Proteasomal regulation of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) stability is part of the complex control of CCA1

Abstract

The circadian (~24 h) clock has an enormous influence on the biology of plants and controls a plethora of processes including growth, photosynthesis, photoperiodic flowering and transcription of more than 30% of the genome. The oscillator mechanism that generates these circadian rhythms consists of interlocking feedback loops. CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) is a single MYB-transcription factor with a key role in the main feedback loop. CCA1 regulation is important for correct oscillator function and may be an important integration point for environmental signals such as temperature and light that entrain the oscillator. Here we show that CCA1 protein stability is controlled by the proteasome and discuss our findings in the context of the different levels of CCA1 regulation.

The circadian system of plants controls a plethora of processes with a period of ~24 h including photosynthesis, growth, shade avoidance, photoprotection, leaf movements, scent production and stomatal opening.¹ Consistent with the role of the circadian clock in the regulation of a wide range of activities, transcription from approximately one third of the genome of Arabidopsis is under circadian control.^2-4 By definition, circadian rhythms persist under constant conditions and are largely insensitive to ambient temperature. In Arabidopsis, as in other organisms, circadian rhythms are generated by an oscillator mechanism comprised of multiple interconnected feedback loops.⁵ This oscillator mechanism is entrained by signals from the environment, primarily changes in light and temperature conditions.

CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) is a single MYB-transcription factor that has been shown to have a key role in the Arabidopsis circadian system. Together with the highly homologous LATE ELONGATED HYPOCOTYL (LHY), and TIMING OF CAB EXPRESSION 1 (TOC1), a member of the PSEUDORESPONSE REGULATOR FAMILY (PRR) family, CCA1 acts in the core oscillator loop. CCA1 and LHY are expressed in the morning. TOC1 represses the expression of CCA1 and LHY and CCA1 and LHY bind the promoter of TOC1 and control its expression.^6-10 CCA1 and LHY also interact with oscillator components in other feedback loops in the oscillator mechanism; for example they repress LUX ARRHYTHMO (LUX), EARLY FLOWERING4 (ELF4), ELF3, PRR7, PRR9, GIGANTEA (GI), JUMONJI DOMAIN CONTAINING5 (JMJD5; also known as JMJD 30)^11-16 and their expression is regulated directly by BROTHER OF LUX ARRHYTHMO (BOA, also known as NOX), PRR5, PRR7 and PRR9^17,18 and, via unknown mechanisms, by GIGANTEA (GI), LUX, PROTEIN ARGININE METHYL TRANSFERASE5 (PRMT5), ELF3 and ELF4.^19-25 In addition to its role in the Arabidopsis oscillator, CCA1 also controls output pathways. CCA1 binding sites (AAAAT/AATCT) know as Evening Elements/CCA1 Binding Sites (EE/CBS) are over-represented in the promoters of circadian controlled genes. Tests have shown that the EE is sufficient to confer rhythms to gene expression and it has been suggested that CCA1 may be directly involved in the regulation of many of the circadian output genes.^26-28 Consistent with the importance of CCA1 in the circadian system, the regulation of CCA1 levels in the cell is crucial for the correct function of the circadian system; mutations in CCA1 cause a shortened period and overexpression of CCA1 results in arrhythmia.^29-31

In this paper we examine whether CCA1 protein levels in the cell are regulated by the 26S proteasome. In previous experiments we have transformed a CCA1-null mutant with epitope and fluorescence-tagged CCA1 under the control of its own promoter (CCA1_pro:CCA1-HA-YFP)³² and showed that the construct restores nomal circadian rhythms to the mutant. Here we have used these CCA1_pro:CCA1-HA-YFP cca1–1 plants to study CCA1 stability in vivo and in vitro. Figure 1A shows that in an in vitro degradation assay on two-week-old CCA1_pro:CCA1-HA-YFP cca1–1 plants, CCA1-HA-YFP was rapidly degraded at room temperature. However, the proteasome inhibitors MG132 and ALLN inhibited the degradation process (Fig. 1B). In parallel experiments, we examined the stability of CCA1-HA-YFP in vivo. CCA1_pro:CCA1-HA-YFP cca1–1 plants were grown for two weeks in LD and then at ZT 2.5, vacuum infiltrated with DMSO or DMSO + MG132 and kept in the light (LL) or transferred to the dark (DD). Figure 2A shows that the levels of CCA1-HA-YFP were not significantly affected by light or dark (Fig. 2A). However, two hours after vacuum infiltration, the control, DMSO-treated, plants had significantly lower levels of CCA1-HA-YFP protein than the MG132-treated plants (Fig. 2B).

