**5. FT/TFL1 gene family**

**Figure 7.** Activities of chimeric *FT* genes in Arabidopsis. (A) ALSV vectors used in this analysis. CnFT, chimeric *FT* genes. (B) Amino acid sequences of AtFT and MdFT1, and the three fragments (f1, f2 and f3) defined in this analysis. (C) Combinations of FT fragments in chimeric FT proteins. The fragments derived from AtFT are grey, and the fragments derived from MdFT1 are white. (D) Arabidopsis plants infected by ALSV vectors. NI, non-inoculated plant. Juvenile plants with three true leaves were inoculated by particle bombardment of viral RNA. Viral infection was confirmed by RT-PCR (reverse transcription-polymerase chain reaction) analysis. Photographs were taken 30 days after inoculation.

80 Plant Engineering

*FT* is a member of the FT/TFL1 gene family (also called PEBP, Phosphatidylethanolaminebinding protein). This gene family consists of five subgroups in plants: FT (Flowering locus T), TFL1 (Terminal flower 1), CEN (Centroradialis), BFT (Brother of FT) and MFT (Mother of FT). All Arabidopsis genes (*AtFT*, *AtTFL1*, *AtATC*, *AtBFT* and *AtMFT*) and apple genes belonging to FT/TFL1 family are displayed in a phylogenetic tree of protein sequences, together with some representative genes from other plant species (**Figure 8**). Names of apple genes and their Genbank/the International Nucleotide Sequence Database Collaboration (INSDC) accessions are as follows: MdFT1 (BAD08340.1), MdFT2 (NP\_001280810.1), MdTFL1-1 (NP\_001280887.1), MdTFL1-2 (NP\_001280794.1), MdCENa (NP\_001280813.1), MdCENb (NP\_001280940.1), MdBFTa (XP\_008376539.1), MdBFTb (NP\_001280770.1), MdMFTa (XP\_008374830.1) and MdMFTb (NP\_001281044.1).

Among the five subgroups of FT/TFL1 gene family, FT and TFL1 regulates initiation (induction) of floral organ development and then regulates the time of flowering. FT protein is produced before the time of flowering and carried to shoot apices. As already described, FT positively regulates (increases) the expression of genes such as *SOC1* (*SUPPRESSOR OF OVEREXPESSION OF CO 1*) and *AP1* (*APETALA1*) to induce flowering. In contrast, TFL1 is expressed at shoot apices and negatively regulates (decreases) gene expression to suppress

**Figure 8.** Phylogenetic tree of FT/TFL1 family genes. Five subfamilies are indicated by sectors.

flowering [37]. Thus, the balance between FT and TFL1 expressions have important roles in determining the time of flowering, and determining the parts of plant shoots where the flowers are generated. For example, the mutant of strawberry *FvKSN* gene and the mutant of rose *RoKSN* gene (the TFL1 family genes) can both flower in any growing season (continuous flowering), whereas ordinary strawberry and rose plants generate flowers only at one specific season of the year (seasonal flowering) [38]. The subgroup CEN is represented by the snapdragon *AmCEN* gene. This subgroup does not regulate flowering time, but does regulate the architecture of inflorescence (flower clusters at shoot apices). Snapdragon usually generates 'indeterminate' inflorescence, where many flowers are repeatedly generated on the side of the inflorescence, and the inflorescence continues to elongate without generating a flower at the very apex of the inflorescence. On the other hand, a snapdragon mutant in the *AmCEN* gene has 'determinate' inflorescence, where a limited number of flowers are generated on the side of the inflorescence, and the inflorescence stops elongating with a flower generated at the very apex of the inflorescence (called terminal flower). Thus, CEN negatively regulates flowering at the apex of the inflorescence to generate indeterminate inflorescence [39].

As apparent in the phylogenetic tree (**Figure 8**), apple has two copies of each five FT/TFL1 subgroup genes. The functions of these apple genes are not completely clear, but there are several reports on their activities and expression patterns. *MdFT1* and *MdFT2* are both expressed in apple plants. *MdFT1* is suggested to regulate the flowering time of apple plants [35], but direct evidence has not yet been found. Expression levels of *MdCENa* are much greater than those of *MdCENb* [40]. Considering its classification into the CEN subgroup and high expression levels, *MdCENa* may regulate the inflorescence architecture of apple plants. Thus, apple plants generate clustered flowers at the apices of newly developing branches. The size of the flower clusters may be smaller in the apple mutants of the *MdCENa* gene. *MdTFL1-1* and *MdTFL1-2* share similar expression patterns. Their expression is reduced when floral buds are initiated in July [40]. According to the observations explained in the next section, *MdTFL1-1* regulates the flowering time of apple plants.
