Jurgen Engelberth, Deptartment of Biology, University of Texas at San Antonio, San Antonio, TX

February, 2011

Introduction

In contrast to the major plant hormones auxins, cytokinins, gibberellins, ethylene, abscisic acid, and brassinosteroids, whose biological functions are well established, the oxylipins have come to our attention only recently as important regulators of many plant responses, in particular those related to biotic stresses caused by pests and pathogens. A diverse array of induced defense responses directed towards the containment of damage provoked by these hostile organisms strongly depends on the rapid activation of distinct oxylipins. Accordingly, plants limited in their ability to produce oxylipins are strongly impaired in their defense responses and usually fail to survive. But while many oxylipins appear to be important for the regulation of the plant immune system by providing direct and indirect protection against pests and pathogens, they also play a significant role in other aspects of plant physiology; for example, in development, pollen maturation, seed germination, and as inducer of tuber and trichome formation in potato and tomato, respectively.

What are oxylipins?

Oxylipins are fatty acid-derived compounds, primarily made from linoleic and linolenic acid. The characterizing and name-giving step in the biosynthesis of these compounds is the oxygenation by a group of enzymes called lipoxygenases (LOX). By adding molecular oxygen to these fatty acids they produce a reactive hydroperoxygenated intermediate, which can feed into at least 6 different biosynthetic pathways (Figure 1). LOX can be grouped in two categories, 9-LOX and 13-LOX. The number in this designation refers to the preferred position of oxygen insertion into the fatty acid. LOX are non-heme iron-containing dioxygenases and consist of a single polypeptide chain.

Figure 1   Major oxylipin pathways in plants. Circled are the bottleneck enzymes for the three major pathways leading to the production of green leaf volatiles (HPL), jasmonic acid (AOS), and the divinyl ether (DES). HPL and AOS utilize predominantly 13-hydroperoxide products starting with linolenic acid (18:3), while the DES pathway uses 9-hydroperoxy linolenic acid. Only 13-hydroperoxy linolenic acid is shown as an example for typical LOX products. (HPL, hydroperoxide lyase; AOS, allene oxide synthase; DES, divinyl ether synthase; LOX, lipoxygenase; ROS, reactive oxygen species; αDOX, α-dioxygenase; EAS, epoxy alcohol synthase; POX, peroxygenase.)

While some LOX can be found in the cytosol of plant cells, most LOX have been localized in plastids.

As mentioned above, LOX catalyze the insertion of molecular oxygen into polyunsaturated fatty acid. The resulting 9- or 13-hydroperoxy fatty acids are then fed into the different oxylipin pathways (Figure 1), and products from these pathways can add up to the approximately 150 different known oxylipins like hydroxy-, oxo-, or keto-fatty acids, divinyl ether, green leaf volatiles (GLV), traumatin, and the jasmonates. Additionally, non-enzymatic conversion into phytoprostanes may occur, further contributing to the diversity of these compounds. Although free fatty acids appear to be the preferred substrates of LOX, they can also catalyze this reaction with esterified fatty acids (e.g., membrane lipids).

The jasmonate pathway

While the described pathways can produce a diverse array of oxylipins, the jasmonates have been in the focus of research for almost 50 years. In particular, jasmonic acid (JA), but also its methyl ester, have been intensely studied and are now generally accepted as important plant hormones. The main interest for JA arose from its predominant role in the regulation of plant defense responses as it is described in Chapter 23. However, JA is also an important developmental signal and, for example, regulates pollen development and maturation.

JA is produced from linolenic acid through the octadecanoid signaling pathway. After incorporation of molecular oxygen by a 13-LOX, the resulting 13-hydroperoxy linolenic acid (13-HPLA) is converted to an unstable allene oxide by the allene oxide synthase (AOS), which represents the bottleneck enzyme for this pathway. The allene oxide undergoes a rapid cyclization by the allene oxide cyclases (AOC). This step also establishes the correct stereochemistry of the resulting 9S, 13S-12-oxo phytodienoic acid (or cis-OPDA). Cis-OPDA is an important intermediate of the pathway, for it has been demonstrated to exhibit its own JA-independent biological activity. This is partially attributed to a distinct structural feature of the molecule, which contains an α,β-unsaturated carbonyl moiety. This makes it a potential target for nucleophilic attack by –NH₂ or –SH groups, thereby forming a stable Michael adduct. This form of protein modification has been shown in the animal system to significantly alter the biochemical properties of enzymes. However, it is unclear if this form of protein modification occurs in plants as a means of biosynthetic regulation.

