Plant and Fungal Oxylipins

While plants lack an immune system in the sense that it exists in animals, they do possess mechanisms that are functionally equivalent in that they recognize potential pathogens and stress vectors and then initiate defence responses. They have pattern recognition receptors in plasma membranes that identify pathogen-associated molecule patterns and activate a rapid defense response as well as an effector-triggered immunity, which is activated following identification of pathogen-secreted effector proteins by the intracellular receptors.

It has become evident that various types of oxygenated fatty acids, collectively termed ‘oxylipins’, are involved in responses to physical damage by animals, insects, abiotic stresses (e.g., freeze-thawing) and attack by pathogens. Among these, the jasmonates have a special importance, and they are present ubiquitously in land plants. These lipid mediators in many ways resemble the eicosanoids derived from arachidonate in animals, which have so many varied functions but mainly in inflammatory processes, but they are also phytohormones, which are intimately involved in the growth and development of plants. Related oxylipins function in fungi, yeasts and mosses. Some plant oxylipins act directly by being distasteful to insect predators, some are sufficiently volatile that they can alert neighbouring plants, while others can communicate the information on cell damage over long distances within a plant to coordinate a comprehensive response. These operate in tandem with many other lipids and lipid enzymes such as phospholipases.

1.   Introduction to Plant Oxylipin Biosynthesis

There are few definitive reports of the presence of arachidonic acid in higher plants, although it is present in some algae and lower plants, so plant oxylipins are derived from linoleic acid or more often α‑linolenic acid, released from their lipid-bound forms by acyl hydrolases (lipases) of various kinds. In Arabidopsis thaliana, which has been the model plant for many studies of oxylipin biosynthesis and function, the core phospholipase A family consists of ten members, some of which can hydrolyse both phospholipids and galactolipids.

In brief, a first step in the biosynthesis of almost all plant oxylipins is the action of lipoxygenases, while cytochrome P450 and pathogen-induced oxygenases have subsidiary roles, and depending on the source of the enzyme, lipoxygenases (EC (LOX) catalyse the oxidation of α‑linolenic acid into either 9- or 13‑hydroperoxy-octadecatrienoic acid 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, each with a range of distinct biological activities.

Oxylipin production from linoleate and linolenate

Those enzymes and their products illustrated appear to be ubiquitous in higher plants, whereas some others can be specific to certain species. Of these, the allene oxide synthase produces short-lived intermediates that rearrange in various ways, for example by cyclizing to form compounds such as oxo-phytodienoic acid and thence the jasmonates, i.e., most of the non-volatile oxylipins in plants, while closely related hydroperoxide lyases produce the fragmented and volatile fatty acid/aldehyde derivatives. The jasmonates in particular are viewed as central to plant development and responses to biotic and abiotic stresses, although there is extensive crosstalk with other oxylipins and plant hormones.

Additional oxidative enzymes to those listed include an α-dioxygenase, which generates 2(R)-hydroperoxy fatty acids, and some caleosins, which produce epoxy fatty acids. Although some red algae such as Gracilaria species contain prostaglandins and are known to have a cyclooxygenase gene, the function of these oxylipins in the organisms is not known. In view of the numerous enzymes and reactions, many of which are dependent on species, it is not surprising that new plant oxylipins and functions continue to be identified.

While many lipid mediators are synthesised by these enzymatic processes, others such as phytoprostanes and aldehydes are produced adventitiously by reaction with reactive oxygen species (ROS), and together, they form a redox regulatory network that couples ROS generation with subsequent stress signalling pathways in plants.

2.   Plant Lipoxygenases and Hydroperoxide Formation

Hydroperoxides are generated in plants primarily by the action of lipoxygenases, i.e., non-heme iron-containing dioxygenases that are widely distributed in fungi, plants and animals. That from soybean was among the first to be studied in detail, including its three-dimensional structure, and the knowledge gained assisted greatly our understanding of the analogous animal enzymes. Plant LOXs consist of a single polypeptide chain with a molecular mass of 94 to 104 kDA with the carboxy-terminal domain harbouring the catalytic site of the enzyme, which contains a non-heme iron atom that is coordinated with five amino acids - three histidines, one asparagine and the carboxyl group of the carboxy-terminal isoleucine; the amino-terminal domain may be involved in membrane or substrate binding. The properties of lipoxygenases in general are discussed in our web page dealing with hydroxyeicosatetraenes (HETE), but here the plant enzymes only are discussed.

