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

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.


Plant Materials
S. viminalis clones were provided by the willow-planting resources of the Tree Physiology Group of the Chinese Academy of Forestry. Young leaf tissue (0.1 g) was frozen in liquid nitrogen and stored at −80 • C for the cloning of SvMBD5.

Molecular Cloning of SvMBD5 by Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA of Salix viminalis leaf tissue was isolated using an EASYspin Plus Plant RNA Kit (Aidlab, Beijing, China). A PrimeScript 1st strand cDNA synthesis kit (Takara, Japan) was used for the synthesis of first-strand cDNA from the RNA. The cDNA was amplified using specific primers (Forward: 5'-ATGTCATTGTCAGCAACTCC; Reverse: 5'-TCAACGCTTTCTGTTACCAT) designed based on the SvMBD5 sequence obtained from transcriptome data for Salix viminalis L.

Conserved Motif and Phylogenetic Analyses of SvMBD5
MEME and Pfam programs were used for the motif analysis of SvMBD5 [37]. A phylogenetic tree was constructed based on protein sequence alignment of MBD5 proteins from S. viminalis, Populus trichocarpa L., S. lycopersicum, Paeonia suffruticosa L., Vitis vinifera L., Gossypium arboretum L., Morus notabilis L., Triticum aestivum L., Helianthus annuus L., and Arabidopsis thaliana L. with the PHYML program [38]. Phylogenetic trees of the MBD protein sequences were constructed using MEGA6 [38].

Subcellular Localization of SvMBD5
The coding sequence (CDS) of SvMBD5 without the terminator codon was cloned into the pEarleyGate101 vector fused with YFP (yellow fluorescent protein) using Gateway Technology (Life Technologies, Carlsbad, CA, USA) ( Figure 1). The Gateway primers for the subcellular localization of SvMBD5 were as follows: Forward: 5'-GGGGACAACTTTGTACAAAAAAGTTGGAATGTCATTGTCAGCAACTCC and Reverse: 3'-GGCGGCCGCACAACTTTGTACAAGAAAGTTGGGTAACGCTTTCTGTTACCAT. The constructs were transiently expressed in Arabidopsis protoplasts as described by Miao et al. [39]. YFP fluorescence was imaged under a confocal laser-scanning microscope (Leica TCS SPII, Leica Microsystems, Wetzlar, Germany).

Generation of SvMBD5-Expressing Arabidopsis Lines
The CDS of SvMBD5 was cloned into plant expression vector pMDC32 using Gateway Technology ( Figure 2). The Gateway primers for the subcellular localization of SvMBD5 were as follows: Forward: 5'-GGGGACAACTTTGTACAAAAAAGTTGGAATGTCATTGTCAGCAACTCC and Reverse: 5'-GGCGGCCGCACAACTTTGTACAAGAAAGTTGGGTATCAACGCTTTCTGTTACCAT. Arabidopsis plants (ecotype Col-0) were transformed with this construct by the floral dip method [40]. Seeds were sowed and plants were cultivated according to Wang et al. [41].

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Arabidopsis plants (ecotype Col-0) were transformed with this construct by the floral dip method 134 [40]. Seeds were sowed and plants were cultivated according to Wang et al. [41].   LR reaction

Transgenic Plant Detection and Phenotype Analysis
Genomic DNA was isolated from Arabidopsis leaves using a MiniBEST Plant Genomic DNA Extraction Kit (TaKaRa, Japan). Transgenic plants were identified by polymerase chain reaction (PCR) analysis using the primers used for cloning. Three independent lines per construct were used for phenotypic analysis. The flowering time of A. thaliana was measured based on the number of rosette leaves as described by Song et al. [42]. Student's t-test was used for the analysis of significant differences relative to control wild-type plants (ecotype Col-0).

Semiquantitative RT-PCR and Quantitative RT-PCR
Total RNA was isolated from the samples using an EASYspin Plus Plant RNA Kit (Aidlab, China). First-strand cDNA was synthesized as described above (2.2.1). The expression of SvMBD5 in Arabidopsis was detected by semiquantitative RT-PCR, and ACTIN8 expression was used as the internal control. The semiquantitative RT-PCR primers were listed in Table 1. Table 1.
Primers used for reverse transcription-polymerase chain reaction (RT-PCR) and reverse-transcription quantitative polymerase chain reaction (qRT-PCR).

Identification and Sequence Characteristics of SvMBD5
We cloned and identified full-length sequences of the coding regions of putative MBD5 homologs from S. viminalis (Figure 3a). The CDS of SvMBD5 was 645 bp long (Supplementary Materials S1), encoding a 214 amino acid protein (Figure 3b). The molecular weight and theoretical pI of SvMBD5 was 23,627.20 Da and 9.32, respectively.

