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BioMed Central
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BMC Plant Biology
Open Access
Research article
Genome-wide identification and analyses of the rice calmodulin and
related potential calcium sensor proteins
Bongkoj Boonburapong and Teerapong Buaboocha*
Address: Department of Biochemistry, Faculty of Science, Chulalongkorn University, Payathai Road, Patumwan, Bangkok 10330, Thailand
Email: Bongkoj Boonburapong - b.bongkoj@gmail.com; Teerapong Buaboocha* - Teerapong.B@Chula.ac.th
* Corresponding author
Abstract
Background: A wide range of stimuli evoke rapid and transient increases in [Ca
2+
]
cyt
in plant cells
which are transmitted by protein sensors that contain EF-hand motifs. Here, a group of Oryza sativa
L. genes encoding calmodulin (CaM) and CaM-like (CML) proteins that do not possess functional
domains other than the Ca
2+
-binding EF-hand motifs was analyzed.
Results: By functional analyses and BLAST searches of the TIGR rice database, a maximum number
of 243 proteins that possibly have EF-hand motifs were identified in the rice genome. Using a
neighbor-joining tree based on amino acid sequence similarity, five loci were defined as Cam genes
and thirty two additional CML genes were identified. Extensive analyses of the gene structures, the
chromosome locations, the EF-hand motif organization, expression characteristics including
analysis by RT-PCR and a comparative analysis of Cam and CML genes in rice and Arabidopsis are
presented.
Conclusion: Although many proteins have unknown functions, the complexity of this gene family
indicates the importance of Ca
2+
-signals in regulating cellular responses to stimuli and this family of
proteins likely plays a critical role as their transducers.
Background
Ca
2+
is an essential second messenger in all eukaryotic
cells in triggering physiological changes in response to
external stimuli. Due to the activities of Ca
2+
-ATPases and
Ca
2+
-channels on the cellular membrane, rapid and tran-
sient changes of its cytosolic concentrations are possible.
In plant cells, a wide range of stimuli trigger cytosolic
[Ca
2+
] increases of different magnitude and specialized
character [1,2], which are typically transmitted by protein
sensors that preferably bind Ca
2+
. Ca
2+
binding results in
conformation changes that modulate their activity or their
ability to interact with other proteins. For the majority of
Ca
2+
-binding proteins, the Ca
2+
-binding sites are com-
posed of a characteristic helix-loop-helix motif called an
EF hand. Each loop, including the end of the second
flanking helix, provides seven ligands for binding Ca
2+
with a pentagonal bipyramid geometry. Ca
2+
-binding lig-
ands are within the region designated as +X*+Y*+Z*-Y*-
X**-Z, in which * represents an intervening residue. Three
ligands for Ca
2+
coordination are provided by carboxylate
oxygens from residues 1 (+X), 3 (+Y) and 5 (+Z), one from
a carbonyl oxygen from residue 7 (-Y), and two from car-
boxylate oxygens in residue 12 (-Z), which is a highly con-
served glutamate (E). The seventh ligand is provided
either by a carboxylate side chain from residue 9 (-X) or
from a water molecule.
Published: 30 January 2007
BMC Plant Biology 2007, 7:4 doi:10.1186/1471-2229-7-4
Received: 5 August 2006
Accepted: 30 January 2007
This article is available from: http://www.biomedcentral.com/1471-2229/7/4
© 2007 Boonburapong and Buaboocha; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
BMC Plant Biology 2007, 7:4 http://www.biomedcentral.com/1471-2229/7/4
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In plants, three major groups of Ca
2+
-binding proteins
that have been characterized include calmodulin (CaM),
Ca
2+
-dependent protein kinase (CPK), and calcineurin B-
like protein (CBL) [3-5]. Recently, Reddy ASN and col-
leagues have analyzed the complete Arabidopsis genome
sequence, identified 250 genes encoding EF-hand-con-
taining proteins and grouped them into 6 classes [6].
CaM, a unique Ca
2+
sensor that does not possess func-
tional domains other than the Ca
2+
-binding motifs
belongs to group IV along with numerous CaM-related
proteins. CaM is a small (148 residues) multifunctional
protein that transduces the signal of increased Ca
2+
con-
centration by binding to and altering the activities of a
variety of target proteins. The activities of these proteins
affect physiological responses to the vast array of specific
stimuli received by plant cells [7]. In plants, one striking
characteristic is that numerous isoforms of CaM may
occur within a single plant species. A large family of genes
encoding CaM and closely related proteins from several
plants has been identified, however, with the exception of
Arabidopsis, families of genes encoding CaM and related
proteins have not been extensively conducted in a whole-
genome scale. In addition, a very limited number of stud-
ies on individual rice CaMs has been published [8-10].
With the completion of the genomic DNA sequencing
project in Oryza sativa L., all sequences belong to a multi-
gene family such as CaM and related proteins can be iden-
tified. Preliminary searching of Oryza sativa L. databases
revealed numerous genes encoding CaM-like proteins. In
Arabidopsis, McCormack and Braam [11] have character-
ized members of Groups IV and V from the 250 EF-hand
encoding genes identified in the Arabidopsis genome. Six
loci are defined as Cam genes and 50 additional genes are
CaM-like (CML) genes, encoding proteins composed
mostly of EF-hand Ca
2+
-binding motifs. The high com-
plexity of the CaM and related calcium sensors proteins in
Arabidopsis suggests their important and diverse roles of
Ca
2+
signaling. It would be interesting to know how this
family of proteins exists in the genome of rice which is
considered a model plant for monocot and cereal plants
because of its small genome size and chromosomal co-lin-
earity with other major cereal crops. In this study, we
identified genes encoding proteins that contain EF-hand
motifs and are related to CaM from the rice genome. Anal-
yses of the identified gene and protein sequences includ-
ing gene structures, chromosomal locations, the EF-hand
motif organization and expression characteristics as well
as comparison with Arabidopsis Cam and CML genes were
carried out.
Results and Discussion
Identification and phylogenetic analysis of EF-hand-
containing proteins
To identify EF-hand-containing proteins, firstly, we func-
tionally searched the Oryza sativa L. genome at The Insti-
tute for Genomic Research (TIGR) [12] for Interpro
Database Matches by five different methods including
HMMPfam, HMMSmart, BlastProDom, ProfileScan and
superfamily as described in the "Methods" section. Sec-
ondly, we searched the rice database using the amino acid
sequences of rice CaM1 [10] and CBL3 [13] as queries in
the programs BLASTp and the protein sequences that were
not found by the domain searches were added to the list.
