Download Comparative Analysis of Plant and Animal Calcium Signal

Comparative Analysis of Plant and Animal Calcium Signal Transduction
Element Using Plant Full-Length cDNA Data
Toshifumi Nagata,* Shigemi Iizumi,* Kouji Satoh,* Hisako Ooka,* Jun Kawai, Piero Carninci, Yoshihide Hayashizaki, Yasuhiro Otomo,à Kazuo Murakami,à
Kenichi Matsubara,à and Shoshi Kikuchi*
*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 àFoundation for Advancement of
International Science; 586-9 Akatsuka-Ushigafuchi, Tsukuba, Ibaraki, 305-0062 Japan
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, Ca21ATPase, Ca21/Na1 -K1 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), Ca21/phospholipid-binding domains, and
calcium storage proteins.
Introduction
Ca21 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 Ca21 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 Ca21 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 Ca21 ionregulated 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 Ca21 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
Key words: calcium transport protein, calcium-binding protein, fulllength cDNA.
E-mail: [email protected].
Mol. Biol. Evol. 21(10):1855–1870. 2004
doi:10.1093/molbev/msh197
Advance Access publication June 9, 2004
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 , 102100 (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.
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 fulllength 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
Molecular Biology and Evolution vol. 21 no. 10 Ó Society for Molecular Biology and Evolution 2004; all rights reserved.
1856 Nagata et al.
Table 1
Calcium Transport and Calcium-Binding Protein Homologs of Human, Rice and Arabidopsis
Type
H. sapiens O. sativa A.thaliana
.Transporter
protein
channel
1.VDCC
2.ROCC
-
a) LVA
b) HVA
a) CNGC
b) Glu R
pump
e) Ryanodine receptor
f) IP3 receptor
g) LCT
c) P-type Ca2+ ATPase
transporter
d) Ca2+ exchanger
T
L
N
P/Q
R
3
0
0
6+1(TPC1) 2(TPC1) 1(TPC1)
1
0
0
1
0
0
1
0
0
4
0
0
4
0
0
8
0
0
1
0
0
4
8
20
AMPA
5
8
20
NMPA
0
0
0
KININ
0
0
0
8
0
0
3
0
0
0
1
1
A
8
5
3
B
11
10
8
2+ +
Ca ,H
0
5
8
Ca2+,Na+
3
0
0
Ca2+,Na+,(K+)
5
3
5
2+
.Ca binding
protein
1.EF-hand protein
mono type
A. internal cell a) calbindin
b) calcineurin B
c) calmodulin
d) caltractin(centrin)
e) myosin regulatory light
chain kinase2
f) parvalbumin
g) recoverin
h) Penta-EF-hand
i) S100
j) troponin C
B. ER golgi a) ERC55
b) calumenin
complex type
A. outside of
a) BM-40
cell
b) thrombospondin(1,4)
c) NADPH oxidase
B. internal cell a) actinin
b) fimbrin
c) calpain
3.γ-carboxyglutamic acid
4.Lipid containing protein
5.Ca2+ storage protein
6.others
0
7
23
4
0
10
54
2
1
0
0
1
1
2
18
2
1
2
0
0
2
0
0
0
1
0
0
2
0
0
0
1
1
0
0
2
4
4
5
11
1
1
1
4
6
4
0
0
0
8
0
1
2
1
0
0
3
3
3
20
4
0
12
0
2
1
1
0
0
3
2
3
34
4
annexin
19
13
8
phospholipaseC
osteocalcin
proteinC
casein
phosvitin
calsequestrin
calreticulin
endoplasmin
allergen
13
1
1
5
4
2
2
1
0
2
0
0
0
0
0
5
0
8
6
0
0
0
0
0
5
0
0
d) diacylglycerol kinaseα
e) EGF receptor substrate15
f) EH-domain
h) CCaMK
i) CaMK
j) CDPK
k) CRK
2.Ca2+/Phospholipid binding
protein
3
5
30
5
large subunit
small subunit
NOTE.—VDCC: voltage-dependent calcium channel; ROCC: receptor-opened calcium channel; LVA: low voltage-activated calcium channels; HVA: high voltageactivated calcium channels; Human: Homo sapiens; ory: Oryza sativa; Ara: Arabidopsis thaliana.
