Anatomical Analysis by Two Approaches Ensure the Promoter Activities of Apple AFL Genes

Abstract

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.

Overexpression of Arabidopsis FT gene in apple leads to perpetual flowering

“…MdMADS-NB was isolated from apple flowers at our research station and is an AGAMOUS homolog in apple, which is considered to function as a class C gene. AFL2 is a LEAFY ortholog in apple (Flachowsky et al 2010;Wada et al 2002Wada et al , 2007Wada et al , 2009, which functions in integration of flowering signals.…”

“…After subcloning and sequencing of the AtFT cDNA, AtFT:GFP under the control of the 35S or rolC promoter vector was constructed and designated pSM35S::AtFT:GFP or pSMrolC::AtFT:GFP ( Figure 1). Transformation of apple was performed in accordance with the method described previously (Wada et al 2009). Leaflets from cultured shoots of JM2 were infected by Agrobacterium tumefaciens strain LBA4404 bearing each vector.…”

“…paradisiaca Schneid. 'M9') was cultured in vitro and leaflets were used in transformation experiments as described previously (Wada et al 2009). …”

“…RT-PCR reactions with 1.0 µg extracted RNA were performed as described previously (Wada et al 2009). Specific primers for the endogenous flowering-related genes MdFT1 (AB161112), MdFT2 (AB458504), MdMADS5 (DQ782960), MdMADS13 (AJ251116), MdMADS-NB (AB370211), and AFL2 (AB056159) were used for real-time RT-PCR analysis (Table 1) with the Power SYBR Green Master Mix (Applied Biosystems Japan, Tokyo, Japan).…”

Effects of the plant growth regulators on expression of MdTFL1 promoter fused β-glucuronidase (GUS) reporter gene in apple (Malus spp.) tissues in vitro

“…The PCR products were digested with KpnI, and each was then cloned in-frame into the KpnI-SmaI sites of the pSMAK343-M2GUS binary vector (modified pSMAK341-M2GUS) upstream of GUS, yielding two constructs; MdTFL1-1pro::GUS/pSMAK343-M2GUS and MdTFL1-2pro:: GUS/pSMAK343-M2GUS (Figure 1). Transformation of apple was performed according to the method described in Wada et al (2009). The root stock cultivar 'Japan Morioka 2: JM2' (M. prunifolia (Wild.)…”