CLOCK · Mechanisms of The Timing of CAB expression 1 (TOC1) regulation and role of casein kinase 2 (CK2) in the Arabidopsis circadian clock

Final Activity Report Summary - CLOCK (Mechanisms of The Timing of CAB expression 1 (TOC1) regulation and role of casein kinase 2 (CK2) in the Arabidopsis circadian clock)

The research project had as a main goal the identification of mechanisms that regulate TOC1 function. Our objective was the elucidation of the role of the casein kinase 2 (CK2) in the Arabidopsis circadian system. The project was successfully developed with a number of interesting results that have been published in research articles. In the first part of our studies, we have identified CKB4, a CK2 regulatory subunit, as a component of the Arabidopsis circadian system. We have demonstrated that the nuclear-localised CKB4 protein exists in vivo as different isoforms, resulting from phosphorylation on serine residues. Our findings show that the phosphorylated isoforms are the preferred substrate for ubiquitination and degradation by the proteasome pathway. We provide evidence of the involvement of the biological clock in the circadian regulation of CKB4 protein abundance, which itself is important for an accurate control of circadian period by the clock. Over-expression of CKB4 results in elevated CK2 overall activity and period-shortening of clock-controlled genes peaking at different phase angles. We hypothesised that restriction of CKB4 protein phosphorylation and/or degradation to specific phases within the circadian cycle might provide the cell with a fine-tuning mechanism to selectively regulate the CK2 phosphorylation activity on specific substrates. Our studies have allowed us to define a new regulatory loop based on the circadian regulation of CKB4 protein abundance which itself is important for clock function. The results obtained during the first year of the grant have been accepted for publication in - The Plant Journal (Perales et al., 2006, The Plant Journal 46:849-60). In a second part of our studies, we have examined the effects of CKB4 over-expression (CKB4-ox) in a number of clock outputs that are modulated by day-length or photoperiod. We have found a phase-shift in gene expression, shortening of hypocotyl elongation and earlier than wild-type initiation of flowering under short-day conditions. Our studies have shown that the earlier expression phases of the floral induction genes GIGANTEA (GI), FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1) and CONSTANS (CO) correlated with higher abundance of the FLOWERING LOCUS T (FT) transcript under short-days. Matching the period of the external light/dark cycles relative to the endogenous short-period of the CKB4-ox oscillator restores the phase of gene expression and the flowering sensitivity to day-length indicating that a clock defect is responsible for the CKB4-ox phenotypes. Our studies suggest a function for CKB4 very close to the oscillator as the expression of the core components TIMING OF CAB EXPRESSION1 (TOC1) and CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is also altered in CKB4-ox plants. Furthermore, adding several inhibitors of the CK2 activity (e.g. DRB) led to a clear alteration of TOC1::LUC and CCA1::LUC expression indicating a role for CK2 in the regulation of oscillator expression. Based on our results, we propose that oscillator dysfunction is responsible for the period defect of CKB4-ox plants that leads to clock dissonance with the environment and reduced sensitivity to day-length. The results of all these studies have been already accepted for publication in - The Plant Journal (Portolés and Más, The Plant Journal 2007, In press).

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