**3. Discussion**

A phylogenetic tree analysis, including other MADS-box genes such as SVP and SOC1, revealed that both MdFLC1 and MdFLC3 belong to the same FLC group as VvFLC [30] and PtFLC [26], which has been reported to function as a floral repressor (Figure 2). Therefore, MdFLC1 and MdFLC3 were further investigated as apple FLCs in this study. FLC is one of the MADS-box proteins, which are transcription factors having a highly conserved region of approximately 60 amino acids, MADS-box, that is involved in DNA binding and dimer formation. Many MADS-box proteins in plants are classified as MIKC-type, contain MADS-, I-, K-, and C-domains [31], and form dimers and higher multimers to function [32,33]. MdFLC1c and MdFLC3 contained these four conserved domains.

Three cDNA sequences of *MdFLC1* were searched in the GDR database, and *MdFLC1* was found to correspond to MD05G1037100. *MdFLC1c* mRNA contained all exons, whereas *MdFLC1a* and *MdFLC1b* mRNA contained the sequences of the fourth and third introns, which were not removed in splicing, respectively (Figure 6). Therefore, the deduced amino acid sequence of MdFLC1c contained MADS-, I-, K-, and C-domains, whereas the sequences of MdFLC1a and MdFLC1b lacked K- and C- domains because of the stop codons in the intron sequences of their mRNA (Figure 1). K- and C-domains are important for protein–protein interactions and other functions in MIKC-type MADS proteins [34]. These results sugges<sup>t</sup> that *MdFLC1c* plays the role of *MdFLC1* and that *MdFLC1a* and *MdFLC1b* are

expected to be non-functional. However, since the regulation of expression by selective splicing has been reported in plant response to environmental stress [35], expression analysis was performed in the three splicing variants of *MdFLC1*. The *MdFLC3* cDNA sequence was consistent with that of MD10G1041100 in the GDR database, and its deduced amino acid sequence contained the four domains of an MIKC-type MADS protein (Figure 1), suggesting that MdFLC3 functions as an MIKC-type MADS protein.

**Figure 6.** Transcript structures of *MdFLC1* splice variants, *MdFLC1a*, *MdFLC1b*, and *MdFLC1c*. E and I with numbers indicate exons and introns, respectively. Numbers under the bar correspond to their sizes, in base pairs.

In the present study, the expression levels of *MdFLC1a*, *MdFLC1b*, and *MdFLC1c* decreased during the period of flowering induction in a seasonal expression pattern in the adult trees. In the annual growth cycle of apple, *MdFT1*, which is a floral integrator, shows high expression, and the expression level of *MdTFL*, which is a floral repressor, decreases during the period of flowering induction [9,27]. Therefore, the expression of *MdFLC1a*, *MdFLC1b*, and particularly *MdFLC1c*, which is expected to translate functional proteins, is likely involved in suppression of flowering in the annual growth cycle of adult apple trees, as is *MdTFL*. In contrast, the expression level of *MdFLC3* did not decrease during the period of flowering induction, suggesting that *MdFLC3* is not involved in flowering induction in the annual growth cycle of adult trees.

Because phase change is observable in individuals, seedlings can be used for expression analysis of phase transition [36]. Five sites in the seedlings used in this study show phase transition based on juvenile characteristics such as flower bud formation, leaf size, and leaf serration, and were used for *MdFLC* expression analysis [27]. Although expression of *MdFLC1a* was not detected, the expression levels of *MdFLC1b* and *MdFLC1c* were high in the juvenile phase and low in the transitional and adult phases. The expression level of *MdFLC3* was similarly high in the juvenile phase. The expression pattern of *MdFLC3* suggests that it does not play a role in flowering in the annual growth cycle but acts specifically in juvenility. A similar seasonal expression pattern was observed in *MdFLC3* within buds in the adult trees and confirmed the specific role of MdFLC3 (Figure S3, Supplementary Materials). Furthermore, the expression of *MdFLC1c* in the juvenile phase of seedlings is significantly lower than the expression in the adult trees (approximately 1/6, data not shown). Therefore, we focused on this specific role of *MdFLC3* and subjected *MdFLC3* to functional analysis by the transformation of *Arabidopsis*. The results showed that *MdFLC3* is a floral repressor, confirming its role in the juvenility of apple. Since the result for phase transition was obtained in crossed seedlings, a genotype-specific effect

could not be excluded. Further study using other several genotype combinations and apomixis will be necessary to confirm the findings of the present study.

