Quantification of Stilbene Compounds in Transgenic Leaves

Stilbene levels in the transgenic leaves were measured as described previously^{44 (link)} with minor modifications. The leaf samples were ground in liquid nitrogen, homogenized and extracted with 80% methanol, and then 20 μl of each sample was quantified by high pressure liquid chromatography (Shimadzu Corp, Kyoto, Japan) with a detection wavelength of 306 nm. The mobile phase was 0.5% (v/v) formic acid and acetonitrile (ACN). The mobile phase elution procedure was carried out as follows: 0–8 min, 10–18% ACN; 8–10 min, 18% ACN; 10–15 min, 18–25% ACN; 15–18 min, 25–35% ACN; 18–25 min, 35% ACN; 25–30 min, 35–70% ACN. The sample peaks and standard chemical peaks were calculated using trans-resveratrol and trans-piceid (Sigma-Aldrich Inc., http://www.sigmaaldrich.com/) as external standards. We obtained the cis-piceid standard by the photoisomerization of trans-piceid under UV irradiation. The 50% ethanol solution containing 400 μM of trans-piceid was irradiated at 366 nm for 3 h, and the resulting cis-piceid standard was stored in total darkness.^{37 (link)}