Alternative splicing of the FLOWERING LOCUS C-like gene MaMADS33 is associated with endodormancy in mulberry.

Alternative splicing (AS) is an important post-transcriptional process that generates multiple mRNA isoforms. FLOWERING LOCUS C (FLC) is a pivotal gene in both the vernalization and autonomous pathways of flowering plants, and MaMADS33 is one of the FLC homologs in white mulberry (Morus alba). Recent studies have revealed that MaMADS33 is involved in endodormancy, but the underlying molecular mechanism remains to be characterized. Here, a comparison of MaMADS33 expression among three mulberry cultivars with different degrees of dormancy revealed a positive association between MaMADS33 expression and dormancy. Further 3' and 5' rapid amplification of cDNA ends (RACE) analyses led to identifying four MaMADS33 isoforms derived from AS and designated MaMADS33-AS1-4. Analysis of their coding potential revealed that MaMADS33-AS1 was a long non-coding RNA. Expression profiling and splicing-efficiency analyses showed that cold stress during endodormancy induced AS of MaMADS33, resulting in a predominance of truncated isoforms, especially MaMADS33-AS1. MaMADS33-AS2 expression was upregulated during both endodormancy and ecodormancy, whereas MaMADS33-AS3 and MaMADS33-AS4 were endodormancy-associated isoforms that were upregulated during endodormancy and then downregulated during ecodormancy. MaMADS33-AS4 was used as bait for a yeast two-hybrid screen because its gene expression was higher than that of MaMADS33-AS3, and mulberry winter-accumulating 18 kDa protein (MaWAP18) was identified as an MaMADS33-AS4 interaction partner. The interaction between MaWAP18 and MaMADS33-AS4 was confirmed by a bimolecular fluorescence complementation assay. These findings offer insight into the role of FLC homologs in the endodormancy of woody plants.