Abstract
A tobacco calcium/calmodulin-binding protein kinase (NtCBK1) was isolated and identified. The predicted NtCBK1 protein has 599 amino acids, an N-terminal kinase domain, and shares high homology with other calmodulin (CaM)-related kinases. Whereas NtCBK1 phosphorylates itself and substrates such as histone IIIS and syntide-2 in the absence of CaM, its kinase activity can be stimulated by tobacco CaMs. However, unlike another tobacco protein kinase designated NtCBK2, NtCBK1 was not differentially regulated by the different CaM isoforms tested. The CaM-binding domain of NtCBK1 was located between amino acids 436 and 455, and this domain was shown to be necessary for CaM modulation of kinase activity. RNA in situ hybridization showed that NtCBK1 was highly regulated in the transition to flowering. Whereas NtCBK1 mRNA was accumulated in the shoot apical meristem during vegetative growth, its expression was dramatically decreased in the shoot apical meristem after floral determination, and in young flower primordia. The expression of NtCBK1 was up-regulated to high levels in floral organ primordia. Fluctuations in NtCBK1 expression were verified by analysis of tobacco plants expressing green fluorescent protein under the control of the NtCBK1 promoter, suggesting a role of NtCBK1 in the transition to flowering. This conclusion was confirmed by overexpressing NtCBK1 in transgenic tobacco plants, where maintenance of high levels of NtCBK1 in the shoot apical meristem delayed the switch to flowering and extended the vegetative phase of growth. Further work indicated that overexpression of NtCBK1 in transgenic tobacco did not affect the expression of NFL, a tobacco homologue of the LFY gene that controls meristem initiation and floral structure in tobacco. In addition, the promotion of tobacco flowering time by DNA demethylation cannot be blocked by the overexpression of NtCBK1.
Highlights
Calcium (Ca2ϩ) plays important roles as a second messenger in plant developmental and physiological processes via a group of Ca2ϩ target proteins, including CaM and Ca2ϩ-dependent
Whereas purified NtCBK1 showed basal kinase activity in the absence of added Ca2ϩ or CaM, it could be fully activated in the presence of tobacco CaMs
chimeric CaM-dependent protein kinases (CCaMKs) has a calcium-binding, visinin-like domain with three EF-hand motifs and binds CaM, whereas rice OsCBK shows high CaM binding affinity, but CaM is not required for full activation of this kinase [9]
Summary
Duration of the vegetative phase is independent of day length, and regulated by as yet unidentified developmental signals [21, 22]. Protein kinases have been shown to be involved in many cellular processes, including regulation of metabolism, transcription, cell cycle progression, cytoskeletal rearrangement and cell movement, apoptosis, differentiation, and responses to a variety of stimuli [25, 26]. Several protein kinases, such as protein kinase CK2 and never in mitosis A (NIMA)-like kinase, have been reported to be involved in the regulation of flowering plants. The expression of NtCBK1 in the shoot apical meristem is highly regulated during the transition to flowering as shown by both RNA in situ hybridization and transgenic analyses with GFP driven by the NtCBK1 promoter. Further work indicated that overexpression of NtCBK1 did not affect flowering by influencing either the expression of NFL, a tobacco homologue of LFY that controls meristem initiation and floral structure, or by blocking demethylation, a treatment that promotes flowering in tobacco
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