Comparative Analysis of Plant and Animal Calcium Signal Transduction Element Using Plant Full-Length cDNA Data

http://www.100md.com 分子生物学进展 2004年第10期

     * Department of Molecular Genetics, National Institute of Agrobiological Sciences, 2-1-2 Kannon dai, Tsukuba, Ibaraki, 305-8602 Japan; RIKEN Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045 Japan; and Foundation for Advancement of International Science; 586-9 Akatsuka-Ushigafuchi, Tsukuba, Ibaraki, 305-0062 Japan

    E-mail: skikuchi@nias.affrc.go.jp.

    Abstract

    We obtained 32K full-length cDNA sequence data from the rice full-length cDNA project and performed a homology search against NCBI GenBank data. We have also searched homologs of Arabidopsis and other plants' genes with the databases. Comparative analysis of calcium ion transport proteins revealed that the genes specific for muscle and nerve calcium signal transduction systems (VDCC, IP3 receptor, ryanodine receptor) are very different in animals and plants. In contrast, Ca elements with basic functions in cell responses (CNGC, iGlu receptor, Ca2+ATPase, Ca2+/Na+ -K+ ion exchanger) are basically conserved between plants and animals. We also performed comparative analyses of calcium ion binding and/or controlling signal transduction proteins. Many genes specific for muscle and nerve tissue do not exist in plants. However, calcium ion signal transduction genes of basic functions of cell homeostasis and responses were well conserved; plants have developed a calcium ion interacting system that is more direct than in animals. Many species of plants have specifically modified calcium ion binding proteins (CPK, CRK), Ca2+/phospholipid-binding domains, and calcium storage proteins.

    Key Words: calcium transport protein ? calcium-binding protein ? full-length cDNA

    Introduction

    Ca2+ is the two-ionic-charge ion that is used most widely in animals and plants; it is used not only to generate membrane voltage but also to control many signal transduction systems once it has entered the cell. Many studies of animal calcium signal transduction have indicated that numerous genes control these systems. Calcium signal transduction systems in plants have also been studied. For examples, touch stimulation response, stress resistance (to cold and dryness), and stomatal opening control are regulated by Ca2+ ions (Braam and Davis 1990; MacRobbie et al. 1998; Kudla et al. 1999; Allen et al. 2001). The results of electrophysiological studies and molecular analyses indicate the existence of many species of Ca2+ ion transport proteins (White 2000; White et al. 2002). The generation of slow action potentials resembling those observed in muscles and nerves of animals and Ca2+ ion-regulated systems for endocytosis and exocytosis have been reported (Miedema et al. 2001; Camacho and Malho 2003). However, despite these analyses, plants do not have the muscles, nerves, or cell immigration systems that are specifically controlled by Ca2+ ions in animals. Therefore, one would expect large differences between plant and animal calcium transduction systems.

    Recent whole-genomic sequence analyses have made it possible to confirm the existence of homologous genes by computer data analysis. In the absence of individual gene analysis, these methods can reveal the overall patterns of gene network systems. In plants, whole-genomic sequence analyses have been completed in Arabidopsis and rice (Arabidopsis Genome Initiative. 2001; Goff et al. 2002). However, although whole-genome information is available, gene annotation programs are not yet sufficiently accurate. We have mapped the full-length cDNA clones to the rice genome sequences and indicate that the genome sequence alone could not correctly identify the gene structure (Kikuchi et al. 2003). Therefore, full-length cDNA sequence data are useful for more precious analysis of genes. We searched for homology with known animal calcium signal genes by using the 32K full-length cDNA data for rice and the Arabidopsis and rice (indica) genomic sequence data (Arabidopsis Genome Initiative 2001; Goff et al. 2002; Yu et al. 2002). We used the BlastX program to search for sequence homologies at the amino acid level. We downloaded sequence data from NCBI's GenBank and checked the alignment pattern of the query sequences at a similarity threshold of E < 10–100 (table 1). Comparative analysis of calcium ion transport proteins revealed that the voltage-dependent calcium channel (VDCC), inosiol-1,4,5-trisphosphate (IP3) receptor, and ryanodine receptor (RYR) are markedly different between animals and plants. In contrast, other transport proteins (the receptor-opened calcium channel [ROCC], calcium pump, and calcium transporter) are conserved between plants and animals. Calcium-binding protein analyses revealed that many of the muscle- and nerve-tissue–specific genes are not present in plants. However, calcium-dependent protein kinase (CDPK), CDPK-related kinase (CRK), and many plant-specific modified genes occurred in plant intracellular signal transduction systems.

    Table 1 Calcium Transport and Calcium-Binding Protein Homologs of Human, Rice and Arabidopsis

    Materials and Methods

    Blast Searches

    Rice: The BlastX program was used to search for homology at the amino-acid level. We downloaded sequence data from NCBI's GenBank and used 32K full-length rice (japonica) cDNA clones as query sequences. For the genes that were negative in homology searches, we also searched the indica rice genome data (92.5% of the whole-genomic information) to attempt to confirm the existence of a homolog. To check the alignment pattern of the query and subject sequences, we used a similarity threshold of E < 10–100.

    Arabidopsis: In accordance with the MIPS data service (http://mips.gsf.de/proj/thal/proj/proj_overview.html), the BLASTX program was used to search for homologies at the amino acid level under the same search conditions as used for rice.

    Human: Genew = Human Gene Nomenclature Database Search Engine (www.gene.ucl.ac.uk/cgi-bin/nomenclature/searchgenes.pl) was used to search for homologies at the amino acid level under the same search conditions as used for rice.

    InterPro Search

    The numbers of calcium signal transduction genes in rice were classified by using the InterPro database at standard conditions.

    Results

    Calcium Transport Proteins

    The Ca2+ concentrations on both sides of the cell membrane are precisely regulated by transport proteins (channels, pumps, and transporters exchangers) to make a 10,000-fold concentration gradient at the cell membrane. Therefore, the opening of the calcium channel gate creates a flood of Ca2+ ions that rapidly switches the calcium signals without any energy supplementation. Some of the plant's cells may have more severe conditions to regulate their extracellular calcium concentrations than animals. In plants, it is thought that cytosolic Ca2+ is homeostatically maintained in the face of different Ca2+ concentration gradients across the cell membrane. The Ca2+ concentration outside root cells is subjected to vagaries in the soil Ca2+ concentration, and fluctuations in the xylem Ca2+ concentration affect that of leaf cells. Nevertheless, the maintenance of an inwardly directed Ca2+ electrochemical gradient occurs in plants too (White et al. 2002). In animals, transport of the Ca2+ ion into cells is controlled by three types of channels: VDCCs, ROCCs, and mechanical-stimulation–gated channels. Electrophysiological analyses have revealed the existence of VDCC and ROCC in plants, (Miedema et al. 2001; Sanders et al. 2002), but molecular analyses have found few plant Ca2+ channel genes homologous to those in animals. To judge whether homologous genes do exist or whether the same functional genes have changed dramatically at a sequence level, we searched for calcium transport protein homologs (table 1) and performed comparative analyses at putative functional domains (figs. 1 and 2).