From amino acid-sequence studies of enzymes from many plant sources, it is evident that there are two main families of lipoxygenases, designated ‘type-1’ (mainly extra-plastidial) and ‘type-2’ (mainly plastidial), and in A. thaliana, lipoxygenase activity is located mainly in the plastidial envelope and stroma of leaf chloroplasts. Many different iso-enzymes exist that are dependent on plant species, and they include both soluble cytoplasmic and membrane-bound enzymes, with eight different isoforms of soybean lipoxygenase, for example. There are suggestions that isoforms in different subcellular regions may provide different pools of hydroperoxy fatty acids, which serve as substrates for alternative metabolic pathways and physiological functions.

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; A. thaliana contains two genes encoding for 9-LOXs and four that encode 13‑LOXs. Physiological conditions can affect this positional specificity (regiospecificity), and under conditions of low oxygen concentration, 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, but 9-LOX metabolites are more abundant in potato.

Action of lipoxygenase on linolenate

Free acids are the preferred substrates for 9-LOX, and under conditions of stress in plants, lipases or unspecific acyl-lipid hydrolases are activated that rapidly break down the complex lipids, another analogy with animal systems, and initiate oxylipin formation, but it is evident that 13-LOX can react with esterified fatty acids in lipids also and perhaps disrupt the cellular membranes, as in stress situations. The reaction then proceeds in three stages as illustrated above for α‑linolenic acid, with the first step the stereospecific abstraction of a hydrogen atom from the methylene group between the double bonds. The resulting delocalized free radical undergoes an allylic rearrangement to form a trans-cis conjugated diene system before the oxygen molecule adds to form the hydroperoxide.

Formulae of colneleic and colnelenic acidsSubsequent steps to produce the wide range of oxylipins found in plants, such as the jasmonates, are specific for either the 9-LOX or 13‑LOX products. Among other oxylipins that could be described, colneleic and colnelenic acids originating from 18:2- and 18:3-derived hydroperoxides, respectively, via the action of 9‑LOX, are divinyl ether fatty acids produced quickly by the subsequent action of divinyl ether synthases of the CYP74 family in leaves of potato plants infected by bacteria, fungi or viruses. They are believed to have a defensive role against potato blight especially. Tomato plants, which like potato are of the Solanum family, produce these oxylipins as a defence response to attack by nematodes, such as the root-knot Meloidogyne javanica. Other plant species can produce stereo-isomers of colnelenic acid and a related oxylipin from oleate.

Algae differ from higher plants in that they can contain significant amounts of higher polyunsaturated fatty acids such as eicosapentaenoic (EPA or 20:5(n‑3)) and docosahexaenoic (DHA or 22:6(n-3)) acids. Hydroxy-EPA derivatives, such as 15‑HEPE, are produced by lipoxygenases in marine algae as a defence mechanism against bacteria and other predators (see below).

3.   Jasmonates and Related Compounds

The jasmonates are 12-carbon cyclic fatty acids and their derivatives, which are produced from α-linolenic acid and have signalling functions in algae and higher plants for plant stress responses, growth and development. A key structural feature is a cyclopentanone ring resembling that in mammalian prostaglandins (surely no coincidence). Upon stimulation by a stress condition, biosynthesis is initiated by the release of α‑linolenic acid from position sn-1 of the galactolipids (or phospholipids) of plastid membranes by a galactolipase (glycerolipase (or phospholipase) A1), though the specific enzyme involved may depend on the nature and site of the stimulus (16:3(n-3) can be a jasmonate precursor in some species). The unesterified α-linolenic acid is then acted upon by a 13‑lipoxygenase to yield 13(S)‑hydroperoxy-9c,11t,15c-octadecatrienoic acid specifically as the first intermediate.

