Eicosapolyenoic acids

Abstract

Thirty years ago arachidonic (AA; 20:4 Δ^5,8,11,14) and eicosapentaenoic (EPA; 20:5 Δ^5,8,11,14,17) acids were identified as elicitors from the late blight pathogen, Phytophthora infestans, capable of triggering the dramatic shifts in isoprenoid metabolism, defense reactions and cell death associated with the hypersensitive response of potato to incompatible races of the pathogen.¹ Among plant pathogens, the capacity for eicosapolyenoic acid synthesis appears to be largely restricted to oomycetes, primitive fungi (e.g., zygomycetes and chytrids) and nematodes. AA and EPA, precursors to eicosanoids that mediate inflammatory responses and serve as critical signals for immune and central nervous system functions in mammals, continue to be compelling molecules for study in plants because of what they may reveal about lipid-based signaling and induced immunity in plant-microbe interactions and possible mechanistic parallels as conserved signaling molecules across eukaryotic kingdoms. In spite of the intriguing cross-kingdom connections in AA/EPA signaling, there has been relatively little research to resolve eicosapolyenoic acid perception and action in plants, in part because of experimental limitations of systems where these fatty acids display strong activity. However, this state of affairs may change with our recent discovery that Arabidopsis responds to AA and that plants engineered to express very low levels of eicosapolyenoic acids (EP plants) have remarkably altered phenotypes to biotic challengers.

In Savchenko et al.,² we demonstrate that (1) AA reciprocally impacts salicylate and jasmonate signaling networks in a manner consistent with the observed outcomes to various biotic challengers and associated gene expression, (2) AA's effects in Arabidopsis are dependent upon jasmonic acid and (3) direct application of AA to Arabidopsis or tomato leaves results in a generalized rapid stress response, manifested as enhanced tolerance to Botrytis infection. These responses, triggered by AA, but not by less-unsaturated eicosenoic acids or fatty acids common to the plant, support the notion that as in animals AA potentiates transcriptional regulation of a selected group of stress responsive genes in plants with the ultimate outcome of enhanced tolerance of plants to a range of biotic stresses. The enabling experimental materials, including plants engineered with a genetic trio for synthesis of eicosapolyenoic acids³ and a collection of Arabidopsis mutants and reporter constructs in oxylipin and stress network signaling,^4–6 will serve as a vehicle to examine the regulatory role of eicosapolyenoic acids as novel microbial associated molecular patterns (MAMPs) in oomycete-plant defense signaling networks.

The rich history to eicosapolyenoic acids as elicitors has been informative about regulation of isoprenoid metabolism as well as suggestive of the role of redox and reactive oxygen species (ROS) in plant-microbe interactions. Furthermore, these studies provided the platform for the most recent findings that specifically exploited AA's elicitor activity to identify a selected group of AA-responsive genes within the stress signaling networks. In potato disk assays, the structural features in fatty acids required for elicitor activity were identified, establishing the critical importance of a minimum chain length of 20 carbons and cis-unsaturation at the 5-position for optimal activity.⁷ Together with inhibitor studies, these findings provided early support for a central role of 5-lipoxygenase (LOX) in the action of eicosapolyenoic acids.^8,9 Subsequent studies found AA/EPA and their LOX products to elicit programmed cell death^10,11 and to induce resistance against various pathogens.¹² Although the functional significance of LOX is unclear, in Arabidopsis we observed that the six LOX isoforms responded differentially to the presence of AA, suggesting a degree of specificity at this level.

The early studies also demonstrated that following elicitor treatment there is a specific and rapid redirection of metabolism from triterpenoids (i.e., steroid glycoalkaloids) to sesquiterpenoid phytoalexins, coordinated in part by differential regulation of genes encoding key enzymes in the mevalonate pathway, specifically isoforms of HMG-CoA reductase, sesquiterpene cyclase and squalene synthase.^13–15 Microautoradiography provided direct evidence for the release of eicosapolyenoic acids from spores and hyphae of the pathogen during infection, where they were detected as common fatty acids covalently linked to phospho- and glycerol-lipids, ceramides and other cellular components, including complex lipoglycoprotein fractions.^16–18 Interestingly, in all cases, removal of any esterified eicosapolyenoic acids from active fractions abolishes elicitor activity of these fractions. A particularly intriguing and as yet unresolved question is the potential interaction(s) between eicosapolyenoic acids with branched β-1,3-glucans, major carbohydrate polymers in oomycete cells that function as MAMPs in some plants.¹⁶ These highly purified glucans do not have inherent elicitor activity in potato, yet dramatically enhance the sensitivity of potato tissue to AA/EPA by up to several orders of magnitude.¹⁹

As low molecular weight, lipophilic elicitors, eicosapolyenoic acids occupy a niche that is different from most microbial elicitors (note: current nomenclature refers to these as pathogen or microbial associated molecular patterns, i.e., PAMPs or MAMPs). As inducers of resistance and a generalized set of defense responses (e.g., PAMP-triggered immunity) that are present in both pathogenic and nonpathogenic microbes, they certainly meet the criteria to be considered as MAMPs.²⁰ However, plant perception of AA/EPA very likely occurs in a different manner from that of well characterized MAMP/receptor complexes in Arabidopsis, such as bacterial flagellin and EF-Tu and their cognate LRR-receptor kinases, FLS2 and EFR, respectively. Lipid profiles of the engineered Arabidopsis plants examined in our recent study together with radiolabelling experiments with exogenous ¹⁴C-AA indicate that plants can readily incorporate eicosapolyenoic acids into phospho- and glycero-lipids as well as oxidize them to as yet uncharacterized products.¹⁹ Thus, any model for signal-receptor-response coupling will need to resolve if and how modification of the fatty acid profile together with AA/EPA metabolism contribute to elicitor action. The additional complexity presented by glucans suggests that there is a coordination of different receptors in eicosapolyenoic acid action, perhaps akin to the heteromerization that occurs among receptors upon activation for other MAMPs.²⁰

As many of the most important oomycete diseases affect roots and crowns of plants, we have developed hydroponic systems with Arabidopsis in parallel with tomato to provide tractable experimental systems for comparative studies of Phytophthora infection and eicosapolyenoic action in plants.²¹ With the resurgence in interest in elicitors/MAMPs and the genomic and metabolomics tools available for receptor discovery and mode of action studies, renewed research on eicosapolyenoic acid action may identify additional surveillance options in plants against oomycetes. These studies will be complementary to the ongoing and elegant work on oomycete effectors, which might act to dampen eicosapolyenoic acid elicitor activity.²² Importantly, these findings could have implications for identification of novel targets for enhanced disease resistance against this challenging group of plant pathogens.

Addendum to: Savchenko T, Walley JW, Chehab EW, Xiao Y, Kaspi R, Pye MF, et al. Arachidonic acid: an evolutionarily conserved signaling molecule modulates plant stress signaling networks. Plant Cell. 2010;22:3193–3205. doi: 10.1105/tpc.110.07385.