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).