The Lipid Library

PLANT OXYLIPINS

Chemistry and Biology

1.   Introduction

Plants lack an immune system in the sense that it exists in animals, but they possess mechanisms that recognize potential pathogens and initiate defense responses. It has become evident that various types of oxygenated fatty acids, collectively termed ‘oxylipins’ or sometimes ‘octadecanoids’, are involved in responses to physical damage by animals or insects, stress and attack by pathogens. These compounds are similar in many ways to the eicosanoids derived from arachidonate in animals, which have so many varied functions but especially in the inflammatory process. There are very few definitive reports of arachidonic acid in higher plants, and oxylipins are derived from linoleic and more importantly α-linolenic acids with a first key step being the action of lipoxygenases, although cytochrome P450 and pathogen-induced oxygenases have lesser roles. For example, depending on the source of the enzyme, lipoxygenases (EC 1.13.11.12) (LOX) catalyses the oxidation of α-linolenic acid into either 9- or 13-hydroperoxy-octadecatrienoic acids, or a mixture of both. Such compounds are highly reactive, and they are quickly metabolized by various enzymes into series of oxylipins, as summarized in the figure below, with a range of distinct biological activities.

2.   Lipoxygenases

Lipoxygenases are non-heme iron-containing dioxygenases that are widely distributed in plants and animals. That from soybean was among the first to be studied in great detail, including its three-dimensional structure, and the knowledge gained assisted greatly with the understanding of the analogous animal enzymes. Plant LOX consist of a single polypeptide chain with a molecular mass of 94 to 104 kDA. From amino acid-sequence studies of enzymes from a number of plant sources, it is evident that there are two main families of lipoxygenases, designated ‘type-1’ and ‘type-2’, but many different iso-enzymes exist depending on the particular species. Soybean lipoxygenase exists in eight different isoforms, for example. They are soluble cytoplasmic enzymes. The properties of lipoxygenases in general are discussed in the Introductory web page. Here, those properties characteristic of the plant enzymes are discussed.

Lipoxygenases catalyse the addition of molecular oxygen to polyunsaturated fatty acids containing a (cis,cis)-1,4-pentadiene system to yield an unsaturated fatty acid hydroperoxide. Oxygen can be added to either end of the pentadiene system with high stereospecificity, and in the case of linoleic and α-linolenic acids, this leads to either the 9(S)- or 13(S)-hydroperoxy derivatives or both depending on the specific iso-form of the enzyme. Physiological conditions can also affect this positional specificity (regiospecificity), and under conditions of low oxygen concentrations, for example, the soybean LOX-1 produces equal amounts of the two isomers, though normally the 13(S) isomer predominates. Photosynthetic tissues tend to produce mainly 13(S)-hydroperoxides.

The reaction proceeds in three stages as illustrated above for α-linolenic acid, with the first step the (antarafacial) stereospecific abstraction of a hydrogen atom from the methylene group between the double bonds. The resulting delocalized free radical undergoes an allylic rearrangement before the oxygen molecule adds to form the hydroperoxide. Subsequent steps are specific for either the 9-LOX or 13-LOX products.

Free acids appear to be the preferred substrates, and under conditions of stress in plants, phospholipases are activated that rapidly break down the complex lipids – another analogy with animal systems. However, it is also evident that lipoxygenases can react with esterified fatty acids in lipids and perhaps disrupt the cellular membranes.

3.   Jasmonates and Related Compounds

The jasmonates are 12-carbon cyclic fatty acids derived from linolenic acid that have important signalling functions in plants. Allene oxide synthase, an enzyme of the cytochrome P450 family, catalyses the first key step from the LOX product, 13(S)-hydroperoxy-9c,11t,15c-octadecatrienoic acid, as the substrate as illustrated below. The product is the allene oxide 12,13(S)-epoxy-9c,11t,15c-octadecatrienoic acid. However, this compound is highly unstable, and it can be hydrolysed rapidly to α- and γ-ketols, or it can cyclize spontaneously to form 12-oxo-10,15c-phytodienoic acids (12-oxo-PDA), i.e. with a prostaglandin-like structure, in two of the four possible stereoisomeric forms. However, the enzyme allene oxide cyclase produces only 12-oxo-9(S),13(S)-phytodienoic acid (the cis-(+)-enantiomer), which is the biologically important isomer. Not only is it the precursor of the jasmonates but it also has distinctive signalling functions of its own. Both the allene oxide synthase and the cyclase are located in the plastids, and they probably operate in concert; they may be even be linked physically in some form of complex, although no direct evidence exists for this.

The product is transferred by an as yet unknown mechanism to the peroxisomes, where a specific 12-oxo-phytodienoate reductase reduces the double bond in position 10, i.e. in the cyclopentenone ring, to 3-oxo-2-(pent-2'-enyl)-cyclopentane-1-octanoic acid. This is a key step in directing the metabolism towards jasmonic acid, as this compound only is able to undergo the three cycles of β-oxidation required to give the 12-carbon (-)-7-iso-jasmonic acid. Although these oxidation steps are also presumed to occur in peroxisomes, much remains to be learned of the process.

