2.3. Enzyme and sugar extraction and assay
Total proteins were prepared by the method of Yamada et al. (2007). The invertase activity was assayed by the method of Yamada et al. (2006). The assay mixture consisted of an aliquot of dialyzed extract, 100 mM sucrose and 100 mM acetate buffer (pH 5.0). The amount of glucose produced in the assay mixture during a 30 min reaction was measured using the Glucose CII-Test (Wako Pure Chemical Industries Ltd., Japan). The soluble sugars in petals were extracted and measured by a liquid chromatography (LaChrom Elite, Hitachi High-Technologies Corporation, Japan) with a column (Shodex SUGAR SP0810, Showa Denko K.K., Japan) according to the method of Yamada et al. (2007).