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.