    FIG. 1.— Comparison of channel proteins in plants and animals. (A) Voltage-dependent calcium channel (VDCC). Topology models of putative VDCCs in plants (TPC1) and animals (TPC2 and VDCC, L-type). I: I subunit; II: II subunit; ?: ? subunit; : subunit; : subunit; PKC: protein-kinase-C-binding site; G: G-protein-binding site; +: Voltage sensitive site. (B) Ionotropic glutamate receptor (iGluR). S(GlnH): glutamate-binding domain; M: membrane-localized domain. (C) Cyclic nucleotide-gated calcium channel (CNGC). CaM: calmodulin binding site, CNB: cyclic nucleotide binding site.

    FIG. 2.— Comparison of pump and transporter proteins in plants and animals. (A) Ca2+ ATPase. Topology models of putative Ca2+ ATPases in plants and animals. Ca2+ ATPase IIA and IIB. (B) Ca2+ transporter. Ca2+/H+ antiporter. Na+/Ca2+-K+ exchanger.

    Channels

    In animals, various types of VDCC complex are controlled by the cell membrane voltage. Polarization or depolarization of the membrane voltage opens the gate subunits and permits a flood of calcium ions through the cell membrane. The central structure of the animal VDCC is an -1 subunit protein that has 24 transmembrane domains, which are grouped into four repeating units; there are many tissue-specific types (Catterall 2000; Serysheva et al. 2002; Yamakage and Namiki 2002). No VDCC -1 subunit homolog exists at the whole-structural level in plants, and only one species of a partly homologous protein (two-pore putative calcium channel [TPC1]) has been detected in both Arabidopsis and rice. TPC1 is half the size of the animal VDCC -1 subunit; it has 12 transmembrane domains (six groups of two units), and the S4 domain is the voltage-sensitive domain (Furuichi, Cunningham, and Muto 2001). Molecular analyses have revealed that TPC1 belongs to the L-type of depolarization-activated calcium channels of animals, as does the yeast plasma membrane Ca2+ channel (Fischer et al. 1997; Ishibashi, Suzuki, and Imai 2000). Although plant VDCCs have retained the calcium-calmodulin binding domain (EF-hand), plant VDCCs seem to have lost the domains for the ryanodine receptor domain, VDCC? subunit, VDCC subunit, and kinase binding sites (figs. 1A and 1-A-S), and the amino acid sequences of the pore-forming domain show considerable diversity. These differences suggest the possibility of a difference in calcium flooding velocity between plants and animals. Furthermore, according to physiological analyses, plants actually have hyperpolarization-activated calcium channels (Miedema et al. 2001), but there are no homologs in plants. Therefore, VDCC systems in animals and plants have diverged dramatically.

    The fundamental structures of ROCCs (cyclic nucleotide-gated channels [CNGCs] and iGlu receptors) seem to have been conserved in plants and animals (figs. 1B, 1-B-S, 1C and 1-C-S).

    Ionotropic glutamate receptors (iGluR) form nonselective (Ca2+-permeable) cation channels as ligand-gated ion channels, which bind glutamate that has been released from a companion cell, thereby allowing charged ions (Na+, Ca2+) to pass through the channels. In animals, this flow of ions results in depolarization of the plasma membrane and generation of an electrical current that is propagated down the processes (dendrites and axons) of one neuron to the next.

    iGluR of animals are constructed in multimeric assemblies of four or five subunits and are subdivided into two groups of receptors—AMPA (activated by -amino-3-hydroxy-5-methyl-4-isoxazole propionate or kainite) and NMDA (activated by N-methyl-D-aspartate)—in light of their pharmacological and structural similarities (Chiu et al. 1999). According to structural and sequence similarity, plant iGluRs are similar to the AMPA type in animals, with four predicted transmembrane regions (M1-M4), two potential glutamate-binding domains, and a long N terminus with similarity to both extracellular calcium sensors and glutamate and -aminobutyric acid receptors. AMPA receptor channels are impermeable to calcium, a function controlled by the iGluR2 subunit that results from post-transcriptional editing of the iGluR2 mRNA, which changes a single amino acid in the TMII region from glutamine to arginine.

    Overall, plant iGluRs are only 50% to 60% homologous to animal iGluRs, and this similarity is even lower for the M2 regions. Furthermore, plant iGluRs lack the ionic selectivity of animal iGluRs (the QRN site), which determines Ca2+ permeability and is blocked by Mg2+ (figs. 1B and 1-B-S). Thus, it is difficult to predict the selectivity of plant iGluR simply from the protein structure. The existence of plant iGluRs has been confirmed by physiological analyses (Dennison et al. 2000). Therefore, glutamate receptor regulating systems in animals and plants have diverged dramatically both at the functional level and in their post-transcriptional regulation mechanisms.

    Cyclic nucleotide (3',5'-cyclic AMP [cAMP]) and 3',5'-cyclic GMP [cGMP])-gated channels (CNGCs) are a recently identified family of plant ion channels. They show a high degree of similarity to Shaker-type voltage-gated channels and contain a C-terminal cyclic nucleotide-binding domain with an overlapping calmodulin-binding domain. In animals, CNGCs in photoreceptor and olfactory neurons are well characterized. In plants, CNGCs are involved in gibberellic acid-induced signaling in aleurone development, phytochrome signaling, pollen tube tip growth, root development, and plant cell cycle progression (Bowler et al. 1994; Penson et al. 1996; Durner, Wendehenne, and Klessig 1998; Ehsan et al. 1998; Moutinho et al. 2001; Tsuruhara and Tezuka 2001; White et al. 2002). Plant CNGCs have six transmembrane domains, S1-S6, with a pore domain (P loop) between S5 and S6 and C-terminal Cyclic nucleotide-binding (CNB) and CaM-binding domains. Overall homology between human CNGCs is less than 50% and the S4-S5 domains, which form the Ca2+ ion transport area and calmodulin-binding domain, show low levels of homology (<30%). In animal CNGCs, a calmodulin-binding area exists at the N-terminus, and a cyclic-nucleotide-binding area occurs at the C-terminus. However, both areas are found in the C-termini of plant CNGCs (Hirschi et al. 1996; Kohler and Neuhaus 2000; figs. 1C and 1-C-S). This overlapping of the CaM-binding region with the CNB domain suggests that the binding of CaM to the C-terminus of plant CNGCs might interfere with cyclic nucleotide binding and thus channel activation, thereby implying a different mechanism for the CaM-associated regulation of plant CNGCs than those described for olfactory and rod CNGCs in animals. The P loop is another domain that sets plant CNGCs apart from other known ion channel subunits. The region that is believed to form the selectivity filter differs markedly from the pores of animal CNGCs, which are nonselective regarding cations, as well as those of K+-selective channels (Talke et al. 2003).