SvMBD5
CTGTCCATCCACCTTCCCTT TAACAGGAGTTACAGGCACAAT TGAACTTTGGAAACGACGTAAC  A phylogenetic tree was constructed based on the sequences of 24 MBD proteins from nine plant species for analysis of the phylogenetic relationships among MBD homologs ( Figure 4). The result showed high levels of conservation between MBD5 and MBD6 in different species. SvMBD5 belongs to the same subfamily as AtMB5 and AtMBD6 and shares the closest evolutionary relationship with PtMBD5. Sequence comparisons with other MBD homologs were performed to validate the identification of SvMBD5 (Figures 3b and 4). There is one conserved methyl-CpG-binding domain in SvMBD5. We detected five motifs among all MBD proteins ( Figure 4 and Table 2). Most MBD5 proteins from the nine species contained highly conserved motifs (motif 1, motif 2, motif 3, and motif 4) ( Figure 4). Motif 1 and motif 2, which were found in the methyl-CpG-binding domain, are located at the N-termini of MBD proteins. Motif 1, which was the core area of the conserved MBD domain, was discovered in all MBD proteins ( Figure 4). Motif 3 and motif 4 were located in the C termini of various MBD proteins (Figure 3b), specifically all MBD5 and MBD6 subfamily members. Motif 5 was only present in AtMBD1, AtMBD2, AtMBD3, AtMBD4, and AtMBD12. proteins from the nine species contained highly conserved motifs (motif 1, motif 2, motif 3, and motif at the N-termini of MBD proteins. Motif 1, which was the core area of the conserved MBD domain, was discovered in all MBD proteins ( Figure 4). Motif 3 and motif 4 were located in the C termini of

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We investigated the expression of SvMBD5 in the leaves and shoot apical meristems (SAMs) of

Expression Analysis of SvMBD5
We investigated the expression of SvMBD5 in the leaves and shoot apical meristems (SAMs) of plants at different developmental stages ( Figure 5). The expression of SvMBD5 in leaves and SAMs exhibited similar dynamic changes during development. SvMBD5 was expressed at relatively low levels during vegetative development, and its expression increased significantly at the floral initiation stage and floral organ development stage. These results suggest that SvMBD5 might play important roles in floral initiation and development.

Subcellular Localization of SvMBD5 Protein
To determine the subcellular localization of SvMBD5, we constructed a YFP-SvMBD5 fusion expression vector, transiently transformed it into Arabidopsis protoplasts, and observed the expression of YFP. Yellow fluorescence from the empty 35S::YFP vector was distributed in the nucleus and cell membrane ( Figure 6F,J), while yellow fluorescence from the 35S::SvMBD5-YFP recombinant expression vector was only observed in the nucleus ( Figure 6A,E). These results indicate that SvMBD5 protein localizes in the nucleus.

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We investigated the expression of SvMBD5 in the leaves and shoot apical meristems (SAMs) of

Heterologous Expression of SvMBD5 Promotes Flowering in A. thaliana
We subjected three transgenic lines (L1, L3, and L6) to phenotypic analysis. We analyzed the expression of SvMBD5 in the transgenic lines by semiquantitative RT-PCR using AtACTIN8 as an internal reference gene. The expression levels of SvMBD5 in transgenic lines L1, L3, and L6 were similar to that of AtACTIN8 (Figure 7a). The flowering time of all three transgenic lines was significantly earlier than that of wild-type Arabidopsis (Figure 7b). These findings suggest that expression of SvMBD5 promotes flowering in Arabidopsis (Figure 7c). expression of SvMBD5 in the transgenic lines by semiquantitative RT-PCR using AtACTIN8 as an internal reference gene. The expression levels of SvMBD5 in transgenic lines L1, L3, and L6 were significantly earlier than that of wild-type Arabidopsis (Figure 7b). These findings suggest that expression of SvMBD5 promotes flowering in Arabidopsis (Figure 7c).

