Calcium Signaling Network in Plants

Abstract

Calcium ion (Ca²⁺) is one of the very important ubiquitous intracellular second messenger molecules involved in many signal transduction pathways in plants. The cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) have been found to increased in response to many physiological stimuli such as light, touch, pathogenic elicitor, plant hormones and abiotic stresses including high salinity, cold and drought. This Ca²⁺ spikes normally result from two opposing reactions, Ca²⁺ influx through channels or Ca²⁺ efflux through pumps. The removal of Ca²⁺ from the cytosol against its electrochemical gradient to either the apoplast or to intracellular organelles requires energized ‘active’ transport. Ca²⁺-ATPases and H⁺/Ca²⁺ antiporters are the key proteins catalyzing this movement. The increased level of Ca²⁺ is recognised by some Ca²⁺-sensors or calcium-binding proteins, which can activate many calcium dependent protein kinases. These kinases regulate the function of many genes including stress responsive genes, resulted in the phenotypic response of stress tolerance. Calcium signaling is also involved in the regulation of cell cycle progression in response to abiotic stress. The regulation of gene expression by cellular calcium is also crucial for plant defense against various stresses. However, the number of genes known to respond to specific transient calcium signals is limited. This review article describes several aspects of calcium signaling such as Ca²⁺ requiremant and its role in plants, Ca²⁺ transporters, Ca²⁺-ATPases, H⁺/ Ca²⁺-antiporter, Ca²⁺-signature, Ca²⁺-memory and various Ca²⁺-binding proteins (with and without EF hand).

Introduction

Calcium plays a fundamental role in plant's growth and development. Many extracellular signals and environmental cues including light, abiotic and biotic stress factors, elicit change in the cellular calcium levels, termed as calcium signatures.¹ Ca²⁺ has been well established as a second messenger and the concentration of Ca²⁺ is delicately balanced by the presence of ‘Ca²⁺ stores’ like vacuoles, endoplasmic reticulum, mitochondria and cell wall. Ca²⁺ is present in milimolar concentrations in the cell wall and vacuoles and is released whenever required by the cell.^{1, 2} Recently, Xiong et al. (2006)³ have demonstrated that the organelles surrounded by a double membrane (e.g. mitochondria, chloroplasts and nuclei) are also equipped to generate calcium signal on their own, which is delimited by a double membrane.

Ca²⁺ ion represents an important signaling molecule and a convergence point of many disparate signaling pathways. Facing the environmental challenge, plant cells reprogram their cellular set up by triggering a network of signaling events that start with stress perception at the membrane level of the cell and ends with a cellular response (Fig. 1). A generic signal transduction pathway has following steps:

Figure 1.

Generic pathway for plant response to stress. The extracellular stress signal is first perceived by the membrane receptors and then activate large and complex signaling cascade intracellularly including the generation of secondary signal molecules. The signal cascade results into the expression of multiple stress responsive genes, the products of which can provide the stress tolerance directly or indirectly. ABA, abscisic acid; LEA, late embryogenesis abundant; InsP, inositol phosphate; ROS, reactive oxygen species.

(a) Perception of the signal by the membrane receptors; (b) Generation of second messengers; (c) A cascade of protein phosphorylation/dephosphorylation events that may target transcription factors controlling specific set of stress regulated genes; (d) Stress tolerance, plant adaptation and other phenotypic responses.

The extracellular stress signal transduces inside the nucleus to induce multiple stress responsive genes, the products of which ultimately lead to plant adaptation to stress tolerance directly or indirectly (Fig. 1).¹ Overall, the stress response could be a coordinted action of many genes, which may cross-talk with each others. Signal transduction requires the proper spatial and temporal coordination of all signaling molecules. In plant cell, many molecules acting as second messenger in signaling pathways have been reported; these include Ca²⁺, lipids like IP₃ and cyclic GMP (cGMP). However no single messenger except cytosolic Ca²⁺ has been demonstrated to be involved in diverse pathways and respond to numerous stimuli. A generic pathway for calcium regulated expression of stress responsive genes is shown in Figure 2. The elevated level of Ca²⁺ can be recognized by calcium sensors or Ca²⁺-binding proteins, which together can activate protein kinases.^{1, 2} These activated protein kinases can phosphorylate many regulatory proteins including transcription factors, which regulate the gene expression level of regulatory proteins resulting in the altered metabolism followed by phenotypic response of stress tolerance (Fig. 2). The response could also be growth inhibition or cell death, which will depend upon how many and what kinds of genes are getting upregulated or down-regulated in response to the elevated calcium.

Figure 2.