    We also investigated whether the other ROCCs (ryanodine and IP3 receptors) exist in plants. In animals, these receptors, whose activity is dependent on flooding of the Ca2+ ion (oscillation system), increase the calcium signal by exposing calcium ions stored in the endoplasmic reticulum (ER) (Berridge 1993; Laurent and Claret 1997). There are no homologous proteins in plants (table 1). Electrophysiological analyses have revealed the existence of calcium oscillation in many tissues and stages of plants (Evans, McAinsh, and Hetherington 2001). Therefore, calcium oscillation systems in animals and plants have diverged dramatically. Because plants, therefore, also alter signal oscillation genes, delaying or accelerating the calcium flood changes the signal transduction velocity. In addition, the changing of signal oscillation precludes alterations in the strength and frequency of the electrical signals for signal transduction to the neighboring cell.

    Pumps and Transporters

    The fundamental structures of Ca2+ ATPase pump proteins and calcium transporters (Ca2+/H+ ion exchanger and Ca2+/Na+ ion exchanger) are well conserved in plants and animals (fig. 2).

    P-type ATP-powered calcium transport proteins, which use ATP hydrolysis to pump Ca2+ ions, transport Ca2+ out of the cell against the electrical gradient. There are two types of Ca2+ ATPases in animals: the plasma-membrane (PM) type (exists in prokaryotes and eukaryotes) and the ER type (exists only in eukaryotes). Plant calcium ATPases also are grouped into two types: type IIA (SERCA; analogous to the ER type in animals) and type IIB (PMCA; analogous to the PM type).

    The transmembrane domains of types IIA and IIB Ca2+ ATPases show high homology (50% to 80%) at the amino acid level. The IIA type is similar between the animal and plant forms at the transmembrane, phosphorylation, and ATP-binding domains. Therefore, Ca2+ ATPases thought to be regulated by the PM-bound protein are well conserved in bacteria, archea, and eukarya. In contrast, the phosphorylation, ATP-binding, and calmodulin-binding regions of type IIB proteins differ between plants and animals (Geisler et al. 2000; figs. 2A and 2-A-S). Therefore, for CaM-stimulated Ca2+ ATPases, the structure of transmembrane domains is conserved well, but the regions that interact with internal factors of the cell have evolved differently in plants and animals. Thus, the calcium interacting and regulatory regions of Ca2+ ATPases that were evolutionally acquired at the eukaryotic level differ in plants and animals.

    Transporters are membrane proteins that convey at least two ions in coupled movement (i.e., symporter, the ions are transported in the same direction; antiporter, the solutes are transported in opposite directions). The Ca2+/H+ ion exchanger is an H+-coupled Ca2+ antiporter that is driven by a proton electrochemical gradient. It is found in yeast, fungi, and bacteria and does not exist in animals. In yeast, the Ca2+/H+ ion exchanger is located at the vacuolar membrane and regulates vacuolar Ca2+/H+ exchange. The plant Ca2+/H+ ion exchanger has total homology with its yeast counterpart, except in the N- and C- terminal regions (Guerini 1998). Therefore, the Ca2+/H+ ion exchange system is well conserved in bacteria, fungi, yeast, and plants.

    The Na+/Ca2+ ion exchanger proteins are electrogenic transporters that can use the Na+ electrochemical gradient to exchange three extracellular Na+ ions for one intracellular Ca2+ ion. Na+/Ca2+ ion exchangers are present in a wide variety of animal tissues and mitochondria (Dunn et al. 2002). Two groups of the exchangers can be distinguished: those that neither require nor transport potassium (e.g., NCX family) and those that require and transport potassium (Na+/Ca2+ -K+ ion exchangers, e.g., NCKX family) (Szerencsei et al. 2000). The structure of the Na+/Ca2+ ion exchangers and Na+/Ca2+ -K+ ion exchangers are similar to each other. Both of the exchangers have a uniform pattern of two large hydrophilic loops and two sets of transmembrane spanning segments. C-terminus transmembrane spanning segments have especially high similarities. All of the plant Na+/Ca2+ ion exchangers are NCKX type Na+/Ca2+ -K+ ion exchangers. There are two homolog types (high and low similarities) in plant Na+/Ca2+ -K+ ion exchangers. We collected homologs of the Na+/Ca2+ -K+ ion exchangers from plants and found that the amino acid sequence of the transmembrane domain and basic structure of the protein are fundamentally conserved (50% to 60% overall). The -1 repeat (which controls the Ca2+ ion exchange) and alternative splice site of the animal Na+/Ca2+ -K+ ion exchanger show low levels of homology (region deleted or homology <50%) with the rice forms (figs. 2B and 2-B-S). There are low levels of homology with the rice and Arabidopsis forms (figs. 2B and 2-B-S). Therefore, only Arabidopsis (dicot) might have exactly homologs of Na+/Ca2+ -K+ ion exchanger in plants. In animals, NCKX type Na+/Ca2+ -K+ ion exchanger specifically exists in photoreceptors, ganglions, and various parts of brains, and it plays a critical role in calcium homeostasis in the special cells. Thus, the NCKX type Na+/Ca2+ -K+ ion exchanger homolog may control plant-specific phenomena. There are no homologs of NCX type Na+/Ca2+ ion exchangers in plants. On the other hand, Ca2+/H+ exchanger has a similarity to NCX type Na+/Ca2+ ion exchangers. Therefore, plants may have modified calcium antiporter systems from Na+/Ca2+ ion exchangers to a Ca2+/H+ exchanger. Nevertheless, we have confirmed the existence of transporter systems of Ca2+/H+ and Ca2+/Na+ in plants.

