Arabidopsis circadian clock protein, TOC1, is a DNA-binding transcription factor

Abstract

The first described feedback loop of the Arabidopsis circadian clock is based on reciprocal regulation between TIMING OF CAB EXPRESSION 1 (TOC1) and CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY). CCA1 and LHY are Myb transcription factors that bind directly to the TOC1 promoter to negatively regulate its expression. Conversely, the activity of TOC1 has remained less well characterized. Genetic data support that TOC1 is necessary for the reactivation of CCA1/LHY, but there is little description of its biochemical function. Here we show that TOC1 occupies specific genomic regions in the CCA1 and LHY promoters. Purified TOC1 binds directly to DNA through its CCT domain, which is similar to known DNA-binding domains. Chemical induction and transient overexpression of TOC1 in Arabidopsis seedlings cause repression of CCA1/LHY expression, demonstrating that TOC1 can repress direct targets, and mutation or deletion of the CCT domain prevents this repression showing that DNAbinding is necessary for TOC1 action. Furthermore, we use the Gal4/UAS system in Arabidopsis to show that TOC1 acts as a general transcriptional repressor, and that repression activity is in the pseudoreceiver domain of the protein. To identify the genes regulated by TOC1 on a genomic scale, we couple TOC1 chemical induction with microarray analysis and identify previously unexplored potential TOC1 targets and output pathways. Taken together, these results define a biochemical action for the core clock protein TOC1 and refine our perspective on how plant clocks function.ost organisms that experience day/night cycles have a circadian clock that phases cellular processes and behavior to specific times of day while also anticipating daily diurnal changes to confer a fitness advantage (1). The basic molecular architecture of most clocks consists of negative-feedback loops where positive and negative components control each other's expression to generate oscillations with an approximate 24-h period (1).At the core of the Arabidopsis clock, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) (2) and LATE ELONGATED HY-POCOTYL (LHY) (3) are morning-expressed Myb transcription factors that directly bind the evening element (4, 5) in the promoters of evening-expressed genes and act as transcriptional repressors. CCA1 and LHY have redundant functions (6, 7), are often coexpressed, and make up the negative arm of the first described feedback loop in Arabidopsis by binding the promoter of TIMING OF CAB EXPRESSION 1 (TOC1) (4). Genetically, TOC1 is the positive component of this feedback loop because CCA1/LHY morning reactivation is dependent on TOC1 (4), yet TOC1 overexpression also results in lower CCA1/LHY expression (8, 9), confusing our understanding of TOC1's role in the core clock feedback loop. TOC1 is an evening-expressed protein (10) that is part of a five-member family called the PSEUDO-RESPONSE REGULATORS (PRRs) that are expressed in succession from the morning to the night in the order PRR9, PRR7, PRR5, PRR3, and then TOC1 (11). Sequence similari...

PSEUDO RESPONSE REGULATORs stabilize CONSTANS protein to promote flowering in response to day length

“…This elegant system directly couples CO stability and function in photoperiodic flowering to cycling circadian‐clock components, ensuring that CO only binds to and activates FT transcription at specific times during the day dependent on day length. Since CO and PRRs contain similar CCT DNA‐binding domains (Matsushika et al , 2000; Gendron et al , 2012), PRRs might also bind along with CO to the FT promoter and contribute to the control of FT transcription, as was recently shown for the interaction between PRRs and PIF transcription factors on their target genes (Soy et al , 2016; Zhu et al , 2016). Morning FT expression is more pronounced when plants are exposed to shade (Wollenberg et al , 2008), suggesting that specific PRRs might also control this process by coupling a distinct flowering signal to CO protein stability or FT induction in the morning.…”

LNK genes integrate light and clock signaling networks at the core of the Arabidopsis oscillator

“…The plant circadian clock is based on interlocking transcriptional feedback loops in which the morning clock factors CCA1 and LATE ELONGATED HYPOCOTYL (LHY) repress the expression of evening clock genes such as TOC1/PRR1 (28). In addition, CCA1 and LHY also promote the expression of PRR9 and PRR7 (29), which, sequentially with PRR5 and TOC1/PRR1, repress CCA1 and LHY expression throughout the remaining of the day and early night (30)(31)(32).…”

“…CCA1 and LHY then promote the expression of PRR9 and PRR7 (29), whereas LNK1 and LNK2 act later during the day to activate clock genes with peak expression in the afternoon, such as PRR5 and ELF4. Simultaneously, members of the TOC1/PRR family repress these morning genes throughout the afternoon and beginning of the night (30)(31)(32)40). Finally, the progressive reduction in TOC1/PRR levels leave CCA1, LHY, and the LNK genes poised to respond again to light signals that reset the clock every morning (Fig.…”

Spatiotemporal Analysis of Localized Circadian Arrhythmias in Plant Roots

“…Some Arabidopsis pseudo-response regulators (APRRs) respond to cytokinin (Brandstatter and Kieber, 1998;D'Agostino et al, 2000) and are involved in control of the circadian clock (Nakamichi et al, 2005). The C-terminal motif of APRRs is involved in recognition of certain specific DNA for clock regulation Gendron et al, 2012). Moreover, the response regulators (RRs) accumulate near the lateral roots, where they regulate cytokinin signaling (Mason et al, 2004;Kushwah et al, 2011).…”