Metabolite Extraction and Analysis of Leaf Samples

Leaf samples (1 g), ground with liquid nitrogen, were sequentially extracted with hexane, diethyl ether, and methanol. For the determination of trehalose, raffinose, and myo-inositol, the residue obtained after extraction in diethyl ether was further extracted with methanol and dried under vacuum at 90 °C. The pellets were resuspended in 0.5 cm³ pyridine, and the metabolites were derivatized by adding 0.05 cm³ N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane. Extracts were analyzed with an HP 6890 Gas Chromatograph equipped with a mass selective detector MSD 5973 (Agilent Technologies, Santa Clara, CA, USA), with an electronic autosampler 7693A ALS system, electronic pressure control, and split/splitless injector (Agilent Technologies, Santa Clara, CA, USA). The injector worked in a split 1:50 mode at 250 °C. The volume of the sample introduced into the injector was 1 μL. The transfer line temperature was 280 °C. Separation was performed on HP-5ms (30 m × 0.25 mm; 0.25 μm film thickness) fused silica column, with a helium flow rate of 1 cm³ min^–1 [1 (link)].