Octanoic acid

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.

Authentic standards were co-analyzed and used for confident metabolite identifications [31 (link)]. Authentic standards used were 1-butanol, 1-pentanol, 1-hexanol, 2-pentylfuran, (Z)-3-hexenyl acetate, (E,E)-2,4-heptadienal, (E)-2-octen-1-ol, β-linalool, nonanoic acid (TCI America, Portland, OR, USA); decanal, decanoic acid, β-ionone, 3-pentanone, butyl acetate, benzaldehyde (Sigma-Aldrich Inc., St. Louis, MO, USA); 2,3-butanedione, 1-penten-3-ol, hexanal, myrcene, p-cymene, D-limonene, 1-octanol (Fluka Chemical Co., Milwaukee, WI, USA); isoprene, 1-penten-3-one, pentanal, 2-ethylfuran, 3-methyl-1-butanol, (Z)-2-penten-1-ol, (E)-2-hexenal, 2-ethylthiophene, heptanal, (E,E)-2,4-hexadienal, (E)-2-heptenal, hexanoic acid, 1-octen-3-ol, octanal, benzeneacetaldehyde, (E)-2-octenal, nonanal, 4-oxoisophorone, (E)-2-nonenal, octanoic acid, 2-nonen-1-ol, (E,E)-2,4-nonadienal, (E)-2-decenal, N,N-dibutylformamide (Aldrich Chemical Co., Milwaukee, WI, USA); p-menth-1-en-4-ol (Wako Chemical Co., Richmond, VA, USA); and 1-octen-3-one (Alfa Aesar, Ward Hill, MA, USA). 3-Pentanol (Fluka Chemical Co.) was used as an internal standard. The internal standard was chosen due to its stability, reproducibility, and absence in alfalfa plants.

Sodium citrate tribasic dihydrate (Na₃(C₆H₅O₇)·2H₂O, ≥99.0% pure) (Na₃Cit), anhydrous potassium phosphate dibasic (K₂HPO₄, ≥99.0% pure), sodium carbonate (Na₂CO₃, ≥99.0% pure), calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O, ≥99.0% pure), and methyl jasmonate (C₁₃H₂₀O₃, 95.0%, racemic), 1 propanol, 2-methyl-1-propanol, 3-methyl-1-butanol, 1-hexanol, E-3-hexen-1-ol, β-phenyl-ethanol, ethyl-acetate, 3-methyl-1-butanol-acetate, ethyl-caprate, ethyl-hexanoate, ethyl-octanoate, ethyl- dodecanoate, diethyl-succinate, ethyl-tetradecanoate, ethyl-hexadecanoate, nerolidol, citronellol, linalool, octanoic acid, hexanoic acid, 3-methyl-thio-1-propanol, 2-octanol and 4-methyl-2-pentanol were supplied by Sigma Aldrich (Madrid, Spain). NaCl, NaOH 0.1N, and ethanol 96% were from Panreac (Barcelona, Spain). Ultrapure water came from a Milli-Q system (Millipore Corp., Bedford, MA, USA). Tartaric acid from Laffort (Bordeaux, France) was used to adjust model wine. The TDI enzymatic kit was used for L-Malic acid analysis (Tecnología Difusion Ibérica S.L., Gavá, Spain)

All chemicals were used as received: lead(ii) acetate tri-hydrate (Aldrich, 99.999%), oleic acid (OA, Aldrich, 90%), nonanoic acid (Alfa Aesar, 97%), octanoic acid (Aldrich, >99%), heptanoic acid (Aldrich, >99%), hexanoic acid (Aldrich, >99%), tri-octylphosphine (TOP; ABCR, 97%), 1,2-diiodoethane (DIE; Aldrich, 99%), poly (methyl methacrylate) in anisole (Allresist, AR-P 632.06), methyl isobutyl ketone (MIBK, Aldrich, >99%), 2-propanol (ACS, >99.7), and chlorobenzene (Aldrich, >99.5%).

Pure reference standards of methyl-2-methylbutanoate (98.0%), 2-hexanol (97.0%), 3-methylbutanoic acid (97.5%), methyl butyl acetate (98.0%), methyl-2-methylpentanoate (99.5%), gamma-butyrolactone (98.0%), dimethyl malonate (97.0%), ethyl-2,3-dimethylbutanoate (99.5%), methyl-2-methyl acetoacetate (99.5%), methyl-2-hydroxy-2-methylbutanoate (98.0%), methyl hexanoate (99.5%), methyl-3-(methylthiol)-propanoate (99.5%), hexanoic acid (97.0%), trans-β-ocimene (98.0%), methyl-2-methylhexanoate (99.5%), ethyl hexanoate (98.0%), δ-lactone (98.0%), 2,5-dimethyl-4-hydroxy-3(2H)-furanone (99.5%), methyl-3-hydroxyhexanoate (99.5%), 2,5-dimethyl-4-methoxy-3(2H)-furanone (98.0%), methyl octanoate (99.5%), octanoic acid (97.0%), y-octalactone (98.5%), δ-octalactone (98.0%), copaene (97.0%), methyl decanoate (99.5%), 2-methyl-4-vinyl phenol (99.5%), decanoic acid (97.0%), y-farnesene (98.0%), germacrene (98.0%), globulol (98.0%), spathulenol (98.0 5), (Z)-7-tetradecenal (97.0%), and octadecanal (99.5%) were purchased from Aldrich, Steinheim, Germany. Gamma-lactone (98.0%) and methyl dodecane (99.5%) were obtained from Parchem, New Rochelle, NY and Achemica Corp. Aigle, Switzerland, respectively. The n-alkane standard (C₇–C₃₀) was obtained from Sigma-Aldrich Chemicals Co. (St. Louis, MO). Other chemicals were of analytical grade.

The calibration curves of alpha-farnesene (Merck, Darmstadt, Germany) and Octanoic acid (Merck, Darmstadt, Germany) were carried out in a concentration range from 100 ppb to 1 ppm. The reproducibility and the recovery of the method were determined by depositing the standard solutions directly on the apple, then conserving the apple at 4 °C for one day, followed by extraction with the strip. Quality control samples (QC) containing spiked standards were also acquired before and after the analysis.