Comparative Analysis of Calcium Systems 1857
existence of a homolog. To check the alignment pattern of
the query and subject sequences, we used a similarity
threshold of E , 102100.
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 ¼ 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 Ca21 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 Ca21 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 Ca21 is homeostatically
maintained in the face of different Ca21 concentration
gradients across the cell membrane. The Ca21 concentration outside root cells is subjected to vagaries in the soil
Ca21 concentration, and fluctuations in the xylem Ca21
concentration affect that of leaf cells. Nevertheless, the
maintenance of an inwardly directed Ca21 electrochemical
gradient occurs in plants too (White et al. 2002). In
animals, transport of the Ca21 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 Ca21
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).
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 a-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 a-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 a-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
Ca21 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, VDCCb subunit, VDCCc 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 (Ca21-permeable) cation channels as ligandgated ion channels, which bind glutamate that has been
released from a companion cell, thereby allowing charged
ions (Na1, Ca21) 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 a-amino-3hydroxy-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 c-aminobutyric acid receptors. AMPA
receptor channels are impermeable to calcium, a function
controlled by the iGluR2 subunit that results from posttranscriptional 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 Ca21 permeability and is blocked by
Mg21 (figs. 1B and 1-B-S). Thus, it is difficult to predict the
1858 Nagata et al.
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). aI: aI subunit; aII: aII subunit; b: b subunit; c: c subunit; d: d subunit; PKC: proteinkinase-C-binding site; G: G-protein-binding site; 1: 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.
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 (39,59-cyclic AMP [cAMP]) and
39,59-cyclic GMP [cGMP])-gated channels (CNGCs) are
Comparative Analysis of Calcium Systems 1859
FIG. 2.—Comparison of pump and transporter proteins in plants and animals. (A) Ca21 ATPase. Topology models of putative Ca21 ATPases in
plants and animals. Ca21 ATPase IIA and IIB. (B) Ca21 transporter. Ca21/H1 antiporter. Na1/Ca21-K1 exchanger.
a recently identified family of plant ion channels. They
show a high degree of similarity to Shaker-type voltagegated channels and contain a C-terminal cyclic nucleotidebinding 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 Ca21 ion transport area and calmodulinbinding 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.
1860 Nagata et al.
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 K1-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 Ca21 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 Ca21 ATPase pump
proteins and calcium transporters (Ca21/H1 ion exchanger
and Ca21/Na1 ion exchanger) are well conserved in plants
and animals (fig. 2).
P-type ATP-powered calcium transport proteins,
which use ATP hydrolysis to pump Ca21 ions, transport
Ca21 out of the cell against the electrical gradient. There
are two types of Ca21 ATPases in animals: the plasmamembrane (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
Ca21 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, Ca21
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 Ca21 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 Ca21
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 Ca21/
H1 ion exchanger is an H1-coupled Ca21 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 Ca21/H1 ion exchanger is located at the vacuolar
membrane and regulates vacuolar Ca21/H1 exchange. The
plant Ca21/H1 ion exchanger has total homology with its
yeast counterpart, except in the N- and C- terminal regions
(Guerini 1998). Therefore, the Ca21/H1 ion exchange
system is well conserved in bacteria, fungi, yeast, and plants.
The Na1/Ca21 ion exchanger proteins are electrogenic transporters that can use the Na1 electrochemical
gradient to exchange three extracellular Na1 ions for
one intracellular Ca21 ion. Na1/Ca21 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 (Na1/Ca21 -K1 ion exchangers, e.g.,
NCKX family) (Szerencsei et al. 2000). The structure
of the Na1/Ca21 ion exchangers and Na1/Ca21 -K1
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 Na1/
Ca21 ion exchangers are NCKX type Na1/Ca21 -K1 ion
exchangers. There are two homolog types (high and low
similarities) in plant Na1/Ca21 -K1 ion exchangers. We
collected homologs of the Na1/Ca21 -K1 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
a-1 repeat (which controls the Ca21 ion exchange) and
alternative splice site of the animal Na1/Ca21 -K1 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 Na1/
Ca21 -K1 ion exchanger in plants. In animals, NCKX type
Na1/Ca21 -K1 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 Na1/Ca21 -K1 ion exchanger
homolog may control plant-specific phenomena. There
are no homologs of NCX type Na1/Ca21 ion exchangers in
plants. On the other hand, Ca21/H1 exchanger has a
similarity to NCX type Na1/Ca21 ion exchangers. Therefore, plants may have modified calcium antiporter systems
from Na1/Ca21 ion exchangers to a Ca21/H1 exchanger.
