Figure 3.

The silk gland extract suppressed formation of green leaf volatiles through conversion of fatty acid hydroperoxide into its keto derivative. The mulberry leaf powder was homogenized in the presence of silk gland extract equivalent to 0–58.8 mg wet weight of each part of the silk gland (per 1 g fresh weight of leaves) and incubated for 10 min to facilitate enzyme reaction to form volatiles. (A) Silk gland used for extraction. (B) The amounts of (E)-2-hexenal and (E)-2-hexen-1-ol formed in the absence or presence of the silk gland extract that was obtained from the anterior part of the silk gland (consisting of the anterior silk gland and the anterior part of middle silk gland, ASG + MSG-A). Averages with error bars (SE, n = 3, technical replicate) are shown. The lowest amount of the silk gland extract (0.59 mg) corresponded to 0.016 [ASG + MSG-A] equivalent of that derived from one silkworm. Different letters above the bars indicate significant differences among treatments for each extract (P < 0.05, GLM following Holm’s P-value adjustment). (C) Overlay of UV spectra of the reaction mixture consisting of the silk glands with 20 µM of 13S-HPOT. The reaction was scanned from 220 to 320 nm at 0, 10, 20, 30, and 40 sec after substrate addition. (D) HPLC-MS/MS analyses of the products formed by the silk gland extract from 13S-HPOT. Chromatograms with absorption at 280 nm (blue) and 234 nm (red) (upper chromatogram) and total ion chromatogram obtained with enhanced MS mode (lower chromatogram) are shown. The peak with an asterisk had a spectrum with λmax at 280 nm. The precedent peak is 13S-HPOT. (E) MS profile obtained with the peak with asterisk in (D) with enhanced product ion mode with the collision energy of −45 V with the molecular ion of m/z 291.2. The possible fragmentation patterns are also shown. Proposed reaction of the enzyme to form 13-oxo-(9Z,11E,15Z)-octadecatrienoic acid (13-OTE) from 13S-HPOT is shown in inset of d.