PMC

Schematic depiction of alternatively spliced AtCBL10 (A) and AtCIPK3 (B) transcripts. The white-boxed regions refer to untranslated mRNA sequences, whereas black boxed domains indicate translated exon sequences. Introns are presented as black lines.

Comparative expression analysis of AtCBL1 and AtCBL9 in response to abiotic stresses. Semiquantitative RT-PCRs were performed with gene-specific primers using cDNAs synthesized from RNA samples isolated from tissues harvested at the indicated time points of cold, salt, or drought treatments. For experimental details, see “Materials and Methods.”

Intron-exon structure of the Arabidopsis CBL (A) and CIPK (B) genes. Introns located within the coding region of the genes are schematically depicted as triangles. Gray triangles refer to introns located between two codons (phase 0), whereas white triangles indicate introns positioned between the first and second nucleotide of a codon (phase 1). Introns, which are conserved within one gene family, are framed.

Chromosomal distribution and schematic representation of segmental chromosomal duplications affecting CBL (A) and CIPK (B) loci. Roman numerals indicate the chromosome numbers, whereas vertical bars labeled with Arabic numerals represent the respective Arabidopsis CBL or CIPK genes. Checkered boxes refer to the chromosomal regions analyzed in detail to identify duplication events. Duplications are indicated by lines connecting the affected loci. Horizontally striped boxes label duplication events, which were followed by subsequent CBL or CIPK gene losses. Centromeric regions are indicated by ellipses.

Phylogenetic relationships of Arabidopsis and rice CIPKs with related SNF-like kinases. Alignment of full-length protein sequences and phylogenetic analyses were performed as described in “Materials and Methods.” The sub-group of CIPK not harboring intron sequences is depicted on gray background. The accession numbers of the Arabidopsis and rice CIPKs are presented in Tables and . Related SNF-like or cAMP-dependent protein kinases from Arabidopsis (AtSnRK2-1, accession no. NP_196476; AtSnRK1-1, accession no. NP_566130; AtKIN11, accession no. T52633), yeast (ScSNF1, accession no. A26030), and rat (RnAMPK2, accession no. Q09137) were included in the analyses as the most closely related non-CIPK-type protein kinases.

Phylogenetic relationships of Arabidopsis and rice CBLs with related calcium-binding proteins. Alignment of full-length protein sequences and phylogenetic analyses were performed as described in “Materials and Methods.” The accession numbers of the Arabidopsis and rice CBLs are presented in Tables and . Arabidopsis CaM 1 (AtCaM1, accession no. NP_198594), human CaM 1 (HsCaM1, accession no. AAA51918), yeast CNB (ScCNB, accession no. P25296), human NCS 1 (HsNCS1, accession no. NP_NP055101), and human recoverin (HsRCV, accession no. BAA19460) were included in the analyses as the most closely related non-CBL-type calcium-binding proteins. Stars indicate myristoylated AtCBLs and potentially myristoylated OsCBLs.

Comparative yeast two-hybrid interaction analysis of AtCBL1 and AtCBL9 with all Arabidopsis CIPKs. The yeast strain SMY3 containing the indicated plasmid combinations were grown on SC medium -L and -W to an A₆₀₀ of 2. Ten microliters of 10-fold dilution series was spotted onto selective (-H, -L, and -W) and nonselective (-L and -W) media. Decreasing cell densities in the dilution series are illustrated by narrowing triangles. LW depicts a representative dilution series on nonselective plates. The observed interaction intensities are illustrated at the right. XX indicates strong interaction (growth detectable to at least 10^-4 dilution). X indicates weak interaction (growth detectable at 10^-3 dilution; –, no interaction).

Analyses of EF hand motif distribution and composition. A, Schematics of the domain and motif organization of AtCBLs. The numbering of the EF hands is depicted on the top of the figure. Short jagged lines indicate experimentally verified myristoylation sites and longer jagged lines point to potential palmitoylation motifs. Gray boxes represent EF hands with a conserved canonical amino acid composition, whereas horizontally striped boxes indicate EF hands with single or double non-oxygen-containing amino acid substitutions. Vertically striped boxes depict EF hands with single or double basic amino acid substitutions and diagonally striped boxes stand for EF hands with basic amino acid and non-oxygen-containing amino acid substitutions. B, Detailed comparisons of EF hand motif sequences of the AtCBLs with EF hands from other calcium-binding proteins. The canonical EF hand consensus sequence and coordinates are depicted in the left bottom corner of the figure. Amino acid residues, which match this consensus sequence within one distance unit, are black shaded. The EF hand consensus sequence was calculated based on the calcium-binding EF hands of human CNB (HsCNB, accession no. AAB08721), GCAP-2 (HsGCAP2, accession no. Q9UMX6), yeast NCS-1 (HsNCS, accession no. NP_055101), and Arabidopsis CaM 1 (AtCaM1, accession no NP_198594) and all canonical calcium-binding EF hands of the AtCBLs.