Allene oxide synthase is one of a family of related cytochrome P450 mono-oxygenases collectively termed the CYP74 subfamily, which are not typical of the common P450 monooxygenases in that they do not require molecular oxygen nor NAD(P)H-dependent cytochrome P450-reductase. Instead, the new carbon–oxygen bonds are formed by using an acyl hydroperoxide both as substrate and oxygen donor, in essence acting as a dehydratase. This enzyme catalyses the next step (2) as illustrated below, and the product is the allene oxide 12,13(S)‑epoxy-9c,11t,15c-octadecatrienoic acid. As this compound is highly unstable, it can cyclize spontaneously to form 12-oxo-10,15c-phytodienoic acids (‘OPDA’), i.e., with a prostaglandin-like structure, in two of the four possible stereoisomeric forms (or it can be hydrolysed rapidly to α- and γ-ketols). However, reaction with the enzyme allene oxide cyclase (3) produces only 12‑oxo‑9(S),13(S)-phytodienoic acid (the cis-(+)-enantiomer - 12‑oxo‑PDA), which is the primary isomer of biological relevance. Not only is this the precursor of the jasmonates and a component of the arabidopsides (see below), but it has distinctive signalling functions of its own.

Biosynthesis of jasmonic acid

Both the allene oxide synthase and the cyclase are located in the chloroplasts, and they probably operate in concert; they may even be linked physically in some form of complex, as suggested by the high stereochemical purity of the product given the reactivity of the intermediates. Fungi have a novel allene oxide cyclase that can utilize linoleic acid to form jasmonates.

Further reactions occur in peroxisomes, cytosol, endoplasmic reticulum and vacuoles. A protein designated JASSY is involved in the transfer of 12‑oxo‑PDA from the chloroplast outer membrane into the cytosol, before this intermediate is transferred by an ATP-binding cassette transfer protein (designated 'CTS1' or 'PAX1') from the cytosol to the peroxisomes, where a flavin-dependent 12‑oxo-phytodienoate reductase (OPR3) reduces the double bond in position 10, i.e., in the cyclopentenone ring, to produce 3-oxo-2-(pent-2'-enyl)-cyclopentane-1-octanoic acid. This is a necessary step in directing the metabolism towards jasmonic acid, as this compound only (after conversion to its CoA ester) can undergo three cycles of β‑oxidation catalysed by the multifunctional enzyme complex acyl-CoA oxidase to give the 12‑carbon 7‑iso‑jasmonic acid. Biosynthesis of jasmonic acid is blocked by several inhibitors, including acetylsalicylic acid (aspirin), which directly inhibits the expression of the relevant genes.

While (-)-7-iso-jasmonic acid is the main isomer isolated from plant tissues and was long thought to be the active metabolite, it is now recognized that (+)-7-iso-jasmonic acid or cis-(epi)-jasmonic acid is in fact the active isomer. As both side chains are on the same side of the 3R,7S-cyclopentanone ring and the keto group at C-6 can tautomerize to an enol, the more thermodynamically stable (3R, 7R) isomer ((-)- or trans-jasmonic acid is usually isolated as the main product (90% of the equilibrium mixture).

Structural formula of jasmonoyl-isoleucineAfter transfer of the jasmonic acid to the cytosol, jasmonoyl-isoleucine is then formed rapidly from jasmonic acid, and this is now known to be a central element in hormone signalling by jasmonates. The enzyme jasmonoyl isoleucine conjugate synthase 1 (JAR1) in the cytosol catalyses the final step in the formation of this bioactive compound, although it is believed that at least one alternative enzyme to JAR1 is present in leaf tissue. As with the free acid, the common isomer (‑)‑7‑jasmonoyl-L-isoleucine is not the active isomer, but rather the much less abundant (+)-7-epimer. pH changes promote conversion of the (+)-7-epimer to the inactive (-)-7- form, suggesting that this may be a simple if minor mechanism to regulate jasmonate activity. cis-(+)-12-Oxo-phytodienoic acid (cis‑(+)‑OPDA), the biosynthetic precursor of jasmonic acid, has also been found in conjugation with isoleucine.

A further 11 classes of jasmonate metabolites are formed by methylation, reduction, decarboxylation and glucosylation, including 12-hydroxy-jasmonoyl-isoleucine, cis-jasmone and methyl jasmonate, and many of these have biological functions of their own, both as isoleucine conjugates and in free form after hydrolysis of the conjugate. On the other hand, some metabolites produced by hydroxylation of the pentenyl side chain, e.g., sulfation of the hydroxylated derivatives, are inactive and switch off jasmonic acid signalling. An ω‑oxidation pathway, in which jasmonoyl-isoleucine is oxidized by cytochrome P450 enzymes, mainly CYP94, to a 12-hydroxy metabolite and then further via a 12-aldehydo intermediate to dicarboxy-jasmonoyl-isoleucine, together with deconjugation by amidohydrolases, is now recognized as a major route for catabolism and deactivation of the hormone, and this can lead to the biosynthesis of further metabolites. A comparable mechanism operates with jasmonoyl-phenylalanine.