Nevertheless, we have confirmed the existence of transporter systems of Ca21/H1 and Ca21/Na1 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
Comparative Analysis of Calcium Systems 1861
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.
and animals. This difference may have evolved in animals
because of the need to develop rapid and multiple cell-tocell signal transduction systems in muscles and nerves.
Calcium-Binding Proteins
After flooding into the cell, the Ca21 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 Ca21 ion binding EF-hand proteins (e.g.,
calmodulin, calcineulin, centrin, fimbrin, calmenin, calpain) (fig. 4 and table 1). Ca21ion/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), c-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
Ca21-binding protein family, S100 homolog, was also not
found. S100 proteins are characterized by two distinct EFhand calcium-binding motifs with different affinities. The
proteins regulate intracellular processes such as cell growth
and motility, cell cycle regulation, transcription, and dif-
ferentiation. Thus, these small Ca21-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.
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 helixloop-helix conformation and has the ability to surround the
FIG. 4.—Ca21 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.
1862 Nagata et al.
Ca21 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 EFhand 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 Nterminal 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).
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 plantspecifically 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 Ca21-binding proteins and function as Ca21 sensors in neurons. The calmodulin-binding
domain of CCaMK is very similar to CaM kinase II. Ca21
binding to the C-terminal visinin-like domain leads
to autophosphorylation of the kinase. Unlike that by
CDPKs, substrate phosphorylation by CCaMK requires
both Ca21 and CaM. The interaction between CCaMK and
CaM is modulated by the Ca21-stimulated autophosphorylation. CaM-dependent protein kinases in invertebrates
and vertebrates require Ca21/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 welldefined EF-hands for Ca21 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 Ca21
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 Ca21 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 Ca21- and calmodulindependent 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 Ca21-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 (Jörg et al. 1999; Halfter, Ishitani, and Zhu 2000;
Comparative Analysis of Calcium Systems 1863
Kim et al. 2003). In animals, calcineurin controls apoptosis,
memory, exocytosis, channel (K1) 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.
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
Ca21-binding domains linked by a flexible tether, which is
capable of binding two Ca21 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 filamentbundling protein, which has two ABP-120-like actinbinding 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 Ca21dependent folding and maturation of secretory proteins in
the ER lumen.
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) (¼ calmodulin-like domain
protein kinase [CPK]). (F) CDPK-related protein kinase (CRK).
1864 Nagata et al.
FIG. 6.—Comparisons of dephosphatase and cell skeleton protein.
(A) Calcineurin. (B) Centrin (caltractin). (C) Fimbrin.
Penta-EF-hand (PEF) proteins comprise a family of
Ca21-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 Ca21-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 (Ca21dependent 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 Ca21-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 Ca21 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 proteinprotein interaction domain present in a growing number of
proteins from yeast to mammals (Santolini et al. 1999). A
FIG. 7.—Comparison of endocytosis proteins. (A) Calumenin. (B)
Penta-EF-hand protein (peflin and ALG-2 type). (C) EH-domaincontaining protein.
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 domaincontaining 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
Comparative Analysis of Calcium Systems 1865
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 Ca21 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 l- 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 transmembranecontaining (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 Ca21-binding EF-hand
domain, but it lacks two EF-hand domains at the Cterminal 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.
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 Cterminal 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 gp91ph8x. This domain contains two Ca21-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 Ca21 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.
Calcium21/Phospholipid Binding and
Ca21 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 1,4,5trisphosphate (InsP3), a universal calcium-mobilizing second messenger, and diacylglycerol (DAG), an activator of
protein kinase C. There are four b-, two c-, and four disoforms, and numerous spliced variants have been described in mammals. Those found in yeasts, slime molds,
filamentous fungi, and plants closely resemble mammalian
d. Animal d PLCs contain a string of modular domains
organized around a catalytic a/b-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 d 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.
