**2. Results**

#### *2.1. cDNA Isolation and Phylogenetic Tree Analysis of MdFLC*

Nine apple expressed sequence tag (EST) sequences were obtained by a BLAST search in DNA Data Bank of Japan (DDBJ, http://blast.ddbj.nig.ac.jp) using the amino acid sequence of the MADS region of *Arabidopsis FLC*. The contig sequences corresponding to these EST sequences were searched in GDR (https://www.rosaceae.org), a Rosaceae genome database, to obtain four contig sequences. Six EST or contig sequences remained after excluding duplicate sequences. A phylogenetic tree was prepared based on the amino acid sequences of these six genes and MADS-box genes from *Arabidopsis* and apple, and three of the six sequences were assigned to the same group as *Arabidopsis FLC*. Next, one of the three sequences was detected by PCR using cDNA from the juvenile phase of apple seedlings with primers specific to the three sequences and designated as *MdFLC1* (MD05G1037100) [23]. Three kinds of mRNA sequence were obtained by the RACE method and designated as splice variants *MdFLC1a* (accession number LC550081), *MdFLC1b* (LC550082), and *MdFLC1c* (LC550083).

PCR was performed using cDNA from the juvenile phase of apple seedlings with degenerate primers in the MADS region, and 10 kinds of MADS box-like sequences were obtained. One sequence among them was found to be juvenile phase-specific and homologous to *Arabidopsis FLC*; it was designated as *MdFLC3* (MD10G1041100) [23].

Figure 1 shows the amino acid sequence alignment of MdFLC1, MdFLC3, and AtFLC (AF537203) from *Arabidopsis*. MdFLC1c, which was the longest of the *MdFLC1* mRNA variants, and MdFLC3 contained MADS-, K-, I-, and C-domains with AtFLC. A phylogenetic tree of the amino acid sequence of MdFLC1c, MdFLC3, and FLC homologous proteins from other plants was prepared with other MADS-box proteins based on [27] (Figure 2). FLC, SVP, SOC1, AP1, and SEP groups were formed, and MdFLC1c and MdFLC3 were included in the FLC group.


**Figure 1.** Amino acid sequence alignment of MdFLC and AtFLC. The MADS-, K-, I-, and C-domains are underlined. Identical amino acids for five and less proteins are shown in black and gray boxes, respectively.

**Figure 2.** A phylogenetic tree based on the amino acid sequences of FLC, SVP, AP1, SEP, and SOC1 homologs from various species. The tree was constructed by the neighbor-joining method after sequence alignment using the ClustalW program. Branch numbers refer to percentage of replicates that support the branch using the bootstrap method (1000 replicates). The scale bar corresponds to 0.1 amino acid substitutions per residue. The accession numbers of the proteins added to construct the phylogenetic tree are as follows: PtFLC (EU497676), VvFLC (GU133630), PpFLC (KP164015), BrFLC1 (DQ866874), BrFLC2, (DQ866875), BrFLC3 (DQ866876), MdJOINTLESS (DQ402055), AtSVP (AF211171), PsSVP (AY830919), PaJOINTLESS (EU332978), VvAP1 (GU133634), MdAP1 (EU672877), AtAP1 (BT004113), BdAP1 (HQ588324), MdMADS1 (U78947), MdMADS3 (U78949), MdMADS4 (U78950), MdMADS7 (AJ000760), MdMADS8 (AJ001681), FvSOC1 (FJ531999), MdSOC1 (DQ846833), GhSOC1 (JF701982), NtSOC1 (JQ686938), AtSOC1 (AY093967).

#### *2.2. Expression Analysis of MdFLC in the Adult Trees*

Seasonal changes in the expression of *MdFLC* were examined in the adult trees. Flowering induction occurs between late June and mid-July [27]. The expression levels of *MdFLC1a*, *MdFLC1b*, and *MdFLC1c* were high in early June and decreased in early July during the period of flowering induction (Figure 3a–c). On the other hand, the expression level of *MdFLC3* did not change from early June to early July, but increased in August (Figure 3d). FLC suppresses the expression of *FT* in leaves as mentioned above [17]. *FT* generally produces mobile floral signals in leaves [28], and FT signal movement is also reported in Rosaceae fruit trees [29]. Therefore, leaves were used for analysis in the present study.

