Chrysanthemum lavandulifolium homolog ClMAD1 modulates the floral transition during temperature shift

Flower initiation and development in chrysanthemum requires accurate cell division and expansion, which are regulated by a complex network involved with many cell cycle regulators. Mitotic arrest deficiency 1 (MAD1) is a key component of spindle assembly checkpoint, which was reported to regulate floral transition in Arabidopsis through the interaction with Suppressor of FRIGIDA 4 (SUF4) to modulate Flowering Locus C (FLC) transcription. So far, no MAD1, SUF4 and FLC homologous genes, and related studies were reported in chrysanthemum. Here we first isolated and clone ClMAD1 and ClSUF4 homologs in Chrysanthemum lavandulifolium. Both ClMAD1 and ClSUF4 localized in nuclei, and their expression varied in different tissues. Overexpression of ClMAD1 in Arabidopsis promoted endopolyploidization in leaves, and restored cell cycle and flowering phenotypes of mad1 mutants to different extents. Overexpression of ClSUF4 in Arabidopsis resulted in significantly late flowering, which was correlated with enhanced transcription of FLC and reduced transcription of FT gene. ClMAD1 was predicted to have three coiled-coil (CC) domains, which is one more than that in Arabidopsis. Similar to AtSUF4, ClSUF4 was predicted to have two Zn-C2H2 domains. Protein-protein interaction assays revealed that ClSUF4 could interact with AtMAD1, and the third coiled-coil domain of ClMAD1 not the full-length protein. All these data suggest that both ClMAD1 and ClSUF4 are functional homologs. We further isolated their downstream signaling component ClFLC-like (ClFLCl) genes. ClFLCl had high expression in leaves at vegetative growth stage, and significantly reduced after the bolting suggesting a negative role in the floral transition. Long-term cold treatment significantly repressed the transcription of ClFLCl genes. Temperature shift promoted floral transition of chrysanthemum, which is correlated with the fluctuation of ClMAD1, ClSUF4 and ClFLCl transcription. Taken together, we reported a novel machinery on the regulation of floral transition in chrysanthemum that ClMAD1 interacts with ClSUF4 to modulate ClFLCl-mediated floral transition during the temperature shift.