    Homologs to other calcium transporters (LCT1, etc.) also exist in plants but show low affinity and/or specificity for calcium ion transport. Comparative analyses of plant and animal calcium transport proteins (channels, pumps, and transporters) revealed that individual internal signal transduction systems, such as ROCCs, calcium pumps, and calcium transporters, are basically conserved (fig. 3). However, cell-to-cell signal transduction systems, such as VDCC signal transduction systems and oscillation controlling receptors, are dramatically different between plants and animals. This difference may have evolved in animals because of the need to develop rapid and multiple cell-to-cell signal transduction systems in muscles and nerves.

    FIG. 3.— Schematic representation of homology identified in calcium transport in plant cells. IP3: inositol 1,4,5-trisphosphate receptor; RyR: ryanodine receptor. Channels identified at a molecular level but whose locations are hypothetical are indicated in red. Compared with that of animals, the cell membrane of plants seems to have fewer species of VDCC. At the molecular level, there are no IP3R or RyR homologs in plants.

    Calcium-Binding Proteins

    After flooding into the cell, the Ca2+ ion binds to specific calcium-binding proteins to switch individual signal transduction pathways. We performed comparative analyses of calcium-binding proteins in plants and animals, including Ca2+ ion binding EF-hand proteins (e.g., calmodulin, calcineulin, centrin, fimbrin, calmenin, calpain) (fig. 4 and table 1). Ca2+ion/phospholipid-binding proteins (phospholipase C, annexin) and calcium storage proteins (calreticulin) were common to plants and animals. However, muscle- and nerve-tissue–specific EF-hand proteins (e.g., calbindin, myosin regulatory light chain kinase, parvalbumin, recoverin, troponin C, BM-40, diacylglycerol kinase alpha), -glutamic-acid–containing proteins (e.g., osteocalcin, protein C), and lipid-containing proteins (e.g., casein, phosvitin) were not found in plants (table 1). One of the largest (18 members) subgroup in the human EF-hand Ca2+-binding protein family, S100 homolog, was also not found. S100 proteins are characterized by two distinct EF-hand calcium-binding motifs with different affinities. The proteins regulate intracellular processes such as cell growth and motility, cell cycle regulation, transcription, and differentiation. Thus, these small Ca2+-binding protein signal transduction systems were developed only in animals. On the other hand, the CDPK and CRK signal transduction systems exist only in plants.

    FIG. 4.— Ca2+ and calcium-binding protein signal transduction pathways in the cell. Genes are indicated by three colors according to their level of specificity: red, animal-specific; green, plant-specific; blue, common to animals and plants.

    Most calcium-modulated proteins contain two to eight copies of the EF-hand, or calmodulin fold. The domain consists of 29 amino acids arranged in a helix-loop-helix conformation and has the ability to surround the Ca2+ ion. Computer analysis of genomic data estimated that 250 EF-hand genes exist in Arabidopsis (Day et al. 2002). Using full-length cDNA data and genomic data analyses, we have found about 180 EF-hand genes in rice. From the 70 subfamilies of EF-hand genes, we focused on the most frequent and/or functionally important proteins.

    Calmodulin and Phosphorylases

    Calmodulin- and calcium/calmodulin-dependent phosphorylases (CCaM kinase, CaM kinase, CDPK, CRK) accounted for about 40% of all of the plant EF-hand genes (fig. 4-E-S). CCaM kinase, CaM kinase, CDPK, and CRK belong to the CDPK–SnRK superfamily, which consists of seven types of serine-threonine protein kinases: calcium-dependent protein kinase (CDPKs), CDPK-related kinases (CRKs), phosphoenolpyruvate carboxylase kinases (PPCKs), PEP carboxylase kinase-related kinases (PEPRKs), calmodulin-dependent protein kinases (CaMKs), calcium- and calmodulin-dependent protein kinases (CCaMKs), and SnRKs (Hrabak et al. 2003). CDPK and three related protein kinases (CCaMK, CaMK, and CRK) have a conserved kinase domain in the N-terminal region. In contrast, a distinguishing structure of the plant genes is a C-terminal calcium-binding (regulatory) domain: CDPK has four EF-hands (calmodulin-like domain); CaMK has no EF-hand domain; CCaMK has three EF-hands (visinin-like domain); and CRK has degenerate EF-domains. Calmodulin, CaM kinase, and CDPK may be evolutionally related (Zhang and Choi 2001; fig. 5-A, -A-S).

    FIG. 5.— Comparison of calmodulin and phosphorylases in plants and animals. (A) Schematic diagrams of comparatively analyzed calmodulin and phosphorylases. Domain structures of calmodulin and calcium signal controlling kinases. The putative evolutionary pathway is also indicated. (B) Calmodulin. (C) Calmodulin-binding kinase (CaMK). (D) Calcium-calmodulin–dependent protein kinase (CCaMK). (E) Calcium-dependent protein kinase (CDPK) (= calmodulin-like domain protein kinase [CPK]). (F) CDPK-related protein kinase (CRK).

    Calmodulin is the most common calcium-binding protein in eukaryotic organisms and is constructed in four EF-hand domains. There are many types of EF-hand domains, most of which are highly homologous in animals and plants and some of which are animal- or plant-specifically modified. The plant stimulation response genes (TCH2, 3) and stress response regulation gene (SOS3) are included in the calmodulin gene family (Braam and Davis 1990; Ishitani et al. 2000). Our comparative analysis of calmodulin genes revealed some calmodulin groups unique to rice and Arabidopsis (fig. 5-B-S).

    CCaMK is characterized by a serine-threonine kinase domain, an autoinhibitory domain that overlaps with the calmodulin-binding domain, and a C-terminal visinin-like domain with three calcium-binding sites. Visinin-like proteins are high-affinity Ca2+-binding proteins and function as Ca2+ sensors in neurons. The calmodulin-binding domain of CCaMK is very similar to CaM kinase II. Ca2+ binding to the C-terminal visinin-like domain leads to autophosphorylation of the kinase. Unlike that by CDPKs, substrate phosphorylation by CCaMK requires both Ca2+ and CaM. The interaction between CCaMK and CaM is modulated by the Ca2+-stimulated autophosphorylation. CaM-dependent protein kinases in invertebrates and vertebrates require Ca2+/CaM for autophosphorylation (Sathyanarayanan, Cremo, and Poovaiah 2000). The CCaM homolog of plants conserved whole sequences of animal CCAMKs, and calmodulin-binding domain is very highly conserved (>80%) at the amino acid level (figs. 5C and 5-C-S).

