DNA methylation, an important epigenetic modification, regulates the expression of genes and is therefore involved in the transitions between floral developmental stages in flowering plants. To explore whether DNA methylation plays different roles in the floral development of individual male and female dioecious plants, we injected 5-azacytidine (5-azaC), a DNA methylation inhibitor, into the trunks of female and male basket willow (Salix viminalis L.) trees before flower bud initiation. As expected, 5-azaC decreased the level of DNA methylation in the leaves of both male and female trees during floral development; however, it increased DNA methylation in the leaves of male trees at the flower transition stage. Furthermore, 5-azaC increased the number, length and diameter of flower buds in the female trees but decreased these parameters in the male trees. The 5-azaC treatment also decreased the contents of soluble sugars, starch and reducing sugars in the leaves of the female plants, while increasing them in the male plants at the flower transition stage; however, this situation was largely reversed at the flower development stage. In addition, 5-azaC treatment decreased the contents of auxin indoleacetic acid (IAA) in both male and female trees at the flower transition stage. These results indicate that hypomethylation in leaves at the flower transition stage promotes the initiation of flowering and subsequent floral growth in Salix viminalis, suggesting that DNA methylation plays a similar role in vegetative–reproductive transition and early floral development. Furthermore, methylation changes during the vegetative–reproductive transition and floral development were closely associated with the biosynthesis, metabolism and transportation of carbohydrates and IAA. These results provide insight into the epigenetic regulation of carbohydrate accumulation.

Methyl-CpG-binding domain (MBD) proteins have diverse molecular and biological functions in plants. Most studies of MBD proteins in plants have focused on the model plant Arabidopsis thaliana L. Here we cloned SvMBD5 from the willow Salix viminalis L. by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed the structure of SvMBD5 and its evolutionary relationships with proteins in other species. The coding sequence of SvMBD5 is 645 bp long, encoding a 214 amino acid protein with a methyl-CpG-binding domain. SvMBD5 belongs to the same subfamily as AtMBD5 and AtMBD6 from Arabidopsis. Subcellular localization analysis showed that SvMBD5 is only expressed in the nucleus. We transformed Arabidopsis plants with a 35S::SvMBD5 expression construct to examine SvMBD5 function. The Arabidopsis SvMBD5-expressing line flowered earlier than the wild type. In the transgenic plants, the expression of FLOWERING LOCUS T and CONSTANS significantly increased, while the expression of FLOWERING LOCUS C greatly decreased. In addition, heterologously expressing SvMBD5 in Arabidopsis significantly inhibited the establishment and maintenance of methylation of CHROMOMETHYLASE 3 and METHYLTRANSFERASE 1, as well as their expression, and significantly increased the expression of the demethylation-related genes REPRESSOR OF SILENCING1 and DEMETER-LIKE PROTEIN3. Our findings suggest that SvMBD5 participates in the flowering process by regulating the methylation levels of flowering genes, laying the foundation for further studying the role of SvMBD5 in regulating DNA demethylation.