Oleic acid

L-amino acid standards, norvaline, sarcosine, sodium dihydrogen phosphate (NaH₂PO₄), hydrochloric acid, sodium hydroxide, guanidine hydrochloride, 2,4-dinitrophenylhydrazine, iron (II) chloride, iron (III) chloride, ammonium thiocyanate, ortho-phthalaldehyde, 9-fluorenylmethoxycarbonyl chloride, iodoacetic acid, 3-mercaptopropionic acid, boric acid (H₃BO₃), trichloroacetic acid, and MS-grade ammonium acetate were from Sigma-Aldrich (Darmstadt, Germany). Standards for fatty acid analysis included Supelco 37 Component FAME mix (Supelco, St. Louis, MO, USA), 68D (Nu-Check-Prep, Elysian, MN, USA), and GLC-490 (Nu-Check-Prep, Elysian, MN, USA). Standards for the lipid class analysis were oleic acid, oleoyl monoacylglycerol, dioleoyl diacylglycerol, triolein, dioleoyl phosphatidylcholine, cholesteryl oleate and ethyl docosahexaenoic acid (Larodan, Solna, Sweden). All solvents used were at least HPLC grade. Water used was ultra-pure water (ELGA LabWater, High Wycombe, UK).

The solutions were prepared by dissolving uniformly ¹³C labelled oleic acid (> 95% purity, Larodan AB, Solna, Sweden) in 0.05% surfactant solution (Glucopon 215 UP/Mb; BASF, Ludwigshafen, Germany) at a final concentration of 167 μM (equiv. to 50 mg L⁻¹). Solutions were vortexed for at least 3 min immediately after preparation and again for 3 min immediately before application to the fruit surface. Donor solutions were always prepared fresh on the day of use.
The solution was applied as described earlier (Si et al., 2021a (link),b (link)). Briefly, polyethylene tubes (25 mm height, 14 mm diameter) with a tapered tip and a minute hole in the tip were mounted in the equatorial region of the apple fruit using a non-phytotoxic silicon rubber (SE 9186 RTV; Dow Toray, Tokyo, Japan). A volume of 400 μL of donor solution was injected through the hole in the tip of the tube, and the hole was sealed using silicone rubber to prevent drying of the donor solution (Figure 1A). Feeding was terminated after 7 d when the tubes were removed. The original footprint of the tube was then marked with a permanent marker and the marked area was rinsed with deionized water. Fruits were sampled either 14 d after the termination of feeding or at commercial maturity.