Functional characterization of OfFD and its alternative splicing in floral transition of Osmanthus fragrans

Alternative splicing (AS) is an important mechanism for regulating gene transcripts through the combination of different exon and intron. FLOWERING LOCUS D (FD), a key flowering gene, is alternatively spliced into two isoforms decided by first intron retention. FD plays a vital role in flowering by interacting with FLOWERING LOCUS T (FT) to form the florigen activation complex (FAC), which in turn activates flowering genes such as APETALA1 (AP1). While key genes like FT are well-studied, the alternative splicing (AS) of the FD gene remains to be further explored, and the functions of its various isoforms remain poorly understood. Here, we cloned the FD homologs, OfFD, isolated from the continuous-flowering cultivar Osmanthus fragrans ‘Sijigui’ and found two abundant transcript variants (OfFDα and OfFDβ) which are caused by AS. First intron retention introduced premature termination codon into OfFDβ, resulted in loss of serine alanine proline (SAP) motif. The expression levels of both FD isoforms showed similar trends under flowering-inhibiting and flowering-promoting temperature. The ratio of OfFDα and OfFDβ is not regulated by ambient temperature. The tissue expression patterns of OfFDα and OfFDβ revealed that OfFDβ is predominantly expressed in the meristem, while OfFDα is enriched in mature tissues. Both FD isoforms localize to the nucleus and have the capacity to promote flowering. FT homologues, OfFTa can interact with OfFDα but not with OfFDβ. Furthermore, OfFDα must form a complex with OfFTa to activate the OfAP1a (AP1 homologues) promoter, whereas OfFDβ can directly activate it. Overall, our work provides an understanding of the function of alternative splicing in FD gene and a deeper view of flowering mechanism in O. fragrans.