The vernalization-induced long non-coding RNA VAS functions with the transcription factor TaRF2b to promote TaVRN1 expression for flowering in hexaploid wheat.

Vernalization is a physiological process in which prolonged cold exposure establishes flowering competence in winter plants. In hexaploid wheat, TaVRN1 is a cold-induced key regulator that accelerates floral transition. However, the molecular mechanism underlying the gradual activation of TaVRN1 during the vernalization process remains unknown. In this study, we identified the novel transcript VAS (TaVRN1 alternative splicing) as a non-coding RNA derived from the sense strand of the TaVRN1 gene only in winter wheat, which regulates TaVRN1 transcription for flowering. VAS was induced during the early period of vernalization, and its overexpression promoted TaVRN1 expression to accelerate flowering in winter wheat. VAS physically associates with TaRF2b and facilitates docking of the TaRF2b-TaRF2a complex at the TaVRN1 promoter during the middle period of vernalization. TaRF2b recognizes the Sp1 motif within the TaVRN1 proximal promoter region, which is gradually exposed along with the disruption of a loop structure at the TaVRN1 locus during vernalization, to activate the transcription of TaVRN1. The tarf2b mutants exhibited delayed flowering, whereas transgenic wheat lines overexpressing TaRF2b showed earlier flowering. Taken together, our data reveal a distinct regulatory mechanism by which a long non-coding RNA facilitates the transcription factor targeting to regulate wheat flowering, providing novel insights into the vernalization process and a potential target for wheat genetic improvement.