A generic pathway for calcium regulated gene expression and the stress response. The calcium level increases in response to the stimuli. The increased level of Ca²⁺ is recognised by some Ca²⁺-sensors or calcium-binding proteins, which can activate many calcium dependent protein kinases. These kinases regulate the function of many genes including stress responsive genes, resulted in the phenotypic response of stress tolerance.

Overall, in response to calcium several genes are reported to be upregulated. An analysis of transcriptome changes revealed 230 calcium-responsive genes, of which 162 were upregulated and 68 were downregulated. These include known early stress-responsive genes as well as genes of unknown function.⁴ Recently, a blue light receptor phototropins, which regulate growth and development of plants, has also been shown to involved in calcium signaling in higher plants.⁵ Ca²⁺ signaling pathway can also regulates a K⁺ channel for low-K response in Arabidopsis. Calcium is also reported to be an essential component of the sucrose signaling pathway that leads to the induction of fructan synthesis.⁶ Calcium signaling is also involved in the regulation of cell cycle progression in response to abiotic stress. Understanding the immense significance of Ca²⁺ ions, this review has been solely dedicated to the salient features associated with calcium signaling. Various aspects regarding the Ca²⁺ requirements of plant, Ca²⁺ deficiency, Ca²⁺ transporters, efflux pumps, Ca²⁺/H⁺ antiporters, Ca²⁺ signatures, Ca²⁺ memory, Ca²⁺ sensor and transducer proteins have been briefly covered in this review.

Role of calcium in plants

1. Calcium is an essential plant nutrient required for growth and development of plant, especially the root and shoot tip. The tips are meristmatic and cell division occurs by mitosis. Ca²⁺ helps in the formation of microtubules and microtubules in turn are essential for the anaphasic movement of chromosomes.

2. Ca²⁺ is an important divalent cation and is required for structural roles in the cell wall and membranes where it exists as Ca²⁺ pectate. Ca²⁺ accumulates as calcium pectate in the cell wall and binds the cells together.

3. It is also required as a counter-cation for inorganic and organic anions in the vacuole and as an intracellular messenger in the cytosol.¹ Externally supplied Ca²⁺ reduces the toxic effects of NaCl and ameliorates stress.

4. Ca²⁺ is required for pollen tube growth and elongation.⁸

The calcium requirement of plants and its uptake from soil

Calcium was first described as an essential macro nutrient element in plants more than a century ago.⁹ Ca²⁺ is taken up by roots from the soil solution and delivered to the shoot via the xylem. Ca²⁺ may traverse the root either through the cytoplasm of the cells, linked together by plasmodesmata (the symplast) or through the spaces between the cells (the apoplast). The cytosolic free Ca²⁺ concentration is maintained typically at 200 nM.¹⁰ However, the Ca²⁺ content of the cytosol is far higher than this because of the high affinity of Ca²⁺ to a range of Ca²⁺ binding proteins. Ca²⁺ mobility within the cell is very low as a consequence of its low free concentration and rapid chelation. It moves predominantly apoplastically rather than symplastically.

Plants vary markedly in their Ca²⁺ content and requirements. Ca²⁺ content can very between 0.1 and >5.0% dry wt.¹¹ for different plants and organs. Soil Ca²⁺ also varies widely from <0.01% calcium in acid laterites to very high abundance in chalky soils.⁹ Ecologists have classified plant species into calcifuges and calcicoles. Calcifuges are plants occurring on acidic soils with low Ca²⁺. The term calciole is used to describe plants, which can grow on calcareous/Ca²⁺ rich soils. These soils are generally Ca²⁺ and base-rich soils. However, plants vary in their Ca²⁺ requirements and the ability to extract Ca²⁺ from complex soil environments. In particular, monocots require less Ca²⁺ for optimal growth than do the dicots.¹² Ca²⁺ enters plant cells through Ca²⁺-permeable ion channels in their plasma membranes. Ca²⁺ competes with other cations both for these sites and for the uptake from the soil. The presence of high levels of Ca²⁺ is known to ameliorate the effects of the uptake of toxic cations (Al³⁺ and Na⁺) from the soil while the presence of high levels of other cations (K⁺, Mg²⁺) may reduce Ca²⁺ uptake. Calcium uptake and requirements for optimal growth are thus strongly dependent on the presence of other cations.⁹

Calcium deficiency is rare in nature, but may occur on soils with low base saturation and/or high levels of acidic deposition. Calcium deficiency may occur because of competition by other cations, or because of low transpiration, where the xylem flow is inadequate to supply the calcium requirements of rapidly growing tissue. Calcium deficiency results in stunted root growth and altered leaf appearances.¹¹ More severe symptoms generally result from the failure of cell membrane integrity and include bitter-pit in apple fruit, blossom end rot in tomato and tip burn in lettuce. Deficiency symptoms develop first in the regions of plant most distal to the region of Ca²⁺ uptake.