In addition, we reviewed literature on reports of EF-hand-
containing proteins in rice that have been identified by
various methods. All of these protein sequences were
again analyzed for EF hands and other domains using
InterProScan [14]. InterProScan is a protein domain iden-
tifying tool that combines different protein signature rec-
ognition methods from the consortium member
databases of the Interpro [15]. As a result, domain
searches identified 245 proteins but six sequences did not
have an EF hand identifiable by InterProScan using
default settings, so they were eliminated from further
analysis. BLAST searches have found four more EF-hand-
containing proteins and literature review has yielded no
additional proteins. Totally, a maximum of 243 putative
EF-hand-containing proteins in rice have been identified
[see Additional file 1]. Nearly half of these proteins con-
tain no other identifiable domains predicted by InterPro-
Scan. It should be noted that 24 proteins contain a single
EF-hand motif that was identified by only one prediction
program and could be false positives.
Next, sequences of all the proteins identified by the Inter-
ProScan as containing an EF-hand motif were aligned
using Clustal X [16] [see Additional file 2]. Tree construc-
tion using the neighbor-joining method and bootstrap
analysis was performed. Figure 1 shows the tree outline
illustrating the numbers of EF hands predicted by Inter-
ProScan for each protein on the right without any gene
identifiers. As a result, proteins that do not possess func-
tional domains other the Ca
2+
-binding EF-hand motifs
were found distributed across the tree but most were con-
centrated in the top half. Conversely, most proteins in the
bottom half contain additional domains that give clues to
their functions which include transcription factor, ion
channel, DNA- or ATP/GTP-binding protein, mitochon-
drial carrier protein, protein phosphatase and protein
kinase. Two known major groups of EF-hand-containing
proteins: calcineurin B-like (CBL) [13] and Ca
2+
-depend-
ent protein kinase (CPK) proteins [17] are separately
grouped as shown in Figure 1. We observed that most of
the proteins containing four EF-hand motifs are either in
the CPK group or located at the top of the tree surround-
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ing the typical CaM proteins. With the exception of two,
all proteins indicated by "CaM & CML" share at least 25%
amino acid identity with OsCaM1 and were selected for
further analyses. This list should contain rice proteins that
are related to CaM or has functions based on Ca
2+
-binding
mode similar to CaM. Existence of these genes and their
deduced amino acid sequences were confirmed using
another annotation database, the Rice Annotation Project
Database (RAP-DB) [18].
Rice CaM proteins
The full-length amino acid sequences of the selected pro-
teins were subjected to phylogenetic analysis. Tree con-
struction using the neighbor-joining method and
bootstrap analysis performed with ClustalX [see Addi-
tional file 3] generated a consensus tree which is depicted
in Figure 2. This analysis led us to separate these proteins
into six groups: 1–6. What defines a "true" CaM and dis-
tinguishes it from a CaM-like protein that serves a distinct
role in vivo is still an open question. Different experimen-
tal approaches including biochemical and genetic analy-
ses have been taken to address this question [19]. In this
study by phylogenetic analysis based on amino acid
sequence similarity, five proteins in group 1 that have the
highest degrees of amino acid sequence identity (≥ 97%)
to known typical CaMs from other plant species were
identified. Because of these high degrees of amino acid
identity, we classified them as "true" CaMs that probably
function as typical CaMs. They were named OsCaM1-1,
OsCaM1-2, OsCaM1-3, OsCaM2 and OsCaM3. Their
characteristics are summarized in Table 1.
OsCam1-1; OsCam1-2 and OsCam1-3 encode identical
proteins, whereas OsCam2 and OsCam3 encode a protein
of only two amino acid differences and their sequences
share 98.7% identity with those of OsCaM1 proteins.
Multiple sequence alignment of the OsCaM amino acid
sequences with those of typical CaMs from other species
shown in Figure 3 indicates their high degree of sequence
conservation. It should be mentioned that OsCaM1
amino acid sequences are identical to those of typical
CaMs from barley (H. vulgare) and wheat (T. aestivum)
reflecting the close relationships among monocot cereal
plants. On average, OsCaM amino acid sequences share
about 99%, 90% and 60% identity with those from
plants, vertebrates and yeast, respectively. Hydrophobic
residues contributing to hydrophobic interaction in the
mechanism of CaM-target protein complex formation
which are critical to CaM function are highly conserved.
All of the conserved eight methionine (M) and nine phe-
nylalanine (F) residues among plant CaMs are present in
all OsCaMs. Conservation of these residues is maintained
between plant and vertebrate CaMs, with the exception of
the methionine residues at position 145–146 in plants
CaMs, which are displaced one residue compared with the
vertebrate proteins. Due to its considerable conforma-
tional flexibility [20] and being weakly polarized, methio-
nine residues which are estimated to contribute nearly
half of the accessible surface area of the hydrophobic
patches of CaM allow it to interact with target proteins in
a sequence-independent manner [21]. Sequence conser-
vation related to functionality of plant CaMs also includes
lysine (K) at position 116 which is assumed to be trimeth-
ylated. All OsCaM proteins possess a lysine residue at this
position. Lysine 116 trimethylation is believed to be a
posttranslational modification that helps regulate CaM
activity. EF-hand motifs will be discussed later in the
"number and structure of EF hand" subsection.
The presence of multiple CaM isoforms is a defining char-
acteristic of CaMs in plants. Even though the explanation
of gene redundancy still cannot be ruled out, accumulat-
ing evidence suggests that each of the Cam genes may have
distinct and significant functions. Previous reports have
shown that highly conserved CaM isoforms actually mod-
ulate target proteins differently [22]. Induced expression
of some but not all of the multiple CaM isoforms in a
plant tissue in response to certain stimuli has been
reported [10,23] thus, competition among CaM isoforms
for target proteins may be found. It is fascinating that the
OsCam1-1, OsCam1-2, and OsCam1-3 genes encode iden-
tical proteins. How these protein sequences have been
maintained with the natural selection pressure through-
out evolution has no clear answer yet but it is likely that
each of these genes has physiological significance.
Rice CaM-like (CML) proteins
The remaining proteins from the phylogenetic analysis in
Figure 2 were named CaM-like or CML according to the
classification by McCormack and Braam [11]. Like CaM,
these proteins are composed entirely of EF hands with no
other identifiable functional domains. A summary of their
characteristics is shown in Table 1. They were named
according to their percentages of amino acid identity with
OsCaM1 which were calculated by dividing the number of
identical residues by the total number of residues that had
been aligned to emphasize the identical amino acids.
