The two FLOWERING LOCUS T (FT)-like genes of apple (Malus x domestica Borkh.), MdFT1 and MdFT2, have been isolated and characterized. MdFT1 and MdFT2 were mapped, respectively, on distinct linkage groups (LGs) with partial homoeology, LG 12 and LG 4. The expression pattern of MdFT1 and MdFT2 differed in that MdFT1 was expressed mainly in apical buds of fruit-bearing shoots in the adult phase, with little expression in the juvenile tissues, whereas MdFT2 was expressed mainly in reproductive organs, including flower buds and young fruit. On the other hand, both genes had the potential to induce early flowering since transgenic Arabidopsis, which ectopically expressed MdFT1 or MdFT2, flowered earlier than wild-type plants. Furthermore, overexpression of MdFT1 conferred precocious flowering in apple, with altered expression of other endogenous genes, such as MdMADS12. These results suggest that MdFT1 could function to promote flowering by altering the expression of those genes and that, at least, other genes may play an important role as well in the regulation of flowering in apple. The long juvenile period of fruit trees prevents early cropping and efficient breeding. Our findings will be useful information to unveil the molecular mechanism of flowering and to develop methods to shorten the juvenile period in various fruit trees, including 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.

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 (Malusxdomestica 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 beta-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.

The 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene is a member of the ACS gene family that is involved in apple ( Malus x domestica Borkh.) fruit ripening. Presence of an allele ( Md-ACS1-2) of this gene is associated with low internal ethylene concentration in some apple cultivars. In this study, inheritance of Md-ACS1 was determined for 50 apple cultivars/advanced selections and 101 F(1) seedlings from five populations. Following this, the softening pattern of apples stored at 20 degrees C for up to 40 days was examined using 35 fruiting cultivars/selections of defined Md-ACS1 status. Md-ACS1 is inherited in a Mendelian fashion and was found to be linked to fruit softening. Maturity season of genotypes also significantly affected fruit softening. Late-season genotypes in the Md- ACS1-2/2 class had the slowest rate of softening, while early-season Md- ACS1-1/1 genotypes had the most rapid softening rate. The implications of these results are discussed in relation to parental selection and breeding for storage ability in apple.

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. DDBJ accession nos. AB531019 (MdTCP2a mRNA), AB531020 (MdTCP2b mRNA), AB531021 (MdTCP4a mRNA), AB531022 (MdTCP4b mRNA) and AB531023 (MdVOZ1a mRNA).