Ca²⁺ Transporters/ Efflux Pumps in the Cellular Membranes

Plant cells, like animal cells, maintain a low regulated free Ca²⁺concentration ranging from 30 nM to 400 nM in higher plants. Maintaining the low cytosolic Ca²⁺ concentration observed in plant cells requires active transport of Ca²⁺ from the cytosol. Active efflux pumping is a prerequisite for the restoration of low levels cytosolic calcium after the signaling event. The removal of Ca²⁺ from the cytosol against its electrochemical gradient to either the apoplast or to intracellular organelles requires energized ‘active’ transport. Ca²⁺-ATPases and H⁺/Ca²⁺ antiporters are the key proteins catalyzing this movement.

By removing Ca²⁺ from the cytosol several important functions are performed by these enzymes.¹³

1. They maintain a low [Ca²⁺]_cyt in the resting or the unstimulated cell which is appropriate for the cytoplasmic metabolism.

2. They restore [Ca²⁺]_cyt levels to the resting normal levels following a [Ca²⁺]_cyt perturbation, thereby influencing the magnitude kinetic and subcellular location of [Ca²⁺]_cyt signals.

3. They replenish intracellular and extracellular Ca²⁺ stores for subsequent [Ca²⁺]_cyt signals and permit the generation of localized [Ca²⁺]_cyt oscillations through their interplay with Ca²⁺ channels.¹⁴

4. They provide Ca²⁺ in the ER for the secretory system to function.

5. They remove some divalent cations, such as Ni²⁺, Zn²⁺, Mg²⁺ and Mn²⁺ from the cytosol to prevent mineral toxicity.¹³

Hirschi (2001)¹³ suggested that the Ca²⁺-ATPases, which have high affinity (Km = 1–10 µM) but low capacity for Ca²⁺ transport, are responsible for maintaining [Ca²⁺]_cyt homeostasis in the resting cells. Whereas the H⁺/Ca²⁺-antiporters, which have lower affinities (Km = 10–15 µM) but high capacities for Ca²⁺ transport, are likely to remove Ca²⁺ from the cytosol during [Ca²⁺]_cyt signals and thereby modulate [Ca²⁺]_cyt perturbations. This hypothesis is supported by the fact that H⁺/Ca²⁺ antiporter, but not the vacuolar Ca²⁺-ATPase, resets [Ca²⁺]_cyt in yeast following hypertonic shock.¹⁵

Plant Ca²⁺-ATPases belongs to two major families (a) The P-type ATPase II A family and (b) The P-type ATPase II B family.¹⁶

(a) The P-type ATPase II A family.

Nucleotide specificity of these pumps is broad (30–60% activity achieved with GTP and ITP). The pumps are inhibited by erythrosine B (IC₅₀ ≤ 1 µM) and estimate Ca²⁺affinity is in the range of 0.4–12 µM. The first family (The P-type ATPase II A family) lacks N-terminal auto regulatory domain. Four members of this family have been identified in the Arabidopsis genome (termed AtECAs 1 to 4 by Axelsen and Palmgren, (2001).¹⁶ They are likely to be present in the plasma membrane, tonoplast ER and the Golgi apparatus.

(b) The P-type ATPase II B family.

The second family of plant Ca²⁺-ATPases (the P-type ATPase IIB family) is characterized by an auto inhibitory N-terminal domain that contains a binding site for Ca-CaM and in addition a serine-residue phosphorylation site. Their catalytic activity can be modulated by [Ca²⁺]_cyt either through activation upon binding CaM or by inhibition following phosphorylation by Ca²⁺-dependent protein kinases (CDPK).¹⁷ Since CaM binding sites are generally quite diverse, each type-II B Ca²⁺-ATPase may have different affinity for CaM or may bind a different CaM isoform. Ten members of the type-II B, Ca²⁺-ATPase family have been identified in the Arabidopsis genome (termed at ACAS1, 2 and 4 and ALACAs 7 to 13 by Axelsen and Palmgren, (2001).¹⁶ These Ca²⁺-ATPases reside on various cellular membranes including the plasma membrane (AtACA8), the tonoplast (AtACA4), and the plastid inner membrane (AtACA1). The relative molecular mass of type II B Ca²⁺-ATPase pumps has been estimated to be between 115,000 Da and 135,000 Da.¹⁸

The abundance of Ca²⁺-ATPase isoforms suggest that individual isoforms are functionally distinct and may respond differentially to distinct cellular processes involving specific Ca²⁺ signals. They also imply a requirement for CaM-independent and CaM-dependent regulation of Ca²⁺-ATPase activities in the modulation of [Ca^2t]_cyt perturbations during cell signaling. The expression of many Ca²⁺-ATPases is increased upon exposure to high salinity or high [Ca²⁺]_cyt and some Ca²⁺-ATPase genes are expressed only under stress conditions.¹⁹ This may reflect a role in maintaining [Ca²⁺]_cyt homeostasis or in reducing Na⁺ influx to the cytosol in saline environments.

