Functional validation of two B-class MADS-box genes HmPI and HmAP3 from Hemerocallis middendorffii

Hemerocallis middendorffii (daylily) is a flowering plant widely used in gardens with high ornamental value and application prospect. However, studies on the flowering regulation mechanism of B-class genes in daylily are limited. As members of B-class MADS-box gene family, PISTILLATA (PI) and APETALA3 (AP3) genes play key roles in the development of petals and stamens. To better understand the molecular mechanisms of PI and AP3 genes in daylily and verify their function during the flowering process, HmPI and HmAP3 genes were isolated and functionally characterized. Subcellular localization analysis showed that HmPI and HmAP3 are nuclear proteins. Quantitative real-time PCR analysis showed that the highest expression of HmPI gene was observed in stamen of daylily, being 2.46- to 2.74-fold of those in leaf, petal, calyx, and pistil. However, the highest expression of HmAP3 gene was found in the leaf, being 1.65-fold higher than that in pistil, followed by petal. Furthermore, Arabidopsis with overexpression of HmPI and HmAP3 genes exhibited earlier flowering time (3 to 4 and 5 to 6 d advanced, respectively) and varying extents of deformation in floral organs, specifically increased or decreased numbers of petals and stamens, with bicarpels and sepals turning to petals. Yeast two-hybrid experiments showed an interaction between HmPI and HmAP3 proteins, as well as between them and the flowering suppressor TERMINAL FLOWER 1 (TFL1) protein. These results suggested that both HmPI and HmAP3 genes are involved in the construction of floral organ morphology, laying a foundation for further studies on the regulatory mechanism of flower development in daylily.