Time-dependent sequestration of RVE8 by LNK proteins shapes the diurnal oscillation of anthocyanin biosynthesis

Significance The circadian clock plays a crucial role controlling a wide variety of physiologic and metabolic processes in plants. However, the components and mechanisms involved in such regulation remain to be fully elucidated. Here we show that a protein complex composed of the clock components REVEILLE8/LHY-CCA1-LIKE5 (RVE8/LCL5) and the NIGHT LIGHT–INDUCIBLE AND CLOCK-REGULATED (LNK) proteins has a dual regulatory function. The complex acts as a repressor of the clock-controlled anthocyanin biosynthetic pathway at midday but it switches to an activating function in the control of evening-expressed clock genes. The interaction with LNKs impedes RVE8 binding to the promoters of the anthocyanin genes but not to the clock genes, which provides a mechanism by which LNKs antagonize RVE8 function to repress anthocyanin accumulation. Circadian clocks sustain 24-h rhythms in physiology and metabolism that are synchronized with the day/night cycle. In plants, the regulatory network responsible for the generation of rhythms has been broadly investigated over the past years. However, little is known about the intersecting pathways that link the environmental signals with rhythms in cellular metabolism. Here, we examine the role of the circadian components REVEILLE8/LHY-CCA1-LIKE5 (RVE8/LCL5) and NIGHT LIGHT–INDUCIBLE AND CLOCK-REGULATED genes (LNK) shaping the diurnal oscillation of the anthocyanin metabolic pathway. Around dawn, RVE8 up-regulates anthocyanin gene expression by directly associating to the promoters of a subset of anthocyanin biosynthetic genes. The up-regulation is overcome at midday by the repressing activity of LNK proteins, as inferred by the increased anthocyanin gene expression in lnk1/lnk2 double mutant plants. Chromatin immunoprecipitation assays using LNK and RVE8 misexpressing plants show that RVE8 binding to target promoters is precluded in LNK overexpressing plants and conversely, binding is enhanced in the absence of functional LNKs, which provides a mechanism by which LNKs antagonize RVE8 function in the regulation of anthocyanin accumulation. Based on their previously described transcriptional coactivating function, our study defines a switch in the regulatory activity of RVE8–LNK interaction, from a synergic coactivating role of evening-expressed clock genes to a repressive antagonistic function modulating anthocyanin biosynthesis around midday.