The apple (Malus × domestica Borkh.) is one of the commercially important fruit crops in the worldwide. The apple has a relatively long juvenile period (up to 4 years) and a long reproductive period between the flower initiation and the mature fruit (14-16 months), which prevent the fruit breeding. Therefore, the understanding of the flowering system is important to improve breeding efficiency in the apple. In this study, to examine the temporal and spatial expression patterns of the floral genes, MdTFL1, MdAP1 (MdMASD5), AFL2, and MdFT, we conducted in situ hybridization analysis in the apple shoot apex. In vegetative phase, MdTFL1 was expressed on the rib meristem zone. When vegetative meristem began converting into inflorescence meristem, the expression level of MdTFL1 was drastically decreased. At the early stage of inflorescence meristem, the expression levels of AFL2, MdFT, and MdAP1 were up-regulated in the leaf primordia and the upper region of cell layers on the shoot apex. In late stage, the expression levels of AFL2 and MdAP1 were up-regulated in the young floral primordia. At a more advanced stage, high expression of MdAP1 was observed in the inflorescence primordium through the inner layer of sepal primordia and the outer layer of receptacle primordia and floral axis. Our results suggest that AFL2, MdFT, and MdAP1 affect to convert from the vegetative meristem into the inflorescence meristem after the decline of MdTFL1 expression. After that, AFL2 and MdAP1 promote the formation of the floral primordia and floral organs.

The apple AFL (Apple FLORICAULA LEAFY) 1 and 2 gene promoter linked β-glucuronidase (GUS) clearly displayed staining at the meristems of the shoot apexes, lateral axils, and leaf primordia in apple trees. The GUS staining of AFL1 promoter revealed that the AFL1 gene was also expressed at a vegetative meristem, and the staining patterns of AFL2 promoter were almost the same in the culture shoots and two-year-old trees. Quantitative RT-PCR analysis also showed that both genes were expressed at floral buds, where the expression of each was higher than the vegetative expression individually and increased with floral developments. These results showed the similarity in the expression patterns of the two AFL genes, and this expression was not affected by the vegetative growth condition, but was regulated at floral development. In situ hybridization of AFL genes supported the results of the GUS staining. The specific probe of each AFL gene showed almost the same expression pattern at the meristem of shoot apexes, lateral axils, and leaf primordia from the seedling, culture shoots, and watersprouts. These results indicated that the promoter GUS analysis and the in situ hybridization can be combined for the analysis of AFL gene expression. The cooperative usage of the GUS analysis and in situ hybridization suggested that they are powerful tools for apple promoter analysis.

Transgenic apple plants that overexpressed Arabidopsis FLOWERING LOCUS T (FT) under the control of the rolC promoter showed early flowering, while the introduction of FT driven by the 35S promoter induced flower development directly from transgenic apple callus in vitro, but vegetative growth was not maintained and the explant died. GFP-FT fusion proteins were detectable in transgenic apple tissues but never caused early flowering in the transformants. Under the control of the rolC promoter, the fused protein was localized in vascular tissues and fluorescence was detectable in companion cells in the stem and petiole. The transgenic apple lines that expressed AtFT driven by the rolC promoter showed differences in inflorescence architecture and floral organ number from those typical of nontransformed apple. Flowers of transgenic apple lines often contained more numerous petals, fewer stamens, and no pistils, and the pollen grains were incapable of germinating. The transgenic apple lines showed perpetual flowering without the requirement for low temperature and obvious photoperiodism. The expression patterns of six floral meristem identity genes and floral organ genes in flowers of transgenic apple lines were investigated. Among the floral meristem identity genes, expression of MdFT2 was suppressed and that of AFL2 was dramatically enhanced in the transformants. Of the floral organ genes, expression of MdMADS13, which functions as a class B gene, was unchanged from that of the flower of wild type one, whereas expression of MdMADS-NB, a possible class C gene, was suppressed.