    Calmodulin (CaM)-binding protein kinases (CaMKs) contain an N-terminal domain of variable length and sequence, a protein kinase catalytic domain, a CaM-binding domain, and a C-terminal domain of variable length and sequence. However, unlike CDPKs, CaMKs lack well-defined EF-hands for Ca2+ binding at their C-termini (Zhang and Lu 2003). Compared with the frequency in animals, plants have few CaMK homologs (Arabidopsis, 3; rice, 2). Therefore, CaMK-regulating systems are not typical of plants. The plant homologs were well conserved (60% to 80%) in the kinase and CaM-binding domains, but the C-terminal regions were strange (figs. 5D and 5-D-S).

    CDPK is a plant-specific calcium signal gene that has EF-hand and CaM kinase regions. It is activated by Ca2+ ion binding and phosphorylates target proteins. CDPK is thought to have evolved from CAMK. However, CDPK and CDK are highly conserved in plants, and the homology between CDPK and animal calmodulin EF-hand is low. Therefore, a change in the calcium-binding domain seems necessary for CDPK to react with the Ca2+ ion and become activated. Addition of the CDPK gene to the EF-hand family changes the numbers and proportions of members of the EF-hand gene family group. Therefore, we assessed the types and proportions of EF-hand genes in plants (fig. 5-E-S). CDPKs accounted for 17% to 18% of the EF-hand family in rice and Arabidopsis. Levels of calmodulin genes, the largest single group of EF-hand gene families, were similar in both types of plant. The genes down-regulated by CDPK and CRK include not only plant-specific genes (sucrose synthesis, stress response) but also genes involved in basic physiologic processes (transport regulation, cell skeleton) (Cheng et al. 2002). Therefore, CDPK genes seem to have originated from half of the calmodulin proteins, which control many aspects of signal transduction.

    CDPK-related kinase (CRK) is discriminated from CDPK through its degenerate sequence in the CaM-like domain including the four EF-hands. CRK is plant-specific but calcium-independent (Furumoto et al. 1996). We analyzed the sequence homologies of CDPK and CRK between Arabidopsis and rice (figs. 5E, 5-E-S, 5F and 5-F-S). Both genes were well conserved between monocot and dicot. In addition, our comparative analyses of CaMK, CCaMK, and CRK revealed the high conservation of these genes. Therefore, plants have CCaM, CaMK, and calmodulin systems as do animals, but plants have also developed CDPK and CRK systems.

    Phosphatase and Cytoskeleton Proteins

    Calcineurin is a eukaryotic Ca2+- and calmodulin-dependent serine/threonine protein phosphatase. This heterodimeric protein consists of a catalytic subunit (calcineurin A), which contains an active site with a dinuclear metal center, and calcineurin B, which is the Ca2+-binding subunit (Rusnak and Mertz 2000). Calcineurin A exists in plants at a low level of homology, and the calcineurin B (Kudla et al. 1999) homolog (CBL) contains plant-specific sequences in the N- and C-terminals and CAN reactive regions (figs. 6A and 6-A-S). In Arabidopsis, the calcineurin B homolog regulates abscisic acid and cold signal transduction (J?rg et al. 1999; Halfter, Ishitani, and Zhu 2000; Kim et al. 2003). In animals, calcineurin controls apoptosis, memory, exocytosis, channel (K+) activation, and other processes (Lin et al. 1998). Therefore, changes in the biological roles of calcineurin between animals and plants might have led to differences in the sequences.

    FIG. 6.— Comparisons of dephosphatase and cell skeleton protein. (A) Calcineurin. (B) Centrin (caltractin). (C) Fimbrin.

    Centrin (caltractin) is a member of the calmodulin subfamily of EF-hand proteins that is an essential component of microtubule-organizing centers in yeast, algae, and animals, and homologs in plants exist (Cordeiro et al. 1998). The protein contains two homologous EF-hand Ca2+-binding domains linked by a flexible tether, which is capable of binding two Ca2+ ions. Centrin is an essential component of the centrosome, which mediates chromosome segregation during mitosis and is required for proper cell division. The gene sequences were well conserved (>80%) among eukaryotes (figs. 6B and 6-B-S). Therefore, centrosome components and systems for chromosome segregation during mitosis are well conserved with regard to their calcium-binding proteins.

    Fimbrin, a 67-kDa monomeric actin filament-bundling protein, which has two ABP-120-like actin-binding motifs (i.e., two pairs of calponin-homology [CH] domains) and two EF-hands, exists in both animals and plants (McCurdy and Kim 1998). Therefore, actin-binding and cell structure proteins are conserved between plants and animals. The basic structures (EF-hand, CH domain) were conserved at 60% to 70% homology between the amino acid sequences. The plant centrin and fimbrin homologs both have the characteristic conserved sequences at the calcium-binding domain and flanking sequences. Therefore, some of the calcium-binding proteins that control basic cell function systems are conserved (figs. 6C and 6-C-S). However, we did not find homologs to other cytoskeleton proteins (e.g., actinin, caldesmon, spectorin, synapsin, myosin) in plants (table 1).

    Endocytosis Genes

    Calumenin contains an N-terminal signal sequence and six EF-hand motifs and shows homology with reticulocalbin, Erc-55, and Cab45. (Yabe et al. 1997). It is involved in the ER and Golgi apparatus, in controlling ER retention signal transduction, and in systems for transport through the cell membrane. Rice and Arabidopsis contained calmenin homologs (figs. 7A and 7-A-S), which have not only six EF-hands but also the C-terminal HDEF sequence present in animal forms. The HDEF sequence is responsible for retention of calmenin in the ER. Therefore, rice has specifically improved calmenin for Ca2+-dependent folding and maturation of secretory proteins in the ER lumen.

    FIG. 7.— Comparison of endocytosis proteins. (A) Calumenin. (B) Penta-EF-hand protein (peflin and ALG-2 type). (C) EH-domain-containing protein.

