Anatomical analysis by two approaches ensure the promoter activities of apple AFL genes.

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.