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

Abstract

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-REGU-LATED 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 upregulation 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 eveningexpressed clock genes to a repressive antagonistic function modulating anthocyanin biosynthesis around midday.circadian clock | anthocyanin accumulation | transcriptional regulation | protein-protein interaction | Arabidopsis thaliana C ircadian clocks are broadly present in nature and allow organisms to anticipate and prepare for the predictable changes that occur during the day/night cycles (1). Synchronization by the environmental signals ensures proper coordination of metabolism and physiology in many organisms, including plants (2). In Arabidopsis, the molecular architecture depends on a complex regulatory network, in which the morning-expressed single Myb-like transcription factors, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) (3) and LATE ELONGATED HYPOCOTYL (LHY) (4) repress the expression (5) of the evening-phased pseudoresponse regulator, TIMING OF CAB EXPRESSION 1 (TOC1/ PRR1) (6, 7). TOC1 in turn represses CCA1 and LHY (8, 9) as well as the other members of the PRR family (PRR9, 7, and 5) (10) that in turn act as repressors of CCA1 and LHY expression (11). TOC1 also represses LUX ARRHYTHMO (LUX) and EARLY FLOWERING 4 (ELF4) (9), whose protein products interact with EARLY FLOWERING 3 (ELF3) to form the socalled Evening Complex (EC) (12).Chromatin changes at the promoters of the core oscillator genes also play an important role modulating clock gene expression and function (13,14). The single Myb-like transcription factor REVEILLE8/LHY-CCA1-LIKE5 (RVE8/LCL5) (15) antagonizes CCA1 repressing function in the regulation of Histone3 acetylation at TOC1 promoter (16). RVE8 overexpressio...

Circadian Clock and Photoperiodic Flowering in Arabidopsis: CONSTANS Is a Hub for Signal Integration

“…Its function is partially dependent on LNKs ; however, it remains unknown how LNKs act as co-activators due to the lack of functional domains. Intriguingly, LNKs can also bind to CCA1 and LHY , and LNKs act as repressors for anthocyanin biosynthesis genes and work antagonistically with RVE8 (Pérez-García et al, 2015). Given that LNKs act downstream of phyB signaling (Rugnone et al, 2013), LNKs also have roles in the light signaling pathway.…”

The Plant Circadian Clock: From a Simple Timekeeper to a Complex Developmental Manager

“…Another small family of morning-expressed genes that plays an important role in the circadian oscillator is comprised by the NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED (LNK1, 2, 3, and 4) components (Rugnone et al 2013). LNKs interact with the RVEs and have been shown to be able to either antagonize or coactivate RVE8 (Xie et al 2014;Pérez-García et al 2015).…”

MYB-related transcription factors function as regulators of the circadian clock and anthocyanin biosynthesis in Arabidopsis

“…32 Currently, a study has revealed that 2 circadian components NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED (LNK) and RVE8 collaborate to control the anthocyanin metabolic pathway. 33 The MYB-related transcription factor RVE8 can bind directly to the promoter of TIMING OF CAB EXPRESSION1 (TOC1) and facilitate this gene expression by increasing the acetylation level of histone H3, whereas CCA1 inhibits TOC1 expression via the reduction of the histone acetylation level. 34 In addition, the study by Perez-Garcia et al 33 revealed that RVE8 can directly associate with the promoters of genes involved in anthocyanin biosynthesis and regulate the expressions of these genes in response to diurnal oscillations.…”

“…33 The MYB-related transcription factor RVE8 can bind directly to the promoter of TIMING OF CAB EXPRESSION1 (TOC1) and facilitate this gene expression by increasing the acetylation level of histone H3, whereas CCA1 inhibits TOC1 expression via the reduction of the histone acetylation level. 34 In addition, the study by Perez-Garcia et al 33 revealed that RVE8 can directly associate with the promoters of genes involved in anthocyanin biosynthesis and regulate the expressions of these genes in response to diurnal oscillations. From these findings, RVE8 can be considered a circadianrelated regulator of anthocyanin biosynthesis in plants.…”