**Figure 3.** Seasonal changes in the expression levels of *MdFLC1a* (**a**), *MdFLC1b* (**b**), *MdFLC1c* (**c**), and *MdFLC3* (**d**) in the leaves of adult trees. Total RNA was prepared for the expression analysis of *MdFLC1* and *MdFLC3* on May 1, June 1, July 1, and August 1, 2016. Relative expression was determined using triplicate measurements taken from three independent biological replicates. The relative expression levels were normalized against *MdACTIN* with standard errors, and the maximum level of the transcripts was set at 1.0. The values with different letters for each gene significantly differed between days at *p* < 0.05, according to a Tukey test.

#### *2.3. Expression Analysis of MdFLC During Phase Transition*

The expression level of *MdFLC* during phase transition was performed in apple seedlings. While *MdFLC1a* expression was not detectable, the expression levels of *MdFLC1b* and *MdFLC1c* were high during the juvenile phase and low during the transitional and adult phases (Figure 4). The expression level of *MdFLC3* during the juvenile phase was also high compared to that in the transitional and adult phases, and it was 7.4 times the level in the adult phase.

**Figure 4.** Changes in the expression levels of *MdFLC1b* (**a**), *MdFLC1c* (**b**), and *MdFLC3* (**c**) during phase transition in apple seedlings. The expression level of *MdFLC1a* was undetectable. Total RNA was prepared from the juvenile phase (A), transition phase (B and C), and adult phase (D and E) in early July 2009 as described in [27]. Relative expression was determined in triplicate measurements taken from three independent biological replicates. The relative expression levels were normalized against *MdACTIN* with standard errors, and the maximum level of the transcripts was set at 1.0. The values with different letters for each gene significantly differed between positions at *p* < 0.05 according to a Tukey test.

#### *2.4. Transformation of Arabidopsis with MdFLC3 cDNA*

Delays in bolting were observed in more than three transgenic plants from individual seeds (T1) obtained by *Agrobacterium* in planta vacuum infiltration transformation. Detailed analysis was performed on their progenies, lines FOX1 and FOX2. The growth of FOX1 and FOX2 lines was observed and expression analysis of *AtFT* was then performed (Figure 5; Figure S1a,b, Supplementary Materials). The number of days from sowing to bolting was 31.8 in the wild-type (WT) control, and that in FOX1 and FOX2 was 35.5 and 35.7 days, respectively. The number of rosette leaves at the time of bolting was 14.4 in the wild-type and 17.5 and 18.2 in FOX1 and FOX2, respectively. A lower expression level of *AtFT* was observed with late flowering in the FOX lines. A very high expression level of *MdFLC3* was confirmed in the FOX lines, whereas only a trace expression of endogenous *FLC* (*AtFLC*) was found in the FOX lines and wild-type (Figure S2, Supplementary Materials). The average value of transformants derived from individual seeds, which are different from FOX1 and FOX1 lines, at the beginning of transformation showed delayed bolting, supporting the above result (Figure S1c,d).

**Figure 5.** The overexpression of *MdFLC3* and phenotypic analysis in *Arabidopsis*. Flowering phenotypes 32 days after sowing (**a**), days to bolting from sowing (**b**), the number of rosette leaves at bolting time (**c**), and the expression levels of *AtFT* (**d**) in the *MdFLC3* transgenic (FOX1, 2) and wild-type (WT) plants grown under a 16 h photoperiod. The values with \*\* significantly differed between FOX and WT plants at *p* < 0.01, according to the Dunnett test. The values in (**b**,**<sup>c</sup>**) indicate means and standard errors (n = 30 or 31). The relative expression of *AtFT* was determined from triplicate measurements of three independent biological replicates 15 days after sowing (**d**). The relative expression levels were normalized against *AtACTIN* with standard errors and the maximum level of the transcripts was set at 1.0.