OPDA is produced in the chloroplast and has to be transferred to the peroxisome for further processing. While the export system from the chloroplast has not yet been identified, import into the peroxisome is facilitated by an ABC transport system. There the olefinic bond in the pentacyclic system is reduced by the enzyme 12-oxo phytodienoate reductase (OPR) with NADPH as a cofactor. Interestingly, plant genomes contain several different homologues (Zea mays 8 OPR, Arabidopsis 6 OPR), but usually only one of these OPR genes is involved in the JA-biosynthetic pathway. A potential function for the other OPRs may be the more general reduction of the olefinic bond in α,β-unsaturated carbonyls as they occur in many other oxylipin-derived compounds like traumatin, E-2-hexenal, and certain phytoprostanes.

After being reduced the resulting 12-oxo phytoenoic acid undergoes 3 cycles of β-oxydation, eventually yielding (+)- iso jasmonic acid (or cis (epi) jasmonic acid). While this was long thought to be the active component, it is now established that the free acid has, in fact, very little biological activity. JA rather needs to be conjugated to an amino acid, for example isoleucine, by JAR proteins, and it is this conjugate that is recognized by its receptor and activates JA-related gene expression. Aiding in the discovery of this signaling pathway was coronatine, a cytotoxin from the plant pathogenic bacteria Pseudomonas syringae. Coronatine, a conjugate of coronafacic acid and coronamic acid, exhibits many, if not all, of the activities previously attributed to JA, and early-on a mutant line was identified in Arabidopsis that did not respond to this toxin. This mutant, named coronatine-insensitive 1 (or coi1), did not respond to coronatine, and was also found to be JA-insensitive. But only after the identification of JAR proteins as those that perform the conjugation of JA to an amino acid, and JAZ proteins as suppressors of JA-induced gene expression, was COI1 found to represent the actual receptor for JA-isoleucine.

Jasmonates-inducible co-regulation of metabolic pathway genes

The mechanisms by which jasmonic acid signals massive reprogramming of gene expression are beginning to resolve (Pauwels et al. 2009, and references therein). It is clear from those and other studies that JA does not act alone, but is rather integrated into an elaborate signaling network and many interactions with other hormone signaling pathways like those for salicylic acid, ethylene, and auxin have been described. In the context of plant defense responses, JA provides a main switch that shuts down growth, a process usually regulated by auxin, and activates those genes that provide attack-specific protection, often in combination with other signaling compounds like ethylene. For example, in response to insect herbivore damage JA not only induces the synthesis of a diverse array of proteinase inhibitors, which negatively affect insect performance, but also other plant-specific metabolic pathways leading to the production of toxic or deterring secondary metabolites like terpenes, alkaloids, phenylpropanes, and glucosinulates. But although these pathways are evolutionary quite distant, it is characteristic for JA to co-regulate the complete biosynthetic pathway genes instead of activating just a single bottleneck enzyme (for illustration, see Figure 2).

Figure 2   Model of jasmonic acid-regulated gene expression. Jasmonic acid often induces whole biosynthetic pathways in plants. While the gene regulatory mechanisms appear to be conserved, the pathways regulated by this mechanism can be very diverse and are often species-specific.

Consequently, complex mixtures of secondary metabolites can be produced; for example, volatile mono- and sesquiterpenes in corn and lima beans, diverse glucosinulates in Arabidopsis and other members of the Brassicaceae family, or complex alkaloids in Eschscholtzia californica. Often, several pathways for secondary metabolites exist within one plant species and can differentially be activated by JA. In Arabidopsis JA can activate several classes of secondary metabolites including phenylpropanoids, glucosinulates, anthocyanines, and isoprenoids. The induction of either pathway or combinations of several depends on the context in which JA accumulates or is exogenously applied. For example, a cell culture of Arabidopsis responds differently to JA treatment than young seedlings growing on an artificial substrate. Interestingly, this co-regulatory activity of JA also includes the genes for its own biosynthetic pathway. Common to all those activated pathways is that they are induced very early upon JA perception, usually within the first 4h, and that those activated pathways are most important for the successful defense. As mentioned above, these pathways are regulated through transcriptional cascades, which suggests the existence of common regulatory elements. Currently, the best-characterized trans element in this context is the transcription factor MYC2, which appears to initiate many of the above described processes. Consequently, this transcriptional regulation also requires common cis elements among the JA-regulated genes, meaning that similar regulatory sequences within the respective promoter regions exist. These sequential functional similarities are not limited to one species, but must have evolved in almost all plant species with regard to JA-activated metabolic pathways and appear to be quite conserved. In fact, functional orthologs of the activator MYC2 and its corresponding suppressor JAZ1 have been identified in Arabidopsis, tomato, tobacco, and periwinkle. In contrast to this, the recruitment and assembly of the metabolic pathway-specific enzyme-coding genes to such JA-orchestrated regulons has to be considered as a series of events that occurred repeatedly and independently in different plant lineages, as it is expressed in the diversity of the produced secondary metabolites. It is probably this capacity of JA to co-regulate whole metabolic pathways leading to the production of species-specific secondary metabolites that makes it so effective in the orchestrations of specific defenses against attacking organisms.

