalco, Bender, & Snedden, 2010; Reddy, Ali, & Reddy, 2002). These CBPs changes conformation upon binding with Ca2+; acting as a sensor. The most common calcium binding motif is EF hand, which usually occurs in pairs and binds calcium cooperatively with high affinity. Three largest groups of these proteins are the CaMs (calmodulins) and the CMLs (calmodulin like proteins), the CDPKs (Ca2+ dependant protein kinases) and the CBLs (calcineurin-B like proeteins). Out of these three classes, only CDPKs act as responders; capable of transmitting signals via their catalytic activity while others are only

sensors. CaMs are found in most of all eukaryotes, while CMLs, CDPKs, CBLs are restricted to plants only.

Figure 1. The Calcium signaling overview. The nature of stimulus can vary from chemicals (salt, aluminum etc.), temperature (heat or cold) to other stressors (like pathogen, touch, light, CO2 etc.). The intracellular compartment is predicted to be either vacuoles or endoplasmic reticulum. CIPKs; CBL interacting protein kinase. Modified from (DeFalco et al., 2010).

Evolution of Ca2+ signaling All life forms utilize ATP as currency for energy. Using ATP for energy ensure that there are inorganic phosphate metabolism in most of all cellular compartment. Further, some enzyme, transcription factors, transporters activation/deactivation also requires phosphorylation/dephosphorylation (Sanders & Brownlee, 1999). So in early life form, maintaining low intracellular Ca2+, may have been constraint, because Ca2+ forms insoluble compound with phosphate (Sanders & Brownlee, 1999). This in turn, may have life form required to evolve mechanism to export Ca2+ out of the cell as well as endo-membrane sequestration compartment. These sequestration and transport system to keep low Ca2+, provided environment to evolve Ca2+ signaling (Dodd et al., 2010; Sanders & Brownlee, 1999). CaMs and CMLs CaMs are highly conserved protein among eukaryotes. Typically in animal genome, there are few CaM genes, where as plant genome has multiple CaM genes and encode similar CaMs with slight variations. Arabidopsis contains seven different CaM genes (CaM1 to CaM7) (Kim et al., 2003). All of these are highly conserved, indicating importance of CaMs in Arabidopsis. Structurally CaMs are small protein (~140-150 amino acids) containing two globular domain each contains two EF hand motif (see Figure 2). Upon binding with calcium CaM undergoes conformational change, exposing the internal hydrophobic surface which binds to various CaM binding proteins. Reddy et al., 2002, used screening of expression libraries form Arabidopsis and revealed 20 different CaM binding proteins. This CaM binding proteins are impressive as they includes kinases, cytoskeleton proteins, ion channels, pumps, transcription factors enzymes and many other with unknown function (Reddy et al., 2002). Besides CaMs, plants also have

CMLs. In Arabidopsis genome, there are 50 genes predicted to encode for CMLs. They vary in similarity with CaM and length from 83 to 330 amino acids (Reddy et al., 2002). It is predicted that these CMLs have different affinity toward calcium because of divergence from consensus EF-hand motif. But this statement does not have any experimental evidence since unlike CaMs, CMLs are not extensively studied (DeFalco et al., 2010).

Figure 2. Domains of different calcium binding protein. Size and different motif of calcium binding protein are indicated with dark areas. CDPKs The Arabidopsis CDPKs have four domain (see Figure 2) N-terminal variable domain, kinase domain (serine/threonine), auto-inhibitory domain and CaM like domain (Li et al., 2008). These proteins are believed to be evolved from fusion of CaM interacting kinase and CaM and vary in size from 40 to 90 kDa. The function of auto-inhibitory domain is to act as pseudo-

substrate and keep protein inactive in absence or low concentration of calcium (Li et al., 2008). Two models for activity of these proteins are predicted. In first model, calcium binding to CaM like domain displaces the auto-inhibitory domain and exposes kinase domain to interact with its substrate (Li et al., 2008). Alternative model suggests that at low basal level of calcium, Cterminus lobe is already bound with calcium, but for activation N-terminus binding to calcium is required at higher concentration (Li et al., 2008). CBLs and CIPKs CBLs are another group of calcium sensors unique to plants and to some extent protozoa. They show limited sequence similarity with neuronal calcium sensors and calcineurin B from animals (Batistic, Waadt, Steinhorst, Held, & Kudla, 2010). But they are demonstrated to be regulatory subunits for plant specific serine/threonine protein kinases (CIPKs). Further subcellular localization has also been studied for CBLs-CIPKs, and found to reside in cytoplasm, tonoplast, plasma membrane and nucleus (Batistic et al., 2010). Coupling spacial specificity with variable affinity to bind calcium vastly increases the ability of these proteins to responds differently to different calcium signatures (Batistic et al., 2010). Early Ca2+-responsive genes in Arabidopsis Cytoplasmic calcium burst has been shown to change the expression of 230 genes. These are the early responsive genes to transient calcium burst (Kaplan et al., 2006). The upregulated and down regulated genes numbered 162 and 68 respectively, which included genes related to transcription factors, signaling, transporter, defense, protease, photosynthesis, cell structure and metabolism (see figure 3).

Figure 3. Pie chart of functional classification of calcium responsive genes. Courtesy (Kaplan et al., 2006)

References Batistic, O., Waadt, R., Steinhorst, L., Held, K., & Kudla, J. (2010). CBL-mediated targeting of CIPKs facilitates the decoding of calcium signals emanating from distinct cellular stores. The Plant journal: for cell and molecular biology, 61(2), 211-22. doi:10.1111/j.1365313X.2009.04045.x DeFalco, T. a, Bender, K. W., & Snedden, W. a. (2010). Breaking the code: Ca2+ sensors in plant signalling. The Biochemical journal, 425(1), 27-40. doi:10.1042/BJ20091147 Dodd, A. N., Kudla, J., & Sanders, D. (2010). The language of calcium signaling. Annual review of plant biology, 61, 593-620. Annual Reviews. doi:10.1146/annurev-arplant-070109104628 Kaplan, B., Davydov, O., Knight, H., Galon, Y., Knight, M. R., Fluhr, R., & Fromm, H. (2006). Rapid transcriptome changes induced by cytosolic Ca2+ transients reveal ABRE-related sequences as Ca2+-responsive cis elements in Arabidopsis. The Plant cell, 18(10), 2733-48. doi:10.1105/tpc.106.042713 Kim, K.-nam, Cheong, Y. H., Grant, J. J., Pandey, G. K., & Luan, S. (2003). CIPK3 , a Calcium Sensor Associated Protein Kinase That Regulates Abscisic Acid and Cold Signal Transduction in Arabidopsis. Society, 15(February), 411-423. doi:10.1105/tpc.006858.2 Reddy, V. S., Ali, G. S., & Reddy, A. S. N. (2002). Genes encoding calmodulin-binding proteins in the Arabidopsis genome. The Journal of biological chemistry (Vol. 277, pp. 9840-52). doi:10.1074/jbc.M111626200 Sanders, D., & Brownlee, C. (1999). Communicating with calcium. The Plant Cell Online, 11(April), 691-706. Retrieved from http://www.plantcell.org/content/11/4/691.short Li, A.-L. L., Zhu, Y.-F. F., Tan, X.-M. M., Wang, X., Wei, B., Guo, H.-Z. Z., Zhang, Z.-L. L., Chen, X.-B. B., Zhao, G.-Y. Y., Kong, X.-Y. Y., Jia, J.-Z. Z., and Mao, L. (2008). Evolutionary and functional study of the CDPK gene family in wheat (triticum aestivum l.). Plant molecular biology, 66(4):429-443.