Dinor-oxo-phytodienoic acid is a related metabolite derived from hexadecatrienoic acid (16:3(n-3)) and has signalling functions in itsown right, as well as being a precursor of jasmonic acid. In addition, a number of further metabolites of jasmonic acid are formed in plants and have biological activity including methyl jasmonate, the formation of which is catalysed by S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase. A number of amino acid conjugates of jasmonic acid have been found in plants, and those with leucine and isoleucine have especial importance. The tuber-inducing factor in potatoes, tuberonic acid or its glucopyranosyl derivative, is derived from jasmonic acid.

At least five different mono- and digalactosyldiacylglycerols containing 12-oxo-phytodienoic acid and/or dinor-oxo-phytodienoic acids in position sn-1, and termed ‘arabidopsides A to E’, have been isolated from stressed plants of Arabidopsis thaliana. For example, in plants challenged by a bacterial pathogen, a monogalactosyldiacylglycerol containing two 12-oxo-phytodienoate and one dinor-oxo-phytodienoate acyl chain (arabidopside E) accumulated in amounts up to 8% of the total lipids. It was shown to have anti-bacterial properties in vitro. However, such compounds were not be detected in species other than Arabidopsis in one systematic study.

4.   Other Oxylipins

Allene oxide synthase is one of a family of related cytochrome P450 mono-oxygenases collectively termed the CYP74 subfamily. These produce three further types of oxidation products of biological importance. Starting again with the 13(S)-hydroperoxy intermediate from the lipoxygenase reaction, oxylipins of various kinds result as summarized in the figure below. These reactions all appear to proceed via first free radical and then epoxide intermediates, though many aspects of the mechanisms require clarification or confirmation.

For example, the enzyme hydroperoxide lyase converts the epoxy intermediate into a vinyl ether, which is rapidly hydrolysed to generate cis-3-hexenal and 12-oxo-cis-9-dodecenoic acid, though the positions and geometry of the double bond may change by chemical or enzymatic isomerization. Traumatin or 12-oxo-trans-10-dodecanoic acid, a plant wound hormone, is produced in this way. The epoxy intermediate can also be converted by an epoxyalcohol synthase into an epoxyhydroxy fatty acid, while another related enzyme synthesises a divinylether fatty acid.

In alternative pathways, the 13(S)-lipoxygenase product can be reduced to a hydroxy acid, while a peroxygenase or pathogen-induced oxygenase pathway is another route to the biosynthesis of epoxy-hydroxy fatty acids. Di- and trihydroxy-octadecanoids are also produced in some plant species.

Fungi and yeasts produce a variety of oxylipins, from saturated and unsaturated fatty acids, including arachidonic acid. For example, 8R-hydroxy-octadeca-9,12-dienoic and 5S,8R-dihydroxy-octadeca-9,12-dienoic acids are produced by Aspergillus species, while the prostaglandin metabolites PGF₂ and PGF₂-lactone, derived from arachidonic acid, have been detected in yeasts of the Lipomycetaceae family. In fungi, these oxylipins function as hormone-like signals that regulate the development of spores. However, much remains to be learned of the biosynthetic enzymes involved and of the functions of these compounds.

5.   Plant Isoprostanes (Phytoprostanes)

Plants utilize linolenic acid to produced C₁₈-isoprostanoids (dinor isoprostanes or phytoprostanes) via a non-enzymatic, free radical-catalysed pathway similar to the isoprostane pathway in animals. As they are derived from linolenic acid, they differ from the animal isoprostanes in the number of double bonds and the lengths of the side-chains. In the twenty or so 20 plant species plant species analysed to date, various regioisomers of free phytoprostanes of the A₁, B₁, D₁, E₁, F₁ and deoxy-J₁ series have been detected, occasionally at remarkably high levels (using the same nomenclature as for animal prostanoids). The phytoprostane PPE₁ is illustrated as an example. There is some evidence that phytoprostanes, like the jasmonates, are mediators of defense reactions in response to oxidative stress in plants.

6.   Biological Activity

When plants are attacked by bacterial of fungal pathogens, lipases are activated that release the unsaturated fatty acids and trigger the synthesis of a range of oxylipins with diverse roles. Some of these have direct antimicrobial or anti-insect functions, while others, especially the jasmonates and their precursors the oxo-phytodienoic acids, are potent regulators of defense mechanisms, for example by stimulating proteinase inhibitors or by promoting the accumulation of antimicrobial secondary metabolites (phytoalexins).

