Trans 2 hexenal

L-Glutathione (reduced), glutathione oxidized, L-methionine, trans-2-hexenal, acetonitrile, sodium borohydride (NaBH₄), ethanol, N-(tert-butoxycarbonyl)-L-cysteine (Boc-Cys-OH), triethylamine, sodium sulfate, triethylsilane, trifluoroacetic acid, 4-methylpent-3-en-2-one (mesityl oxide), mesityl oxide-d₁₀, sodium carbonate, acetic acid, hydrochloric acid, (+)-catechin, (−)-epicatechin, (−)-epicatechin gallate, procyanidin B1 and procyanidn B2 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Diethyl ether, pyridine, dichloromethane, 1,4-dioxane, pentane and ethyl acetate were obtained from Merck (Darmstadt, Germany). Formic acid (LC-MS, Fluka), methanol (LC-MS, Chromasolv, Sigma-Aldrich) and ultra-pure water of Milli Q gradient (EMD Millipore, Billerica, MA, USA) were used for chromatography. For grape grinding under cryogenic conditions (−196 °C) a M20 mill from IKA (Staufen, Germany) was used.

Emulsions were prepared using canola oil purchased from a local supermarket. Davisco Foods International, Inc. (Eden Prairie, Minnesota, USA) provided β-lactoglobulin with a purity above 95%. Sodium azide and phosphoric acid were obtained from Daejung Chemicals & Metals Co., Ltd. (Siheung, Gyeonggi, Republic of Korea). Four volatile organic compounds (VOCs) of different physicochemical properties (Table 1) were used: ethyl acetate, 1-penten-3-one, trans-2-hexenal and nonanal (Sigma-Aldrich, St. Louis, Missouri, USA). The purity of all VOCs was above 95%.Table 1

Physicochemical properties of selected volatile organic compounds (VOCs)

Volatile flavour compound	Chemical formula	Boiling point (°C)	Log Pa	Vapour pressure (mmHg) 25 °C	Water solubility (g/L) 25 °C
ethyl acetate	C4H8O2	77	0.7	73	88
1-penten-3-one	C5H8O	103	1.0	31	20
trans-2-hexenal	C6H10O	146	1.8	4.6	5.3
nonanal	C9H18O	191	3.5	0.5	0.1

Lide (2005 ) and TGSC (2009 )

^aPartition coefficient between octanol and water

All the dyes (Table S1) were purchased from Sigma-Aldrich (Merck KGaA, St. Louis, MI, USA). Anhydrous ethanol (≥99.5%) and ethyl acetate were purchased from Sinopharm. Trans-2-hexenal and benzaldehyde were purchased from Sigma-Aldrich. Hexyl acetate, (+)-limonene, β-myrcene, 3-carene, and isoamyl acetate were purchased from Aladdin Biochemistry Technology Co., Ltd (Shanghai, China).

The cellular effects of VvHPL1 products were examined in different systems expressing a GFP fusion of the actin-binding domain 2 of Ath fimbrin 1. Both cis-3-hexenal (50% purified in triacetine, Sigma-Aldrich) and trans-2-hexenal (98% purified, Sigma-Aldrich) were tested for their putative role in signalling and defence responses. In the case of transgenic tobacco BY-2 (Sano et al., 2005 (link)) and transgenic V. vinifera cv. ‘Chardonnay’ (Guan et al., 2014 (link)), cells were collected at day 3 after subcultivation on 0.8% MS agar, and 1 µl of cis-3-hexenal, trans-2-hexenal, or the solvent triacetin/water was placed on 10 mm sterile filter paper discs in the centre of the MS agar plate and incubated for 10 min. After incubation, cells were transferred to MS medium. Cells were then followed over time by spinning disc microscopy as described above. In the case of transgenic grapevine plants (Guan et al., 2014 (link)), fully expanded leaves (leaf 4 from the top) were used in two orientations to view both the abaxial and the adaxial side of the leaf.

The following chemicals and reagents were used for the analyses: benzyl alcohol, benzaldehyde, β-cyclocitral, dihydroactinidiolide, eugenol, geraniol, trans-2-hexenol, trans-2-hexenal, cis-3-hexenol, cis-3-hexenal, β-ionone, jasmine lactone, linalool, methyl salicylate (MeSA), MeJA, 2-phenylethanol, phenylacetaldehyde, and phenylacetonitrile (Sigma–Aldrich, Germany).

Humulene, 2-penten-1-ol, 2-hexen-1-ol, (E)-3-hexen-1-ol, 1-hexanol, p-cymen-8-ol, 2-vinyloxy)-ethanol, 3-methyl-1-butanol, 1,3,8-p-menthatriene, allo-ocimene, 2-carene, α-phellandrene, 3-carene, terpinolene, 1,3-cyclohexadiene, 1-methyl-4-(1-methylethyl), isovaleraldehyde, 3-hexenal, 2,4-dimethyl-benzaldehyde, heptanal, trans-2-heptenal, trans-2-pentenal, 1-nonanal, decanal, citral, 1-penten-3-one, 2-cyclohepten-1-one, 3-methylcyclohex-3-en-1-one, 6-methyl-5-hepten-2-one, ethyl-propionate, ethyl cyclopropanecarboxylate, ethyl crotonate, isoamyl acetate, tetraethyl orthosilicate, ethyl butyrate, and γ-octanoic were purchased from TCI (Tokyo, Japan); and linalool, α-pinene, β-pinene, β-myrcene, D-limonene, p-cymene, β-ocimene, hexanal, γ-terpinene, and trans-2-hexenal were purchased from Sigma (St. Louis, MO, USA). All of the chemical standards were of GC quality.

Dichloromethane, anhydrous sodium sulfate, n-hexane were purchased from Yifengtiancheng Scientific Instruments Co. Ltd. (Beijing, China); n-Alkanes (C7~C30) and 4-methyl-2-pentanol were purchased from Sigma-Aldrich (St Louis, MO, USA); Nitrogen (99.9992% purity) was purchased from Beijing Haipu Beifen Gas Industry Co. Ltd. (Beijing, China).
Standards of volatile compounds, hexanal, trans-2-hexenal, (Z)-3-hexenyl acetate, trans-2-heptenal, 6-methyl-5-hepten-2-one, (Z)-3-hexen-1-ol, acetic acid, 2-octanone, and 1-octen-3-one were purchased from Sigma-Aldrich Co. Ltd. (Santa Clara, CA, USA).