Unveiling previously undescribed circadian clock regulators and action mechanisms via TurboID-based profiling of the LWD1 interactome in Arabidopsis thaliana.

The synchronization of the internal circadian clock with the external 24-h diel cycle is critical for plant growth and fitness. LIGHT-REGULATED WD proteins (LWDs) are essential for sustaining a precise and robust circadian clock in Arabidopsis [Y. Wang et al., Plant Cell 23, 486-498 (2011); J. F. Wu et al., Nat Commun. 7, 13181 (2016)]. However, the action mechanisms by which LWDs orchestrate the expression of clock genes remain largely elusive. We systematically identified 219 LWD1-interacting protein partners at three Zeitgeber times (ZTs) using a TurboID-mediated proximity labeling approach. Selected interacting proteins were experimentally verified as new regulators of the Arabidopsis circadian clock. Our data also support that LWD1-PSEUDO RESPONSE REGULATORS (PRRs)-TOPLESS (TPL) complex represses the morning gene CIRCADIAN CLOCK ASSOCIATED (CCA1), shaping the expression trough of CCA1 in the late afternoon/early evening under diel cycles. Our results revealed a new corepressor role of LWD1, working in conjunction with epigenetic regulators and transcription factors to underpin the precise operation of the circadian clock.