Spider mite-induced (3S)-(E)-nerolidol synthase activity in cucumber and lima bean. The first dedicated step in acyclic C11-homoterpene biosynthesis

Abstract

Many plant species respond to herbivory with de novo production of a mixture of volatiles that attracts carnivorous enemies of the herbivores. One of the major components in the blend of volatiles produced by many different plant species in response to herbivory by insects and spider mites is the homoterpene 4,8-dimethyl-1,3(E), 7-nonatriene. One study (J. Donath, W. Boland [1995] Phytochemistry 39: 785-790) demonstrated that a number of plant species can convert the acyclic sesquiterpene alcohol (3S)-(E)-nerolidol to this homoterpene. Cucumber (Cucumis sativus L.) and lima bean (Phaseolus lunatus L.) both produce 4,8-dimethyl-1,3(E),7-nonatriene in response to herbivory. We report the presence in cucumber and lima bean of a sesquiterpene synthase catalyzing the formation of (3S)-(E)-nerolidol from farnesyl diphosphate. The enzyme is inactive in uninfested cucumber leaves, slightly active in uninfested lima bean leaves, and strongly induced by feeding of the two-spotted spider mite (Tetranychus urticae Koch) on both plant species, but not by mechanical wounding. The activities of the (3S)-(E)-nerolidol synthase correlated well with the levels of release of 4, 8-dimethyl-1,3(E),7-nonatriene from the leaves of the different treatments. Thus, (3S)-(E)-nerolidol synthase is a good candidate for a regulatory role in the release of the important signaling molecule 4,8-dimethyl-1,3(E),7-nonatriene.

Figure 1

Schematic representation of the release of 4,8-dimethyl-1,3(E),7-nonatriene, a volatile homoterpene released by many plant species after herbivory. Donath and Boland (1995) demonstrated the ability of leaves and flowers of several species to convert nerolidol to 4,8-dimethyl-1,3(E),7-nonatriene (2). We investigated the induction by herbivory in cucumber and lima bean of nerolidol synthase (1), the enzyme that should precede the sequence of reactions reported by Donath and Boland (1995).

Figure 2

Headspace analysis of cucumber and lima bean leaves. A, Undamaged cucumber leaves; B, cucumber leaves infested with spider mites for 5 d; C, cucumber leaves mechanically damaged with carborundum powder at 0 and 5 d before sampling; D, uninfested lima bean leaves; and E, lima bean leaves infested with spider mites for 5 d. Headspace samples were analyzed by GC-MS. The peaks are: (Z)-3-hexen-1-yl acetate (1); (E)-β-ocimene (2); 4,8-dimethyl-1,3(E),7-nonatriene (3); methyl salicylate (4); and 4,8,12-trimethyl-1,3(E),7(E),11-tridecatetraene (5).

Figure 3

Radio-GLC analysis of radiolabeled products formed from 10 μm [³H]FDP (60-min assay in buffer B) by enzyme preparations of cucumber leaves. A, Flame ionization detection (FID) signal of co-injected, unlabeled standards of (Z)-nerolidol (1), (E)-nerolidol (2), and (E,E)-farnesol (3). B to D, Radiotraces showing radiolabeled products of assays with undamaged cucumber leaves (B), cucumber leaves infested with spider mites for 5 d (C), and cucumber leaves mechanically damaged with carborundum powder 0 and 5 d before sampling (D).

Figure 4

Radio-GLC analysis of radiolabeled products formed from 50 μm [³H]FDP (30-min assay in buffer B) by enzyme preparations of lima bean leaves. A, FID signal of co-injected, unlabeled standards of (Z)-nerolidol (1), (E)-nerolidol (2), and (E,E)-farnesol (3). B to C, Radiotraces showing radiolabeled products of assays with undamaged lima bean leaves (B) and lima bean leaves infested with spider mites for 5 d (C).

Figure 5

Time-dependent formation (dpm per 100-μL assay) of nerolidol (•, right y axis) and total hexane-soluble products (left y axis) for undiluted (○) and 2-fold-diluted (▵) enzyme preparations of spider mite-infested cucumber (A) and lima bean (B). Enzyme extracts of spider mite-infested leaves were desalted to assay buffer B supplemented with 5 mm sodium orthovanadate, and assayed with 50 μm [³H]FDP as a substrate. Total hexane-soluble products were determined in duplicate 100-μL assays, and nerolidol formation was analyzed by radio-GLC on 1-mL enzyme assays run for 30, 60, and 90 min.

Figure 6

Radio-GLC analysis of sesquiterpene synthase activities in spider mite-infested cucumber leaves using heptakis(6-O-tert-butyldimethylsilyl-2,3-di-O-methyl)-β-cyclodextrin (50% in OV17, w/w) as the enantioselective stationary phase. A, FID signal of co-injected, unlabeled (3R)- and (3S)-(Z)-nerolidol (1 and 2, elution order of the two enantiomers is unknown), (3R)-(E)-nerolidol (3), and (3S)-(E)-nerolidol (4). B and C, Radiotraces showing radiolabeled product formed from 50 μm [³H]FDP (30-min assay in buffer B) by enzyme preparations from cucumber leaves infested with spider mites for 5 d (B) and lima bean leaves infested with spider mites for 5 d (C).

Figure 7

Proposed enzymatic mechanism of the formation of (3S)-(E)-nerolidol from FDP.