Epicuticular waxes and IWs were isolated from the 5th leaf of WW control, day 8 of WD, and day 10 of RE as Zhang et al. (2020) (link). Delipidated gum arabic was dissolved in water to a final concentration of 90% (w/v), and aqueous solution was evenly applied to leaf surface to form a film. Dry polymer film was peeled off and collected, then extracted in 21 ml of chloroform/water (2:1, v/v) containing 75 μg of internal standard n-tetracosane (Sigma-Aldrich, St. Louis, MO, United States). After vigorous agitation and phase separation, the organic phase was collected into a new glass tube. The residue was extracted one more time in 4.5 ml extraction buffer; the organic phase was combined and evaporated in a rotary evaporator (Labconco, Kansas, MO, United States) to obtain EWs. The adaxial EW was removed first, followed by abaxial EW.
After EW removal from both surfaces, IWs were extracted with chloroform rinsing. The adaxial IWs and the abaxial IWs were subsequently extracted. The collected chloroform solution was evaporated dry to obtain adaxial and abaxial IWs.
After EW removal from both surfaces, IWs were extracted with chloroform rinsing. The adaxial IWs and the abaxial IWs were subsequently extracted. The collected chloroform solution was evaporated dry to obtain adaxial and abaxial IWs.
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