The annexins are reversible Ca21-dependent intracellular membrane–binding proteins. Annexin is an atypical
membrane channel protein, as it does not contain transmembrane domains; the predicted a-helices are too small
to span the membrane. The proteins bind to the surfaces of
phosphatidylserine-containing phospholipid bilayers, either in the presence of Ca21 or under conditions of low pH
(pH 5 to 6). Then they undergo major conformational
changes to obtain their integral transmembrane channel
1866 Nagata et al.
FIG. 8.—Comparison of apoptosis and active oxygen species signal transduction. (A) Calpain. (B) NADPH oxidase (gp91phox subunit).
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 Ca21/phospholipid binding site. They have been
subdivided into tetradcores with short N-termini, tetradcores with long N-termini, and octadcores with short Ntermini. 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 calciumbinding 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, Ca21ion/phospholipid-binding proteins are
conserved in plants and animals, with modification of their
structures.
Plants lacked homologs for striated-muscle-tissue–
specific Ca21 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 Ca21-binding chaperone residing in the ER lumen. This protein binds Ca21 with high
Comparative Analysis of Calcium Systems 1867
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 Ca21
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 Ca21 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 Ca21 and Na1
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 (IP3binding 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
FIG. 9.—Comparison of Ca21/phospholipid-binding protein, Ca21
storage protein, and allergen. (A) Phospholipase C. (B) Annexin. (C)
Calreticulin. (D) Allergen.
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, Ca21/Na1 ion
exchanger, iGlu receptor, and Ca21 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 Ca21 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
1868 Nagata et al.
FIG. 10.—Schematic representation of calcium transport systems in
animal and plant cells. In the animal cell, the many types of voltagedependent 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
difference associated with the location. Therefore, the
conservation or translocation of the ligand and/or ionbinding region might have been decided accidentally.
Ca21/H1 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 Ca21/H1 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 Ca21 ion that comes from membrane pores, and switch
on individual signal pathways, thereby controlling cellspecific 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 Ca21-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 plantspecific 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
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.
Comparative Analysis of Calcium Systems 1869
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 Ca21 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 calciumbinding 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 Ca21 and phosphorylate target protein.
Changing the system from Ca21 ion ! calmodulin !
kinase to Ca21 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
Ca21 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 EFhand 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 calciumbinding protein species than does the same process in
plants. The Ca21 ion cannot carry the specific message of
the biological signal by itself. Without information
regarding membrane voltage strength and patterns, the
acceptor of the Ca21 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).
Literature Cited
Allen, G. J., S. P. Chu, C. L. Harrington, K. Schumacher, T.
Hoffmann, Y. Y. Tang, E. Grill, and J. I. Schroeder. 2001. A
defined range of guard cell calcium oscillation parameters
encodes stomatal movements. Nature 411:1053–1057.
Arabidopsis Genome Initiative. 2001. Analysis of the genome
sequence of the flowering plant Arabidopsis thaliana. Nature
408:796–815.
Banfi, B., G. Molnar, A. Maturana, K. Steger, B. Hegedus, N.
Demaurex, and K. H. Krause. 2001. A Ca (21)-activated
NADPH oxidase in testis, spleen, and lymph nodes. J. Biol.
Chem. 276:37594–37601.
Berridge, M. J. 1993. Inositol trisphosphate and calcium signaling. Nature 361:315–325.
Bowler, C., G. Neuhaus, H. Yamagata, and N. H. Chua. 1994.
Cyclic GMP and calcium mediate phytochrome phototransduction. Cell 77:73–81.
Braam, J., and R. W. Davis. 1990. Rain-, wind-, and touchinduced expression of calmodulin and calmodulin-related
genes in Arabidopsis. Cell 60:357–364.
Camacho, L., and R. Malho. 2003. Endo/exocytosis in the pollen
tube apex is differentially regulated by Ca21 and GTPases.
J. Exp. Bot. 54:83–92.
Catterall, W. A. 2000. Structure and regulation of voltage-gated
Ca21 channels. Annu. Rev. Cell Dev. Biol. 16:521–555.
Cheng, G., Z. Cao, X. Xu, E. G. van Meir, and J. D. Lambeth.
