2.5 Non-structural carbohydrate analysis
The NSC concentrations were measured according to the method by Wong (Wong, 1990) as modified by Hoch et al. (Hoch et al. , 2002). NSC refers to the sum of mobile sugars (mainly glucose, fructose and sucrose) and starch. First, 10-12mg sample powder was boiled in 2 ml distilled water for half an hour. Then, 200μl aliquot mixed with Invertase (Sigma-Aldrich, Buchs, Switzerland) were extracted to degrade sucrose to glucose and fructose. After centrifugation, glucose hexokinase and phosphogluconate isomerase (Sigma-Aldrich, Buchs, Switzerland) were added. The concentration of sugars was obtained as the total amount of glucose that was determined by 340nm photometry (HR 7000, Hamilton, Rone, NE, USA) in a 96-well microplate photometer (Sigma-Aldrich, Buchs, Switzerland). 500μl extract were taken from the sample aliquot and reacted with amyloglucosidase (Sigma-Aldrich, Buchs, Switzerland) for 15h at 49°C, to break down starch to glucose, and to measure the total NSC concentration. The starch concentration was calculated as total NSC minus soluble sugars. NSC concentrations are expressed on a dry mass (d.m.) basis.