**1. Introduction**

Fruit trees, which are perennial woody plants, have a long juvenile period after germination and before flowering and fruit set. Because fruit quality cannot be evaluated during this juvenile phase, it is necessary to address the long juvenile period of fruit trees with regard to fruit tree breeding. Lengths of juvenile periods di ffer among species, and apple requires six to eight years of juvenility [1]. Although some cultivation techniques to shorten the juvenile period have been proposed, such as plant hormone treatment, grafting to dwarf rootstocks, suppression of dormancy, and adjustment of cultivation conditions [2–4], little is known about the molecular mechanisms of juvenility. It is more di fficult to elucidate the mechanism underlying the control of juvenility in fruit trees than in herbaceous plants because fruit trees take longer to grow and are larger in size. Apple is one of the

earliest plants whose genome sequences have been reported as woody plants and fruit tree [5], and is utilized as a model because of its extensive genome information [6,7]. Therefore, apples are useful for studying phenomena unique to woody plants such as juvenility and annual flowering.

In general, juvenility is stronger in young seedlings and gradually weakens as age progresses. There is also known to be a gradient of juvenility in individual trees; that is, juvenility is stronger at the base of trunks and branches and becomes weaker approaching the tip [8]. Genes that are potentially related to juvenility have been reported in studies of homologs of flowering-related genes from *Arabidopsis*, which is a model herbal plant. Previous studies focused on the relationship between juvenility and the apple homologs of *TERMINAL FLOWER 1* (*TFL1*), *FLOWERING LOCUS T* (*FT*), *LEAFY* (*LFY*), and *APETALA1* (*AP1*), which are floral meristem identity-related genes in *Arabidopsis*. The expression of apple *TFL1* homolog *MdTFL* is high during the juvenile phase, and the expression of apple *FT* homolog *MdFT* is high during the adult phase [9]. The overexpression of apple homologs of *LFY* and *AP1* in *Arabidopsis* promotes flowering, and the overexpression of *MdTFL* delays flowering [10–12]. In citrus, the overexpression of *LFY* or *AP1* of *Arabidopsis* reportedly decreases the juvenile period from seven years to two years [13], and the methylation of the *LEAFY* homolog is thought to be involved in juvenility [14]. However, most of these genes are located relatively downstream of the flowering pathway, and in order to clarify the molecular mechanism of juvenility, it is necessary to analyze genes farther upstream.

There are several flowering pathways, including the photoperiod, vernalization, gibberellin, and autonomous pathways [15]. The photoperiod and vernalization-dependent pathways are controlled by environmental factors, and the gibberellin-dependent pathway is comprised of a group of genes related to the synthesis and signal transduction of gibberellin. In contrast, the autonomous pathway is dependent on endogenous growth-related factors. Juvenility is not reduced by environmental factors such as temperature and day length, but is reduced by growth over several years, which suggests that juvenility is controlled by endogenous growth-related factors. Some genes in the autonomous pathway are known to induce flowering by suppressing the expression of *FLOWERING LOCUS C* (*FLC*) [16]. *FLC* plays a role as a key regulator of the autonomous and vernalization pathways and inhibits flowering by suppressing the expression of floral induction genes *SOC1* and *FT* in *Arabidopsis* [17–19]. Since *FLC* is a key gene for the flowering pathways, including the autonomous pathway, it can be expected to play a role in the suppression of flowering in the juvenile phase.

There have been some reports on *FLC* homologs in fruit trees. *FLC* homologs have been identified in apple, and divergent functions have been suggested based on their nucleotide sequences [20]. Two of these exhibit increased expression during dormancy and decreased expression with dormancy release, which suggests that they repress flowering as described in *Arabidopsis*. In fact, the possibility that the *FLC*-like gene is a candidate gene is mentioned in the genome-wide association mapping of the flowering period in apple [21]. In other apple *FLC*-like genes, the repression of bud outgrowth during dormancy and the promotion of flowering under non-chilling conditions have been reported [22,23]. Among other Rosaceae fruit trees, the *FLC* homolog in peach (*Prunus persica*) has not been reported to be associated with dormancy, and its role has not been elucidated [24]. No *FLC* homolog has been found in Japanese apricot (*Prunus mume*) either [25]. In fruit trees other than Rosaceae, changes in the expression and splicing of an *FLC*-like gene were reported in trifoliate orange (*Poncirus trifoliata*) [26]. Although these reports provide some information about the role of *FLC* in fruit trees, information about the relationship between juvenility and *FLC* homologs is still lacking. Therefore, to investigate the physiological roles of apple *FLC* homolog *MdFLC* at the molecular level, sequence and expression analyses and transgenic experiments were performed.
