Analysis of EF-hand-containing proteins in Arabidopsis

Abstract

Background: In plants, calcium (Ca2+) has emerged as an important messenger mediating the action of many hormonal and environmental signals, including biotic and abiotic stresses. Many different signals raise cytosolic calcium concentration ([Ca2+]cyt), which in turn is thought to regulate cellular and developmental processes via Ca2+-binding proteins. Three out of the four classes of Ca2+-binding proteins in plants contain Ca2+-binding EF-hand motif(s). This motif is a conserved helix-loop-helix structure that can bind a single Ca2+ ion. To identify all EF-hand-containing proteins in Arabidopsis, we analyzed its completed genome sequence for genes encoding EF-hand-containing proteins.

Results: A maximum of 250 proteins possibly having EF-hands were identified. Diverse proteins, including enzymes, proteins involved in transcription and translation, protein- and nucleic-acid-binding proteins and a large number of unknown proteins, have one or more putative EF-hands. Phylogenetic analysis identified six major groups that contain some families of proteins.

Conclusions: The presence of EF-hand motif(s) in a diversity of proteins is consistent with the involvement of Ca2+ in regulating many cellular and developmental processes. Thus far, only 47 of the possible 250 EF-hand proteins have been reported in the literature. Various domains that we identified in many of the uncharacterized EF-hand-containing proteins should help in elucidating their cellular role(s). Our analyses suggest that the Ca2+ messenger system is widely used in plants and that EF-hand-containing proteins are likely to be the key transducers mediating Ca2+ action.

Figure 1

(a) A comparison of the number of genes encoding putative EF-hand proteins (green) in different species and their percentage of the total number of genes (blue). At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Sc, Saccharomyces cerevisiae, Hs, Homo sapiens. (b) The number of Arabidopsis proteins having 1, 2, 3, 4, 5 or 6 EF-hands.

Figure 2

Schematic diagrams of representative EF-hand proteins. The number of amino acids is given at the end of each diagram. Domain names are written above the domain except as given in the key. PI-PLC-X(Y) and C2, phosphatidylinositol-specific phospholipase C subdomains. ABI1, ABA-insensitive 1; APC1, Arabidopsis pollenCa²⁺-binding protein; AtCP1, Arabidopsis thaliana Ca²⁺-binding protein; AtFIM1, Arabidopsis thaliana fimbrin 1; AtPLC1, Arabidopsis thaliana phosphatidylinositol-specific phospholipase C; CAM2, calmodulin 2; CaBP22, 22 kd Ca²⁺-binding protein; CBL/SOS3, calcineurinB-like, salt-overly-sensitive protein; CH, calponin homology; CLO1, caleosin1; CPK, Ca²⁺-dependent protein kinase; CRK, CPK-related kinase; GDH2, NAD(H)-dependent glutamate dehydrogenase; GTPase, small GTPase-like protein (At3g63150); KCO1, potassium channel outwardly rectifying protein 1; KIC, KCBP-interacting CCD-1-like protein; MCP, mitochondrial carrier protein (At5g61810); PM129, protein isolated from plasma-membrane enriched library; PPA, protein phosphatase 2A-like protein (At1g03960); PYR, pyridine nucleotide-disulfide oxidoreductase (At2g20800); RbohA, respiratory burst oxidase homology; TCH2 and TCH3, touch-induced proteins. // indicates a break in the protein.

Figure 3

Phylogenetic tree showing the overall relatedness of the EF-hand proteins. All EF-hand proteins were aligned using MEGALIGN (DNAstar) and analyzed using a heuristic method in PAUP 4.08a. Numbers represent the number of times the branch appeared in 100 saved trees. The tree was reduced by hand to show a few representative proteins for each major group. The expanded groups are shown in Figures 4,5,6,7,8,9.

Figure 4

Group I tree showing all proteins included in this group. None has been published in the literature.

Figure 5

Group II tree showing all proteins included in this group. Proteins published in the literature are in color.

Figure 6

Group III tree showing all proteins included in this group. Proteins published in the literature in color.

Figure 7

Group IV tree showing all proteins included in this group. Proteins published in the literature are in color.

Figure 8

Group V tree showing all proteins included in this group. CPKs are in red and CRKs are in green. Subgroups are named as in Figure 10.

Figure 9

Group VI tree showing all proteins included in this group. Proteins published in the literature are in color.

Figure 10

Phylogenetic analysis of CPK, CRK and CCaMK. The EF-hand domains of CPKs, CRKs and other plant and protist protein kinases were aligned in MEGALIGN (DNAstar) and analyzed using a bootstrap method. Numbers are the percentage of bootstrap replicates showing the branch. The accession numbers are listed here in brackets for EtCDPK (CAA96439; 332-487 amino acids), TgTPK4 (AAC02532; 355-501 amino acids), LiCCaMK (AAC49008; 339-520 amino acids) and NtCCaMK (AAD28791; 336-517 amino acids). Et, Eimeria tenella; Li, Lilium longiflorum; Nt, Nicotiana tabacum; Tg, Toxoplasma gondii.