Analysis of multiple calmodulin isoforms in Arabidopsis thaliana
Gawienowski, Margaret C.
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Permalink
https://hdl.handle.net/2142/21990
Description
Title
Analysis of multiple calmodulin isoforms in Arabidopsis thaliana
Author(s)
Gawienowski, Margaret C.
Issue Date
1994
Doctoral Committee Chair(s)
Zielinski, Raymond E.
Department of Study
Crop Sciences
Discipline
Crop Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Plant Physiology
Language
eng
Abstract
Signal transduction in plant cells is in part mediated by fluctuations in cytosolic calcium levels. These fluctuations are found to alter the activity of cellular proteins that are sensitive to the calcium levels. The exact nature of signal transduction from external stimuli through calcium is not known, but variations in calcium-binding proteins may play a role in this process. One extensively characterized calcium-binding protein is calmodulin (CaM), a 17 kDa protein which binds four calcium ions. Three calmodulin isoforms have previously been characterized in Arabidopsis thaliana, and in this study three additional cDNA sequences encoding calmodulin isoforms have been isolated. These sequences (ACaM-4, -5, and -6) represent members of the Arabidopsis calmodulin gene family distinct from the three DNA sequences previously reported. While ACaM-4 and -6 encode full-length copies of calmodulin mRNAs, the ACaM-5 sequence encodes only a partial length copy lacking 10 amino acids at the amino-terminus of mature calmodulin. Most of the amino acid sequence substitutions in the isoforms occurred in the fourth calcium-binding domain.
The six different calmodulin cDNA sequences each hybridize with unique EcoRI restriction fragments in genomic Southern blots of Arabidopsis DNA, indicating that these sequences were derived from distinct structural genes. The expression of each gene was monitored by polymerase chain reaction amplification assays with gene-specific primers, and by northern slot blot hybridization. The expression levels of each CaM isoform mRNA were found to vary according to the particular cellular environment or after particular environmental cues.
Three CaM isoform cDNAs and two ACaM-6 amino-terminus mutant cDNAs were cloned into pET5a expression vectors for production of proteins in E. coli. The purified isoform proteins showed differential epitope recognition by CaM monoclonal antibodies, most likely resulting from the different structural properties of the isoforms. Each protein isoform was found to decrease mobility in the presence of EGTA, indicating that the isoform proteins alter their conformation upon binding calcium. The functional activity of each CaM isoform was analyzed by activation of NAD kinase. The ACaM-2, -4, and -6 proteins each showed approximately the same activation level of NAD kinase, but an ACaM-6 amino-terminus mutant required ten-fold higher concentrations of protein to activate NAD kinase to a half-maximal rate. This demonstrates the importance of the interaction of the amino-terminus of CaM with NAD kinase in the activation of the enzyme. This CaM isoform sequence analysis, isoform mRNA expression studies, and biochemical characteristics have increased the understanding of the nature of the CaM multigene family in Arabidopsis.
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