These proteins are small proteins consisting of 145 to 250
amino acid residues and sharing amino acid identity
between 30.2% to 84.6% with OsCaM1. All the CML pro-
teins in group 2 share more than 60% of amino acid
sequence identity with OsCaM1. The CML proteins in
groups 3, 4, and 5 have identities with OsCaM1 that aver-
age 48.2%, 46.9%, and 43.8%, respectively. By the boot-
strapped phylogenetic tree based on amino acid sequence
similarity of these proteins, group 6 CML proteins were
separated into five subgroups: 6a-6e. These proteins share
identities no more than 40.7% with OsCaM1 that average
at 35.6% with the exception of OsCML10 (45.6%). All
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Phylogenetic tree showing the overall relatedness of EF-hand-containing proteins in riceFigure 1
Phylogenetic tree showing the overall relatedness of EF-hand-containing proteins in rice. Alignment of full-length
protein sequences and phylogenetic analysis were performed as described in the "Methods" section. The numbers of EF hands
predicted by InterProScan for each protein are shown as black blocks on the right with their heights proportional to their
numbers of motif. With the exception of two proteins, all proteins indicated by the vertical line labelled "CaM & CML" at the
right share more than 25% amino acid identity with OsCaM1 and were selected for further analyses. Positions of CBL and CPK
members are also shown along the tree to emphasize their separation.
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Table 1: Characteristics of OsCam and OsCML genes and the encoded proteins
Name Locus
1
Chr
2
cDNA length
3
Amino Acids
4
EF hands
5
% of Met
6
Identity to OsCaM1(%)
7
Cys 27
8
Lys 116
9
Prenylation
10
Myristoylation
11
References
OsCam1-1 LOC_Os03g20370 3 450 149 4 6.0 100.0 + + [10]
OsCam1-2 LOC_Os07g48780 7 450 149 4 6.0 100.0 + +
OsCam1-3 LOC_Os01g16240 1 450 149 4 6.0 100.0 + +
OsCam2 LOC_Os05g41210 5 450 149 4 6.0 98.7 + + [10]
OsCam3 LOC_Os01g17190 1 450 149 4 6.0 98.7 + +
OsCML1 LOC_Os01g59530 1 564 187 4 4.3 84.6 + [8,9,10]
OsCML2 LOC_Os11g03980 11 552 183 4 4.9 70.3 +
OsCML3 LOC_Os12g03816 12 552 183 4 4.9 68.9 +
OsCML4 LOC_Os03g53200 3 465 154 4 6.5 68.9 + +
OsCML5 LOC_Os12g41110 12 501 166 4 4.8 62.2 + +
OsCML6 LOC_Os11g37550 11 513 170 4 6.5 53.9 +
OsCML7 LOC_Os08g02420 8 447 148 2 2.8 47.7 +
OsCML8 LOC_Os10g25010 10 576 191 4 5.2 47.0
OsCML9 LOC_Os05g41200 5 468 155 1 3.2 46.1
OsCML10 LOC_Os01g72100 1 558 185 4 4.3 45.6 +
OsCML11 LOC_Os01g32120 1 636 211 4 1.4 44.1
OsCML12 LOC_Os01g41990 1 750 249 4 2.8 43.9
OsCML13 LOC_Os07g42660 7 510 169 4 5.3 43.6
OsCML14 LOC_Os05g50180 5 522 173 4 4.6 43.3
OsCML15 LOC_Os05g31620 5 606 201 4 4.0 40.7
OsCML16 LOC_Os01g04330 1 546 181 4 3.9 40.5
OsCML17 LOC_Os02g39380 2 495 164 4 4.9 37.7 +
OsCML18 LOC_Os05g13580 5 477 158 4 5.7 37.7 +
OsCML19 LOC_Os01g72550 1 441 146 3 7.5 37.2
OsCML20 LOC_Os02g50060 2 525 174 4 4.0 35.3 +
OsCML21 LOC_Os05g24780 5 594 197 3 4.6 35.3
OsCML22 LOC_Os04g41540 4 753 250 4 3.6 35.2
OsCML23 LOC_Os01g72540 1 456 151 3 7.9 35.1
OsCML24 LOC_Os07g48340 7 594 197 3 3.0 33.9
OsCML25 LOC_Os11g01390 11 450 149 3 6.7 33.6
OsCML26 LOC_Os12g01400 12 450 149 3 6.7 33.6
OsCML27 LOC_Os03g21380 3 573 190 3 3.2 33.3
OsCML28 LOC_Os12g12730 12 519 172 4 4.8 33.1 +
OsCML29 LOC_Os06g47640 6 513 170 3 4.1 33.1
OsCML30 LOC_Os06g07560 6 711 236 4 2.1 32.8
OsCML31 LOC_Os01g72530 1 456 151 3 5.3 31.6
OsCML32 LOC_Os08g04890 8 591 196 3 2.6 30.2
1
The Institute of Genomics Research (TIGR) gene identifier number.
2
Chromosome number in which the gene resides.
3
Length of the coding region in base pairs.
4
Number of amino acids of the deduced amino acid sequence.
5
Number of EF hands based on the prediction by InterProScan.
6
Percentage of methionine (M) residues in the deduced amino acid sequence.
7
Number of identical residues divided by the total number of amino acids that have been aligned expressed in percentage.
8
Presence of a cysteine equivalent to Cys26 of typical plant CaMs at residue 7(-Y) of the first EF-hand.
9
Presence of a lysine equivalent to Lys115 of typical plant CaMs.
10
Presence of a putative prenylation site.
11
Presence of a putative myristoylation site.
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Neighbor-joining tree based on amino acid similarities among OsCaM and OsCML proteinsFigure 2
Neighbor-joining tree based on amino acid similarities among OsCaM and OsCML proteins. Tree construction
using the neighbor-joining method and bootstrap analysis was performed with ClustalX. The TIGR gene identifier numbers are
shown and the resulting groupings of CaM and CaM-like proteins designated as 1–6 are indicated on the right. Schematic dia-
grams of the OsCaM and OsCML open reading frames show their EF hand motif distribution.
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members of groups 6b and 6e contain three EF-hand
motifs though with different configurations.
