Methyl laurate

Wheat bran (WB) (0.5–2 mm) was a generous gift by the company BioWanze (Wanze, Belgium).
Acetonitrile (>99.9%) was purchased from Carlo Erba Reagents (Dasit Group S.p.A, Cornaredo, Italy). 2-methylbutan-2-ol (2M2B, 99%), pentan-1-ol (>99%), lauric acid (99%), methyl laurate (99%), molecular sieves (3 and 5 Å) and the enzyme Novozym 435 (immobilized lipase on acrylic resin from Candida antarctica) were purchased from Sigma-Aldrich Corp. (St. Louis, United States). CellicCtec2 enzyme was a generous gift from Novozymes.

To analyze the quantity and composition of intact plant emitted volatiles, a dynamic headspace trapping system was employed. In order to minimize emissions from the soil, the plant pots were carefully wrapped in aluminum foil. The collection vessels used were sealed 10-liter glass containers equipped with two connector plugs on the top. Prior to introduction into the vessels, the air was passed through Teflon tubing and a charcoal filter to ensure its cleanliness, and it was then introduced at a controlled rate of 400 ml/min. The outlet of the system was connected to a glass GC inlet liner containing 1g of Tenax TA resin (60-80 mesh; SuperQ). For the mechanical damage treatment, flowering plants of the transgenic chrysanthemum were rapidly punctured 50 times with entomological pins on the flower stems and then immediately placed in the volatile collection vessels. Headspace collection was carried out for 24 hours, with 12 hours during the light period and 12 hours in darkness. To elute the volatiles from the liner, 1 ml of hexane (HPLC grade) containing methyl laurate (7.8 ng/μl, Sigma-Aldrich, Co., LLC., USA) as an internal standard was used. Finally, the samples were subjected to analysis by GC-MS using the same program employed for the analysis of hexane extraction of fresh tissues.

Precultures were grown overnight (180 rpm, 28 °C) in YPD. The cells were centrifuged, washed with YNB, and resuspended at an OD600 of 1. Successive tenfold dilutions were performed (10⁰–10^–4), and 10 µl of each dilution was spotted onto YNB plates containing the indicated FAs and lipids. The following FAs were used in our study: mC10, methyl decanoate (SAFC, 99%); mC12, methyl laurate (Sigma Aldrich, 98%); mC14, methyl myristate (SAFC, 98%); mC16, methyl palmitate (SAFC, 97%); tributyrin (ACROS, 98%); triolein (Fluka, 65%); and C8, octanoic acid (Aldrich, 98%). The minimal YNB medium contained 0.17% (w/v) yeast nitrogen base (without amino acids and ammonium sulfate, YNBww), 0.5% (w/v) NH₄Cl, and 0.2% (w/v) glucose; it was supplemented with 0.5% (w/v) erythritol for induction. To complement strain auxotrophy, uracil (0.1 g/L) and lysine (0.2 g/L) were added as required. The media were buffered with 50 mM phosphate buffer at pH 6.8. Stock solutions of the methyl esters of the FAs and of the lipids were subjected to sonication three times for 1 min in the presence of Tween 40 (Sigma) and used at a final concentration of 0.4%. Solid media were created by adding 1.6% agar. The plates were incubated at 28 °C. The drop tests were conducted twice, with two subclones of each specific genotype. Pictures were taken every 24 h. Representative images are shown.

For the first stage of the process, lauric acid (≥ 98%) and methanol (99.9%) were purchased from Thermo Scientific and RCI Labscan, respectively. Amberlyst 15 was obtained from Alfa Aesar and used without any modification. For the second stage of the process, sucrose was purchased from Merck and DMSO (≥ 99%) was obtained from Loba Chemical. Amberlyst 21 from Alfa Aesar was used as a catalyst without any pretreatment. Methyl laurate (≥ 98%) and sucrose monolaurate (98%) were obtained from Sigma-Aldrich for analytical purposes.