Oxidation and further metabolism of jasmonoyl-isoleucine

The glucopyranosyl derivative of tuberonic acid, derived from jasmonic acid after hydroxylation at C-12, is synthesised in the leaves of potatoes and transported down to the stolons where it induces tuber formation indirectly by influencing gibberellic acid signalling. Another metabolite of jasmonic acid with biological activity is methyl jasmonate, the formation of which is catalysed by S-adenosyl-L-methionine:jasmonic acid carboxyl methyltransferase. Decarboxylation is the route to the volatile cis-jasmone.

As well as being a primary precursor of (-)-jasmonic acid, 12-oxo-phytodienoic acid has signalling functions that regulate growth processes and certain jasmonate-responsive genes in plants, and it activates and fine-tunes defence and stress responses (see below). Dinor-oxo-phytodienoic acid (dinor-OPDA) is a metabolite related to jasmonic acid but derived from hexadecatrienoic acid (16:3(n-3)), and it has its own signalling functions, such as the response to wounding. A C20 analogue of OPDA has been detected in a liverwort and has comparable biological activities. In a separate metabolic pathway in maize, 9‑lipoxygenase generates hydroperoxides from linoleate that are cyclized with the ultimate formation of jasmonate-like molecules, such as 10‑oxo‑11-phytoenoic acid and metabolites, which have been termed "death acids". These are induced by fungal infections, such as southern leaf blight, and are an important element in plant defence. In the roots of cereals, the allene oxide synthase pathway produces a range of novel oxylipins (dioic acids) termed "graminoxins".

4.   Other Products of Enzymes of the CYP74 Subfamily

The CYP74B subfamily of fatty acid hydroperoxide-transforming cytochromes P450 enzymes, including the allene oxide synthase, generate several further types of oxidation product of biological relevance. Most start again with the 13(S)‑hydroperoxy intermediate from the lipoxygenase reaction, and oxylipins of various kinds are synthesised in plants as summarized in the figure below. These reactions all proceed first via free radical and then unstable epoxide intermediates, which can either rearrange spontaneously or undergo enzymic reactions of various kinds, though many aspects of the mechanisms require clarification or confirmation. It is of interest from an evolutionary standpoint, that two key enzymes in this family, hydroperoxide lyase and divinylether synthase, differ only in a few amino acid residues, and there are other close relationships between enzymes of the CYP74 family. Analogous enzymes are found in bacteria and lower animals.

Conversion of lipoxygenase products into epoxy acids, divinyl ethers and aldehydes

After the formation of an epoxyallylic radical, the enzyme hydroperoxide lyase, which should be more accurately termed a hemiacetal synthase, converts the epoxy intermediate into a vinyl ether and thence to a short-lived hemiacetal, which spontaneously decomposes into two short chain fragments, i.e., cis-3-hexenal and 12‑oxo-cis-9-dodecenoic acid, though the positions and geometry of the double bonds may change by chemical or enzymatic isomerization. The enzyme is found in various subcellular locations, depending on plant species. The unsaturated aldehydes, such as hex-3-enal, are volatile and have potent biological effects (as discussed briefly below, although bioactive aldehydes in general have their own web page). Hexenyl acetate is a major product of wounding in Arabidopsis.

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. The epoxyallylic radical intermediate is a precursor for further oxylipins, and for example, it can be converted by an epoxyalcohol synthase into an epoxyhydroxy fatty acid (allene oxides are products of a separate enzymic reaction). 9-Hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (KODA) is synthesised from α-linolenic acid via 9-lipoxygenase (9-LOX) and allene oxide synthase; although the amount is usually low, it aids plants to recover from stress. In a few plant species, the enzyme divinylether synthase is believed to function outwith chloroplasts and convert fatty acid 9- or 13‑hydroperoxides into divinyl ethers such as colneleic, colnelenic or etheroleic acids, which are found both in free and esterified forms and are toxic to many plant pathogens (see their structures above).