    Penta-EF-hand (PEF) proteins comprise a family of Ca2+-binding proteins that has five repetitive EF-hand motifs. Among the eight alpha-helices (alpha1-alpha8), alpha4 and alpha7 link EF2-EF3 and EF4-EF5, respectively. The PEF protein family members also have hydrophobic Gly/Pro-rich N-terminal domains and are translocated Ca2+-dependently to membranes. Based on comparison of amino acid sequences, mammalian PEF proteins are classified into two groups: group I PEF proteins (ALG-2 and peflin) and group II PEF proteins (Ca2+-dependent protease calpain subfamily members, sorcin and grancalcin). The group I genes have also been found in lower animals, plants, fungi, and protists (Maki et al. 2002). Plant penta-EF-hand Ca2+-binding protein homologs belong to the group I (ALG-2 and peflin) type PEF family. Peflin type protein works as a dimer and has the ability to interact with annexin (VII, XI), another peflin. It has a calcium-dependent regulatory role in cell growth, cell death, and exocytosis (figs. 7B and 7-B-S), and the regulating protein network in animals also contains PI3K, the SH3 protein, RyR, and the L-type Ca2+ channel (Maki et al. 2002). Homologs in plants retained the EF-hand and LNT regions. Thus, the regulatory protein interaction network of the group I (ALG-2 and peflin) type PEF protein may have been conserved between plants and animals.

    The EH domain is an evolutionary conserved protein-protein interaction domain present in a growing number of proteins from yeast to mammals (Santolini et al. 1999). A number of cellular ligands of the domain have been identified and demonstrated to define a complex network of protein-protein interactions in the eukaryotic cell. The principal function of the EH protein is to regulate various steps in endocytosis. The EH network is supposed to work as an integrator of signals controlling cellular pathways as diverse as endocytosis, nucleocytosolic export, and ultimately cell proliferation. The EH domain is an 100 amino acid–long protein-protein interaction domain that includes an EF-hand-type calcium-binding motif. Various types of the EH-domain proteins were reported in animals and yeast. In plants, there is a single type of EH domain-containing protein homologous gene (figs. 7C and 7-C-S), which seems to participate in ligand-induced endocytosis. Areas of high homology in the plant homolog are restricted to the P-loop, coiled-coil, and EH domains. There are no other domains of EH domain proteins (e.g., SH3, Src homology 3, RalBP1-binding region) in plant homologs. Therefore, the target protein and/or ligand likely have changed dramatically. Thus, plants may have modified the EH domain network for specific uses.

    Compared to rice, there are few homologs in Arabidopsis for these transport system-controlling genes. Therefore, rice (or monocots in general) may have different endo- and exocytosis systems than does Arabidopsis (dicots). Plants have the ability to synthesize carbohydrates and oxygen (photosynthesis), and there is a waste pool (vacuole) inside the cell. Therefore, plants might have less need for transport systems than do animals. However, rice is a crop, and the crops were artificially selected to evolute in storage carbohydrates in the seed for a long time. Thus, rice may require outer-cell transport systems similar to those of animal cells.

    Cysteine Protease and NADPH-oxidase

    The calpains are cytoplasmic, calcium-dependent cysteine proteases that differ in their Ca2+ requirements for activity. The gene is known to control homeostasis, development, cell migration, learning and memory, and apoptosis systems in animals (Perrin and Huttenlocher 2002). It consists of a large catalytic subunit and a small regulatory subunit, thus forming a heterodimeric molecule. The large subunit of the gene has diversity in animals. In mammals, the best-characterized calpains are the ubiquitously expressed μ- and m-calpains, consisting of a common 30-kDa small S-subunits (domains V and VI) and slightly differing 80-kDa L-large subunits (domains I to IV). These proteins have five EF-hands in the C-terminal domain (thus it belongs to the PEF protein family). However, most of the calpain large subunit of insects, nematodes, and yeast lacks EF-hands. Calpain C of Drosophila has nine transmembrane regions in the N-terminal region (Spadoni et al. 2003). Plant calpain orthologous genes have been identified as phytocalpains. Like their animal counterparts, phytocalpains have significant homology within the catalytic domain but lack the conserved calcium-binding domain IV, and some members (Arabidopsis, rice, maize) have an N-terminal transmembrane receptor-like domain (Rogério and Margis-Pinherio 2003). We confirmed that rice has both the transmembrane-containing (consisting of two protein regions, the transmembrane region and catalytic region) and noncontaining forms of phytocalpains (figs. 8A and 8-A-S). In animals, the small regulatory subunit consists of a glycine-rich domain V and a calmodulin-like domain VI. The plant homolog retains the glycine-rich domain V (the region also contains much prolin) and a Ca2+-binding EF-hand domain, but it lacks two EF-hand domains at the C-terminal site. Thus, the calpain proteins of both the large and small subunits were drastically modified between plants and animals. Data analyses revealed that there are no p53, p21, or other apoptosis-controlling genes in plants. Therefore, the role of the calpain homolog in plants is different than that in animals.

    FIG. 8.— Comparison of apoptosis and active oxygen species signal transduction. (A) Calpain. (B) NADPH oxidase (gp91phox subunit).

    NADPH oxidase catalyses the NADPH-dependent reduction of molecular oxygen to generate superoxide, which can dismute to form secondary metabolites, including hydrogen peroxide and HOCl. The respiratory burst oxidase consists of six subunits, including two plasma membrane-associated proteins (gp91phox and p22phox) that comprise flavocytochrome b558 and four cytosolic factors (p47phox, p67phox, p40phox, and Rac). gp91phox is the catalytic subunit of the respiratory burst oxidase. This subunit is anchored to the membrane through its hydrophobic N-terminal half, which contains a cluster of five predicted transmembrane alpha helices and which is also thought to contain two bound heme groups. The C-terminal half of gp91phox is homologous to known flavoprotein dehydrogenases and contains consensus sequences comprising a putative NAD(P)H binding site (Cheng et al. 2001).

    Genes homologous to NADPH oxidase in plants encode a putative 108-kDa protein, with a C-terminal region that shows pronounced similarity to the 69-kDa apoprotein of the gp91phox subunit of the respiratory burst NADPH oxidase. The plant homologs have a large hydrophilic N-terminal domain that is not present in standard gp91phox. This domain contains two Ca2+-binding EF-hand motifs and has extensive similarity to human RanGTPase-activating protein 1 (Keller et al. 1998; figs. 8B and 8-B-S). The animal gp91phox subunit of the phagocyte NADPH oxidase (NOX5) is the only exception. NOX5 has retained the regions crucial for electron transport (NADPH, FAD, and heme binding sites) and has a unique N-terminal extension that contains three EF-hand motifs. This protein is expressed in pachytene spermatocytes of the testis and in B- and T-lymphocyte-rich areas of spleen and lymph nodes, and in response to elevations of the cytosolic Ca2+ concentration, it generates large amounts of superoxide (Banfi et al. 2001). Like NOX5, the active oxygen signal transduction systems of plants were modified to connect to the calcium signal transduction system.

