The relative growth rate (RGR, % day−1) of the projected total leaf area was obtained by multiplying b by 100. Carbohydrate assay Leaf samples for carbohydrate assay were harvested after 10 h of illumination by different light regimes on the second and fifth day of the treatments. As described for the
Chl fluorescence analysis, only mature leaves, which had existed before starting the experiments, were used for the analysis. After excision, leaves were quickly weighed, frozen in liquid N2, and stored at −80 °C until extraction. Soluble sugars (glucose, fructose and sucrose) and starch were extracted from the leaves as described by Czech et al. (2009). Concentrations of soluble sugars were determined according to Jones et al. (1977). Starch concentration was measured as glucose after enzymatic digestion with α-amylase and amyloglucosidase (Czech et al. 2009). Carbohydrate contents were expressed relative to leaf fresh weight (μmol g−1 selleck FW). Analysis SBE-��-CD purchase of photosynthetic pigments Leaf disks (0.77 cm2) were taken from mature leaves early in the morning on day 0 (before
the treatments) and on day 7 (after 7 days under different light regimes) to analyze photosynthetic pigments. The mature leaves used for sampling on day 7 were those that existed already on day 0. Two samples were collected from each plant: a “dark” sample taken at the end of the night period and a “light” sample taken after exposure of plants to halogen lamps (Haloline; Osram) of ca. 1,000 μmol photons m−2 s−1 for 5 min. The latter condition is comparable with the actinic illumination used for NPQ measurements in the second experiment. Leaf disks were immediately frozen in liquid N2 and stored at −80 °C until pigment extraction. Photosynthetic pigments were extracted by grinding frozen leaf disks in 1 mL acetone. The homogenate was then centrifuged at 13,000 rpm for 5 min and filtered (0.45-μm True Syringe Filter; Alltech Associates) before injection (20 μL) into the HPLC system. Chlorophylls and carotenoids
were separated with an Allsphere ODS-1 column (5 μm, 250 × 4.6 mm; Alltech Associates) at a constant flow rate of 1 mL min−1 Vitamin B12 according to the method modified from Gilmore and Yamamoto (1991). Pigments were detected using a Waters 996 photodiode array detector (Waters Corporation) and the peak area of chromatograms was integrated at 440 nm with the Empower software (Waters Corporation). Western blot analysis Leaf samples for PsbS protein analysis were taken early in the morning on day 0 and day 7 in parallel with the “dark” samples of pigment analysis. The leaves were frozen in liquid N2 and stored at −80 °C. Proteins were extracted by homogenizing frozen leaves in a strongly denaturing buffer (7 M urea, 5 % SDS, 50 mM Tris–HCl (pH 7.6), and 5 % β-mercaptoethanol) followed by centrifugation at 13,000 rpm for 10 min at 4 °C. Samples from three replicate plants were pooled together for each treatment and accession.