Biochem. J. (2010) 425, 27–40 (Printed in Great Britain) doi:10.1042/BJ20091147 27 REVIEW ARTICLE Breaking the code: Ca 2+ sensors in plant signalling Thomas A. DEFALCO 1 , Kyle W. BENDER 1 and Wayne A. SNEDDEN 2 Department of Biology, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Ca 2+ ions play a vital role as second messengers in plant cells dur- ing various developmental processes and in response to environ- mental stimuli. Plants have evolved a diversity of unique proteins that bind Ca 2+ using the evolutionarily conserved EF-hand motif. The currently held hypothesis is that these proteins function as Ca 2+ sensors by undergoing conformational changes in response to Ca 2+ -binding that facilitate their regulation of target proteins and thereby co-ordinate various signalling pathways. The three main classes of these EF-hand Ca 2+ sensors in plants are CaMs [calmodulins; including CMLs (CaM-like proteins)], CDPKs (calcium-dependent protein kinases) and CBLs (calcineurin B-like proteins). In the plant species examined to date, each of these classes is represented by a large family of proteins, most of which have not been characterized biochemically and whose physiological roles remain unclear. In the present review, we discuss recent advances in research on CaMs and CMLs, CDPKs and CBLs, and we attempt to integrate the current knowledge on the different sensor classes into common physiological themes. Key words: calcium, calmodulin (CaM), calcium-dependent pro- tein kinase (CDPK), calcineurin B-like protein, calmodulin-like protein (CML), plant signalling. INTRODUCTION The ability of cells to respond to external stimuli in a timely and specific manner is a recurring theme in biology. In the paradigm of information processing by cells, stimulus perception is followed by signal transduction and culminates in an appropriate physiological response. The propagation and amplification of signals involves rapid changes in subcellular levels of second messengers such as Ca 2+ ions, cyclic nucleotide monophosphates, inositiol polyphosphates, and a host of other small molecules. These messengers relay information to myriad downstream effectors such as enzymes, cytoskeletal proteins, transcription factors, etc. that comprise the cellular machinery responsible for changes in gene and protein expression, metabolic activity and developmental patterning. In eukaryotes, Ca 2+ serves as a universal second messenger whose cytosolic concentration is tightly regulated by Ca 2+ transporters. Resting cytosolic [Ca 2+ ] is kept low (∼100 –200 nM) due to its potential toxicity at elevated levels. Consequently, a steep concentration gradient is established between the cytosol and Ca 2+ stores. Animal cells rely primarily on the ER (endoplasmic reticulum) and mitochondria for intracellular Ca 2+ sequestration, whereas in plant cells the central vacuole is the main intracellular Ca 2+ repository [1–3]. One of the most intriguing aspects of Ca 2+ signalling is the complex spatio-temporal patterns of Ca 2+ influx that are evoked in cells by various stimuli [1,4]. Despite significant correlations between Ca 2+ fluxes and physiological response in plants, relatively little is understood about the role of such signals in regulating downstream events. How does specificity of response derive from such an apparently universal mechanism? The Ca 2+ signature hypothesis suggests that stimulus-specific spatial and temporal Ca 2+ flux patterns encode information that help a cell co-ordinate the appropriate cellular responses [1,5,6]. Although this hypothesis remains somewhat controversial [4,7], it has proven to be a good working model. Certainly, Ca 2+ signals are but one part of the intracellular language used for timely and precise communication in cells. A corollary of encoding information in a Ca 2+ signal is the need to decode such information. For this task, cells employ an array of CBPs (Ca 2+ -binding proteins) that serve as Ca 2+ sensors (Figure 1). In general, CBPs that function as sensors undergo conformational changes upon Ca 2+ binding that allow them to interact with downstream effectors [3,8]. The most common Ca 2+ -binding structural motif in proteins is the EF-hand. Typically, EF-hands occur in pairs and facilitate high-affinity co- operative binding of Ca 2+ . This helix-loop-helix structure has been extensively studied [8–10] and is found in more than 250 proteins encoded in the Arabidopsis genome [11]. Not surprisingly, the majority of EF-hand Ca 2+ sensors remain unstudied in plants. The three largest categories of EF-hand proteins in plants are the CaMs (calmodulins) and CMLs (CaM-like proteins), the CDPKs (Ca 2+ -dependent protein kinases), and the CBLs (calcineurin B-like proteins). Of these, only CDPKs represent ‘responders’ capable of directly transducing a signal via catalytic activity, whereas CaMs/CMLs and CBLs are non-catalytic relay sensors that regulate dowstream targets (Figure 2). Although CaM is found in all eukaryotes, CMLs, CDPKs and CBLs are restricted to plants and some protists. Abbreviations used: ABA, abscisic acid; ABF, ABRE (ABA-responsive element) binding factor; ACA, autoinhibited Ca 2+ -ATPase; ACS, ACC (1-amino- cyclopropane-1-carboxylate) synthase; AM, arbuscular mycorrhizae; CaM, calmodulin; CaMBD, CaM-binding domain; CaML, CaM-like; CaMK/CBK, CaM- binding protein kinase; CAMTA, CaM-binding transcriptional activator; CBL, calcineurin B-like protein; CBP, Ca 2+ -binding protein; CBRLK, CaM-binding receptor-like kinase; CCaMK, Ca 2+ and Ca 2+ /CaM-dependent protein kinase; CDPK, Ca 2+ -dependent protein kinase; CIPK, CBL-interacting protein kinase; CLD, CaM-like domain; CML, CaM-like protein; DWF1, DWARF1; ER, endoplasmic reticulum; GA, gibberellin; HCPro, TEV helper component protease; HR, hypersensitive response; KIC, kinesin-interacting protein; MAPK/MPK, mitogen-activated protein kinase; MKP, MAPK phosphatase; NO, nitric oxide; RBOH, respiratory burst oxidase homologue; rgsCaM, regulator of gene silencing CaM; RNAi, RNA interference; RSG, REPRESSION OF SHOOT GROWTH; SA, salicylic acid; SAUR, small auxin up-regulated RNA; SOS, salt overly sensitive; SuSy, sucrose synthase; TEV, tobacco etch virus; VIGS, virus-induced gene silencing; Y2H, yeast two-hybrid. 1 These authors contributed equally to this work 2 To whom correspondence should be addressed (email wayne.snedden@queensu.ca). c  The Authors Journal compilation c  2010 Biochemical Society www.biochemj.org Biochemical Journal