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.