Ca²⁺/H⁺ Antiporters

In addition to a P-type pump, the presence of a low affinity Ca²⁺/H⁺ antiport mechanism at the plasma membrane level had been suggested. The first plant H⁺/Ca²⁺ antiporter to be cloned and functionally expressed was CAX1 (Calcium exchanger 1).²⁰ The gene was identified by its ability to restore growth on a high Ca²⁺ medium to a yeast mutant defective in vacuolar Ca²⁺ transport.

The H⁺/Ca²⁺-antiporters present in the plasma membrane and tonoplast have been characterized biochemically.⁸ These have a lower affinity for Ca²⁺ than Ca²⁺-ATPases and may also transport Mg²⁺. The stoichiometry of the dominant H⁺/Ca²⁺-antiporter in the tonoplast is apparently 3H⁺/1Ca²⁺. Eleven genes encoding putative H⁺/Ca²⁺ antiporters (AtCAX) have been identified in the genome of Arabidopsis thaliana.¹³ The transporters AtCAX1, AtCAX2 and AtCAX4 are located at the tonoplast.¹³ The AtCAX1 antiporter exhibits both a high affinity and high specificity for Ca²⁺. By contrast, the AtCAX2 transporter is a high-affinity, high capacity H⁺/heavy metal cation antiporter. The AtCAXs have homologues in other plant species and their physiological roles have been investigated using transgenic plants.¹³ Transgenic tobacco overexpressing AtCAX1 exhibits Ca²⁺-deficiency disorders, which includes tip burn, metal-hypersensitivity and susceptibility to chilling that can be reversed by increasing Ca²⁺supply. By raising Ca²⁺ supply the expression of AtCAX1 and AtCAX3 (but not AtCAX2 or AtCAX4) genes was increased.¹³

Ca²⁺/H⁺ antiporters, utilize the H⁺ gradient generated by the tonoplast V-type H⁺-pump and by a proton-pumping pyrophosphatase to sequester Ca²⁺ in the Vacuole.²¹ In many plant cells the vacuole occupy more than 50% of the cell volume, and it is evident that trans tonoplast Ca²⁺ transport makes a very significant contribution to the regulation of cytosolic Ca²⁺ concentrations. Ca²⁺ concentrations within the vacuole range from 0.1 to 10 mM.²²

Ca²⁺-ATPases are estimated to represent only <0.1% of the membrane protein and are thus 30–100 fold less abundant than H⁺-ATPases in the PM (3%) and the endomembranes (5–10%). All Ca²⁺ pumps are inhibited by orthovanadate. H⁺/Ca²⁺ antiporters are efflux transporter and are different from Ca²⁺-ATPases in that they do not require ATP and they are not sensitive to vanadate.

In contrast Ca²⁺ influx to the cytosol is mediated via Ca²⁺ channels. The principal roles of Ca²⁺ permeable channels in the plasma membrane appear to be in all signaling. Ca²⁺ permeable channels have been found in all plant membranes. They have been classified on the basis of their voltage-dependence into depolarization activated (DACC), hyperpolarization-activated (HACC) and voltage independent cation channels (VICC).⁸

The Ca²⁺ Signatures

The cytosolic Ca²⁺ in plant cells increases in response to various environmental challenges like abiotic and biotic stresses and developmental cues. This transient increase in [Ca²⁺]_cyt is considered critical for the production of a physiological response. Elevation of [Ca²⁺]_cyt is considered a universal response to stress. The perturbations in cytosolic calcium levels (termed as [Ca²⁺]_cyt “signature”) elicited by each environmental challenge and developmental cue is unique and results in an appropriate physiological response to a particular stimulus. The uniqueness is manifested in the sub-cellular location and/or the kinetics of magnitude of the [Ca²⁺]_cyt perturbation.²³ An increase in [Ca²⁺]_cyt is effected by Ca²⁺ influx to the cytosol either from the apoplast, across the plasma membrane, or from the intracellular organelles. The Ca²⁺ influx is mediated by Ca²⁺ permeable ion channels, and their type, cellular localization and the abundance influences the spatial characteristics of [Ca²⁺]_cyt perturbations. Since the diffusion of Ca²⁺ within the cytoplasm is low, and the buffering of Ca²⁺ in the cytoplasm is high (0.1 to 1 mM)²⁴ the opening of Ca²⁺ channel produces a local increase in [Ca²⁺]_cyt that dissipates rapidly after the channel has been closed. The subcellular localization of Ca²⁺ channel is therefore critical for the targeting of different cellular processes. Cytosolic calcium “waves” are produced within the cytoplasm by the successive recruitment of particular Ca²⁺ channels to coordinate cellular responses. It was suggested that a local elevation of [Ca²⁺]_cyt might generate soluble second messengers, such as IP₃ or cADPR, that diffuse through the cytoplasm to activate a relay of spatially separated Ca²⁺ channels.²⁴ This theory was supported in the plant cells responding to salt stress.²⁵

