A Tobacco Calcium/Calmodulin-binding Protein Kinase Functions as a Negative Regulator of Flowering

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.

Members of the Plant CRK Superfamily Are Capable of Trans- and Autophosphorylation of Tyrosine Residues

“…Thus, it is possible that both CRK3 and CRK2 play a role in the phosphorylation of these TFs in plants. On the other hand, previous studies have also suggested that some CRKs are involved in the signal transduction of different extracellular stimuli such as salt, low/high-temperature stress, wounding, and phytohormones in Arabidopsis, tobacco, tomato, and pea (61,(63)(64)(65)(66)(67). Phenome analysis based on genetic techniques might provide significant information regarding the role of CRK-dependent Tyr phosphorylation in plants.…”

Recent Advances in Calcium/Calmodulin-Mediated Signaling with an Emphasis on Plant-Microbe Interactions

“…-mediated signals could regulate a broad range of physiological activities related to plant growth, development, and responses to environmental stimuli (Zhang et al, 2002;Vanoosthuyse et al, 2003;Charpenteau et al, 2004;Hua et al, 2004;Liu et al, 2008;Kim et al, 2009a;Yang et al, 2010;Oh et al, 2012). Among all these CaM-regulated kinases, CCaMK has been widely studied because of its role in symbioses, developmental processes, and stress responses.…”

“…The first subgroup is the CaM-binding protein kinases similar to mammalian CaMK, such as apple calmodulin-binding peptide 1, Nicotiana tobacum Ca 2+ /calmodulin-dependent protein kinase, maize Ca 2+ /calmodulin-dependent protein kinase, and Arabidopsis calmodulin binding protein kinase 1 (AtCBK1) to AtCBK3. These kinases are sometimes called CDPKrelated kinases (CRKs, also called CBKs) because they carry a kinase domain in the N terminus and degenerated, nonfunctional EF hands in their C terminus (Zhang et al, 2002;Zhang and Lu, 2003;Hua et al, 2004;Wang et al, 2004). The second subgroup is Ca 2+ and calcium/calmodulin-dependent protein kinases (CCaMKs), found in most of the higher plants (Patil et al, 1995).…”

Inactivation of Plasma Membrane–Localized CDPK-RELATED KINASE5 Decelerates PIN2 Exocytosis and Root Gravitropic Response inArabidopsis

“…All so far known CRKs, including CRK5, share a conserved C-terminal calmodulin (CaM) binding domain, which overlaps with the kinase autoinhibitory domain (Zhang et al, 2002). Selective CaM binding in the presence of Ca 2+ simulates autophosphorylation but leads only to marginal increase of substrate phosphorylation by CRKs, which are equally active without CaM in the presence of either Ca 2+ or the Ca 2+ chelator EGTA (Lindzen and Choi, 1995;Furumoto et al, 1996;Lu et al, 1996;Wang et al, 2001;Zhang et al, 2002;Hua et al, 2003Hua et al, , 2004Wang et al, 2004;Leclercq et al, 2005).…”