    Calcium2+/Phospholipid Binding and Ca2+ Ion Storage Proteins

    Phosphoinositide-specific phospholipase C (PLC) isozymes found in eukaryotes comprise a related group of proteins that cleave the polar head group from inositol phospholipids. Under the control of cell surface receptors, these enzymes hydrolyze the highly phosphorylated lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), generating two intracellular products: inositol 1,4,5-trisphosphate (InsP3), a universal calcium-mobilizing second messenger, and diacylglycerol (DAG), an activator of protein kinase C. There are four ?-, two -, and four -isoforms, and numerous spliced variants have been described in mammals. Those found in yeasts, slime molds, filamentous fungi, and plants closely resemble mammalian . Animal PLCs contain a string of modular domains organized around a catalytic /?-barrel formed from the characteristic X- and Y-box regions. These proteins include a pleckstrin homology (PH) domain, EF-hand motifs, and a single C2 domain that immediately follows the Y-box region (Rebecchi and Pentyala 2000). Plant PLC homologs contain the X, Y, and C2 regions at high homology. However, the EF-hand and N-terminal PH domains (IP3/PIP2-binding region) seem to have been lost (fig. 9-A). Therefore, the regulatory systems of PLC homologs in plants may no longer involve calcium, IP3, or PIP2.

    FIG. 9.— Comparison of Ca2+/phospholipid-binding protein, Ca2+ storage protein, and allergen. (A) Phospholipase C. (B) Annexin. (C) Calreticulin. (D) Allergen.

    The annexins are reversible Ca2+-dependent intracellular membrane–binding proteins. Annexin is an atypical membrane channel protein, as it does not contain transmembrane domains; the predicted -helices are too small to span the membrane. The proteins bind to the surfaces of phosphatidylserine-containing phospholipid bilayers, either in the presence of Ca2+ or under conditions of low pH (pH 5 to 6). Then they undergo major conformational changes to obtain their integral transmembrane channel state (hexamer). All annexins display a conserved core domain consisting of four homologous repeats, each of about 70 residues. Two of these repeat units may comprise a single Ca2+/phospholipid binding site. They have been subdivided into tetradcores with short N-termini, tetradcores with long N-termini, and octadcores with short N-termini. The core of the protein is a 34-kDa C-terminal domain of four repeats (eight repeats in annexin VI). Each 70-residue repeat contains a so-called endonexin fold with its identifying GXGTDE sequence. Although there are many species of annexin in animals (Gerke and Moss 2002), plants have genes homologous to only one type of annexin (annexin V) (figs. 9B and 9-B-S), which is the simplest type and has the characteristic N-terminal regulatory domain and 4 C-terminal repeated calcium-binding domains (endonexin fold). The N-terminus regulatory regions of the plant sequences have been deleted to half the size of those in animals, but the C-terminus structures were well conserved. In animals, type V annexin binds to prolactin (internal cell) and collagen (outside of cell), but the targets likely have changed in plants. Therefore, Ca2+ion/phospholipid-binding proteins are conserved in plants and animals, with modification of their structures.

    Plants lacked homologs for striated-muscle-tissue–specific Ca2+ ion storage protein (calsequestrin). However, they contain high-homology counterparts for the calreticulin located in the ER of smooth muscle and nonmuscle tissue (Michalak, Mariani, and Opas 1998; figs. 9C and 9-C-S).

    Calreticulin is a major Ca2+-binding chaperone residing in the ER lumen. This protein binds Ca2+ with high capacity, and it participates in the folding of newly synthesized proteins and glycoproteins. Therefore, calreticulin is an important component of the calreticulin-calnexin cycle and the quality-control pathways in the ER. The chaperone region (globular N-domain and proline-rich domain) is well conserved between plants and animals (figs. 9D and 9-D-S). However, the C-terminal side of the calcium storage region is diverse. Therefore, the Ca2+ storage capacity and affinity might have changed between plant and animals. Plants also show homologs of centrin, fimbrin, and calmenin, which are involved in control of the ER and cell membrane. Therefore, a Ca2+ ion storage network of the ER also operates in plants.

    The calcium-binding allergens also contain two or three EF-hand motifs (Valenta et al. 1998). Basic structures were conserved in plants and animals, and the proteins in plants show high homology with each other (figs. 9E and 9-E-S). ATP/GTP-binding protein exhibits a high level of homology between plants and animals (data not shown).

    Discussion

    Our comparative analysis of calcium transport proteins revealed that the VDCC calcium channel systems have dramatically changed between animals and plants. Whereas plant ROCCs have simplified calcium pumps and calcium transporters but are basically conserved in plants and animals, plant VDCC proteins are markedly altered. Plants lack animal-type VDCC channel homologs and have only a single type of VDCC; the TPC1 is half the size of the animal VDCC. There is no protein homolog that has 24 transmembrane domains in whole-genomic data (data not shown), and the structures of voltage-dependent sodium ion channels are very similar to those of VDCCs (24-transmembrane structure that are grouped in four repeated structures of six transmembrane regions). Therefore, not only calcium-dependent systems but also many voltage-dependent channels appear to have dramatically changed between plants and animals. Furthermore, plants lack homologs of other subunits that control the VDCC pure subunit. VDCC systems seem to be more simplified in plants than in animals. TPC1 also exists in animals, and its primary structure suggests that it might be a predecessor of the conventional 4-repeat voltage-gated Ca2+ and Na+ channels (Ishibashi, Suzuki, and Imai 2000).

    Regarding channels, plants appear to lack homologs of the IP3 and the ryanodine receptors, even though we searched for homologs by using total and partial (IP3-binding region) sequences as probes. There have been many physiological observations and molecular analyses of IP3 signal transduction systems (White 2000). Thus, IP3 exists in plant cells, but its receptors have changed dramatically. Therefore, the calcium signal oscillation system (the amplification and transduction system of the calcium signals) genes also have changed dramatically. Except for a few examples (e.g., stomata guard cell), plants have fewer of the precise transduction systems for information regarding the membrane voltage's strength and activated length. There are two potential explanations. First, the necessity for cell-to-cell transduction systems is lower in plants than in animals. Plants lack the ability to move (except for a few reactionary movements), and there is no muscle tissue or central nervous system in plants. Therefore, rapid and multiple transmission systems of membrane voltage-dependent information delivery methods might not well developed in plants. Second, plant cells cannot easily control extracellular ionic concentrations, and the membrane potential is substantially more negative in plant cells than in animal cells. These factors might affect the structure of domains that confer ionic selectivity and/or voltage regulation. Therefore, plants might adopt more stable and/or easy-to-control systems.