5.   Other Oxylipins

Di- and trihydroxy-octadecanoids are produced in some plant species, and phaseolic or 2-oxo-5,8,12-trihydroxy-dodecanoic acid was first found in bean seeds and has been shown to stimulate the growth of pea stem segments, to induce α‑amylase synthesis in barley endosperm, and to retarded senescence in barley leaf segments.

Formula of phaseolic acid

In response to fungal infections, additional oxidation reactions occur in leaf oil bodies. An α-dioxygenase, two forms of which exist that are distinct from the lipoxygenases (and related structurally to mammalian COX enzymes), functions in concert with caleosins, i.e., Ca2+‑binding proteins associated with lipid bodies, to generate 2(R)‑hydroperoxy fatty acids such as 2‑hydroxy-linolenic acid. This can decay spontaneously to produce an aldehyde one carbon atom shorter as part of the α-oxidation process in plants.

Biosynthesis of 2-hydroxy-linolenate in oil bodies

Caleosins can react directly with α-linolenic acid to introduce an epoxide group at any of the double bonds, and they can react with the 13‑lipoxygenase metabolite to produce a 13-hydroxy,15,16-epoxy-octadecadienoic acid. Persin or 1-acetoxy-2R-hydroxy-12Z,15Z-heneicosadien-4-one from avocado leaves is a powerful deterrent to animal predation and is toxic to lactating livestock.

6.   Phytoprostanes (Plant Isoprostanes)

Formula of a phytoprostanePlants subjected to biotic and abiotic stresses utilize α-linolenic acid to produce oxygenated fatty acids and C18‑isoprostanoids (dinor-isoprostanes or phytoprostanes) via non-enzymatic, free radical-catalysed pathways as for isoprostane synthesis in animals with singlet oxygen generated in chloroplasts during photosynthesis as the most important reactive oxygen species involved. As phytoprostanes are derived from C18 precursors, they differ from the animal isoprostanes in the number of double bonds and the lengths of the side chains. The phytoprostane PPE1 is illustrated as an example.

The reaction is initiated by the abstraction of a hydrogen radical and is followed by addition of oxygen with formation of a cyclic endoperoxide, before addition of a further oxygen molecule forms the hydroperoxide. Abstraction of the hydrogen atom at C-14 generates phytoprostanes G1 (PPG1) of type I, while hydrogen abstraction at C-11 yields PPG1 of type II. The endoperoxide group is highly unstable, and PPG1 forms rearrange spontaneously or are reduced to D1, E1 and F1 ring analogues of the animal prostaglandins (and named from these), with further dehydration and isomerization to the J1 and other types.

Formation of phytoprostanes G types

Whereas lipoxygenases insert oxygen only at C-9 or C-13 of the precursor fatty acids with defined stereochemistry, reactive oxygen species can produce racemic hydroperoxides at C-10 and C-12 of linoleate and at C-15 and C-16 of linolenate. Indeed, in the twenty or so plant species analysed to date, various regioisomers of free phytoprostanes of the A1, B1, D1, E1, F1, G1 and deoxy-J1 series have been detected, but the series with the hydroxyl group in positions 9 and 16 tend to be most abundant in vivo, especially PPD1 and PPF1. Isofurans formed from linolenate and analogous to those found in animal tissues have also been detected in plants. Like the isoprostanes, phytoprostanes are generated mainly in lipid-bound form in membranes, but they can occur as the free acids, while glutathione conjugates of PPA1 accumulate after pathogen infection in A. thaliana.

Such non-enzymatically produced lipids are formed continuously in healthy plant tissues in the range for each component of 0.01 to 6.7 nmol/g dry weight in tomato leaves. Indeed, the concentrations of esterified phytoprostanes can be an order of magnitude higher than those of the equivalent free jasmonates and two to three orders of magnitude higher than of analogous isoprostanes in animal tissues. If there were not efficient repair mechanisms in place, such high levels of oxidized lipids in membranes might be injurious to the plant. In red and brown macroalgae and microalgae, analogous phytoprostanes are formed together with isoprostanes derived from endogenous arachidonic and eicosapentaenoic acids.

In evolutionary terms, phytoprostanes are likely to have been developed before the oxylipins produced enzymatically, and they have retained a wide range of biological activities in plants. The cyclopentenone-phytoprostanes PPA and PPB up-regulate gene expression for enzymes involved in the response to challenges by foreign organisms or external conditions, while they down-regulate genes involved in cell division and growth. Like the jasmonates, they trigger phytoalexin production (see below). Such studies are at an early stage, and little is known of the effect of specific regioisomers.