2001. Homologs of gp91phox: cloning and tissue expression
of Nox3, Nox4, and Nox5. Gene 269:131–140.
Cheng, S. H., M. R. Willmann, H. C. Chen, and J. Sheen. 2002.
Calcium signaling through protein kinases. The Arabidopsis
calcium-dependent protein kinase gene family. Plant Physiol.
129:469–485.
Chiu, J., R. DeSalle, H. M. Lam, L. Meisel, and G. Coruzzi.
1999. Molecular evolution of glutamate receptors: a primitive
signaling mechanism that existed before plants and animals
diverged. Mol. Biol. Evol. 16:826–838.
Cordeiro, M. C., R. Piqueras, D. E. de Oliveira, and C.
Castresana. 1998. Characterization of early induced genes in
Arabidopsis thaliana responding to bacterial inoculation:
identification of centrin and of a novel protein with two
regions related to kinase domains. FEBS Lett. 434:387–393.
Day, I. S., V. S. Reddy, G. Shad Ali, and A. S. Reddy. 2002.
Analysis of EF-hand-containing proteins in Arabidopsis.
Genome Biol. 3:RESEARCH0056.
Dennison, K. L., and E. P. Spalding. 2000. Glutamate-gated
calcium fluxes in Arabidopsis. Plant Physiol. 124:1511–1514.
Dunn, J., C. L. Elias, H. D. Le, A. Omelchenko, L. V. Hryshko,
and J. Lytton. 2002. The molecular determinants of ionic regulatory differences between brain and kidney Na1/Ca21 exchanger (NCX1) isoforms. J. Biol. Chem. 277:33957–33962.
Durner, J., D. Wendehenne, and D. F. Klessig. 1998. Defense
gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP–ribose. Proc. Natl. Acad. Sci. USA 95:10328–10333.
Ehsan, H., J. P. Reichheld, L. Roef, E. Witters, F. Lardon, D.
Van Bockstaele, M. Van Montagu, D. Inze, and H. Van
Onckelen. 1998. Effect of indomethacin on cell cycle
dependent cyclic AMP fluxes in tobacco BY-2 cells. FEBS
Lett. 422:165–169.
Evans, N. H., M. R. McAinsh, and A. M. Hetherington. 2001.
Calcium oscillations in higher plants. Curr. Opin. Plant Biol.
4:415–420.
Fischer, M., N. Schnell, J. Chattaway, P. Davies, G. Dixon, and
D. Sanders. 1997. The Saccharomyces cerevisiae CCH1 gene
is involved in calcium influx and mating FEBS Lett. 419:
259–262.
Furuichi, T., K. W. Cunningham, and S. Muto. 2001. A putative
two-pore channel AtTPC1 mediates Ca (21) flux in
Arabidopsis leaf cells. Plant Cell Physiol. 42:900–905.
Furumoto, T., N. Ogawa, S. Hata, and K. Izui. 1996. Plant calciumdependent protein kinase-related kinases (CRKs) do not require
calcium for their activities. FEBS Lett. 396:147–151.
Geisler, M., K. B. Axelsen, J. F. Harper, and M. G. Palmgren.
2000. Molecular aspects of higher plant P-type Ca (21)ATPases. Biochim. Biophys. Acta 1465:52–78.
Gerke, V., and S. E. Moss. 2002. Annexins: from structure to
function. Physiol. Rev. 82:331–371.
Goff, S. A., D. Ricke, T. H. Lan et al. (55 co-authors). 2002. A
draft sequence of the rice genome (Oryza sativa L. sp.
japonica). Science 296:92–100.
Guerini, D. 1998. The Ca21 pumps and the Na1/Ca21
exchangers. Biometals 11:319–330.
1870 Nagata et al.
Halfter, U., M. Ishitani, and J. K. Zhu. 2000. The Arabidopsis
SOS2 protein kinase physically interacts with and is activated
by the calcium-binding protein SOS3. Proc. Natl. Acad. Sci.
USA 97:3735–3740.
Hirschi, K. D., R. G. Zhen, K. W. Cunningham, P. A. Rea, and
G. R. Fink. 1996. CAX1, an H1/Ca21 antiporter from
Arabidopsis. Proc. Natl. Acad. Sci. USA 93:8782–8786.
