Abstract
Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.
Highlights
In plants, hormonal and environmental signals control many aspects of growth and development
One group of clones was represented by three independent clones (PCBP4, PCBP7, and PCBP25) of the same gene but differing in their length
The distribution of PCBP-GUS is very similar to NIa-GUS, where GUS is fused to a known nuclear protein (Fig. 8, GUS-NIa). These results suggest that PCBP is a nuclear protein
Summary
CaM, calmodulin; PCBP, potato calmodulin-binding protein; CBP, calmodulin-binding protein; KCBP, kinesin-like calmodulin-binding protein; GUS, -glucuronidase; GA, gibberellic acid; IPTG, isopropyl-1-thio--D-galactopyranoside; DAPI, diamidinophenylindole; NLS, nuclear localization signal(s). Low light intensity delays tuberization even in short day conditions, and as is the case with nitrogen levels, both GA and carbohydrate have been implicated as mediators [30]. The expression patterns of these genes differed in various tissues [16, 37] These studies indicate regulation of tuberization by Ca2ϩ/CaM. Fusion of the Nterminal region of PCBP to a cytosolic reporter gene targeted the fusion protein to the nucleus, indicating a nuclear role for PCBP
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