Flowering is an important signal indicating the transformation from vegetative growth to reproductive growth and has a significant effect on plant biomass. Alfalfa is one of the upmost forage crops worldwide, its yield and quality are closely related to flowering time. The optimum harvest time for alfalfa is during the early flowering stage, which could give the highest yield and the best quality. In the current study, an alfalfa flowering related gene, Medicago sativa High Mobility Group Y (MsHMG-Y), was cloned. The gene structure and expression pattern of MsHMG-Y were studied. Function of MsHMG-Y in alfalfa flowering regulation was analyzed. This work could provide theoretical support for mechanism study underlying flowering regulation.

MsHMG-Y was cloned by homology cloning strategy and the amino acid sequence was analyzed by multiple sequence alignment. The phylogenetic tree was also constructed. qRT-PCR analysis was used to detect the expression level of MsHMG-Y in different tissues and different flowering stages. The expression pattern of MsHMG-Y under light, gibberellin (GA3), salicylic acid (SA) or methyl jasmonate (MeJA) treatment were analyzed. The phenotype of MsHMG-Y-overexpressing alfalfa was analyzed, and the expression levels of flowering activators and suppressors were also analyzed.

Phylogenetic analysis showed that MsHMG-Y has the closest relationship with MtHMG-Y in Medicago truncatula. Spatial expression pattern analysis showed that MsHMG-Y was expressed in flowers, stems and leaves, with the highest expression level in flowers and the lowest expression level in leaves in both paternal and maternal alfalfa. In paternal alfalfa with early flowering phenotype, the expression level of MsHMG-Y was the highest at early flowering stage. The highest expression level of MsHMG-Y was detected at flower bud differentiation stage in maternal alfalfa with late flower phenotype. Photoperiod analysis showed that MsHMG-Y was down-regulated after 16-hour light treatment. After 28 hours of light treatment, the expression level of MsHMG-Y was continuously lower than that in the control group, indicating that MsHMG-Y was down-regulated after light treatment. After 50 μmol·L^-1 GA3, 100 μmol·L^-1 SA or 100 μmol·L^-1 MeJA treatment, the expression level of MsHMG-Y was up-regulated compared with the mock treatment. In detail, the expression level of MsHMG-Y was the highest at 1 h under GA3 treatment, which was 3.5 folds higher than control. Under SA treatment, the expression level of MsHMG-Y was the highest at 6 h, which was 24 folds higher than the mock treatment. The expression level of MsHMG-Y was the highest at 3 h under MeJA treatment, which was 11 folds higher than the control. These results indicated that the expression of MsHMG-Y was inducible by the above three hormones. MsHMG-Y-overexpressing alfalfa has late flowering phenotype. The expression levels of flowering activator genes were down-regulated in MsHMG-Y-overexpressing alfalfa, while the expression levels of flowering inhibitor genes were up-regulated. Among these genes, expression of flowering activator genes MsPHYA, MsGI and MsFTa1 was significantly down-regulated by 4.9 folds, 3.9 folds and 2.8 folds respectively, and the expression level of flowering inhibitor genes MsTEM and MsSVP was increased by 2.5 folds and 1.9 folds, respectively.

The expression of MsHMG-Y is inducible by photoperiod and exogenous hormone treatment, including GA3, SA and MeJA. Overexpression of MsHMG-Y in alfalfa resulted in delayed flowering time. MsHMG-Y plays an important role in regulatory mechanism underlying late flowering in alfalfa.