Hrabak, E. M., C. W. Chan, M. Gribskov et al. (14 co-authors).
2003. The Arabidopsis CDPK-SnRK superfamily of protein
kinases. Plant Physiol. 132:666–680.
Ishibashi, K., M. Suzuki, and M. Imai. 2000. Molecular cloning
of a novel form (two-repeat) protein related to voltage-gated
sodium and calcium channels. Biochem. Biophys. Res.
Commun. 270:370–376.
Ishitani, M., J. Liu, U. Halfter, C. S. Kim, W. Shi, and J. K. Zhu.
2000. SOS3 function in plant salt tolerance requires Nmyristoylation and calcium binding. Plant Cell 12:1667–1678.
Jörg, K., X. Qiang, H. Klaus, G. Wilhelm, and L. Sheng. 1999.
Genes for calcineurin B-like proteins in Arabidopsis are
differentially regulated by stress signals. Proc. Natl. Acad. Sci.
USA 96:4718–4723.
Keller, T., H. G. Damude, D. Werner, P. Doerner, R. A. Dixon, and
C. Lamb. 1998. A plant homolog of the neutrophil NADPH
oxidase gp91phox subunit gene encodes a plasma membrane
protein with Ca21 binding motifs. Plant Cell 10:255–266.
Kikuchi, S., K. Satoh, T. Nagata et al. (74 co-authors). 2003.
Collection, mapping, and annotation of over 28,000 cDNA
clones from japonica rice. Science 301:376–379.
Kim, K. N., Y. H. Cheong, J. J. Grant, G. K. Pandey, and S.
Luan. 2003. CIPK3, a calcium sensor-associated protein
kinase that regulates abscisic acid and cold signal transduction
in Arabidopsis. Plant Cell 15:411–423.
Kohler, C., and G. Neuhaus. 2000. Characterization of
calmodulin binding to cyclic nucleotide-gated ion channels
from Arabidopsis thaliana. FEBS Lett. 471:133–136.
Kudla, J., Q. Xu, K. Harter, W. Gruissem, and S. Luan. 1999.
Genes for calcineurin B-like proteins in Arabidopsis are
differentially regulated by stress signals. Proc. Natl. Acad. Sci.
USA 96:4718–4723.
Laurent, M., and M. Claret. 1997. Signal-induced Ca21 oscillations
through the regulation of the inositol 1,4,5-trisphosphategated Ca21 channel: an allosteric model. J. Theor. Biol. 186:
307–326.
Lin, Q., E. S. Buckler IV, S. V. Muse, and J. C. Walker. 1998.
Molecular evolution of type 1 serine/threonine protein phosphatases. Mol. Phylogenet. Evol. 12:57–66.
MacRobbie, E. A. 1998. Signal transduction and ion channels
in guard cells. Phil. Trans. R. Soc. Lond. B Biol. Sci. 353:
1475–1488.
Maki, M., Y. Kitaura, H. Satoh, S. Ohkouchi, and H. Shibata.
2002. Structures, functions and molecular evolution of the
penta-EF-hand Ca21-binding proteins. Biochim. Biophys.
Acta 1600:51–60.
McCurdy, D. W., and M. Kim. 1998. Molecular cloning of a
novel fimbrin-like cDNA from Arabidopsis thaliana. Plant
Mol. Biol. 36:23–31.
Michalak, M., P. Mariani, and M. Opas. 1998. Calreticulin,
a multifunctional Ca21 binding chaperone of the endoplasmic
reticulum. Biochem. Cell Biol. 76:779–785.
Miedema, H., J. H. Bothwell, C. Brownlee, and J. M. Davies.
2001. Calcium uptake by plant cells—channels and pumps
acting in concert. Trends Plant Sci. 6:514–519.
Moutinho, A., P. J. Hussey, A. J. Trewavas, and R. Malho. 2001.
cAMP acts as a second messenger in pollen tube growth and
reorientation. Proc. Natl. Acad. Sci. USA 98:10481–10486.
Penson, S. P., R. C. Schuurink, A. Fath, F. Gubler, J. V.
Jacobsen, and R. L. Jones. 1996. cGMP is required for
gibberellic acid-induced gene expression in barley aleurone.
