Cis-Regulatory Architecture of PSEUDO-RESPONSE REGULATOR 9 and Its Role in the Integration of the Plant Circadian...

The plant circadian clock synchronizes endogenous rhythms with environmental cues, ensuring optimal growth and development. While the transcription-translation feedback loops (TTFLs) model provides a foundational framework for circadian system, the mechanisms that precisely control the diel and circadian oscillation of key clock genes remain incompletely understood. In particular, the expression of PSEUDO-RESPONSE REGULATOR 9 (PRR9), a dawn-phased core oscillator, needs to be rigorously regulated for proper clock function and environmental responsiveness. Here, we systematically dissected the cis-regulatory architecture of PRR9 promoter using LUC reporter transgenic lines with large-fragment deletions and site-directed mutagenesis. We identified distinct cis-elements that govern PRR9 transcript abundance, rhythmicity and environmental responsiveness. Notably, we discovered a previously uncharacterized 27-bp sequence without any known circadian motifs, which is essential for PRR9 transcriptional rhythmicity, challenging the common view that clock-controlled cis-elements alone determine clock gene expression. These findings refine the TTFLs model and strengthen the link between PRR9 promoter architecture and its role in responding to light and temperature signals to achieve growth advantage.