Genome-Wide Characterization and Seasonal–Circadian Expression Analysis of CCT Family Genes in Populus

Background: The CONSTANS, CONSTANS-like, and TIMING OF CAB EXPRESSION 1 (CCT) domain proteins are key regulators of flowering time and circadian rhythms in annual plants, but their diversity and temporal expression patterns in perennial trees remain poorly understood. Methods: Here, we performed a genome-wide characterization of CCT family genes and analyzed their seasonal and circadian expression dynamics in Populus. Using an HMM-based search, we identified 49 putative CCT genes (PtCCTs) in the Populus genome and classified them into five subfamilies (COL, CMF, PRR, ALSM and ZIM) based on domain composition and phylogeny. Results: Synteny and duplication analyses showed that most PtCCTs arose from segmental duplication and have predominantly evolved under purifying selection. Promoter analyses revealed a rich repertoire of cis-regulatory elements, with a marked enrichment of light- and hormone-responsive motifs, particularly G-box and ABRE elements, in PtPRR and a subset of PtCOL promoters. Transcriptome data indicated that many PtCCTs display distinct tissue-specific expression patterns, with PtPRRs and PtZIMs being strongly enriched in dormant buds. Seasonal transcriptomes from leaves and shoot apices revealed discrete expression profiles associated with growth, bud set, and winter dormancy, and most PtPRRs showed increasing transcript levels from September to December. Diurnal time-series data further identified 19 PtCCTs with significant rhythmic expression, separating COL and PRR members into night- and day-phased groups. Network analysis using STRING indicated that PtPRRs interact with photoperiodic pathway components such as PtGI, and re-analysis of diurnal data from wild-type and lhy-RNAi hybrid aspen showed that several PtPRRs exhibit phase and amplitude changes when LHY expression is reduced. Conclusions: Together, these results provide a comprehensive overview of the CCT gene family in Populus and highlight PtPRRs and specific PtCOLs as promising candidates that link the circadian clock and light signaling to seasonal growth cessation and bud dormancy in perennial trees.