Plant Cell 8:2325–2333.
Perrin, B. J, and A. Huttenlocher. 2002. Calpain. Int. J. Biochem.
Cell Biol. 34:722–725.
Rebecchi, M. J, and S. N. Pentyala. 2000. Structure, function,
and control of phosphoinositide-specific phospholipase C.
Physiol. Rev. 80:1291–1335.
Rogério, M., and M. Margis-Pinheiro. 2003. Phytocalpains:
orthologous calcium-dependent cysteine proteinases. Trends
Plant Sci. 8:58–62.
Rusnak, F., and P. Mertz. 2000. Calcineurin: form and function.
Physiol. Rev. 80:1483–1521.
Sanders, D., J. Pelloux, C. Brownlee, and J. F. Harper. 2002.
Calcium at the crossroads of signaling. Plant Cell 14:401–417.
Santolini, E., A. E. Salcini, B. K. Kay, M. Yamabhai, and P. P. Di
Fiore. 1999. The EH network. Exp. Cell Res. 253:186–209.
Sathyanarayanan, P. V., C. R. Cremo, and B. W. Poovaiah. 2000.
Plant chimeric Ca21/calmodulin-dependent protein kinase.
Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity. J. Biol. Chem.
275:30417–30422.
Serysheva, I. I., S. J. Ludtke, M. R. Baker, W. Chiu, and S. L.
Hamilton. 2002. Structure of the voltage-gated L-type Ca21
channel by electron cryomicroscopy. Proc. Natl. Acad. Sci.
USA 99:10370–10375.
Spadoni, C., A. Farkas, R. Sinka, P. Tompa, and P. Friedrich.
2003. Molecular cloning and RNA expression of a novel
Drosophila calpain, calpain C. Biochem. Biophys. Res.
Commun. 303:343–349.
Szerencsei, R. T., J. E. Tucker, C. B. Cooper, R. J. Winkfein,
P. J. Farrell, K. Iatrou, and P. P. Schnetkamp. 2000. Minimal
domain requirement for cation transport by the potassiumdependent Na/Ca-K exchanger. J. Biol. Chem. 275:669–676.
Talke, I. N., D. Blaudez, F. J. M. Maathuis, and D. Sanders.
2003. CNGCs: prime targets of plant cyclic nucleotide
signaling? Trends Plant Sci. 8:286–293.
Tsuruhara, A., and T. Tezuka. 2001. Relationship between the
self-incompatibility and cAMP level in Lilium longiflorum.
Plant Cell Physiol. 42:1234–1238.
Valenta, R., B. Hayek, S. Seiberler et al. (11 co-authors). 1998.
Calcium-binding allergens: from plants to man. Int. Arch.
Allergy Immunol. 117:160–166.
White, P. J. 2000. Calcium channels in higher plants. Biochim.
Biophys. Acta 1465:171–189.
White, P. J., H. C. Bowen, V. Demidchik, C. Nichols, and J. M.
Davies. 2002. Genes for calcium-permeable channels in the
plasma membrane of plant root cells. Biochim. Biophys. Acta
1564:299–309.
Yabe, D., T. Nakamura, N. Kanazawa, K. Tashiro, and T. Honjo.
1997. Calmenin, a Ca21-binding protein retained in the
endoplasmic reticulum with a novel carboxyl-terminal
sequence, HDEF. J. Biol. Chem. 272:18232–18239.
Yamakage, M., and A. Namiki. 2002. Calcium channels: basic
aspects of their structure, function and gene encoding; anesthetic
action on the channels—a review. Can. J. Anaesth. 49:151–164.
Yu, J., S. Hu, J. Wang et al. (99 co-authors). 2002. A draft
sequence of the rice genome (Oryza sativa L. sp. indica).
Science 296:79–92.
Zhang, L., and Y. T. Lu. 2003. Calmodulin-binding protein
kinases in plants. Trends Plant Sci. 8:123–127.
Zhang, X. S., and J. H. Choi. 2001. Molecular evolution of
calmodulin-like domain protein kinases (CDPKs) in plants
and protists. J. Mol. Evol. 53:214–224.
Takashi Gojobori, Associate Editor
Accepted June 1, 2004