    The ROCC transport proteins (CNGC, Ca2+/Na+ ion exchanger, iGlu receptor, and Ca2+ ATPase pump proteins IIA and IIB) are basically conserved in plants and animals. The transmembrane regions are conserved well, and the main differences in the sequences are located at the inner and/or outer membrane regions. The ligand (calcium, cyclic dNTP, ATP, etc.) binding, phosphorylation, and inhibitor-binding regions of the sequences are conserved, but some had different positions in the sequences. In this regard, the binding of CaM to the C-terminus of plant CNGCs might interfere with cyclic nucleotide binding, suggesting perhaps a different mechanism for regulation. In Ca2+ ATPase IIB (plasma membrane type, ACA), the regulatory domain can occur at either the N- or C-terminal side of the proteins, and there seems to be no functional difference associated with the location. Therefore, the conservation or translocation of the ligand and/or ion-binding region might have been decided accidentally. Ca2+/H+ ion exchangers exist in plants, fungi, and yeast, and their presence in these organisms is related to the existence of vacuoles. The vacuole is an internal organ that collects various ions, phenolics, acids, and a range of nitrogenous wastes, and plants can exchange ions easier through the vacuole than outside of the plasma membrane. The Ca2+/H+ ion exchanger may work to regulate internal pH and calcium ion concentration. Compared with animals, plants don't have well developed waste elimination systems. Thus, they need to maintain a waste dump in the cell. However, overall membrane voltage-independent and various individual internal signal transduction systems, such as ROCCs, are fundamentally conserved between plants and animals.

    Calcium-binding proteins exist inside of cells, bind to the Ca2+ ion that comes from membrane pores, and switch on individual signal pathways, thereby controlling cell-specific variations of signal transduction pathways. Plants lack homologs of S100 (two-EF-hand protein), many types of myosin light chain kinase, phosphorylase kinase (phosphorylase), NO synthetic enzyme (cyclic nucleotide metabolic enzyme), calsequestrin (calcium storage protein), caldesmon, spectrorin, synapsin (cell skeleton protein), EGF receptor, insulin receptor, and IRS-1 (signal transduction gene). However, CDPK and CRK (phosphorylase), calcineulin A and B (dephosphorylase), adenylate cyclase and phosphodiesterase (cyclic nucleotide metabolic enzyme), phospholipase C and annexin (calcium phospholipid binding protein), calreticulin (calcium storage protein), centrin and fimbrin (cell skeleton protein), stress response genes, and hormonal response genes (signal transduction genes) are present in plants.

    Homologs for the most common Ca2+-binding protein, calmodulin, and its related kinases (CCaMK, CaMK) also exist in plants. Furthermore, plants have specifically evolved the kinases CDPK and CRK. In the phosphorylation system, plants have fewer genes for CCaMK and CaMK than do animals. Therefore, the calcium-calmodulin–dependent phosphorylation system has been modified in plants. Dephosphorylase (calcineurin) gene structures are conserved, but some of the regulating phenomena (stress response) have been changed to plant-specific versions. Many endocytosis gene homologs were found in rice and in Arabidopsis. The homologs are of a lesser variety and, therefore, show lower similarities to the animal endocytosis genes. Rice develops much more quickly than Arabidopsis and has a specialized carbohydrate storage system. Therefore, unlike Arabidopsis, rice has a need for systematic transport and has developed transport systems similar to some of those in animals. In plants, the only cytoskeleton protein homologs were those associated with basic roles (e.g., control of cell division), and the calcium storage protein of nonmuscle tissue in animals is conserved. Some of the plant signal transduction systems showed changes in their regulatory ligand. For example, the reactive oxygen species signal transduction gene (NADPH oxidase) is still regulated by the calcium ion, but Ca2+ no longer directly regulates the activity of phospholipase C homologs in plants. As mentioned before, the channel systems have been modified, and the channel-type phospholipid protein (annexin) system in plants is simple.

    Therefore, plants lack homologs of the calcium-binding proteins that are specific to animal tissues (muscle, nerve, bone). However, plants have homologs to most of the fundamental genes as well as the plant-specific calcium-binding proteins CDPK and CRK. CDPK has EF-hand and CaM II kinase regions. Therefore, it has the ability to bind Ca2+ and phosphorylate target protein. Changing the system from Ca2+ ion calmodulin kinase to Ca2+ ion CDPK steps up the process and saves energy for protein synthesis. However, in that case, the selecting and/or regulating step associated with the Ca2+ ion calmodulin step disappears.

    Comparative analysis of plants and animals revealed that animal systems have the benefit of high velocity and multiple selectivity for cell-to-cell signal transduction in tissues like nerve and muscle. In contrast, plants have modified, specialized, and evolved to simplify the calcium signal cascade by skipping the transduction step through the use of CDPK and CRK (fig. 10). The number of EF-hand genes in plants is two or three times that in animals (Day et al. 2002). This result indicates that animal signal transduction operates with a smaller variety of calcium-binding protein species than does the same process in plants. The Ca2+ ion cannot carry the specific message of the biological signal by itself. Without information regarding membrane voltage strength and patterns, the acceptor of the Ca2+ ion requires the diversity of more binding protein species. Therefore, if it has the same numbers of signal transduction signals, plants need more calcium-binding protein species than do animals. These different patterns of calcium signal transduction systems indicate that animals have developed calcium signal transduction for cell-to-cell communication (development, morphogenesis, pattern formation, hormonal regulation, muscle, and nervous system), whereas plants have developed it for internal gene regulation (homeostasis, stress response).

    FIG. 10.— Schematic representation of calcium transport systems in animal and plant cells. In the animal cell, the many types of voltage-dependent channels make it possible to distinguish calcium-flooding signals with regard to membrane voltage strength and patterns. The InsP3 (inositol 1,4,5-trisphosphate) and RyR (ryanodine) receptors accept calcium ions from the surface of the endoplasmic reticulum and expose them to the inside of the membranes. The exposure of the calcium ions rapidly increases the concentration of the calcium in the cell and creates the oscillation pattern of electrical activation at the cell membrane. The active current from the system discharges to neighboring cells by the electrical pattern and/or ligand (IP3) transport. Plant cells have fewer types of voltage-dependent channels and recognition systems for membrane voltage strength and patterns. There are also dramatic changes in the IP3 and ryanodine receptors. The resulting changes in the oscillation system and ligand transport mechanism affect the system for disseminating electrical charge to neighboring cells.

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