Some important CaM functional features were found
existing only in a few CaM-like proteins. The characteristic
cysteine (C) at residue 7(-Y) of the first EF hand, a hall-
mark of higher plant CaM sequences is absent in all CaM-
like proteins with the exception of three highly conserved
CML proteins, which are OsCML4, OsCML5 and
OsCML6. Based on multiple sequence alignment,
OsCML4, OsCML5, OsCML7 OsCML10, OsCML17,
OsCML18, and OsCML28 are the only CaM-like proteins
that contain lysine at a position equivalent to the Lys116
of CaMs. These features may be indicators of proteins that
serve similar in vivo functions with those of CaMs.
OsCML4 and OsCML5 are the only CaM-like proteins that
possess both of these signature characteristics. However,
another important determinant of CaM function, which is
a high percentage of methionine (M) residues, has been
found in most of the OsCML proteins. The average per-
centage of M residues among OsCMLs is 4.6% compared
with 6.0% in OsCaMs. Considering the usually low per-
centage found in other proteins, the Met-rich feature in
CMLs is likely an indication of their relatedness to CaMs
and possibly similar mechanisms of action i.e. exposure
of hydrophobic residues caused by conformational
changes upon Ca
2+
binding. Nonetheless, some newly
attained characteristics specific to CMLs probably allow
them to fine-tune their Ca
2+
-regulated activity to more
specialized functions.
Of these proteins, three OsCMLs contain an extended C-
terminal basic domain and a CAAX (C is cysteine, A is
aliphatic, and X is a variety of amino acids) motif, a puta-
tive prenylation site (CVIL in OsCML1 and CTIL in
OsCML2 and 3). OsCML1, also known as OsCaM61 was
identified as a novel CaM-like protein by Xiao and col-
leagues [8]. The CML protein was reported to be mem-
brane-associated when it is prenylated and localized in
the nucleus when it is unprenylated [9]. A similar protein
called CaM53 previously found in the petunia also con-
tains an extended C-terminal basic domain and a CAAX
motif which are required for efficient prenylation [24].
Similar subcellular localization of CaM53 depending on
its prenylation state was reported. To locate another pos-
sible modification, all proteins were analyzed by the com-
puter program, Myristoylator [25]. As a result, OsCML20
was predicted to contain a potential myristoylation
sequence. No other potential myristoylated glycines either
terminal or internal were found among the rest of the
OsCML proteins. In addition, to determine the possible
localization of the OsCML proteins, their sequences were
analyzed by targetP [26]. OsCML30 was predicted to con-
tain an endoplasmic reticulum signal sequence and
OsCML21 was predicted to be an organellar protein. For
OsCaM protein sequence similarity with CaM from other speciesFigure 3
OsCaM protein sequence similarity with CaM from
other species. Comparison of the deduced amino acid
sequences of OsCaM1, 2, and 3 with those of other plants,
Mus Musculus CaM (MmCaM), and Saccharomyces cerevisiae
CaM (CMD1p). The sequences are compared with OsCaM1
as a standard; identical residues in other sequences are indi-
cated by a dash (-), and a gap introduced for alignment pur-
poses is indicated by a dot (.). Residues serving as Ca
2+
-
binding ligands are marked with asterisks (*).
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OsCaMs and other OsCMLs, no targeting sequence was
present, thus, they are probably cytosolic or nuclear pro-
teins
Number and structure of EF hand
The number of EF hands in the rice EF-hand-containing
proteins varied from 1 to 4. A summary of the number of
proteins having 1, 2, 3, or 4 EF hands is shown in Figure
4a. It turned out that among the 243 proteins identified,
almost all proteins that contain 4 EF hands were included
in our study or are CPK proteins. All five OsCaM proteins
have two pairs of EF hands with characteristic residues
commonly found in plant CaMs. Consensus sequence of
the Ca
2+
-binding site in the EF hands of plant CaMs com-
pared with OsCaM1, OsCaM2, OsCaM3, vertebrate CaM,
and CMD1p from yeast is shown in Figure 4b. Ca
2+
-coor-
dinating residues among OsCaMs are invariable with
those of the plant CaM consensus sequence. Other resi-
dues in the Ca
2+
-binding loop are also conserved with
only the exception of aspartate (D) at residue 11 of the
fourth EF hand in OsCaM3. Among the twenty EF-hand
motifs of OsCaMs, residues 1(+X) and 3(+Y) are exclu-
sively filled with aspartate (D); residues 5(+Z) are aspar-
tate (D) and asparagine (N); and residues 12(-Z) are
glutamate (E) which is invariably found in this position of
most Ca
2+
-binding EF hand motifs. This residue may
rotate to give bidentate or monodentate metal ion chela-
tion. Glutamate provides two coordination sites, favoring
Ca
2+
over Mg
2+
coordination [27]. Residues 7(-Y) are usu-
ally varied; and residues 9(-X) are aspartate (D), asparag-
ine (N), threonine (T), and serine (S) which are all
normally found among plant CaMs.
Schematic diagrams of each protein sequence with the
predicted EF hands represented by closed boxes are shown
in Figure 2. Among all the identified OsCaM and OsCML
proteins, about three fourths of the EF hands that exist in
pairs (59 pairs) are interrupted by 24 amino acids. The
rest are positioned at a similar distance relative to each
other which is between 25–29 amino acids with the
exception of two pairs that are less than 24 amino acids
apart. Most OsCML proteins have either two pairs or at
least one pair of identifiable EF hands except OsCML9
which has a single EF hand and OsCML7 which appears
to have two separate EF hands. OsCML7 and OsCML9 are
interesting because of their high amino acid identities
with OsCaM1 (47.7% and 46.1%) but they possess only
2 and 1 EF hands; and have relatively low methionine (M)
content (2.8% and 3.2%) compared with other OsCML
proteins, respectively. In addition, 10 OsCML proteins
with one pair of identifiable EF hands have an extra EF
hand that does not pair with any other motif. Pairing of
EF-hand motifs in the CaM molecule helps increase its
affinity for Ca
2+
, therefore an unpaired EF hand in these
proteins may bind Ca
2+
with a lower affinity, or may be
non-functional.
