Multi-omics and functional analyses reveal an HcELF4-HcELF3 circadian module underlying flowering time divergence in Hemerocallis citrina

Abstract Breeding cultivars with staggered flowering time is an urgent objective for the sustainable development of the Hemerocallis citrina industry. However, the circadian clock-mediated regulatory mechanisms underlying flowering time divergence in H. citrina cultivars remain poorly understood, limiting the precise regulation of flowering time. To address this, five H. citrina cultivars with clearly differentiated flowering times were subjected to whole-genome resequencing and transcriptome sequencing. Through the integration of selective sweep analysis, graded-pool analysis, differential expression analysis, weighted gene co-expression network analysis, and functional annotation, HcELF4 and HcELF3 were identified as key candidate regulators associated with flowering time divergence among the five H. citrina cultivars. Both genes exhibited clear diurnal transcriptional rhythms, and their encoded proteins were predominantly localized to the nucleus. Heterologous overexpression of HcELF4 or HcELF3 delayed flowering in Arabidopsis thaliana, accompanied by the repression of AtCO, AtFT, and AtSOC1 and the induction of AtTFL1. In H. citrina, transient overexpression of either gene reduced plant height, downregulated HcCO, HcFT, HcAP1, and HcSOC1, and upregulated HcTFL1, whereas virus-induced gene silencing produced opposite effects. Notably, HcELF4 physically interacted with HcELF3, supporting the existence of an interacting circadian module. Collectively, these findings suggest that the HcELF4-HcELF3 module negatively modulates flowering and contributes to flowering time divergence in H. citrina cultivars. HcELF4 and HcELF3 therefore represent promising molecular targets for breeding H. citrina cultivars with staggered flowering times, with potential applications in bridging gaps in the current flowering period and extending the fresh-bud supply period.