E 2 hexenol

EAG was performed on one isolated antenna from each G2 wild-type, heterozygous or homozygous adults, to evaluate the Orco mutation effect on the antennal capacity to detect specific odorants. Mounted between two glass electrodes containing Ringer solution49 and continuously humidified by charcoal-filtered airflow (70 L/h), antennae were stimulated, with a one minute interval, with 10 μg of E-ocimene, Z3-hexenyl-acetate, E2-hexenol, benzyl alcohol and 2-phenyl acetaldehyde diluted in paraffin oil, 10 μg of propionic acid in water (all purchased from Sigma-Aldrich) and 1 μg of the main sex pheromone component Z9,E11-14:Ac (Gift from Martine Lettere, Versailles, France) in hexane (Carlo Erba) (puffs of 500 ms, 10 L/h). EAG amplitudes were calculated by subtracting the solvent response. Analyses were done with Clampfit 10 software (Molecular Devices).

Unless otherwise stated, all the reagents used in this study were analytically pure. The water used for various experiments was purified with a Milli-Q purification system. Tween 80, glucose anhydrous, cupric sulfate, potassium sodium tartrate tetrahydrate, sodium hydroxide, magnesium chloride, acetyl-CoA, linoleic acid sodium salt, nicotinamide adenine dinucleotide, dithiothreitol, Triton X-100, 4-morpholineethanesulfonic acid, polyvinylpolypyrrolidone (PVPP), dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium acetate, acetaldehyde, and butanol were obtained from Yuanye Co., Ltd. (Shanghai, China). BTH, E-2-hexenal, hexanal, Z-3-hexenal, E-2-hexenol, Z-3-hexenol, hexanol, Z-3-hexenyl acetate, hexyl acetate, 2-octanol, linolenic acid, and linoleic acid were HPLC grade and obtained from Sigma-Aldrich (Shanghai, China).

Synthetic hexyl butyrate was obtained from Pherobank (Wageningen, The Netherlands; >99% purity). (E)-2-Hexenyl butyrate (98.6% purity), (Z)-2-hexenyl butyrate (95.0% purity), (E)-2-octenyl butyrate (98.0% purity) and (Z)-2-octenyl butyrate (97.0% purity) were prepared by reaction of butyric anhydride with (E)-2-hexenol (Sigma-Aldrich, St. Louis, MO, USA), (Z)-2-hexenol (TCI, Tokyo, Japan), (E)-2-octenol (TCI, Tokyo, Japan) and (Z)-2-octenol obtained by hydrogenation of 2-octyn-1-ol using Lindlar’s catalyst, respectively, in the presence of pyridine. (E)-4-Oxo-2-hexenal (97.4% purity; 17.5% yield) was synthesized by treating 2-ethylfuran (Alfa Aesar, Heysham, UK) with N-bromosuccinimide and pyridine in aqueous tetrahydrofuran as described by Moreira and Millar [15 (link)]. n-Alkanes (C₁₄-C₁₇), dimethyldisulfide (DMDS) and dodecyl acetate (12:OAc) were purchased from Sigma-Aldrich.

LiChrolut EN cartridges (500 mg, 6 mL) were obtained from Merck (Darmstadt, Germany). All chemicals were of analytical reagent grade unless otherwise stated, and water was obtained from a Milli-Q purification system (MilliporeSigma, Burlington, MA, USA). Folin-Ciocalteu reagent, sodium carbonate, sodium acetate, potassium chloride, sodium chloride, sucrose, methanol (HPLC grade), ethanol (HPLC grade), dichloromethane (HPLC grade) and gallic acid were purchased from SCR^® (Shanghai, China). Eucalyptol (99%), linalool (≥95%), (−)-myrtenol (95%), carveol (97%, mixture of isomers), borneol, (≥99.0%, sum of enantiomers, GC), terpinolene (≥94.0%), β-citronellol (95%), geraniol (98%), ethyl-2-methylbutyrate (99%), ethyl-3-methylbutyrate (98%), (Z)-3-hexenol (98%), (E)-2-hexenol (96%), benzyl alcohol (≥99%), phenylethyl alcohol (≥99%), cuminic alcohol (97%), methyl butanoate (99%), isobutyl acetate (99%), ethyl butanoate (99%), isoamyl acetate (≥99%), methyl benzoate (99%), ethyl benzoate (≥99%), diethyl succinate (99%), methyl salicylate (≥99%), ethyl octanoate (≥99%), ethyl decanoate (98%), methyl vanillate (99%), decanoic acid (≥98.0%), benzaldehyde (≥99%), p-cresol (99%), 4-vinylguaiacol (≥98.0%), vanillin (99%), (E)-asarone (98%), (Z)-asarone (70%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All volatile standards were prepared by dilution with HPLC grade methanol.

According to Yi et al. (2017) (link), the main GLVs from N. tabacum are (Z)-3-hexenal (CAS: 929-96-1, purity of 98%), (E)-2-hexenol (CAS: 928-95-0, purity of 97%), (E)-2-hexenal (CAS: 85761-70-2, purity of 98%), (Z)-3-hexenyl acetate (CAS: 3681-71-8, purity of 98%), and (E)-2-hexenyl acetate (CAS: 2497-18-9, purity of 98%), and all were purchased from Sigma-Aldrich (Shanghai, China). These chemicals were tested individually in a Y-tube experiment.

The synthetic standards of the following EAD-active compounds were used: hexanal (Sigma Aldrich, 99%), (E)-2-hexenol (Aldrich, 96%), benzaldehyde (Sigma Aldrich, 99.5), β-myrcene (Sigma Aldrich, 99%), ocimene (International Flavors and Fragrance, New York, USA, (Z)-β-ocimene = 27%, (E)-β-ocimene = 67% and allo-ocimene = 6%), linalool oxide (Sigma Aldrich, mixture of stereoisomers with furanoid form, 99.5% and 0.5% pyranoid form), indole (Sigma Aldrich, 99%) and 1-octen-3-ol (Fluka Chemica, racemic mixture of R and S 98%).