Ligands for Ca
2+
coordination in the EF-hand motifs of
OsCML proteins are highly conserved. One hundred and
thirteen Ca
2+
-binding sequences were aligned and the fre-
quency at which amino acids were found is tabulated in
Figure 4c. Most residues in the Ca
2+
-binding loops are
conserved among OsCML proteins, thus suggesting that
most of them are functional EF hands. Similar to OsCaMs,
residues 1(+X) are exclusively filled with aspartate (D);
and residues 3(+Y) and 5(+Z) are usually aspartate (D) or
asparagine (N). Even though they are not coordinating
residues, glycine (G) at position 6 is absolutely conserved
and hydrophobic residues (I, V, or L) are always found at
position 8 in all 133 EF hands in OsCaM and OsCML pro-
teins. Residues 12(-Z) are mostly glutamate (E) with the
exceptions of an EF hand in OsCML7, OsCML8, and
OsCML13 which have aspartate (D) instead. While
OsCML8 and OsCML13 have two pairs of EF-hand motifs,
OsCML7 possess two separate EF hands with D at residue
12 in the EF-hand motif at the carboxyl terminus. Cates
and colleagues [27], previously reported that mutation of
E12 to D reduced the affinity of EF hands for Ca
2+
in par-
valbumin by 100-fold and raised the affinity for Mg
2+
by
10-fold. It is likely that these EF hands bind Mg
2+
rather
than Ca
2+
but the physiological significance of Mg
2+
-bind-
ing CaM-like activity is still not known.
Cam and CML gene structures and chromosomal
distribution
The structures of the OsCam and OsCML genes were
mapped by comparing their full length cDNAs with the
corresponding genomic DNA sequences. In cases where
no full length cDNA was available, partial cDNA and EST
sequences were used. Their results were compared and
verified with the annotation at the TIGR database. Out of
37 OsCam and OsCML genes, 13 genes contain intron(s)
in their coding regions in which none of these is found in
group 5 and 6 members. It should be mentioned that by
TIGR annotation OsCam1-2 and OsCML1 genes were
shown to have an alternatively spliced mRNA that
encodes a slightly different protein with little supporting
evidence so they were eliminated from our list. Schematic
diagrams depicting intron-exon structures of the intron-
containing genes are shown in Figure 5. All OsCam genes
contain a single intron which interrupts their coding
regions within the codon encoding Gly26, a typical rear-
rangement of all plant Cam genes.
Interestingly, all of the intron-containing OsCML genes
are also interrupted by an intron at the same location as
OsCam genes. The conservation of this intron position
indicates their close relationships which is consistent with
the fact that these genes encode members of the CML pro-
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teins groups 1-4, closely-related CaM-like proteins to
OsCaMs. OsCML1, OsCML2, and OsCML3 genes contain
an additional intron that resides at the codon correspond-
ing to the last residue of genes encoding conventional
CaMs. These proteins have an extended C-terminal basic
domain and a putative prenylation site. The position of
these introns reflects the separation of functional
domains within these proteins and suggests that the
sequences encoding their carboxyl extensions arose later
in the evolution by the fusion of existing Cam genes to the
additional exons. Similarly, OsCML8 and OsCML13
which encode group 3 proteins have the same gene struc-
ture which is the same intron number (6) and location.
The gene duplication event that led to the existence of
OsCML8 and OsCML13 is also supported by the high
degree of amino acid identity (60%) between OsCML8
and OsCML13. In these proteins, one of the six introns
locates within the sequence encoding the third EF-hand
motif, a location comparable to Gly26 of the first EF-hand
motif. This intron is probably the remnant of a duplica-
tion event that originally gave rise to two EF-hand pairs in
these proteins. Interestingly, OsCML8 and OsCML13 are
Characteristics of EF hands in rice proteinsFigure 4
Characteristics of EF hands in rice proteins. (a) Number of EF-hand-containing proteins containing 1, 2, 3 or 4 EF hands.
(b) Residues in the EF hands #1-4 of OsCaMs compared with those of typical plant CaMs, vertebrate CaM (CaMv) and Saccha-
romyces cerevisiae CaM (CMD1p) using a consensus sequence of plant CaMs as a standard; identical residues in other sequences
are indicated by a dash (-), and a gap introduced for alignment purposes is indicated by a dot (.). (c) Residues in Ca
2+
-binding
loops in 32 OsCML proteins shown as the frequency at which an amino acid (shown at the left) is found in each position
(shown at the top). The amino acids most frequently found are indicated by bold letters and shown below as a consensus
sequence along with the positions of residues serving as Ca
2+
-binding ligands indicated in Cartesian coordinates. Bracketed res-
idues are alternative residues frequently found in each position and "x" is a variety of amino acids. Residues serving as Ca
2+
-
binding ligands are marked with asterisks (*).
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two out of only three OsCMLs that contain aspartate (D)
at residues 12(-Z). These observations suggest that the
mutation of E12 to D in OsCML8 and OsCML13 probably
occurred before the duplication event that led to their
existence.
The chromosomal location of each gene was determined
from the annotation at the TIGR database. OsCam and
OsCML genes were found distributed across 11 chromo-
somes of rice as shown in Figure 6 with chromosome 1
having the most numbers (10) of genes. OsCam1-1 was
mapped in chromosome 3, OsCam1-2 in chromosome 7;
OsCam1-3, and OsCam3 in chromosome 1; and OsCam2
in chromosome 5. Their nucleotide sequences share
between 86–90 % identities which are lower than their
amino acid identities (98–100%). Multiple OsCam genes
encoding nearly identical proteins have been maintained
through natural selection suggesting the functional signif-
icance of each gene. OsCam1-1 and OsCam1-2 which
encode identical proteins were mapped to the duplicated
regions of chromosome 3 and 7, respectively. OsCam1-1
and OsCam2 were also located within duplicated genome
segments of their respective chromosomes. These observa-
tions suggest that these pairs of genes are derived from
segmental duplication. In addition, there are many pairs/
groups of OsCML genes which encode proteins that share
a high degree of amino acid identity (≥ 60%). OsCML2/
OsCML3 (98.9% identical) and OsCML25/OsCML26
(100% identical) are the most closely related pairs.
