Figure 1.

Elicitation and volatile collection procedures. A, M. sexta's OS were collected from third to fourth instar larvae reared on wild-type N. attenuata plants. OS purified by IEX (IOS), which are free of FACs, 2-HOT, and other FA metabolites, were prepared as described previously (Halitschke et al., 2001) using Amberlite IRA-400 resin. OS and IOS extracts were flushed with argon and stored at −20°C until use. B, The first fully expanded leaf (L+1) of rosette-stage N. attenuata plants (randomized; n = 6 biological replicates per treatment) was wounded (W) with a fabric pattern wheel, and 20 μL of one of the different eliciting solutions to test was directly applied to the leaf surface. Volatile emissions strictly induced by the mechanical wounding and/or the alkalinity of M. sexta's OS were assessed by applying a 0.1 m Tris, pH 9, buffer solution (B) containing 0.1% (v/v) Triton X-100 to wounded leaves. This nonionic surfactant was added to the B solution to evaluate its potential eliciting effect, as it was used for the preparation of the FAC solution. Synthetic N-linolenoyl-l-Gln (C18:3-Gln) and N-linolenoyl-l-Glu (C18:3-Glu), the two major FACs, and 2-HOT were diluted in the B solution at concentrations similar to those found in M. sexta's OS. Control plants (CTRL) were left untreated. C, Leaves were enclosed, 1 h after elicitation, in food-quality plastic containers secured with miniature clips. Plant volatiles were collected, using self-packed Super Q traps connected to a manifold vacuum pump, as described by Halitschke et al. (2000), during three consecutive periods of 12 h, the second one occurring during the glasshouse dark time. Background contaminants present in ambient air were also tested using empty trapping containers.