Biologically active derivatives of jasmonic acid

Jasmonic acid is undoubtfully the most important signaling molecule produced by the octadecanoid pathway. However, metabolites of this molecule have also been described to have biological activity. Most prominently is the methyl ester of JA (MeJA). MeJA is formed by the enzyme S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase. MeJA exhibits mostly the same signaling functions when compared to JA. However, because of its volatility it may also serve as a mobile signal between plants as it has been demonstrated for the interaction of sagebrush and wild tobacco in nature. Sagebrush releases significant amounts of MeJA when mechanically damaged, which in turn is perceived by neighboring tobacco plants and boosts their defenses. Additionally, MeJA may also serve as a within-plant signaling molecule. However, MeJA is not released by all plants upon mechanical damage or actual insect herbivory, making it somewhat limited in its effectiveness as a volatile mobile signal.

Another volatile signal derived from jasmonic acid is cis-jasmone, which is produced by decarboxylation and re-establishment of an olefinic bond between carbon 2 and 3. Cis-jasmone was first described as an important semiochemical that either attracts or repels insects. Recently, it was found that it may also serve as a volatile signal between plants. However, its activity is distinctly different from that of jasmonic acid and it appears to be most effective in the induction of defenses against aphids and other phloem-feeding insect herbivores (Matthes et al. 2010).

Hydroxylation at the C-12 of jasmonic acid and subsequent glucosylation produces tuberonic acid-O-glucopyranoside, the tuber-inducing factor in potato. It is synthesized in the leaves and then transported basipetal to the stolons.

As mentioned before, OPDA and dinor OPDA (a C-16 OPDA homolog) are intermediates of the octadecanoid pathway that exhibit their own biological activity. Additionally, they can be found as acyl residues in at least five different mono- and digalactosyldiacylglycerols. These molecules were termed arabidopsides A to E. OPDA and dinor-OPDA were predominantly found in the sn-1 position, with arabidopside E having them in position 1 and 2. To date these compounds have only been identified in Arabidopsis, where they are rapidly mobilized upon mechanical damage and pathogen infection by a site-specific phospholipase A₁.

The hydroperoxide lyase pathway

Another biosynthetic pathway derived from 13-hydroperoxy linolenic acid is catalyzed by the enzyme hydroperoxide lyase (HPL) and results in the production of so-called green leaf volatiles (GLV), the typical “green” smell of plants. Major products of this pathway are Z-3-hexenal, Z-3-hexenol, and Z-3-hexenyl acetate and their respective E-2-enantiomers. Additionally, this pathway also produces 12-oxo-Z-9-decenoic acid, the natural precursor of traumatin, the first wound hormone described for plants (see Figure 1). HPLs, like AOS (and also the divinyl ether synthase; see Figure 1 and below), belong to the family of P450 enzymes and even show a high degree of sequence similarities among each other. In fact, the exchange of just one amino acid in AOS converted the protein into a HPL (Lee et al. 2008). Although the HPL pathway was already characterized 100 years ago, it has only recently gained significance, when it was shown that the volatile products of this pathway serve as potent signals in inter- and intra-plant signaling. Communication between plants through the release of volatiles was first described by Rhoades (1983) and Baldwin and Schultz (1983). They found that plants exposed to volatiles from damaged neighboring plants were less attractive to insect herbivores. More than 15 years later it was found that plants exposed to volatiles from herbivore-infested plants accumulate transcription of defense genes that were previously described to be important in the insect herbivore defense (Arimura et al. 2000). While in all those cases complex blends of volatiles were emitted by the source plants, Bate and Rothstein (1998) demonstrated that GLVs, when applied as pure chemicals, also induced defense-related genes, and it can be assumed that among those volatiles emitted by attacked plants, GLVs play a significant role in inter-plant signaling. But although these examples demonstrate the more direct defensive function of GLVs, their main activity may lie in the preparation of plants against impending herbivore attack as outlined in Web Essay 23.6. This priming against insect herbivore attack by GLVs may give receiver plants a head start in their defense efforts when actually attacked, without taking away resources that are otherwise needed, for example, for growth.

But how do GLVs signal?