Franklin-Tong et al. (2002)²⁶ suggested that repetitive Ca²⁺ influx across the plasma membrane contributed in the [Ca²⁺]_cyt that occur following the application of ABA to guard cells. This transient increase was first close to the plasma membrane and subsequently adjacent to the vacuole.²⁷ These waves are thought to reflect the sequential opening of hyperpolarization-activated Ca²⁺ channels at the plasma membrane and then second-messenger activated Ca²⁺ channels in the tonoplast.²⁸

In addition to these sub cellular waves, “waves” of cells with high [Ca²⁺]_cyt may also propagate through the plant tissue. This can be induced in root tissues by mechanical stimulation²⁹ or saline shock³⁰, in cotyledons by cold shock³¹ and in leaves by chilling plant roots briefly.³² Electrical action potentials, osmotic perturbations or chemical signals may trigger these waves. Although an elevated [Ca²⁺]_cyt is necessary for signal transduction, a prolonged increase in [Ca²⁺]_cyt is lethal. Sustained high [Ca²⁺]_cyt is implicated in apoptosis, both during the normal development and in hypersensitive responses to pathogens.³³

To effect adaptive responses, the [Ca²⁺]_cyt perturbations must be either of low amplitude or transient. Transient increases in [Ca²⁺]_cyt can be single (spike), double (biphasic) or multiple (oscillations). The perturbations generated may differ in their cellular location, role and extent of propagation and on their amplitude during propagation. Calcium signatures may also be tissue specific. For example, within the root, the [Ca²⁺ ]_cyt perturbations induced by mechanical perturbation, salinity, osmotic stress, cold shock or slow cooling differ markedly between cell types.³⁰

Several abiotic challenges result in an immediate, transient increase in [Ca²⁺]_cyt that is restored to basal levels within minutes. Such changes include mechanical perturbations and rapid cooling for brief periods, termed as ‘cold shock’. The duration, periodicity and amplitude of oscillations vary considerably, and their form is often dependent on the strength and combination of specific stimuli.²⁷

Calcium “Memory”

The term “memory” was put forward first by Knight et al. (1996).³⁴ There is considerable evidence that [Ca²⁺]_cyt signatures are modified by previous experience. A diminished [Ca²⁺]_cyt elevation upon repetitive stimulation by the same environmental challenge or a developmental cue is a common observation. Many examples support this. The magnitude of the [Ca²⁺]_cyt perturbation elicited by the wind-induced motion becomes progressively smaller upon repeated stimulation and a refractory period of several minutes is required before a full response is observed again. A second exposure to an elicitor does not influence [Ca²⁺]_cyt for several hours after its initial application.³⁵ For example, plant cells challenged with H₂O₂ fail to respond to H₂O₂ for several hours.³⁶ There is also evidence that the [Ca²⁺]_cyt signatures elicited by one environmental challenge can be modified by prior exposure to a contrasting one. For example, the magnitude of the [Ca²⁺]_cyt perturbations in response to oxidative stress was reduced by prior exposure to hyperosmotic stress and vice-versa was also found to be true. These observations also imply a cross talk between the signaling cascades. The attenuated response of [Ca²⁺]_cyt after repeated stimulation by various elicitors forms a part of cellular memory and the cells are able to retain the previous information. This ’memory’ is significant and helps the cells to respond better to a particular stress without disturbing the delicate balance of Ca²⁺ levels and maintaining cellular [Ca²⁺] homeostasis. It is noteworthy that Nicotiana plumbaginifolia, which could not retain ‘cold memory’ during acclimation of the plant by pretreatment with nonfreezing cold temperature was cold-sensitive whereas Arabidopsis which could retain ‘cold memory’ was more resistant to cold stress.³⁴

Calcium Binding Proteins

Transient increase in the cytoplasmic Ca²⁺ in response to signals is sensed by an array of Ca²⁺ sensors. Ca²⁺ sensors are small proteins that bind Ca²⁺ and change their conformation in a Ca²⁺ dependent manner. Specificity in the signaling pathway is provided by the uniqueness in calcium signatures and also by plethora of Ca²⁺ sensors, which can decode the Ca²⁺ perturbations quite precisely. Once Ca²⁺ sensors decode the elevated [Ca²⁺]_cyt, Ca²⁺efflux into the cell exterior and/or the sequestration into cellular organelles such as vacuoles, ER and mitochondria restores its levels to resting state. The properties of binding proteins are described below:

Properties essential for Ca²⁺-binding proteins (CaBPs) to function as intracellular-Ca²⁺receptor.

