Heterologous Expression of SvMBD5 from Salix viminalis L. Promotes Flowering in Arabidopsis thaliana L.

Abstract

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.