Does CK2 affect flowering time by modulating the autonomous pathway in Arabidopsis?

Abstract

CK2 (Casein kinase II), a ubiquitous Ser/Thr kinase, affects multiple developmental and stress response pathways in Arabidopsis, including flowering time under both long- and short-day conditions through the photoperiod and autonomous pathways. CK2 phosphorylates central clock components, CCA1 and LHY, to modulate circadian clock that regulates flowering time through the photoperiod pathway. However, how CK2 regulates flowering time through the autonomous pathway is still unknown. Analyses of phosphorylation sites using several prediction softwares show that most of the autonomous pathway components have multiple CK2 phosphorylation sites. CK2 might phosphorylate any or all of these components to modulate their activity/stability resulting in altered expression of FLC that drives flowering time through the autonomous pathway.

CK2 (Casein kinase II), a ubiquitous serine/threonine kinase, is implicated to play roles in various physiological processes in plants including circadian rhythms, light responses and hormone signaling. CK2 is encoded by eight genes in Arabidopsis thaliana, four for each of the α (catalytic) and β (regulatory) subunits.¹ The number of genes encoding CK2 kinase as well as the essential nature of CK2 poses a challenge to dissect the roles of individual catalytic and regulatory subunits. In a recent report, we analyzed the roles of three of the four CK2 α subunits (α1, α2 and α3) by characterizing single, double and triple mutants generated by crossing homozygous individual T-DNA insertion mutant lines.²

One of the previously unreported biological functions for CK2 α subunits is their involvement in regulating flowering time. CK2 α triple mutant flowered much later than the wild type under both long- and short-day conditions, while the single and double mutants flowered similar to wild type. The triple mutant responded to both exogenous GA treatment as well as prolonged vernalization, suggesting that the α subunit triple mutant is not defective in these pathways. The triple mutant also showed reduced expression of the floral integrators like FT and SOC1 and higher expression of FLC compared with wild type under both long- and short-day conditions. The reduced expression of FT and SOC1 under long day can be partly explained by a reduced expression of CO, suggesting a defect in the photoperiod pathway. This is supported by another recent report that showed CK2 α triple mutant is also defective in circadian clock.³ However, the higher expression of FLC resulting in reduced expression of FT and SOC1 under both long- and short-day conditions is a hallmark characteristic for the autonomous pathway that regulates flowering time. Taken together, these data suggest that CK2 α subunits regulate flowering time through both photoperiod and autonomous pathways.

The involvement of CK2 in regulating circadian clock and photoperiod pathway is better understood. Both α and β subunits regulate the circadian clock by phosphorylating the central clock components, CCA1 and LHY.^4-6 Also, overexpression of two of the CK2 regulatory subunits (CKB3 and CKB4) in Arabidopsis leads to defect in circadian clock and altered flowering under both long- and short-day conditions.^6,7 The CKB4 level is also regulated post-translationally by the circadian clock resulting in altered flowering time.⁷

Despite the above progresses on involvement of CK2 in regulation of circadian clock and photoperiod pathway, how CK2 subunits control the FLC expression through the autonomous pathway remains unknown. The autonomous pathway in Arabidopsis consists of internal signals that regulate floral induction.⁸ The genes implicated in this pathway are known to act by repressing the FLC levels; however, the underlying molecular mechanisms are not completely elucidated. Of those, FPA, FCA and FLK contain an RNA binding domain, while FLD is a histone demethylase and LD is a homeodomain protein that might bind to both DNA and RNA.^9,10 These genes are not known to be regulated at the level of transcription and are preferentially expressed in shoot and root apical regions.¹⁰ One possibility is that CK2 phosphorylates any or all of these autonomous pathway components and regulate their activity. Interestingly, three different phosphorylation site prediction programs (Scansite 2.0, NetPhosK 1.0 and KinasePhos 2.0) showed that all five proteins have putative CK2 phosphorylation sites (data not shown). Since phosphorylation by CK2 has been shown to alter protein function as well as stability, it is possible that these proteins are substrates of CK2 and are regulated post-translationally (Fig. 1). Regulation of the activity and/or stability of the autonomous pathway components by CK2 mediated phosphorylation may explain the higher expression of FLC in the CK2 α triple mutant compared with wild type, resulting in delayed flowering under both long- and short-day conditions.

Figure 1. Model for a role of CK2 α subunits in autonomous pathway. (Top) In wild-type plants, phosphorylation of the proteins involved in autonomous pathway by CK2 leads to repression of FLC expression resulting in promotion of flowering. (Bottom) In CK2 α subunit triple mutant, lack of phosphorylation of the autonomous pathway components results in higher FLC levels resulting in delayed flowering.

In summary, our data and those of others show involvement of CK2 in multiple pathways in plants. In 2003, more than 300 CK2 substrates have been identified in animal system,¹¹ and more are predicted to be identified. In plants, the number of CK2 substrates is still less than 30.^12-15 Given the range of physiological processes CK2 has been shown to affect, there is a high probability of finding new substrates, which will improve our current understanding of CK2’s diverse roles in plant growth and development.

Mulekar JJ, Bu Q, Chen F, Huq E. Casein kinase II α subunits affect multiple developmental and stress-responsive pathways in Arabidopsis. Plant J. 2012;69:343–354. doi: 10.1111/j.1365-313X.2011.04794.x.