FLC suppresses the expression of *FT* containing the CArG box, which is an FLC-binding sequence, in the promoter region and first intron in *Arabidopsis* leaves [17]. Apples also have su fficient genomic information and such binding sites can be analyzed. Since the *MdFTs* in the database have CArG box-like sequences in the intron and promoter region (Table 1), it can be expected that MdFLC1 and MdFLC3 bind to these sequences to control annual flowering and juvenility. Regarding the molecular mechanism of juvenility of woody plants, including fruit trees, little is known about genes located upstream of the flowering pathway. Our results sugges<sup>t</sup> that the apple homolog of *FLC*, which appears to play a central role relatively upstream of the flowering pathway, could be involved in juvenility as well as in annual flowering. Testing the correlation between MdFLC characteristics and the length of the juvenile phase could provide valuable insight into the function of MdFLC in the regulation of this process in the near future. In addition, if this gene could be used as a marker, it would be possible to breed cultivars with a short juvenile period as well as various useful traits by marker-assisted selection [21,37]. As apples have advanced in genome information and genome editing technology [38], accumulating results as a model will be utilized for other fruit trees.



a The boxes in the introns and promoter regions, the upstream 1500-bp *MdFT* promoter sequences of the start codon were checked in both strands according to Helliwell et al. [17]. b The boxes in the introns and the promoter regions are shown with the positive and negative number of positions, respectively.

#### **4. Materials and Methods**

### *4.1. Plant Materials*

Mature leaves in the juvenile phase of 8-year-old apple (*Malus domestica*) seedlings of a cross between 'Fuji' and 'Himekami' were used for cDNA isolation and sequence analysis of *MdFLC*. Mature leaves from 11-year-old apple trees ('Fuji' grafted onto M.9, *M. prunifolia*) were used as adult trees. For expression analysis in adult trees, mature leaves were sampled from 20-year-old apple trees ('Fuji' grafted onto M.9, *M. prunifolia*) on May 1, June 1, July 1, and August 1, 2016. For expression analysis of phase transition, mature leaves were collected from the juvenile to adult phase in 8-year-old apple seedlings of a cross between 'Fuji' and 'Himekami' [27]. All samples were collected in the Tohoku University (Sendai, Japan) experimental field at 38◦16- N and 140◦52- E.

#### *4.2. cDNA Isolation, Sequence Analysis, and Phylogenetic Tree Analysis of MdFLC*

To isolate *MdFLC1* cDNA, the apple EST sequences were searched using DDBJ and GDR. DDBJ was also used to search homologous genes in other plants. The cetyltrimethylammonium bromide (CTAB) method [27] and a TaKaRa RNA PCR kit (AMV) (Takara-Bio, Kusatsu, Japan) were used for RNA extraction and reverse transcription, respectively. Cloning of *MdFLC3* cDNA was performed by PCR with degenerate primers MdMADSF and MdMADSR (Table S1, Supplementary Materials) based on highly conserved sequences in the MADS-box protein using PROSITE (https://prosite.expasy.org). The restriction sites of *Eco*RI and *Bam*HI were added to the primers in advance. PCR was performed with cDNA from leaves in the juvenile and adult phases. PCR products were electrophoresed on

agarose gel, and amplified fragments of the expected size were collected using TaKaRa RECOCHIP (TaKaRa). pUC18 was digested with *Eco*RI and *Bam*HI, and the PCR fragments were ligated into this vector and then transformed into *Escherichia coli*. The plasmid was purified for sequence analysis. Sequence Alignment by ClustalW (http://align.genome.jp) was used to prepare the sequence alignments and phylogenetic trees.