Each of the various jasmonate derivatives, i.e. the free acid, methyl ester and conjugates with amino acids, has distinct biological effects. Wound response is one of the most-studied pathways of jasmonates in signal transduction with the tomato as the model. In brief, local wounding initiates cleavage of a peptide systemin from prosystemin and in turn stimulates jasmonic acid biosynthesis. This is believed to act as a signal leading to systemic expression of genes encoding proteinase inhibitors, which deter herbivores and in effect immunize the plant against further herbivore attacks. Methyl jasmonate is also important in initiating defensive strategies against both insect predators and herbivores.

The jasmonates also have a role in fertility, for example in pollen maturation, and in such varied processes as fruit ripening, root growth and tendril coiling. They are believed to interact with receptors in the cell to activate signalling pathways both intra- and inter-cellularly that modulate the expression of a number of genes, and thence the synthesis of a many key proteins. The picture emerging is a highly complex one, and many aspects await clarification. For example, there appear to be functional differences between species that have yet to be explained, and the relationship with other defense mechanisms including those based on auxin, ethylene, abscisic acid and salicyate requires further elaboration.

Volatile jasmonate metabolites, such as cis-jasmone, may regulate the behavior of some insects, for example by deterring herbivorous species or attracting their predators. They may even enable communication between plants.

The oxylipins derived from the other branches of the lipoxygenase pathway have characteristic biological activities also. Indeed, even the primary 9(S)- and 13(S)-hydroperoxides have antifungal and anti-microbial properties. They may be part of a short-term local response while the jasmonates operate over a longer time scale. Some of the further metabolites, such as the C₆ volatiles produced by hydroperoxide lyase in damaged tissues may act like methyl jasmonate to elicit defense responses. They also have potent antimicrobial effects and reduce the fecundity of insect pests. As mentioned briefly above, the other product of the enzyme, a C₁₂ fatty acid, is a precursor of traumatic acid and other wound hormones, which also have growth-stimulating effects.

The epoxy and hydroxy derivatives of linoleic acid resulting from the peroxygenase pathway are toxic to fungal pathogens. Similarly, colneleic and colnelenic acids (divinyl ether fatty acids originating from 18:2- and 18:3-derived hydroperoxides, respectively) are produced quickly in leaves of potato plants infected by fungi or viruses, and they are believed to have a defensive role against potato blight especially. Indeed, colneleic and colnelenic acids have been found esterified at the sn-2 position of phospholipids in potato, suggesting the presence of a preformed pool that would be immediately available in response to challenge by pathogens. Other oxylipins may be stored in the same way and 12-oxo-phytodienoic acid is found esterified to position sn-1 of the monogalactosyldiacylglycerol in Arabidopsis, for example.

The 3-hydroxy oxylipins in fungi and yeasts are believed to play a role in cell aggregation and spore release.

Although they do not occur naturally in animal tissues, some jasmonate metabolites and methyl jasmonate in particular have been shown to have pronounced cytotoxic effects against human cancer cell lines in vitro.

7. Analysis

As with the eicosanoids, methods involving gas chromatography allied to mass spectrometry are preferred for the analysis of the plant oxylipins, with high-performance liquid chromatography and UV detection as a useful complementary technique.

Recommended Reading

• Blee, E. Phytooxylipins and plant defense reactions. Prog. Lipid Res., 37, 33-72 (1998).
• Feussner, I. and Wasternack, C. The lipoxygenase pathway. Annu. Rev. Plant Biol., 53, 275-297 (2002).
• Kock, J.L.F., Strauss, C.J., Pohl, C.H. and Nigam, S. The distribution of 3-hydroxy oxylipins in fungi. Prostaglandins Other Lipid Mediators, 71, 85-96 (2003).
• Liavonchanka, A. and Feussner, I. Lipoxygenases: occurrence, functions and catalysis. J. Plant Physiol., 163, 348-357 (2006).
• Mueller, M.J., Mene-Saffrane, L., Grun, C., Karg, K. and Farmer, E.E. Oxylipin analysis methods. Plant J., 45, 472-489 (2006).
• Rosahl, S. and Feussner, I. Oxylipins. In: Plant Lipids: Biology, Utilisation and Manipulation. pp. 328-354 (Ed. D.J. Murphy, Blackwell Publishing, Oxford) (2005).
• Schaller, F. Enzymes of the biosynthesis of octadecanoid-derived signalling molecules. J. Exp. Bot., 52, 11-23 (2001).
• Thoma, I., Krischke, M., Loeffler, C. and Mueller, M.J. The isoprostanoid pathway in plants. Chem. Phys. Lipids, 128, 135-148 (2004).
• Tsitsigiannis, D.I. and Keller, N.P. Oxylipins as developmental and host-fungal communication signals. Trends Microbiol., 15, 109-118 (2007).
• Turner, J.G., Ellis, C. and Devoto, A. The jasmonate signal pathway. Plant Cell, 14, S153-164 (2002).
• Wasternack, C. Jasmonates: An update on biosynthesis, signal transduction and action in plant stress response, growth and development. Annals Botany, 100, 681-697 (2007).

Updated: 17/12/2007 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland