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 XgtlO 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 Ca2+-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.

Cytoplasmic Free Ca2+ in Arabidopsis Roots Changes in Response to Touch but Not Gravity

“…CaM (TCH-1) also belongs to a class of touch-induced ( T C H ) genes, which also encode CaM-like proteins (TCH-2 and TCH-3) (Braam and Davis, 1990;Ling et al, 1991;Braam, 1992;Perera and Zielinski, 1992;Sistrunk et al, 1994;Takezawa et al, 1995) and a xyloglucan endotransglycosylase (TCH-4;Xu et al, 1995). Induction of TCH-3 is blocked when stimulus-evoked changes in [Ca2+], are blocked (Polisensky and Braam, 1996), suggesting one role for touch-induced [Ca2+], changes may be the regulation of gene transcription.…”

Multiple Forms of Calmodulin Genes in Carrot Treated with Fungal Mycelial Walls

“…1B) which are highly conserved in CAM proteins from animal and plant sources. 1,2,11,[16][17][18][19] However, a replacement of an amino acid in the domain was found in cam-203 and cam-205 (Fig. 1B) (Fig.…”

“…9) We have also shown 7,10) that this protein functions in the response-decay processes of cells as well as in the activation of the Ca 2ϩ cascade. Those results strongly suggested that carrot CAM should be composed of several isoforms, although only one CAM gene, cam-1, had been reported 11) from this plant. We have recently isolated several clones of carrot CAM genes and demonstrated 12,13) that the transcriptional activation of the specific CAM genes is induced in carrot cells upon the treatment with either oligogalacturonide elicitor or ethylene.…”

Synthesis and Accumulation of Calmodulin in Suspension Cultures of Carrot (Daucus carota L.)

“…Following addition of EcoRI linkers, the cDNA was cloned in XgtlO. Approximately 3 x 104 unamplified recombinant phage were screened using a barley CaM cDNA (20) as a heterologous probe as described (19). A single positively hybridizing phage was plaque purified and the EcoRI insert subcloned into pBS-SK' plasmid (Stratagene, La Jolla, CA).…”

“…Figure 3A is a restriction map of pCcam-1 and also shows the sequencing strategy; Figure 3B shows the complete nucleotide and amino acid sequence of the pCcam-1 insert. The nucleotide sequence of Ccam-1 shares 70 to 75% identity with those of barley (20), alfalfa (3), and Arabidopsis (19) CaM cDNAs. The derived amino acid sequence is very similar to other plant CaM proteins, with only one conservative amino acid substitution compared with alfalfa (Asp for Glu at position 7) and barley (Ser for Ala at position 10).…”