This question has to date only partially been answered. It seems clear now that for GLVs to fully exhibit their activity a functioning JA signaling pathway is required. However, while in corn and other monocots JA accumulates during the initial exposure to GLV, no such effect has been reported for dicot plants, albeit the fact that they also recognize these signaling compounds and in most cases this recognition primes JA-regulated defense responses. Considering the conserved nature of the GLV signal emission among various plant species it can be hypothesized that common signaling mechanisms exist for the perception of GLVs, but these have yet to be discovered. Nonetheless, GLV signaling appears to be closely associated with the JA signaling pathway.

Other oxylipins

Divinyl ether

Divinyl ether are either 9- or 13-LOX products that were first identified in solanaceous plants like potato, tomato, and tobacco. Two major compounds, colneleic acid and colnelenic acid, were characterized in potato plants infested with Phytophthora infestans, the cause of potato blight disease. Both compounds accumulated rapidly in a more resistant cultivar up to several micrograms per gram fresh weight and it was shown that they have a direct inhibitory effect on the growth of the pathogen as it has also been described for other oxylipins (Prost et al. 2005).

Other 9-LOX products

9-LOX products are much less characterized than their corresponding 13-LOX products. However, there is increasing evidence that 9-LOX derivatives have hormone-like activities and may regulate growth, development, and defense against pathogens and pests. Studies of 9-LOX mutant lines in maize demonstrated effects on seed germination, root growth and senescence, as well as susceptibility or resistance against fungal pathogens (Christensen and Kolomiets 2011).

Phytoprostanes

A rather unusual pathway for the production of oxylipins is initiated by the non-enzymatic incorporation of reactive oxygen species into unsaturated fatty acids like linolenic and linoleic acid. Products of this pathway, that is mainly activated when plants encounter oxidative stress (for example, during pathogen infection) were named phytoprostanes due to their structural resemblance with prostaglandins. Several of these compounds were shown to induce defense and other stress-related genes like glutathion-S-transferase, and caused the accumulation of phytoalexins (Thoma et al. 2003).

References

A detailed description of lipids and their biology can be found at http://lipidlibrary.aocs.org and is highly recommended to those interested in the topic. This website also provided most of the information described in this web essay.

Additional literature

Arimura, G., Ozawa, R., Shimoda, T., Nishioka, T., Boland, W., and Takabayashi, J. (2000) Herbivory-induced volatiles elicit defence genes in lima bean leaves. Nature 406, 412–515.

Baldwin, I.T. and Schultz, J.C. (1983) Rapid changes in tree leaf chemistry induced by damage: evidence for communication between plants. Science 221, 277–270.

Bate, N.J. and Rothstein, S.J. (1998) C6-volatiles derived from the lipoxygenase pathway induce a subset of defense-related genes. The Plant Journal 16, 561–569.

Christensen, S.A. and Kolomiets, M.V. (2011) The lipid language of plant-fungal interactions. Fungal Genetics and Biology 48, 4–14.

Lee, D.S., Nioche, P., Hamberg, M., and Raman, C.S. (2008) Structural insights into the evolutionary paths of oxylipin biosynthetic enzymes. Nature 455, 363–368.

Matthes, M.C., Bruce, T.J.A., Ton, J., Verrier, P.J., Pickett, J.A., and Napier, J.A. (2010) The transcriptome of cis-jasmone-induced resistance in Arabidopsis thaliana and its role in indirect defense. Planta 232, 1244–1254.

Pauwels, L., Inze, D., and Goossens, A. (2009) Jasmonate-inducible gene: what does it mean? Trends in Plant Science 114, 87–91.

Prost, I., Dhondt, S., Rothe, G., Vicente, J., Rodriguez, M.J., Kift, N., Carbonne, F., Griffiths, G., Esquerré-Tugayé, M.T., Rosahl, S., Castresana, C., Hamberg, M., and Fournier, J. (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense against pathogens. Plant Physiology 139, 1902–1913.

Rhoades, D.F. (1983) Responses of Alder and Willow to Attack by Tent Caterpillars and Webworms: Evidence for Pheromonal Sensitivity of Willows. In: Plant Resistance to Insects, Chapter 4, 55–68, ACS Symposium Series, Volume 20.

Thoma, I., Loeffler, C., Sinha, A.K., Gupta, M., Krischke, M., Steffan, B., Roitsch, R., and Mueller, M.J. (2003) Cyclopentenone isoprostanes induced by reactive oxygen species trigger defense gene activation and phytoalexin accumulation in plants. The Plant Journal 34, 363–375.

Weber, H., Chetelat, A., Caldelari, D., and Farmer, E.E. (1999) Divinyl ether fatty acid synthesis in late blight-diseased potato leaves. The Plant Cell 11, 485–493.