Expression of Key Flowering-Related Genes in SvMBD5 Transgenic Plants
FLC, CONSTANS (CO), and FT play important roles in flowering regulation. To investigate the molecular mechanism by which SvMBD5 promotes flowering, we measured the expression levels of key flowering genes in the leaves of Arabidopsis plants heterologously expressing SvMBD5 (Figure 8). In the SvMBD5 transgenic plants, the expression levels of FT and CO were higher than those of wild-type Arabidopsis at 11 and 21 days after sowing. By contrast, the expression level of the flowering inhibitor FLC was significantly lower in the transgenic plants than in the wild type, but only at 21 days after sowing.
We subjected three transgenic lines (L1, L3, and L6) to phenotypic analysis. We analyzed the 216 expression of SvMBD5 in the transgenic lines by semiquantitative RT-PCR using AtACTIN8 as an 217 internal reference gene. The expression levels of SvMBD5 in transgenic lines L1, L3, and L6 were 218 similar to that of AtACTIN8 (Figure 7a). The flowering time of all three transgenic lines was 219 significantly earlier than that of wild-type Arabidopsis (Figure 7b). These findings suggest that 220 expression of SvMBD5 promotes flowering in Arabidopsis (Figure 7c).      Figure 8. The expression patterns of key flowering genes in transgenic and wild-type Arabidopsis. Least significant difference (LSD) tests were used to determine significant differences between wild type and transgenic plants. Data are mean ± SE, n = 3. Different lowercase letters in each column indicate a significant (p < 0.05) difference between samples.

Expression of DNA Methylation-Related Genes in SvMBD5 Transgenic Plants
Plant flowering is often accompanied by large changes in DNA methylation. To investigate the relationship between SvMBD5 and DNA methylation status, we examined the expression of key genes involved in DNA methylation establishment (RDM1, DRM2), maintenance (CMT2, CMT3, MET1), and removal (DME, ROS1, DML2, DML3) by quantitative RT-PCR (Figure 9). In SvMBD5-expressing plants, the expression levels of the DNA methylation establishment-related genes RDM1 and DRM2, as well as the DNA methylation maintenance gene CMT2, appeared to be higher than those in wild-type Arabidopsis at 11 and 21 days after sowing, but these differences did not reach significant levels. However, the expression levels of CMT3 and MET1 in SvMBD5-expressing plants were lower than those in wild-type plants. The expression levels of CMT3 and MET1 in transgenic vs. wild-type Arabidopsis were significantly different (p < 0.05), indicating that expressing SvMBD5 significantly inhibited the expression of CMT3 and MET1. Among the DNA methylation removal-related genes, the expression levels of DME and DML2 (associated with demethylation) appeared to differ slightly between transgenic and wild-type Arabidopsis, but these differences were not significant. The expression levels of ROS1 and DML3 in transgenic Arabidopsis were significantly (p < 0.05) higher than those in wild-type Arabidopsis. These results indicate that expressing SvMBD5 significantly increases the expression of ROS1 and DML3. MET1), and removal (DME, ROS1, DML2, DML3) by quantitative RT-PCR (Figure 9). In SvMBD5-250 expressing plants, the expression levels of the DNA methylation establishment-related genes RDM1 251 and DRM2, as well as the DNA methylation maintenance gene CMT2, appeared to be higher than 252 those in wild-type Arabidopsis at 11 and 21 days after sowing, but these differences did not reach significant levels. However, the expression levels of CMT3 and MET1 in SvMBD5-expressing plants 254 were lower than those in wild-type plants. The expression levels of CMT3 and MET1 in transgenic 255 vs. wild-type Arabidopsis were significantly different (P < 0.05), indicating that expressing SvMBD5 256 significantly inhibited the expression of CMT3 and MET1. Among the DNA methylation removal-257 related genes, the expression levels of DME and DML2 (associated with demethylation) appeared to 258 differ slightly between transgenic and wild-type Arabidopsis, but these differences were not   to methylation-dense sites, and they cooperate with ROS5/IDM2 to recruit ROS1 [31]. MBD6 binds to the histone deacetylase AtHDA6 in the RdDM pathway [32]. In the current study, heterologously  Figure 9. The expression of methylation-related genes in transgenic and wild-type Arabidopsis. Least significant difference (LSD) tests were used to determine significant differences between groups. Data are mean ± SE, n = 3. Different lowercase letters in each column indicate a significant (p < 0.05) difference between samples.

