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.Genes 2020, 11, 285 2 of 13 CHH (where H is A, C, or T) contexts [3,8,9]. The establishment, maintenance, and removal of methyl groups in these three sequence contexts are implemented via different pathways [2]. In plants, RNA-directed DNA methylation (RdDM) is the main pathway of DNA methylation establishment [2,10,11]. The cytosine-DNA-methyltransferases METHYLTRANSFERASE 1 (MET1) and CHROMOMETHYLASE 3 (CMT3) are key proteins for maintenance of CG and CHG methylation, respectively [1,2,12,13]. The methyltransferases DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), CHROMOMETHYLASE 2 (CMT2) and DECREASE IN DNA METHYLATION 1 (DDM1) are responsible for CHH methylation through the RdDM pathway [14][15][16]. REPRESSOR OF SILENCING 1 (ROS1), TRANSCRIPTIONAL ACTIVATOR DEMETER (DME), DEMETER-LIKE PROTEIN 2 (DML2), and DML3 excise 5mC from all cytosine sequence contexts [17][18][19][20][21][22].In the classic epigenetic model, DNA methylation of promoters is responsible for transcriptional silencing [23,24]. Methyl-CpG-binding domain (MBD) proteins recognize DNA methylation and play important roles in mediating the effects of DNA methylation [25,26]. In Arabidopsis thaliana L., there are 13 MBD genes [27], which can be divided into eight subclasses [28]. Bioinformatics analysis showed that MBD5, MBD6 and MBD7 are unique to dicots [22]. Arabidopsis MBD proteins have different DNA binding abilities [28]. Only AtMBD5, AtMBD6, and AtMBD7 specifically bind to methyla...

Revealing the transitory and local effect of zebularine on development and on proteome dynamics of Salix purpurea

“…In addition to the use of methyltransferase inhibitors, further approaches have been employed to study the mechanisms and implications of DNA methylation. In this concern, the availability of multigenerational epimutants (mutants in the DNA methylation patterns) has unveiled the relevance of methylation and epimutation in plant inheritance, with effects on transcriptomic dynamics ( Chodavarapu et al., 2012 ), whereas the heterologous expression of Salix viminalis methyl-CpG-binding domain protein is able to regulate the methylation and the expression of several genes involved in flowering in Arabidopsis thaliana ( Cheng et al., 2020 ).…”

Genome-Wide Characterization of the Methyl CpG Binding Domain-Containing Proteins in Watermelon and Functional Analysis of Their Roles in Disease Resistance Through Ectopic Overexpression in Arabidopsis thaliana

“…Mutation in AtMBD8 or knockdown of AtMBD11 led to a delay in flowering time, while the atmbd9 mutant showed a significantly earlier flowering time ( Berg et al, 2003 ; Peng et al, 2006 ; Stangeland et al, 2009 ). Overexpression of Salix viminalis L. SvMBD5 led to an early flowering phenotype in transgenic Arabidopsis ( Cheng et al, 2020 ). These observations indicate that the MBD proteins play critical roles in regulation of flowering in plants.…”