Metabolite sample processing
Leaves were randomly collected from different treatments and immediately frozen in liquid nitrogen and stored at −80 °C for metabolite extraction. Frozen leaf tissues (~10–35 mg) were ground in liquid N using a mortar and pestle to ensure that the samples are metabolically inactive. Frozen powder was homogenized in ice-cold solution of methanol: chloroform: water (3:1:1), with the addition of ribitol (0.2 mg ml−1 of methanol) as an internal standard. The slurry was mixed for 5 min using a microtube mixer. Approximately 160 μl of distilled water was added to the extraction solution to separate the polar and nonpolar phases. After centrifugation, only the upper layer (polar phase) was used for further analysis. A quality control (QC) sample was prepared by mixing aliquots from each of the samples.