Silencing of hydroperoxide lyase and allene oxide synthase reveals substrate and defense signaling crosstalk in Nicotiana attenuata.

Abstract

The fatty acid hydroperoxide (HP) substrates required for the biosynthesis of jasmonic acid (JA) and green leaf volatiles (GLVs) are supplied by separate lipoxygenases (LOX). We silenced the expression of two genes downstream of the LOX: allene oxide synthase (AOS) and HP lyase (HPL) by antisense expression of endogenous genes (NaAOS, NaHPL) in Nicotiana attenuata, in which the biosynthesis of JA is amplified by herbivore-specific elicitors. We report that these elicitors also amplify wound-induced GLV releases, but suppress the wound-induced increase of NaHPL transcripts, suggesting that substrate flux controls GLV biosynthesis. As expected, silencing of NaHPL and NaAOS reduced GLV release and JA accumulation, respectively. Surprisingly, HPL- and AOS-silenced plants had enhanced JA and GLV responses, suggesting substrate 'crosstalk' between these two oxylipin cascades. Plants with depleted GLVs (as-hpl) were less attractive than wild type (WT) or empty vector control plants in choice-tests with native lepidopteran herbivores. In feeding trials, Manduca sexta larvae developed slower on as-hpl plants. The reduced larval consumption and performance, which was not caused by increases in defense responses in as-hpl plants, could be restored to WT levels by the addition of synthetic GLVs, demonstrating that GLVs function as feeding stimulants. Gene expression profiling by cDNA microarray analysis and characterization of several induced defenses in herbivore-elicited as-hpl and as-aos plants revealed differential involvement of JA and GLVs in defense signaling. Elicitation of volatile terpenoids (an indirect defense) requires JA signaling, where as trypsin protease inhibitor elicitation (a direct defense) requires both functional JA and GLV cascades.