MBD5 Might Be Involved in Demethylation
DNA methylation is one of the most important epigenetic modifications in plants. MBD proteins function as interpreters of DNA methylation. In Arabidopsis, AtMBD5, AtMBD6, and AtMBD7 share a relatively close relationship. Furthermore, MBD5 and MBD6 belong to the same subfamily, as both of these proteins contain only one methyl-CpG-binding domain. However, MBD7 contains three methyl-CpG-binding domains. The different molecular structures of these proteins might contribute to their functional differences. AtMBD7 participates in active demethylation by specifically binding to methylation-dense sites, and they cooperate with ROS5/IDM2 to recruit ROS1 [31]. MBD6 binds to the histone deacetylase AtHDA6 in the RdDM pathway [32]. In the current study, heterologously expressing SvMBD5 significantly increased the expression of ROS1 and DML3 in Arabidopsis; these genes function in the active demethylation pathway. In A. thaliana, four bifunctional 5mC DNA glycosylases, ROS1, DME, DML2, and DML3, can excise 5mC from all cytosine sequence contexts [17][18][19][20][21][22]. Our results suggest that SvMBD5 might be involved in activating the demethylation of DNA via ROS1 and DML3.
In addition, heterologously expressing SvMBD5 significantly reduced the expression of CMT3 and MET1 genes which function in the methylation maintenance pathway. MET1 is a homolog of the DNA methyltransferase gene DNMT1 in mammals and is responsible for maintaining the methylation of CG sites [1,43]. MET1 is also involved in the establishment of DNA methylation in the RdDM pathway in plants [14]. CMT3 can bind to H3K9me2 at CHG sites to maintain CHG methylation [12,44]. This finding suggests that SvMBD5 might also prevent the maintenance of CG and CHG methylation. ROS1 can antagonize the RdDM pathway and prevent DNA methylation [45], suggesting that DNA methylation and demethylation are synergistic processes. These findings indicate that SvMBD5 can promote DNA demethylation but inhibit DNA methylation and methylation maintenance. This finding not only highlights the antagonism of demethylation on the RdDM pathway, but also indicates that the DNA demethylation pathway inhibits the pathway for maintenance of DNA methylation. Our results provide a powerful reference for further revealing the relationship between active DNA demethylation and DNA methylation maintenance.

How Does the Heterologous Expression of SvMBD5 Promote Flowering?
MBD proteins have different effects on plant growth and development. In Arabidopsis, the MBD8 mutant exhibits delayed flowering and reduced FT and SOC1 expression, whereas the expression of FLC is not significantly altered in this mutant [34]. Arabidopsis MBD9 plants show early flowering and significantly reduced FLC expression. The early flowering phenotype of this mutant was eliminated by overexpressing FLC [35]. These findings suggest that MBD8 and MBD9 affect flowering via different pathways. However, Arabidopsis plants showed no significant phenotypic changes when the expression of AtMBD6 and AtMBD7 was inhibited [27]. Therefore, AtMBD5, AtMBD6, and AtMBD7 may share functional redundancy. In the current study, heterologously expressing SvMBD5 in A. thaliana promoted flowering. In the transgenic plants, FLC was significantly downregulated compared to wild-type plants, whereas FT and CO were significantly upregulated. These results indicate that SvMBD5 promotes flowering by influencing the expression of flowering genes (FT and CO) and a flowering inhibitor gene (FLC).
DNA methylation levels tend to increase in plants during the transition from vegetative to floral stages [46][47][48][49]. In Arabidopsis plants heterologously expressing SvMBD5, demethylation genes (ROS1, DML2) were upregulated and methylation maintenance and establishment genes (CMT3 and MET1, respectively) were downregulated, suggesting that SvMBD5 promotes flowering by relieving or inhibiting the methylation of flowering-related genes. However, a previous study examining whole-genome DNA methylation patterns during flower development in Arabidopsis did not detect significant changes in methylation in FT, CO, or FLC [47]. Moreover, Finnegan demonstrated that DNA methylation is not directly involved in regulating FLC expression in the vernalization pathway [50]. These studies suggest the SvMBD5 does not directly regulate the expression of FT, CO, and FLC. However, a recent study showed that the loss of methylation in the CONSTANS-LIKE2D homolog COL2D was associated with its higher expression levels and promoted flowering in cotton (Gossypium hirsutum) [51]. The different results between Arabidopsis and cotton indicate that the role of DNA methylation in flowering regulation varies among species.

Conclusions
The CDS of SvMBD5 is 645 bp long and encodes a 214 amino-acid protein with one MBD domain. SvMBD5 belongs to the same subfamily as AtMBD5 and AtMBD6 in Arabidopsis. SvMBD5 localizes in the nucleus. Heterologously expressing SvMBD5 in Arabidopsis reduced the expression of the flowering inhibitor gene FLC, increased the expression of flowering genes CO and FT, and promoted flowering. SvMBD5 increased the expression of ROS1 and DML3 in the demethylation pathway and inhibited the expression of MET1 and CMT3 in the methylation establishment and maintenance pathway. These findings suggest that MBD5 is involved in the DNA demethylation pathway.