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Ginkgo biloba  has edible, medicinal, and ornamental value. However, the long juvenile phase prevents the development of the  G. biloba  industry, and there are few reports on the identification and functional analysis of genes regulating the flowering time of  G. biloba . EMBRYONIC FLOWER 2 (EMF), an important protein in flower development, functions to promote vegetative growth and repress flowering. In this study, a novel  EMF  gene ( GbEMF2 ) was cloned and characterized from  G. biloba .  GbEMF2  contains a 2,193 bp open reading frame (ORF) encoding 730 amino acids. GbEMF2 harbors conserved VEFS-Box domain by the plant EMF protein. The phylogenic analysis showed that GbEMF2 originated from a polycomb-group (Pc-G) protein ancestor and was a member of the EMF2 protein. The quantitative real-time PCR (qRT-PCR) analysis revealed that  GbEMF2  was expressed in all detected organs, and it showed a significantly higher level in ovulating strobilus and microstrobilus than in other organs. Compared with  emf2  mutant plants, overexpression of  GbEMF2  driven by the CaMV 35S promoter in  emf2  mutant  Arabidopsis  plants delayed flowering but earlier than wild-type (WT) plants. This result indicated that  GbEMF2  repressed flowering in  G. biloba . Moreover, the RNA-seq analysis of  GbEMF2  transgenic  Arabidopsis  plants ( GbEMF2-OE/emf2 ), WT plants, and  emf2  mutants screened out 227 differentially expressed genes (DEGs). Among these DEGs,  FLC ,  MAF5 , and  MAF5-1  genes were related to flower organ development and regulated by  GbEMF2 . In addition, some genes participating in sugar metabolism, such as  Alpha-amylase 1  ( AMY1 ),  BAM1 , and  Sucrose synthase 3  ( SUS3 ) genes, were also controlled by  GbEMF2 . Overall, our results suggested that  GbEMF2  negatively regulates flowering development in  G. biloba . This finding provided a foundation and target gene for shortening the Ginkgo juvenile period by genetic engineering technology.

Functional Characterization of the EMBRYONIC FLOWER 2 Gene Involved in Flowering in Ginkgo biloba