OsCML2 and OsCML3 encode potential Ca
2+
-binding
proteins in group 2 with an absolute conservation of the
C-terminal sequences that contain a prenylation site
(CTIL). OsCML2 and OsCML25; and OsCML3 and
OsCML26 were mapped to the recently duplicated regions
of chromosomes 11 and 12, respectively. Therefore,
OsCML2/OsCML3; and OsCML25/OsCML26 may have
arisen through the segmental duplication event. Other
pairs/groups of closely related CaM-like genes that are
likely to be derived from gene duplication events are
OsCML1/OsCam1-1; OsCML10/OsCML15;OsCML24/
OsCML27; and OsCML19/OsCML23/OsCML31. All mem-
bers in each pair or group have the same number and
positions of EF-hand motifs. The positions of predicted
segmental duplication according to the analyses by TIGR
are illustrated along with the chromosomal locations of
the affected genes in Figure 6. Conversely, OsCML19,
OsCML23 and OsCML31 are arranged in tandem orienta-
tion on chromosome 1 suggesting that they were derived
from tandem duplication. Interestingly, OsCML27 is adja-
cent to OsCam1-1 on chromosome 3 and its duplicated
gene, OsCML24, resides in tandem with OsCam1-2
(OsCaM1-1 and OsCaM1-2 are 100% identical). There-
fore, a local duplication followed by a segmental duplica-
tion possibly occurred.
Comparative analysis of rice and Arabidopsis Cam and
CML genes
The full-length amino acid sequences of rice CaMs and
CMLs and Arabidopsis CaMs and CMLs were subjected to
phylogenetic analysis. Tree construction using the neigh-
bor-joining method and bootstrap analysis was per-
formed with ClustalX [see Additional file 4]. In
Arabidopsis by the neighbor joining tree based on amino
acid similarities, McCormack and Braam [11] divided
CaMs and CMLs into 9 groups. We found that several rice
CaMs and CMLs shared high levels of similarity with Ara-
bidopsis CaMs and CMLs and displayed relationships
among the family members similar to those previously
reported in Arabidopsis as shown in Figure 7. All of
OsCaM proteins in Arabidopsis and rice are highly con-
served (sharing 96.6%–99.3% identity). Interestingly,
both Arabidopsis and rice have three OsCam genes that
encode identical proteins (ACaM2, 3, 5 and OsCam1-1, 1-
2, 1-3). Rice CMLs groups 2, 3, 4, and 5 proteins were
closely related to Arabidopsis CMLs group 2, 5, 3, and 4,
respectively. The more divergent rice CMLs groups 6a to
6e are also distributed among members of Arabidopsis
CML groups 6, 7, 8, 6, and 9, respectively. Apparently,
groups 1 from both species are embedded in groups 2.
These resulted from the arbitrary separation of groups 1
(CaMs) even though group 2 members share very high
degrees of identity (at least 50%) with group 1 proteins.
Because what defines a "true" CaM and distinguishes it
from a CaM-like protein that serves a distinct role in vivo
is still unknown, therefore at the moment, only members
that share extremely high degrees of identity (>97%) were
grouped together to emphasize that they were considered
and are possible "true" CaMs.
Based on amino acid sequence alignments (data not
shown), many of OsCMLs have putative homologues in
Arabidopsis. In group 2, OsCML4 which shares a high
level of identity with AtCML8 and AtCML11 has the same
number (3) and locations of introns except that AtCML11
lacks the first intron. Similarly, AtCML19 and AtCML20
which share a high level of identity with their homologues
(OsCML8 and OsCML13 in group 3) have a similar gene
structure which is the conservation of five out of the six
introns present in their rice counterparts. Interestingly,
AtCML19/20 and OsCML8/13 proteins have aspartate
(D) at residues 12(-Z) in one of their EF hands, though
not on the same hand. AtCML13 and AtCML14, which
were thought to have a common progenitor, have very
high level of identity (74.3% and 70.9%) with group 4
OsCML7 and all have the mutation of E12 to D in an EF
hand corresponding to the third EF hand position. How-
ever, OsCML7 has lost an EF hand corresponding to the
second position while a second E12 to D mutation was
found in AtCML13 and AtCML14. Therefore, similar to
AtCML13 and AtCML14, OsCML7 has only one EF hand
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with canonical amino acids which may result in an
impaired ability to bind Ca
2+
. In OsCML group 5,
OsCML11 is closely similar to AtCML17 and AtCML18
and, interestingly all have a relatively low percentage of
methionine (M) compared with other CML proteins that
share similar levels of identity with CaMs. OsCML11 has
only 1.4% methionine content which suggests that its
mode of action upon Ca
2+
binding is probably different
from the hydrophobic surface exposure upon conforma-
tional changes of CaM.
Previous reports identified 250 EF-hand-containing pro-
teins from the Arabidopsis genome [6]. Seven loci were
defined as Cam genes and 50 additional genes were CML
genes [28]. Here, we identified 243 EF-hand-containing
proteins, five Cam genes and 32 CML genes. Fewer mem-
bers of rice CMLs were identified and several Arabidopsis
CMLs did not fall into any group of the rice proteins prob-
ably because rice OsCML proteins we included in these
analyses had at least 25% identity with typical CaMs com-
pared to 16% in Arabidopsis by McCormack & Braam
(2003). We noticed that all of the Arabidopsis proteins
that did not fall into any group of the rice proteins shared
only 16–30 % identity with typical CaMs. Therefore, both
plants appear to have more or less similar numbers of EF-
hand-containing and CaM-like proteins. Both also have
similar numbers of CPK (34 in Arabidopsis and 29 in rice)
and CBL genes (10 in both Arabidopsis and rice) [13,29].
However, one strikingly different characteristic that we
observed is the three OsCML proteins (OsCML1,
OsCML2, and OsCML3) that have the carboxyl-terminal
CAAX motif for prenylation but none was found in CMLs
from Arabidopsis [11]. It would be interesting to know
what functions these rice proteins possess and how the
prenylation state affects their activity.
Cam and CML expression
Because the presence of cDNA or EST clones indicates
expression of the corresponding genes, we performed
searches against the cDNA/EST rice databases. The
searches revealed that majority of the OsCam and OsCML
genes have corresponding cDNA or EST clones. We have
identified all the EST clones for each of the OsCam and
OsCML genes. Characteristics of their expression can be
inferred according to which libraries the EST clones were
derived from. A summary of the numbers of EST clones
found in different organs is presented in Table 2. Based on
the availability of their EST clones, most OsCam and
OsCML genes are expressed. Some OsCML genes are
highly expressed in specific organs compared with other
genes such as OsCML13 and OsCML18 in floral tissues.
No cDNA or EST clone is available for OsCML6,
OsCML19, OsCML23, and OsCML25. However, it is not
conclusive that these genes do not express relying solely
on the absence of their EST clones. Nonetheless, the avail-
ability of EST clones for the rest of the OsCam and OsCML
genes indicate that they are expressed and indeed are func-
tional genes.