#### *4.3. Expression Analysis by Real-Time PCR*

For expression analysis in the adult trees, RNA extraction and reverse transcription were performed using a Cica Geneus RNA prep kit for Plant (Kanto Kagaku, Tokyo, Japan) and ReverTra Ase qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan), respectively. THUNDERBIRD SYBR qPCR Mix (TOYOBO) was used for the subsequent PCR. For expression analysis with phase transition, RNA extraction and reverse transcription were performed using the CTAB method [27] and QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany), respectively. A QuantiTect SYBR Green PCR Kit (Qiagen) was used for the subsequent PCR. Real-time PCR was performed as described in Ikeda et al. [39], using the primer sets listed in Table S1. Coe fficients of variant for quantification cycle of the reference genes among samples were 1.85%, 2.75%, and 1.01% in Figures 3–5, respectively.

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

The translated region of *MdFLC3* cDNA was amplified by PCR using Q5 DNA polymerase (New England Biolabs, Ipswich" MA, USA) electrophoresed in an agarose gel. It was then collected from the gel and used as an insert. The primers used were MdFLC3InsertF and MdFLC3InsertR, in which *Bam*HI and *Sac*I sites were added to the 5- and 3- ends, respectively (Table S2, Supplementary Materials). The *GUS* sequence in the binary vector pBI121 was excised using *Bam*HI and *Sac*I, and the *MdFLC3* insert was introduced into the vector instead. The resultant vector containing *MdFLC3* cDNA with CaMV *35S* promoter was used for the transformation of *Arabidopsis thaliana* ecotype Columbia (Inplanta Innovation Inc., Yokohama, Japan). Transformed seeds (T1) were selected in a medium containing kanamycin, and a transgene check was performed by PCR using the primer set for *MdFLC3* (Table S2). Expression analysis was performed by real-time PCR using the primer sets for *AtFT*, *MdFLC3*, and *AtFLC*, and the *Arabidopsis* actin primer was set as a reference (Table S2). Seeds (T2) were collected separately from individuals (T1) derived from individual seeds initially obtained by *Agrobacterium* in planta vacuum infiltration transformation and each of them was sown as a line. In the T2 generation, segregation of the transgene was checked in each line and seeds (T3) were collected. Homozygous seeds that did not segregate in T3 were used. The homozygous seeds (T3) were planted and grown in vermiculite/pearlite (1:1) at 22 ◦C under a 16 h photoperiod, and the number of rosette leaves and days after sowing was measured at bolting [40].

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/12/ 4562/s1. Table S1. Primers used for cloning of cDNA encoding *MdFLC* and real-time PCR in apple; Table S2. Primers used for transformation of *Arabidopsis* with *MdFLC3* cDNA and real-time PCR in transgenic *Arabidopsis*; Figure S1. Phenotype analysis in the *MdFLC3* transgenic (FOX) and wild-type (WT) plants as support data for Figure 5; Figure S2. Expression levels of *MdFLC3* and *AtFLC* in the *MdFLC3* transgenic (FOX1, 2) and wild-type (WT) plants; Figure S3. Seasonal changes in the expression levels of *MdFLC3* in the buds of adult trees.

**Author Contributions:** Conceptualization, S.K. and Y.K.; methodology, H.K., T.S., and S.K.; investigation, H.K. and N.I.; formal analysis, H.K., N.I., and T.S.; resources, K.K.; data curation, K.K.; writing—original draft preparation, H.K.; writing—review and editing, K.K. and Y.K.; visualization, H.K.; supervision, S.K. and Y.K.; project administration, Y.K.; funding acquisition, K.K. and Y.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Grants-in-Aid for Scientific Research (24248006), Japan.

**Acknowledgments:** The authors thank Shohei Hano and Mai Asakawa for preliminary experiments and materials used for experiments.

**Conflicts of Interest:** The authors declare no conflict of interest. *Int. J. Mol. Sci.* **2020**, *21*, 4562
