Genome-wide identification and analyses of the rice calmodulin and related potential calcium sensor proteins

Abstract

Background: A wide range of stimuli evoke rapid and transient increases in [Ca2+]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 Ca2+-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 Ca2+-signals in regulating cellular responses to stimuli and this family of proteins likely plays a critical role as their transducers.

Figure 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.

Figure 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 diagrams of the OsCaM and OsCML open reading frames show their EF hand motif distribution.

Figure 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 indicated by a dash (-), and a gap introduced for alignment purposes is indicated by a dot (.). Residues serving as Ca²⁺-binding ligands are marked with asterisks (*).

Figure 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 Saccharomyces 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 Ca²⁺-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 Ca²⁺-binding ligands indicated in Cartesian coordinates. Bracketed residues are alternative residues frequently found in each position and "x" is a variety of amino acids. Residues serving as Ca²⁺-binding ligands are marked with asterisks (*).

Figure 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.

Figure 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.

Figure 7

Phylogenetic relationships among rice and Arabidopsis CaM and CML proteins. Tree construction using the neighbor-joining method and bootstrap analysis was performed with ClustalX based on the amino acid similarities among the proteins. 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.

Figure 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-specific primers as shown in Table 3. The products derived from 250 ng of total RNA were separated in agarose gels and visualized by ethidium bromide staining. The sizes of DNA markers in base pairs are shown on the right.