Phytochromes are red/far-red light receptors in plants involved in the regulation of growth and development. Phytochromes can sense the light environment and contribute to measuring day length; thereby, they allow plants to respond and adapt to changes in the ambient environment. Two well-characterized signalling pathways act downstream of phytochromes and link light perception to the regulation of gene expression. The CONSTITUTIVELY PHOTOMORPHOGENIC 1/SUPPRESSOR OF PHYA-105 (COP1/SPA) E3 ubiquitin ligase complex and the PHYTOCHROME INTERACTING FACTORs (PIFs) are key components of these pathways and repress light responses in the dark. In light-grown seedlings, phytochromes inhibit COP1/SPA and PIF activity and thereby promote light signalling. In a yeast-two-hybrid screen for proteins binding to light-activated phytochromes, we identified COLD-REGULATED GENE 27 (COR27). COR27 and its homologue COR28 bind to phyA and phyB, the two primary phytochromes in seed plants. COR27 and COR28 have been described previously with regard to a function in the regulation of freezing tolerance, flowering and the circadian clock. Here, we show that COR27 and COR28 repress early seedling development in blue, farred and in particular red light. COR27 and COR28 contain a conserved Val-Pro (VP)-peptide motif, which mediates binding to the COP1/SPA complex. COR27 and COR28 are targeted for degradation by COP1/SPA and mutant versions with a VP to AA amino acid substitution in the VP-peptide motif are stabilized. Overall, our data suggest that COR27 and COR28 accumulate in light but act as negative regulators of light signalling during early seedling development, thereby preventing an exaggerated response to light.

Many molecular and physiological processes in plants occur at a specific time of day. These daily rhythms are coordinated in part by the circadian clock, a timekeeper that uses daylength and temperature to maintain rhythms of approximately 24 hours in various clock-regulated phenotypes. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT INDUCIBLE AND CLOCK REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover connections within the clock and between clock elements and other pathways, here we used affinity purification coupled with mass spectrometry to identify time-of-day-specific protein interactors of the RVE8-LNK1/LNK2 complex in Arabidopsis (Arabidopsis thaliana). Among the interactors of RVE8/LNK1/LNK2 were COLD REGULATED GENE 27 (COR27) and COR28, which coprecipitated in an evening-specific manner. In addition to COR27 and COR28, we found an enrichment of temperature-related interactors that led us to establish a previously uncharacterized role for LNK1 and LNK2 in temperature entrainment of the clock. We established that RVE8, LNK1, and either COR27 or COR28 form a tripartite complex in yeast (Saccharomyces cerevisiae) and that the effect of this interaction in planta serves to antagonize transcriptional activation of RVE8 target genes, potentially through mediating RVE8 protein degradation in the evening. Together, these results illustrate how a proteomic approach can be used to identify time-of-day-specific protein interactions. Discovery of the RVE8-LNK-COR protein complex indicates a previously unknown regulatory mechanism for circadian and temperature signaling pathways.

The timing of many molecular and physiological processes in plants occurs at a specific time of day. These daily rhythms are driven by the circadian clock, a master timekeeper that uses daylength and temperature to maintain rhythms of approximately 24 hours in various clock-regulated phenotypes. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT INDUCIBLE AND CLOCK REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover new connections within the clock and between other pathways, here we use affinity purification coupled with mass spectrometry to discover time-of-day-specific protein interactors of the RVE8-LNK1/2 complex. Among the interactors of RVE8/LNK1/LNK2 were COLD REGULATED GENE 27 (COR27) and COR28, which were coprecipitated in an evening-specific manner. In addition to COR27/28, we found an enrichment of temperature-related interactors that led us to establish a novel role for LNK1/2 in temperature entrainment of the clock. We established that RVE8, LNK1, and either COR27 or COR28 form a tripartite complex in yeast and that the effect of this interaction in planta serves to antagonize transcriptional activation of RVE8 target genes through mediating RVE8 protein degradation in the evening. Together, these results illustrate how a proteomic approach identified time-of-day-specific protein interactions and a novel RVE8-LNK-COR protein complex that implicates a new regulatory mechanism for circadian and temperature signaling pathways.