Lipoxygenase-mediated modification of insect elicitors

Abstract

Plants can distinguish mechanical damage from larval folivory through the recognition of specific constituents of larval oral secretions (OS) which are deposited on the surface of leaf wounds during feeding. Fatty acid-amino acid conjugates (FACs) are major constituents of the OS of Lepidopteran larvae and they are strong elicitors of herbivore-induced defense responses in several plant species, including the wild tobacco Nicotiana attenuata. When OS from Manduca sexta larvae is deposited on N. attenuata wounded leaves, the major FAC N-linolenoyl-glutamic acid (18:3-Glu) is modified within seconds by a heat labile process. Some of the major modified forms are oxygenated products derived from 13-lipoxygenase activity and one of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of enhanced JA biosynthesis and differential monoterpene emission in N. attenuata leaves.

HAEs are Specific Elicitors of Plant Responses to Insect Folivory

Upon perception of insect folivores (e.g., Lepidopteran larvae), plant cells activate complex mechanisms to induce specific defense responses that depend on the interacting species of plants and insects. These mechanisms are mediated by the perception of components (HAEs; herbivore associated elicitors) in the insect's oral secretions (OS),^1–3 a particular temporal pattern of mechanical damage^4,5 or a combination of both. Among the HAEs present in the insect's OS, one of the first to be isolated was the fatty-acid amino-acid conjugate volicitin (17-OH-18:3-Gln), which was found in the OS of Spodoptora exigua larvae feeding on maize (Zea mays) plants.³ Glucose oxidase was first identified from the corn earworm Helicoverpa zea,⁶ and it was shown to be active as an elicitor in Nicotiana tabacum,⁷ Nicotiana attenuata,⁸ Medicago truncatula⁹ and Solanum lycopersicum (tomato)¹⁰ among other species. β-glucosidase was first identified in the OS of Pieris brassicae¹¹ and it was shown to be active in Phaseolus lunatus (lima beans),¹² maize¹² and Brassica oleracea (cabbage).¹¹ Inceptins were found in the OS of Spodoptora frudgiperda larvae feeding on cowpea (Vigna unguiculata)¹³ and more recently, sulfur-containing compounds (caeliferins), were isolated from Schistocerca americana OS and shown to induce responses in maize plants.¹⁴

M. sexta's main elicitors to induce insect specific defense responses in Nicotiana attenuata plants are FACs, which are composed predominantly of linoleic acid (18:2) or linolenic acid (18:3) conjugated to Glu or Gln.¹⁵ When applied to wounded N. attenuata leaves, synthetic FACs induce (among other responses) the differential production of jasmonic acid (JA) and ethylene,^15,16 large scale transcriptomic and proteomic changes,^1,17 and the release of herbivore induced plant volatiles (HIPVs).¹⁸ Moreover, when removed from M. sexta OS, the remaining FAC-free OS fraction loses its capacity to elicit insect specific responses in N. attenuata^1,17,18 which can be recovered after reconstitution of the FAC-free OS fraction with synthetic FACs.¹

In contrast to elicitors derived from plant pathogens, the perception of insect elicitors and their mode of action are poorly understood. New insights into the mode of action of FACs have emerged from a recent study showing that one of the major FACs in M. sexta OS, 18:3-Glu, is enzymatically modified within seconds upon contact with wounded N. attenuata leaves and that its metabolism may tailor some defense responses against herbivores in this plant species.¹⁹

Rapid Metabolism of FACs in Wounded N. attenuata Leaves

When synthetic 18:3-Glu or OS from M. sexta larvae were applied onto N. attenuata wounded leaves, more than 70% of the initial amount of this FAC was metabolized within 30 seconds. This heat-labile process was not the result of the hydrolysis of 18:3-Glu into free 18:3 and glutamate but of the rapid generation of metabolites both more and less polar than 18:3-Glu; 13-hydroxy-18:3-Glu, 13-hydroperoxy-18:3-Glu and 13-oxo-13:2-Glu were the major metabolites formed as identified by LC-MS/MS. The formation of these metabolites was largely dependent on the activity of lipoxygenase 2 (NaLOX2); plants silenced in the expression of this gene showed reduced rates of 18:3-Glu oxidation and accumulated only small amounts of the 18:3-Glu oxygenated derivatives compared to control plants. NaLOX2 is a chloroplast-localized 13-LOX involved in the supply of polyunsaturated fatty acids for green leaf volatile biosynthesis²⁰ and the results suggested that upon tissue disruption, NaLOX2 gains access to components in the insect's OS deposited on the wound surface during feeding. Similar to 18:3-Glu, 13-oxo-13:2-Glu also activated the enhanced production of JA in N. attenuata leaves whereas 13-hydroxy-18:3-Glu did not, indicating that the modification of 18:3-Glu generated both an active elicitor and an inactive molecule. Moreover, 13-oxo-13:2-Glu also induced the differential emission of two monoterpene volatiles (β-pinene and an unidentified monoterpene) in plants deficient in NaLOX2 activity. Finally, in addition to the three derivatives described above, at least ten other modified forms of 18:3-Glu were detected on the leaf wound surface by analysis of radiolabeled 18:3-Glu, suggesting the possibility that different modified forms may be active in the differential stimulation of distinct responses to herbivores in N. attenuata (Fig. 1). Whether these responses depend on different receptors is an intriguing question.

Figure 1.

Modification of FACS on N. attenuata during insect folivory. During caterpillar feeding, OS are deposited on the wound surface and FACS get into contact with disrupted leaf tissue. NaLOX2 is probably released from broken chloroplasts and dioxygenates 18:3-Glu to form 13-OOH-18:3-Glu, which is subsequently reduced to 13-OH-18:3-Glu to form an inactive elicitor or cleaved to produce a C₅ molecule and a C₁₃ active elicitor, 13-oxo-13:2-Glu. We hypothesize that additional FACS containing polyunsaturated fatty acids in their structures are also metabolized by LOX activity (dashed arrows). Additional modifications independent of LOX activity are probably also generated (dashed arrows).

The results suggested that the recognition of insect derived elicitors may be at least partially mediated by the modification of components in the OS of feeding larvae by plant enzymes. Thus, plant-mediated conversion of insect elicitors, resulting in the biogenesis of novel elicitors, adds a level of complexity to plant-insect interactions and opens up new directions on how plants may perceive folivorous insects to tailor defense and tolerance responses.

Addendum to: VanDoorn A, Kallenbach M, Borquez AA, Baldwin IT, Bonaventure G. Rapid modification of the insect elicitor N-linolenoyl-glutamate via a lipoxygenase-mediated mechanism on Nicotiana attenuata leaves. BMC Plant Biol. 2010;10:164. doi: 10.1186/1471-2229-10-164.