Quantification of total soluble proteins and chlorophylls
Using 25 mg of ground freeze-dried leaf tissue, total soluble proteins (TSP) were extracted in 500 µl (birch) or 250 µl (spruce) of Tris buffer (100 mM Tris-HCl, 20 mM MgCl2, 10 mM NaHCO3, 1 mM EDTA, and 10% (v/v) glycerol, pH 8.0) by 3 freeze-thaw cycles (Jones, Hare & Compton 1989). The supernatant (centrifugation 13,000 x g for 5 min) was analyzed for total soluble protein content per Bradford (1976) using Bio-Rad protein assay dye reagent (Bio-Rad Laboratories, Hercules, CA). Absorbances were recorded at 595 nm with a Hitachi U2010 spectrophotometer (Hitachi Ltd., Tokyo, Japan; spectral bandwidth 2 nm, wavelength accuracy of +0.3 nm, wavelength setting reproducibility of ±0.1 nm) and analyzed with Hitachi UV Solutions software version 2.0.
Using 3-5 mg of ground freeze-dried leaf tissue, chlorophylla +b were extracted in 1 ml of 95% EtOH at 65°C for 16 hours and detected as described in Minocha et al. (2009). Chlorophylla +b were analyzed with a Hitachi U2010 spectrophotometer (Hitachi Ltd., Tokyo, Japan; spectral bandwidth 2 nm, wavelength accuracy of + 0.3 nm, wavelength setting reproducibility of ± 0.1 nm; with Hitachi UV Solutions software version 2.0) by scanning absorbances in the range of 350-710 nm. Equations from Lichtenthaler (1987) were used to calculate chlorophyll a +b concentrations in the leaf tissue.