Because five OsCam genes encode only three different pro-
teins, whether or not they have different physiological
functions is an interesting question. Here, we experimen-
tally determined whether the expression of each of the
OsCam genes is restricted to specific organs. Total RNA
was isolated from the leaves, roots, flowers, immature
seeds and calli of rice plants and used to perform reverse
transcription and PCR amplification reactions. Primers
selected by computer analysis of the cDNA and EST
sequences corresponding to these genes are given in Table
3. A control RT-PCR reaction without adding reverse tran-
scriptase was done in parallel with each experimental
reaction to ensure that the product obtained could be
attributed to the product of the reverse transcriptase reac-
tion. Figure 8 shows that bands of the expected sizes based
on each of the gene sequences (698, 526, 551, 201, and
520 base pairs for OsCam1-2, OsCam1-2, OsCam1-3,
OsCam2, and OsCam3, respectively) were detected in all
organs or tissues examined including the leaves and roots
of 2-week old seedlings, mature leaves, flowers, immature
seeds and calli. No band was detected in the control RT-
PCR reactions. It should be noted that the RT-PCR condi-
tions used in this study did not allow quantitative deter-
Schematic representation of the OsCam and OsCML genesFigure 5
Schematic representation of the OsCam and OsCML
genes. Boxes represent exons and lines represent introns.
EF-hand motif #1, #2, #3, and #4 are represented by green,
yellow, blue and red stripes at their positions, respectively.
Groupings of the genes are shown on the right.
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mination, therefore comparison of the expression levels
among different organs or different genes can not be
made. Nevertheless, it can be concluded that all of OsCam
genes were expressed in all organs that we examined.
The expression of closely related Cam genes in a single
organ was not surprising. Several similar occurrences in
other plant species have been reported. In tobacco, all 13
Cam closely related genes were expressed in almost all
organs examined with a few exceptions, notably
NtCam13, which was exclusively expressed in the root
[23]. However, NtCam13 encodes a protein of less than
80% identity to typical plant CaMs. Similarly, ACam1-
ACam5 genes which encode nearly identical proteins were
all expressed in the leaves and siliques of Arabidopsis
[30,31]. While Cam expression is ubiquitous among dif-
ferent cells, protein concentrations may vary in specific
cell types. Immunolocalization studies have shown that
root cap cells and meristematic zones have increased CaM
accumulation [32]. In addition, levels of steady state tran-
scripts of Cam genes have been reported to be modulated
at different developmental stages [33,34] and in response
to external stimuli such as salinity, wind, cold, wounding
and pathogen attack [23,35-37]. OsCam1-1 was shown to
be rapidly and strongly increased in leaves under osmotic
stress [10,38]. Modulation of expression in specific organs
of a CaM isoform possibly serves its roles in a timely fash-
ion.
Conclusion
We identified 243 proteins that possibly have EF-hand
motifs and 37 CaMs and related potential calcium sensor
Chromosomal distribution of the OsCam and OsCML genes in the rice genomeFigure 6
Chromosomal distribution of the OsCam and OsCML genes in the rice genome. The chromosome numbers are
shown at the bottom; horizontal lines represent the respective genes; and the centromeric regions appear constricted. Regions
of the predicted segmental duplications are indicated by grey sections in the chromosomes and lines connecting the affected
loci.
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proteins in the rice genome. The functions of most pro-
teins encoded by these genes are still unknown. Nonethe-
less, the complexity of CaM protein family likely reflects
the importance of Ca
2+
signals in regulating cellular
responses to various cellular stimuli and this family of
proteins potentially plays a critical role. The present
results can lead to further studies on each member of this
family which will be invaluable in understanding the
mechanisms of Ca
2+
-regulated signal transduction path-
ways in rice.
Phylogenetic relationships among rice and Arabidopsis CaM and CML proteinsFigure 7
Phylogenetic relationships among rice and Arabidopsis CaM and CML proteins. Tree construction using the neigh-
bor-joining method and bootstrap analysis was performed with ClustalX based on the amino acid similarities among the pro-
teins. Rice protein names are highlighted with colours representing each group as used in Figure 2 for clarity and groupings of
OsCaM and OsCML proteins are indicated accordingly. OsCaM (group 1) and AtCaM portion of the tree was expanded and
shown in the bottom right corner.
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Methods
Database searches and analyses of gene structures and
chromosomal distribution
Searches of the rice genome at The Institute of Genomic
Research (TIGR) [39] for Interpro Database Matches by
five different methods including HMMPfam, HMMSmart,
BlastProDom, ProfileScan, and superfamily were carried
out. Proteins shown to contain an EF-hand motif or in the
family of Ca
2+
-binding proteins which included domains
PF00036, SM00054, PD000012, PS50222, and protein
family SSP47473, respectively by each method were col-
lected. In addition, BLAST searches (blastp) [40] using the
protein sequences of rice CaM1 [GenBank: NP_912914
]
and CBL3 [GenBank: NP_643248
] as query sequences
against the rice genome were conducted. Nucleotide and
amino acid sequences as well as information regarding
each gene of interest were obtained. Gene annotations at
the Rice Annotation Project Database (RAP-DB) [41] were
also used to confirm the existence and sequences of these
genes. Gene structure and locations were determined by a
comparison of cDNA and genomic DNA sequences
obtained from GenBank and searches of the identified
loci at TIGR. Information from EST sequences was used
when any discrepancy was found. Gene duplication was
determined according to the analysis of chromosomal
segmental duplication of the rice genome by TIGR.
Alignments and tree construction
If necessary, predictions of coding regions were verified
using available EST and cDNA sequences. Deduced
sequences of proteins identified by InterProScan as con-
taining an EF hand were subjected to phylogenetic analy-
sis. Alignments were performed by the multiple sequence
alignment program ClustalX [16] using default settings.
Alignments were carried out and protein trees were con-
structed using the neighbor-joining method [42] with
bootstrap analysis by Clustal X (default settings). A com-
parison of OsCaM proteins with those from other species
by multiple sequence alignment was performed by Clus-
talW. GenBank accession numbers for the sequences used
in the alignment are as follows: ACaM2 [GenBank:
AAA32763
]; HvCaM [GenBank: AAA32938]; T-CaM1
[GenBank: AAC49578
]; ZmCaM [GenBank: CAA52602];
SCaM1 [GenBank: AAA34013
]; PCM5 [GenBank:
AAA85155
]; MmCaM [GenBank: NP_033920]; CMD1p
[GenBank: AAA34504
].