1. The Ca²⁺ receptor/sensor must have Ca²⁺binding sites essentially unoccupied in the resting cell (free Ca²⁺ is 10⁻⁷M) and occupied at levels, which are reached upon stimulation (∼10⁻⁷M − 10⁻⁶M). This means that the affinity constants (Ka) for Ca²⁺ should be ∼10⁻⁶M⁻¹.

2. The protein must show selectivity and preference for Ca²⁺ in the presence of other cations like Mg²⁺ and K⁺.

3. After binding to Ca²⁺, a Ca²⁺ sensor must undergo a conformational change that either alters its interaction with other molecules or changes its activity if it is an enzyme. Formation of Ca²⁺ protein complex is reversible.

4. The kinetics of interaction should be very fast so as to correspond with the short lived Ca²⁺ signature.

(b) EF Hand, Structure and Function.

Most of the Ca²⁺ sensors bind Ca²⁺ using a helix-loop-helix motif termed as the ‘EF hand’ or the elongation factor, which binds a single Ca²⁺ molecule with high affinity. The Ca²⁺ sensors utilize the side chain oxygen atoms of the EF hand motif for Ca²⁺ coordination. In 1973 Kretsinger and Nockolds³⁷ first discovered the EF hand structural motif in the crystal structure of parvalbumin. The properties, structure and function of EF hands are described below.

1. The EF hand motifs mostly exist in pairs, which helps in the stabilization of the protein structure. Exceptions to this norm include proteins such as, parvalbumin and Plasmodium falciparum membrane surface protein. Frequently, EF hand pairs interact through antiparallel β-sheets, which allow cooperation in Ca²⁺ binding.

2. The EF hand is a highly conserved 29 amino-acid motif consisting of a α helix E (residue 1–10), a loop (residue 10–21), which binds the Ca²⁺ ion and a second α-helix F (residue 19–29) (Moews and Kretsinger, 1975). The loop consists of 12 residues with the pattern X (D), Y(D), Z (D), −Y (K), −X (D), −Z (E) which participate in binding Ca²⁺ and the intervening residues are represented by an asterisks (*). A representation of the EF hand and loop is shown in (Fig. 3A and B), respectivel.

3. The Ca²⁺ ion is coordinated by an oxygen atom or by a bridging water molecule (−x) of the side chains of residues 10 (X), 12 (Y), 14 (Z) and 18 (−X). The ligand at vertex (−Y) is the carbonyl oxygen at residue 16.³⁸ Position 21 (−Z) is usually Glutamate and is the sixth residue to coordinate Ca²⁺.

4. In most of the functional EF hand motifs, the first amino acid is Aspartate (Asp) and the twelfth is Glutamate (Glu), Glu contributes both its side chain oxygen atoms to the metal ion coordination. Since there are 7 oxygen ligands, the Ca²⁺ coordination exhibits pentagonal bipyramidal symmetry, whose axis is 10(X)–18(−X). Glycine (Gly), present at the apex of the loop is an invariant residue and allows the loop to take a sharp bend. Each loop is flanked on either side by 8–10 residues of alternating hydrophilic and hydrophobic character. This arrangement favors the formation of the outer and inner surface of an alpha helical cylinder respectively.

5. Most of the EF hand proteins are characterized by the relatively high percentage of acidic residues (Troponin C, 29%, CaM, 25%, intestinal Ca²⁺ binding protein 23%, and parvalbumin, 18%). Several isoforms of an EF hand protein may exist in a single organism. The Ca²⁺ binding affinities of the EF hand protein vary substantially (Kd = 10⁻⁴−10⁻⁹M) and depend on the amino acid sequence of the protein, especially with regards to the 12 residue consensus loop that provide all the acids that directly ligate to Ca²⁺ ions. When Ca²⁺ binds to [Ca²⁺]_cyt sensors their structural and/or enzymatic properties change and their subsequent interactions with target proteins can alter enzymatic activities, cytoskeletal orientation, protein phosphorylation cascades and gene expression. It is believed that these changes result in stress tolerance or a developmental switch.

Figure 3.