Figure 1. Stability of CCA1-HA-YFP protein in vitro. Protein degradation assays were performed as previously described.³³ For the experiments with inhibitors, 2% DMSO (the solvent control), 40 µM MG132 or ALLN or 2 mM PMSF were added. Extracts were incubated at room temperature (23°C) for the time described and then degradation reactions were stopped using SDS protein gel loading buffer. Western blotting was performed as previously described.³²

Figure 2. Stability of CCA1-HA-YFP protein in vivo. (A and B) Two-week-old plants grown MS supplemented with 1% sucrose were grown in LD before being vacuum-infiltrated at ZT 2:30 with liquid MS medium supplemented with 3% sucrose and (A) 0.5% DMSO or (B) 0.5% DMSO + 50 µM of MG132. Following the vacuum-infiltration, some of the DMSO-treated seedlings were frozen immediately (0 h, ZT 2:30) and the rest were incubated with gentle shaking (50 rpm) in the medium at room temperature in the light (LL) or dark (DD), before being harvested and frozen in liquid nitrogen at the times indicated. Western blotting was performed as previously described.³² The experiment was repeated four times with similar results.

Our results show that CCA1, in common with several other Arabidopsis oscillator components including LHY, PRR5, PRR7, TOC1 and GI, is degraded by the proteasome.^33-37 However, like LHY, CCA1 stability appears to be unaffected by light and dark, in contrast with most of the other proteasome regulated oscillator components which show differential stability in light and dark.^35,36,38 Although we have not yet investigated the mechanisms regulating CCA1 proteasomal degradation, degradation of LHY by the proteasome is controlled by DE-ETIOLATED (DET1) which has been shown to negatively regulate the expression of a number of genes including some involved in light signaling pathways.³⁷ Intriguingly, recently research on the smallest free-living Eukaryote, Ostreococcus tauri a photosynthetic organism with a relatively tiny genome, has revealed that it has an oscillator mechanism that consists of a feedback loop between OtCCA1 and OtTOC1.³⁹ Degradation of OtCCA1 by the proteasome is an important aspect of Ostreococcus oscillator function; using inhibitors to block proteasome function resulted in circadian rhythms with a longer period. Moreover, Ostreococcus has a DET homolog which may be involved in regulating OtCCA1 stability. Thus it appears that proteasomal regulation of CCA1 stability may be an evolutionarily conserved aspect of the circadian oscillator in photosynthetic organisms.

From the results presented here and work from a number of other laboratories, it is becoming increasingly clear that the regulation of CCA1 from transcription to post-translation is complex. Figure 3 summarizes what is known so far about the different levels of CCA1 regulation and how these are affected by the environmental conditions that entrain the circadian system. So far it appears that light and temperature mostly affect pre-translational steps. CCA1 expression is controlled both by light and the circadian system.²⁹ Following transcription, CCA1 mRNA stability is regulated by light; CCA1 transcripts are unstable in the dark and in far-red light and stable in blue and red light⁴⁰ suggesting a role for phytochromes and cryptochromes in the regulation of CCA1 transcript stability. Alternative splicing of CCA1 transcripts is controlled by temperature. At higher temperatures a splice variant of CCA1, CCA1β, is produced that makes a protein lacking the MYB DNA-binding domain. CCA1 β acts as a dominant negative regulator interfering with the formation by the functional variant CCA1α of homodimers and heterodimers with LHY.⁴¹ The regulation of CCA1 translation by environmental signals has not yet been investigated, however LHY translation is light induced.⁴² The formation of CCA1 heterodimers with LHY and the transport of CCA1 to the nucleus appear not to be light regulated.^32,43 The only post-translational step that has so far been shown to be affected by the environment is CCA1 phosphorylation by CASEIN KINASE2 (CK2). CCA1 phosphorylation is positively temperature dependent^12,44 and the phosphorylation status of CCA1 regulates its binding to several target genes with an increase in phosphorylation causing a decrease in binding.

Figure 3. Levels of CCA1 regulation.

Although more work needs to be done to fully understand the regulation of CCA1, it appears that the environment may affect CCA1 on different levels. It is likely that this multifaceted control ensures that active CCA1 is present at the correct time of day despite seasonal changes of light and temperature in the plant’s environment.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.