Amino acid identity and motif analyses of proteins
Deduced amino acid sequences CaM and CaM-like pro-
teins were aligned with one another by Align [43] and the
percentage of amino acid identity was calculated by divid-
ing the number of identical amino acids by the total
Table 3: Oligonucleotide primers used in this research
Name Sequence
OsCaM1-1F 5'-GAAGCCAGGCTAAGCCCAGC-3'
OsCaM1-1R 5'-GCAAGCCTTAACAGATTCAC-3'
OsCaM1-2F 5'-CTTCGTTGATCCACTCACCC-3'
OsCaM1-2R 5'-ACACAATCTCCTCTGCCTTA-3'
OsCaM1-3F 5'-CCCCTCGCCGCCTCGCCACC-3'
OsCaM1-3R 5'-CCCATAACCAAATGCTGTCA-3'
OsCaM2-F 5'-GAGGAGGGTTCCCATTAAAT-3'
OsCaM2-R 5'-CGCAAGCTAAGCATCACAAT-3'
OsCaM3-F 5'-CCTTCCTCTCTCTCTCGCTC-3'
OsCaM3-R 5'-CCCCCTGTGTTGATCCAAAT-3'
Table 2: ESTs showing OsCam and OsCML expression in different tissues
Cam/CML name Number of ESTs identified Cam/CML name Number of ESTs identified
leaf root panicle seed callus others Total leaf root panicle seed callus others Total
OsCam1-1 106 17 49 6 14 89 281 OsCML15 51 8 - 3 37 54
OsCam1-2 34 13 17 7 11 38 120 OsCML16 2081221 4386
OsCam1-3 21 5 9 - 9 15 59 OsCML17 1 - 10 - - 6 17
OsCam2 35 7 25 5 17 36 125 OsCML18 11 2 88 - 1 17 119
OsCam3 57 5 30 6 23 29 150 OsCML19 -- - - - - 0
OsCML1 30 - 4 1 2 6 43 OsCML20 -- 7 - - 2 9
OsCML2 -- - - - 1 1 OsCML21 -2 - - - - 2
OsCML3 61 - 26 6 6 22 121 OsCML22 4 - 35 - 2 9 50
OsCML4 20 5 2 2 2 7 38 OsCML23 -- - - - - 0
OsCML5 -- - - 1 2 3 OsCML24 10 4 1 - - 16 31
OsCML6 -- - - - - 0 OsCML25 -- - - - - 0
OsCML7 18 - 6 4 3 33 64 OsCML26 1- 1 - - 1 3
OsCML8 13 1 4 - 1 4 23 OsCML27 2127353068
OsCML9 1- - - - 3 4 OsCML28 -- 1 - - 2 3
OsCML10 16 7 19 - 1 30 73 OsCML29 62 1 - - 4 13
OsCML11 7- 1 - 2 - 10 OsCML30 10 13 7 2 2 21 55
OsCML12 31 1 - - 1 6 OsCML31 1155151946
OsCML13 13 4 109 - 4 12 142 OsCML32 -- 2 - - 2 4
OsCML14 -- - - 2 10 12
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number of amino acid residues of the aligned sequences.
All of the protein sequences were analyzed for EF hands
and other domains using InterProScan [44]. Positions of
the EF hands were located using information from the
prediction by InterProScan and by comparing the com-
plete sequences of all proteins with the plant EF-hand
consensus sequence. All identified EF hand sequences
were aligned with ClustalX and a consensus sequence was
generated. To locate sequences for protein modification
and targeting, computer programs: Myristoylator [45] and
targetP [46] were used.
Expressed Sequence Tags
ESTs corresponding to Cam and CML genes were identi-
fied by performing BLAST searches of the Oryza sativa EST
database and by searching UniGene entries corresponding
to all genes at GenBank [47]. Expression characteristics of
all genes were determined based on the types of library
from which ESTs were derived and from literature reviews.
Analysis by Reverse Transcription Polymerase Chain
Reaction (RT-PCR)
Oryza sativa L. tissues were ground in liquid nitrogen
using chilled mortars and pestles. Total RNA was isolated
according to [48] and used in reverse transcription.
Reverse transcription was primed by oligo(dT)
15
primers
and PCR was carried out using forward and reverse oligo-
nucleotide primers (Operon, Germany) as given in Table
3. The numbers of cycles desired before reaching the pla-
teau phase of amplification were determined for each
gene. PCR amplification by Taq polymerase was con-
ducted using a program of 94°C for 2 minutes, 55°C for
1 minute, and 72°C for 2 minute for OsCam1-1; OsCam1-
2; OsCam2; and OsCam3 and a program of 94°C for 2
Expression pattern of the OsCam genesFigure 8
Expression pattern of the OsCam genes. The total RNA isolated from organs indicated was used in RT-PCR assays either
without (-RT) or with (+RT) the addition of M-MLV reverse transcriptase. The cDNAs were amplified by PCR using gene-spe-
cific primers as shown in Table 3. The products derived from 250 ng of total RNA were separated in agarose gels and visual-
ized by ethidium bromide staining. The sizes of DNA markers in base pairs are shown on the right.
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minutes, 58°C for 1 minute, and 72°C for 2 minute for
OsCam1-3. PCR products were separated by agarose gel
electrophoresis and visualized by ethidium bromide
staining and UV fluorescencing. All enzymes and chemi-
cals for RT-PCR were purchased from Promega (Madison,
WI, USA).
Authors' contributions
BB and TB participated in database searches and extensive
analyses of the gene and protein sequences. BB carried out
the laboratory work and prepared figures and tables. TB
performed data analysis and interpretation, and drafted
the manuscript. Both authors read and approved the final
manuscript.
Additional material
Acknowledgements
This work was supported by the National Center for Genetic Engineering
and Biotechnology at the National Science and Technology Development
Agency, Thailand (under grant no. BT-01-RG-09-4711). BB was supported
by Graduate School, Chulalongkorn University through the thesis fund.
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Additional File 1
A list of all identified EF-hand-containing proteins as an Excel spread-
sheet.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2229-7-4-S1.xls]
Additional File 2
The alignment for the phylogenetic tree in Figure 1 as a ClustalX align-
ment file.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2229-7-4-S2.aln]
Additional File 3
The alignment for the phylogenetic tree in Figure 2 as a ClustalX align-
ment file.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2229-7-4-S3.aln]
Additional File 4
The alignment for the phylogenetic tree in Figure 7 as a ClustalX align-
ment file.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2229-7-4-S4.aln]
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