(A) Helix-loop-helix pattern of an EF hand. The EF hand is a highly conserved 29 amino-acid motif consisting of a α helix E (residue 1–10), a loop (residue 10–21), which binds the Ca²⁺ ion and a second α-helix F (residue 19–29). (B) The canonical EF hand consensus sequence of the 12 amino acid Ca²⁺ binding loop. The loop consists of 12 residues with the pattern X (D), Y(D), Z (D), -Y (K), -X (D), -Z (E) which participate in binding Ca²⁺ and the intervening residues are represented by an asterisks (*).

(c) Important calcium sensors in plants.

The major family of Ca²⁺ sensors includes; (1) Calmodulin (CaMs); (2) Calmodulin like proteins; (3) Calcium dependent protein kinases (CDPKs); (4) Calcineurin B-like proteins.

(1) Calmodulin: CaM.

Calmodulin (CaM) is a small (17 KDa), highly conserved, acidic protein with two globular domains each containing two EF hands connected by a flexible α-helical linker.³⁹ CaM is found in the apoplast and in the cytosol, ER and the nucleus of plant cells. Within the cytosol, the estimated CaM concentration is 5 to 40 µM.⁴⁰ Role of CaM has been implicated in many physiological processes like light, gravity, mechanical stress, phytohormones, pathogens, osmotic stress, heat shock and chilling.^23,29,40

The structure of CaM was first solved by Babu et al., 1985, and revealed that all 4 EF hands are saturated by Ca²⁺ ions (4 Ca²⁺). CaM appears to be regulatory protein and induces large changes in inter-helical angles as Ca²⁺ is bound. The affinity of CaM for Ca²⁺ is influenced by the presence of particular target proteins.⁴⁰ CaM can also regulate gene expression by binding to specific transcription factors.⁴¹

(2) CaM-like proteins.

Plants also possess CaM-like proteins, which differ from the CaM in containing more than 148 amino-acid residues and have between one to six EF hand motifs. They possess limited homology to CaM (75% identity with canonical CaM isoforms.³⁹ In Arabidopsis, they include: CaBP-22, TCH2 and TCH3, AtCP1, NADPH oxidases, and Ca²⁺ binding protein phosphatases such as ABI 1 and ABI 2. These proteins have been implicated in cellular responses to diverse environmental, developmental and pathological challenges.

(3) Ca²⁺-dependent protein kinases.

5 types of Ca²⁺ regulated protein kinases have been reported in plants: (i) Ca²⁺ dependent and CaM independent protein kinases (CDPKs); (ii) CDPK-related protein kinases (CRKs); (iii) CaM dependent protein kinases (CaMKs); (iv) Ca²⁺/CaM-dependent protein kinases (CCaMK); (v) SOS3/CBL interacting protein kinases (SIPKs/CIPKs).

(i) Ca²⁺-dependent and CaM-independent protein kinases CDPKs.

The CDPKs are ubiquitous in plants. There are at least 34 genes encoding CDPKs in the Arabidopsis genome⁴² and similar numbers in other plant species. Apart from plants, CDPKs are also found in protozoans and algae. They generally have four EF hands at their C-terminus that bind Ca²⁺ and activate the serine/threonine kinase activity of the enzyme. These kinases require micromolar concentrations of Ca²⁺ for their activity and have no requirement of CaM or lipids. They have a unique structure as N-terminal protein kinase domain is fused with C-terminal auto-regulatory domain and a CaM like domain, which has Ca²⁺ binding EF hand or helix-loop-helix motif. The autoinhibitory domain of CDPKs is a 30 amino acid sequence, which acts as a pseudo-substrate.⁴³ The N-terminal domain of CDPKs is variable and provides specificity to different CDPK isoforms. These enzymes show several fold stimulation with Ca²⁺ and show autophosphorylation. The binding of Ca²⁺ to some of CDPKs, is modulated by lipids or phosphorylation.⁴² Ca²⁺ binding to CDPK effects conformation of the kinase and relieve the inhibition caused by the autoinhibitory region.

CDPKs are implicated in pollen development, control of cell cycle, phytohormone signaling, light-regulated gene expression, gravitropism, thigmotropism, cold acclimation, salinity tolerance, drought tolerance and responses to pathogens.⁴⁴

CDPK-related protein kinases (CRKs).

CRKs are similar to CDPKs expect that the CaM-like region is poorly conserved with degenerate or truncated EF hands that may not be able to bind Ca²⁺. There are at least seven CRKs in Arabidopsis genome, and orthologues of these are present in many plant species. However, the regulation and function of these kinases are not known.⁴⁵

(iii) CaM kinases (CaMKs).

Several CaMKs have been cloned from Arabidopsis and other plants.⁴⁶ Their kinase activity is stimulated by CaM dependent autophosphorylation and their catalytic activity is also modulated by CaM. They are highly expressed in rapidly growing cells and tissues of the root and flower.⁴⁶

(iv) Ca²⁺/CaM-dependent protein kinases (CCaMKs).

