Primary Structures of Arabidopsis Calmodulin Isoforms Deduced from the Sequences of cDNA Clones

Abstract

Complementary DNA (cDNA) clones encoding calmodulin isoforms were isolated from an Arabidopsis leaf lambdagt10 library by screening with cloned barley calmodulin cDNA probes. Two cDNAs, one a 626-base pair partial-length clone (ACaM-1) and one a 1400-base pair full-length clone (ACaM-2), encode calmodulin polypeptides that differ by four conservative amino acid substitutions. None of the amino acid sequence differences occur within the four Ca(2+)-binding domains of the proteins. Whereas the deduced amino acid sequences of the two Arabidopsis calmodulin isoforms share 97% identity, the nucleotide sequences encoding the two isoforms share 87% sequence identity. Most of these nucleotide sequence differences (80%) occur in codon wobble positions. ACaM-1 and ACaM-2 both hybridize with a distinct set of restriction fragments of Arabidopsis total DNA, indicating that they were derived from transcripts of separate genes; these genes are single- or very low-copy in the Arabidopsis genome. Both cDNAs hybridize to messenger RNA (mRNA) species of 0.8 kilobases that are expressed to a greater extent in developing siliques compared with leaves, flowers, and stems. Northern blot and polymerase chain reaction assays both indicate that ACaM-1 mRNA is more highly expressed than ACaM-2 mRNA in developing siliques. The steady-state levels of both isoform mRNAs increase as a result of touch stimulation; the kinetics and extent of increase are comparable for the two mRNAs.