Rapid modification of the insect elicitor N-linolenoyl-glutamate via a lipoxygenase-mediated mechanism on Nicotiana attenuata leaves

Abstract

Background: Some plants distinguish mechanical wounding from herbivore attack by recognizing specific constituents of larval oral secretions (OS) which are introduced into plant wounds during feeding. Fatty acid-amino acid conjugates (FACs) are major constituents of Manduca sexta OS and strong elicitors of herbivore-induced defense responses in Nicotiana attenuata plants.

Results: The metabolism of one of the major FACs in M. sexta OS, N-linolenoyl-glutamic acid (18:3-Glu), was analyzed on N. attenuata wounded leaf surfaces. Between 50 to 70% of the 18:3-Glu in the OS or of synthetic 18:3-Glu were metabolized within 30 seconds of application to leaf wounds. This heat-labile process did not result in free alpha-linolenic acid (18:3) and glutamate but in the biogenesis of metabolites both more and less polar than 18:3-Glu. Identification of the major modified forms of this FAC showed that they corresponded to 13-hydroxy-18:3-Glu, 13-hydroperoxy-18:3-Glu and 13-oxo-13:2-Glu. The formation of these metabolites occurred on the wounded leaf surface and it was dependent on lipoxygenase (LOX) activity; plants silenced in the expression of NaLOX2 and NaLOX3 genes showed more than 50% reduced rates of 18:3-Glu conversion and accumulated smaller amounts of the oxygenated derivatives compared to wild-type plants. Similar to 18:3-Glu, 13-oxo-13:2-Glu activated the enhanced accumulation of jasmonic acid (JA) in N. attenuata leaves whereas 13-hydroxy-18:3-Glu did not. Moreover, compared to 18:3-Glu elicitation, 13-oxo-13:2-Glu induced the differential emission of two monoterpene volatiles (beta-pinene and an unidentified monoterpene) in irlox2 plants.

Conclusions: The metabolism of one of the major elicitors of herbivore-specific responses in N. attenuata plants, 18:3-Glu, results in the formation of oxidized forms of this FAC by a LOX-dependent mechanism. One of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of JA biosynthesis and differential monoterpene emission.

Figure 1

18:3-Glu is rapidly metabolized on wounds of N. attenuata leaves. Three lines of puncture wounds were generated on leaves with a fabric pattern wheel and (a) 0.17 nmoles of synthetic 18:3-Glu, or (b) 10 μL M. sexta OS (containing approximately 0.17 nmoles of 18:3-Glu) were applied onto the wounds. Leaves were extracted at different times and 18:3-Glu levels quantified by LC-MS/MS. Initial 18:3-Glu amounts (T0 ↑) were set at 100%. Bars represent standard errors (± SE, n = 4).

Figure 2

Radio-HPLC chromatograms of wounded N. attenuata leaves treated with ¹⁴C-18:3-Glu. (a) Leaves of rosette stage N. attenuata plants were wounded with a pattern wheel and 0.1 μCi of [1-¹⁴C]18:3-Glu were immediately applied onto leaf wounds. The damaged tissue was harvested immediately (T0) or 2 min after the treatment. After extraction, the radiolabeled metabolites were separated by RP-radio-HPLC. The numbers (1 to 3) indicate the different fractions collected for further analysis. CPM: counts per minute. (b) M. sexta OS was spiked with 0.1 μCi ¹⁴C 18:3-Glu, applied onto wounded leaf tissue, extracted after different time points and separated by TLC (W+OS). Plates were exposed for 24 h to ¹⁴C-sensitive screens. Time 0: ¹⁴C 18:3-Glu was applied onto wounded leaves and immediately extracted. W+: ¹⁴C-18:3-Glu was applied onto wounded leaves and extracted after 2 min. 18:3-Glu^m1 and 18:3-Glu^m2 indicate different modified forms of 18:3-Glu.

Figure 3

MS analysis of the purified 18:3-Glu derivatives. (a, b, c). Proposed structures and MS spectra obtained by LC-ESI-XL-Orbitrap analysis (negative mode) of the three 18:3-Glu derivatives purified from fractions 1, 2 and 3 in Fig. 2. See text for a detailed discussion of the MS spectra.

Figure 4

Accumulation of 18:3-Glu derivatives on wounded WT N. attenuata leaves. WT plants were wounded with a pattern wheel and 0.17 nmoles of 18:3-Glu were applied to the wounds and samples harvested after different times. After extraction, samples were analyzed by LC-MS/MS (n = 3, bars denote ± SE). Numbers between brackets correspond to compounds 1, 2 and 3 in Fig. 2 and 3.

Figure 5

Analysis of 18:3-Glu metabolism in steamed leaves and in LOX deficient plants. (a) 18:3-Glu (0.17 nmoles) was applied to either wounded leaves, wounded-steamed leaves, or unwounded leaves. The 18:3-Glu blank sample denotes an unwounded leaf onto which 18:3-Glu was added after freeze killing the leaf. Leaves were extracted after 5 min of the treatments and 18:3-Glu levels were analyzed by LC-MS/MS. Statistical analysis was performed using a non-parametric Moses extreme test with Bonferroni correction, * indicates P < 0.017, N.S. denotes non-significant differences (n = 3, bars denote ± SE). (b) Leaves of irlox2 and irlox3 plants were wounded, 0.17 nmoles of 18:3-Glu were applied on the wounds, and the samples were harvested after 2 min. After extraction, 18:3-Glu derivatives were analyzed by LC-MS/MS (n = 3, bars denote ± SE). Different letters indicate significant differences (univariate ANOVA 13-oxo-13:2-Glu: F_3,3 = 85.4 P < 0.001, 13-OH-18:3-Glu: F_3,3 = 25.1 P < 0.001 13-OH-18:3-Glu: F_3,3 = 136.1 P < 0.001 followed by a Scheffé post-hoc test, P < 0.05). (c) Turnover of 18:3-Glu on WT and irlox2 plants. Leaves were wounded, 18:3-Glu (0.17 nmoles) were applied onto the wounds, and the samples were harvested after different times. 18:3-Glu levels were analyzed by LC-MS/MS. Initial 18:3-Glu amounts (T0) were set at 100% (n = 4, bars denote ± SE).

Figure 6

Analysis of JA and terpenoid volatiles in WT and irlox2 plants after elicitation. (a) WT plants were wounded and of the wounds immediately treated with either solvent alone, 18:3-Glu (0.17 nmoles), 13-oxo-18:3-Glu or 13-OH-18:3-Glu (see text for a description of the amounts used). Samples were taken after 60 min and JA quantified by LC-MS/MS (n = 3, bars denote ± SE). Different letters denote significant differences (univariate ANOVA F_3,3 = 17.9, P < 0.001 followed by a Scheffé post-hoc test, P < 0.05). (b,c) irlox2 and WT plants were wounded and treated with either 18:3-Glu (0.17 nmoles) or 13-oxo-13:2-Glu (see text for a description of the amounts used), the emitted volatiles were trapped for 8 h after 24 h of the treatment and analyzed by GCxGC-ToF. Injection of a β-pinene standard confirmed the structure of the first compound, while the second monoterpene remained unidentified. The MS spectra of the two compounds are shown in Additional file 3. Asterisks indicate significant difference (P < 0.05, Student's t-test).