These are a group of Ca²⁺-dependent kinases, which in addition to Ca²⁺ also requires CaM for their activity. Thus CaM besides acting directly could also exert its effect by binding to protein kinases and modulating their activity. A Ca²⁺/CaM-dependent protein kinase (CCaMK) was characterized from lily and other plant species.⁴⁷ Sequence analysis revealed the presence of an N-terminal catalytic domain, a centrally located CaM-binding domain and a C-terminal visinin-like domain containing only three EF hands. Biochemical studies of CCaMK established that Ca²⁺ and CaM stimulates CCaMK activity. In the absence of CaM, Ca²⁺ promotes autophosphorylation of CCaMK. The phosphorylated form of CCaMK possesses more kinase activity than the non-phosphorylated form.

(v) SOS3/CBL interacting protein kinases (SIPKs/CIPKs).

Calcineurin B-like proteins were found to interact specifically with a class of serine-threonine protein kinases known as CBL interacting protein kinases (CIPKs).^{1, 2} Recently, a novel CIPK from pea has been reported and found to interact and phosphorylate the pea.⁴⁸

(4) Calcineurin B-like proteins.

Calcineurin B-like proteins (CBLs) are relatively a new class of calcium sensors discovered in Arabidopsis originally, in search for the genes imparting salt tolerance and maintaining cellular ion homeostasis.^{49, 50} Molecular analysis of the sos mutants opened a new chapter in relation to salt stress signalling that led to the discovery of a pathway that transduce a salt stress induced Ca²⁺ signal to reinstate cellular ion homeostasis. Currently 10 CBL and 25 CBL-interacting protein kinases (CIPKs) have been reported from Arabidopsis.² The CBL-CIPK network is also widely distributed among higher plants but except Arabidopsis, the complexity and characterization of this pathway remains largely unrevealed. As different plants vary in their genome complexity, phenotype and physiology therefore, specie specific function or functional diversification can be expected. The essential role imparted by CBL-CIPK genes in stress tolerance, necessitates their detailed characterization from higher plants. In fact, there has been no report on experimental characterization of CBL from any higher plant except Arabidopsis. AtCBL3, in particular has been largely overlooked even in Arabidopsis. Recently, we (Mahajan et al., 2006)⁴⁸ have reported the cloning and characterization of a novel CIPK and its interacting partner CBL from pea. Pea CIPK showed autophosphorylation and could phosphorylate pea CBL. Both pea CBL and CIPK were found to be coordinately upregulated in response to various stresses such as cold, salinity but not to dehydation stress.⁴⁸

(d) Ca²⁺-binding proteins without EF hands.

There are several proteins that bind Ca²⁺ but do not contain EF hand motifs. These include the phospholipase D (PLD), annexins, calreticulin and Pistil-expressed Ca²⁺ binding protein (PCP).

Phospholipase D

The activity of PLD, which cleaves membrane phospholipids into a soluble head group and PA, is regulated by [Ca²⁺]_cyt through a Ca²⁺/phospholipids binding-site termed as the ‘C2 domain’.⁵¹ PLD activity is implicated in cellular responses to ethylene and ABA, α amylase synthesis in aleurone cells, stomatal closure, pathogen responses, leaf senescence and drought tolerance.⁵¹ Plants posses several PLD isoforms that differ in their affinity for Ca²⁺ and their modulation by phosphoinositides, free fatty acids and lysolipids.⁵¹ These biochemical modulators of PLD activity are the substrates or products of PLC, which generates IP₃, DAG, phospholipase A₂ and DAG-kinase, both of which are regulated by CaM. It is suggested that [Ca²⁺]_cyt signaling cascades might coordinate the activities of these diverse enzymes to effect specific responses to the environmental stimuli.⁵²

Annexins

These are a family of proteins in plants and animals that bind phospholipids in a Ca²⁺ dependent manner and contain four to eight repeats of approximately 70 amino acids.⁵³ Although exact function of annexins is not known, plant annexins are implicated in secretory processes and some have ATPase, peroxidase activities.

Calreticulin

It is a Ca²⁺ sequestering protein in the ER and functions as a molecular chaperone.⁵⁴ The function of calreticulin is also implicated in Ca²⁺ homeostasis.⁵⁵

Pistil expressed Ca²⁺-Binding Protein (PCP)

A 19 kDa novel Ca²⁺ binding protein (PCP) expressed in anthers and pistil was isolated.⁵⁶ PCP is high capacity (binds 20 mol of Ca²⁺ per mol of PCP), low affinity Ca²⁺ binding protein. Role of PCP has been implicated in pollen-pistil interactions and/or pollen development.