Nobuhiro Kotoda

Understanding the flowering process in apple (Malus × domestica Borkh.) is essential for developing methods to shorten the breeding period and regulate fruit yield. It is known that FLOWERING LOCUS T (FT) acts as a transmissible floral inducer in the Arabidopsis flowering network system. To clarify the molecular network of two apple FT orthologues, MdFT1 and MdFT2, we performed a yeast two-hybrid screen to identify proteins that interact with MdFT1. We identified several transcription factors, including two members of the TCP (TEOSINTE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL FACTORs) family, designated MdTCP2 and MdTCP4, and an Arabidopsis thaliana VOZ1 (Vascular plant One Zinc finger protein1)-like protein, designated MdVOZ1. MdTCP2 and MdVOZ1 also interacted with MdFT2 in yeast. The expression domain of MdTCP2 and MdVOZ1 partially overlapped with that of MdFT1 and MdFT2, most strikingly in apple fruit tissue, further suggesting a potential interaction in vivo. Constitutive expression of MdTCP2, MdTCP4 and MdVOZ1 in Arabidopsis affected plant size, leaf morphology and the formation of leaf primordia on the adaxial side of cotyledons. On the other hand, chimeric MdTCP2, MdTCP4 and MdVOZ1 repressors that included the ethylene-responsive transcription factors (ERF)-associated amphiphilic repression (EAR) domain motif influenced reproduction and inflorescence architecture in transgenic Arabidopsis. These results suggest that MdFT1 and/or MdFT2 might be involved in the regulation of cellular proliferation and the formation of new tissues and that they might affect leaf and fruit development by interacting with TCP- and VOZ-family proteins.

Recent molecular analyses in several plant species revealed that TERMINAL FLOWER1 (TFL1) and CENTRORADIALIS (CEN) homologs are involved in regulating the flowering time and/or maintaining the inflorescence meristem. In apple (Malus×domestica Borkh.), four TFL1/CEN-like genes, MdTFL1, MdTFL1a, MdCENa and MdCENb, were found and mapped by a similar position on putatively homoeologous linkage groups. Apple TFL1/CEN-like genes functioned equivalently to TFL1 when expressed constitutively in transgenic Arabidopsis plants, suggesting that they have a potential to complement the TFL1 function. Because MdTFL1 and MdTFL1a were expressed in the vegetative tissues in both the adult and juvenile phases, they could function redundantly as a flowering repressor and a regulator of vegetative meristem identity. On the other hand, MdCENa was mainly expressed in fruit receptacles, cultured tissues and roots, suggesting that it is involved in the development of proliferating tissues but not in the control of the transition from the juvenile to the adult phase. In contrast, MdCENb was silenced in most organs probably due to gene duplication by the polyploid origin of apple. The expression patterns of MdTFL1 and MdCENa in apple were also supported by the heterologous expression of β-glucuronidase fused with their promoter regions in transgenic Arabidopsis. Our results suggest that functional divergence of the roles in the regulation of vegetative meristem identity may have occurred among four TFL1/CEN-like genes during evolution in apple.

Fruit trees, such as apple (Malus × domestica Borkh.), are woody perennial plants with a long juvenile phase. The biological analysis for the regulation of flowering time provides insights into the reduction of juvenile phase and the acceleration of breeding in fruit trees. In Arabidopsis, LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is involved in epigenetic silencing of the target genes such as flowering genes. We isolated and characterized twin apple LHP1 homolog genes, MdLHP1a and MdLHP1b. These genes may have been generated as a result of ancient genome duplication. Although the putative MdLHP1 proteins showed lower similarity to any other known plant LHP1 homologs, a chromo domain, a chromo shadow domain, and the nuclear localization signal motifs were highly conserved among them. RT-PCR analysis showed that MdLHP1a and MdLHP1b were expressed constantly in developing shoot apices of apple trees throughout the growing season. Constitutive expression of MdLHP1a or MdLHP1b could compensate for the pleiotropic phenotype of lhp1/tfl2 mutant, suggesting that apple LHP1 homolog genes are involved in the regulation of flowering time and whole-plant growth. Based on these results, LHP1 homolog genes might have rapidly evolved among plant species, but the protein functions were conserved, at least between Arabidopsis and apple.

Because fruit trees such as apple ( Malus × domestica Borkh.) flower and set fruit only after an extended juvenile phase lasting several years, efficient breeding of fruit trees is limited. We previously suggested that MdTFL1 ( Malus × domestica TFL1 ) functions analogously to TERMINAL FLOWER 1 ( TFL1 ) and that MdTFL1 is involved in the maintenance of the juvenile/vegetative phase in apple. To clarify the function of MdTFL1 in apple, we produced transgenic `Orin' apple trees expressing MdTFL1 antisense RNA. One of them flowered only 8 months after the transfer to the greenhouse, whereas the nontransformed control plants have not flowered in nearly 6 years. As expected, the expression of endogenous MdTFL1 was suppressed in the transgenic lines that showed precocious flowering. In addition, the expression level of the transgene was correlated with the reduction of the juvenile phase. These findings confirm that MdTFL1 functions like TFL1 and that MdTFL1 maintains the juvenile and vegetative phase in apple. Flower organs of the transgenic apple trees were normal in appearance, and a precocious flowering transgenic line set fruit and seeds. Interestingly, some flowers of the transgenic apple trees developed without undergoing dormancy. The expression of MdTFL1 in apple may affect flower development as well as flower induction.