Sugar Content Analysis
Immediately after photosynthesis measurement, the same leaf was sampled to determine non-structural carbohydrate content (NSC: starch, sucrose, glucose and fructose). Prior to grinding with a ball grinder (Mixer mill MM 200, Retsch, Germany), the samples were frozen in liquid nitrogen. The sugars were extracted three times from 20 mg samples with 1 ml of 80% ethanol for 30 min at 75°C and then centrifuged for 10 min at 9500 g (Mikro 200, Hettich centrifuge). Soluble sugars (sucrose, glucose and fructose) were contained in the supernatant and starch in the sediment. The supernatant was filtered in the presence of polyvinyl polypyrrolidone and activated carbon to eliminate pigments and polyphenols. After evaporation of solute with Speedvac (RC 1022 and RCT 90, Jouan SA, Saint Herblain, France), soluble sugars were quantified by high-performance ionic chromatography (HPIC, standard Dionex) with pulsed amperometric detection (HPAE-PAD). The sediment was solubilized with 0.02 N NaOH at 90°C for 1hr 30 min and then hydrolyzed with a-amyloglucosidase at 50°C and pH 4.2 for 1 hr 30 min. Starch was quantified as described by Boehringer (Pomeranz and Meloan, 1994) with 5 μl of a mixture of hexokinase and glucose-6-phosphate dehydrogenase (HK/G6P-DH Sigma Aldrich), followed by spectrophotometry of NADPH at 340 nm (spectrophotometer UV/VIS V-530, Jasco Corporation, Tokyo, Japan).