The output pathways are the link between the core oscillator and biological processes regulated by the rhythms generated. In plants, the circadian clock controls a wide range of processes including developmental processes from germination to reproduction and cellular process such as stomatal opening, leaf movement, photosynthesis, and stress responsiveness (Yakir et al., 2007). One of the best understood examples of a circadian clock output pathway is regulation of
Chapter 1 Genetic control of flowering time
photoperiodic control of flowering time through CO.
CO mRNA shows rhythmic expression under both LD and SD and
accumulates to maximum level during the night. However, an additional evening peak is present only in the LD which is thought to be critical to induce flowering of
Arabidopsis in LD (Figure 1.4). The CO mRNA abundance is regulated by a
transcriptional repressor CYCLING DOF FACTOR 1 (CDF1). CDF1 bind directly to the Dof binding sites (AAAG) in the CO promoter to repress CO transcription (Imaizumi et al., 2005; Fornara et al., 2009). This CDF1-mediated repression of CO can be destabilised by FKF1-GI complex and hence release CO for transcription (Sawa et al., 2007). As mentioned before, FKF1 and GI are both part of the circadian clock where FKF1 is a blue light photoreceptor involved in light dependent
degradation and GI is a component of the core oscillator. The transcription of FKF1, GI and CDF1 are all regulated by circadian clock in which FKF1 and GI expressed 8-12h after dawn and CDF1 expression peak at dawn. The LD-specific peak of CO mRNA can be explained by light dependent interaction of FKF1 and GI. It is because this complex will only form in the late afternoon in LD when FKF1 and GI are expressed in the light. This complex will then target CDF1 for ubiquitin-proteasome degradation and release CO for transcription to produce LD-specific daytime peak of CO expression. Nevertheless, ELF3 might act as repressor to CO transcription
independent to CDF1 mechanism. In elf3 mutant, FKF1 and GI expression is elevated and so does CO expression (Kim et al., 2005). It is possible that ELF3 repress CO expression through down regulating FKF1 and GI expression (Yu et al., 2008). On the other hand, in SD, FKF1 and GI expressions peak at night and FKF1-GI complex is unable to form due to absence of light (Imaizumi et al., 2005; Sawa et al., 2007). This will eventually result in CO mRNA only has a night time peak in SD (Figure 1.4).
CO mRNA CO protein LD SD CDF1 FKF1 GI CDF1 PHYB RFI2? PHYA CRY2 CDF1 COP1 SPA1
COP1 SPA1 PHYB COP1 SPA1
RFI2?
Figure 1.4 CO mRNA and protein regulation by daylength in Arabidopsis. CO mRNA level shows diurnal expression rhythms in response to LD and SD. CDF1 act as a transcriptional repressor of CO in the early morning of both LD and SD. FKF1 protein abundance exhibits diurnal rhythm and peak in the mid day. In LD, FKF1 is induced by light to form a FKF1-GI complex to target CDF1 for degradation which eventually releases CO transcriptional inhibition by CDF1 and facilitate the formation of LD-specific daytime peak for CO mRNA. On the other hand, CO protein is stable in light and degraded in the darkness. In the daytime, CO protein is destabilised by PHYB while stabilised by PHYA and CRY2. In the night time, CO protein is degraded by COP1-SPA1 complex but COP1 protein is inhibited by PHYA and CRY2 in the day time. PHYB inhibit CO protein in the daytime through COP1-independent mechanism and maybe via RFI2. In SD, FKF1 expression peaks in the evening which fails to be induced by light and result in only the night time peak of CO mRNA. The translated CO protein in SD is degraded by COP1-SPA1 mediated degradation in the night.
Besides transcriptional regulation of CO mRNA, post-translational regulation of CO protein by light is also essential for control of flowering. CO protein is only stable in the presence of light and rapidly degraded in the dark (Suarez-Lopez et al., 2001). The CO protein stability has been shown to be regulated by photoreceptors. There is an increased abundance of CO protein when flowering is promoted in far-red and blue light which is perceived by PHYA and CRY1/CRY2 respectively (Cerdan and Chory, 2003). However, PHYB is known to delay flowering and no accumulation of CO protein is detected in the red light. Consistent with this idea, phyA and cry1cry2
Chapter 1 Genetic control of flowering time
mutants are late-flowering (Johnson et al., 1994; Devlin and Kay, 2000; Imaizumi et al., 2003) mutants and showed to reduce in CO protein level in the morning and evening of LD. Conversely, phyB early-flowering mutant showed to have increase CO protein abundance (Valverde et al., 2004). Recently, CONSTITUTIVE
PHOTOMORPHOGENIC 1 (COP1) (Jang et al., 2008) and SUPPRESSOR OF PHYA 1 (SPA1) (Ishikawa et al., 2006) has been shown to form an ubiquitin-ligase complex to ubiquitinate CO protein and promote CO degradation in the darkness. This
ubiquitin-mediated degradation of CO protein is inhibited by light through repression of COP1 by PHYA, CRY1, and CRY2 (Wang et al., 2001; Liu et al., 2008b). However, PHYB has shown to promote degradation of CO protein in the morning via
COP1-SPA1 independent mechanism but the underlying mechanism is unclear. RED AND FAR-RED INSENSITIVE 2 (RFI2) encodes for a protein contains RING-finger domain which was normally found in protein involved in protein degradation suggests RFI2 might affects CO protein stability (Chen and Ni, 2006b, a). The rfi2 mutant has increased CO expression and RFI2 expression is reduced in phyB mutant when compared to wild-type plants. This suggests RFI2 might have a role in transcriptional regulation of CO via PHYB. As a result of post-transcriptional regulation of CO, CO protein accumulate in the evening of LD. Reciprocally, no CO protein is produced in SD because CO transcript peak in the night and the translated CO protein is degraded by COP1-SPA1 ubiquitin-ligase complex (Figure 1.4).
The regulation of CO can be explained as a result of external coincidence model in which the circadian clock acts in concert with light to transcriptionally and post-translationally regulate CO activity. As a LD plant, the circadian clock in
Arabidopsis regulates the oscillations of most of the genes involved in monitoring CO mRNA and CO protein to ensure they coincide with the light inducible phase in LD
but not SD (Suarez-Lopez et al., 2001). This also emphasises the importance of circadian clock in the photoperiod pathway of flowering by synchronising endogenous signals (CO protein level) with external signals (light).