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.