Phytoprostanes may be good biomarker for oxidative degradation of plant foods such as occurs during improper storage. As they are present in vegetable oils and have been detected in plasma, the biological properties of phytoprostanes in terms of potential effects on human cells are under investigation in vitro at least, and the initial reports are that they may have useful anti-inflammatory properties. On the other hand, F1‑phytoprostanes, which are present in remarkably high concentrations in pollen, may stimulate pro-inflammatory agents associated with allergic responses.

7.   Aldehydes

Other natural products of fatty acid oxidation with potent biological activities include unsaturated aldehydes (alkenals), such as trans‑3-hexenal and trans‑3-hexenol - sometimes termed the 'leaf aldehyde and alcohol', respectively. The primary precursors are hydroperoxides of lipid-bound fatty acyl moieties, and these can be generated by lipoxygenases or non-enzymatically (autoxidation) by the action of reactive oxygen species (ROS), which include hydrogen peroxide (H2O2), superoxide anions (O2•-), hydroxyl radicals (OH), nitric oxide (NO) and peroxyl radicals (LOO) produced by enzymic and other means under conditions of oxidative stress in plant cells, together with singlet oxygen (1O2 or O=O) in photosynthetic tissues.

Fission of hydroperoxides can then occur spontaneously or by the action of the enzyme hydroperoxide lyase (as discussed above), and the products can be isomerized or further oxidized to produce many different bioactive compounds, including the volatile aldehydes. The formation and biological activities in relation to mammalian systems of such aldehydes are discussed in greater detail in a separate web page, although mechanistic aspects of their formation in plants and their reactions with proteins are very similar. In plants, there is a more restricted range of unsaturated fatty acid precursors than in animals, and as α-linolenate is the main fatty acid in leaf tissue, C6 products predominate, although C9 products are produced but from linoleate mainly.

Some aldehydes produced by oxidative fission in plants

The other fission products, aldehydo-acids, remain attached to the glycerolipid precursors, and such 'oxidatively truncated lipids' can have biological activity by disrupting membranes to increase their permeability and through signalling activities. In this manner, traumatin or 12‑oxo‑trans-10-dodecanoic acid, a plant wound hormone, is produced by isomerization of 10‑oxo-cis-9-octadecanoic acid, and this can in turn undergo autoxidation to produce 'traumatic' acid, i.e., 10(E)-dodeca-1,12-dicarboxylic acid. The latter is a wound healing agent in plants that stimulates cell division near a trauma site to form a protective callus over the damaged tissue, but it is a growth hormone in algae. Fatty acid fragments such as pimelic and azelaic acids (C7 and C9 dicarboxylic acids, respectively) are generated from hydroperoxy fatty acids at the same time, and these may be involved in the defence against pathogens.

As high concentrations of such aldehydes can cause irreversible damage to plant cells membranes and ultimately lead to cell death, their damaging effects and the mechanisms of detoxification are of appreciable importance. Defence mechanisms include the activities of enzymes such aldehyde dehydrogenases, aldo/keto reductases, 2-alkenal reductases and glutathione transferases, together with the effects of antioxidants, such as tocopherols and carotenoids.

Several unsaturated aldehydes, but especially (2E)-hexenal, have antimicrobial properties and have defence roles against fungi, bacteria and arthropods to which they are toxic; they can reduce the fecundity of insect pests. When leaves are wounded, this volatile compound is formed rapidly and is able to diffuse rapidly through damaged or infected tissues before release transiently into the air. It can then participate in a complex signalling system as a warning to other plants and insect predators. (2E)‑4‑Hydroxynonenal produced from (3Z)‑nonenal in plants has anti-fungal properties. By inducing the expression of stress-associated genes, volatile aldehydes are involved in abiotic stress responses. Malondialdehyde generated in leaves non-enzymatically from hydroperoxides is an indicator of cellular damage when in excess, but it may have a more positive role by interacting with plant proteins and modifying gene expression to activate regulatory genes involved in plant defence to provide cellular protection under conditions of oxidative stress.

Other effects result from lipoxidation, i.e., covalent binding of electrophilic aldehydes to proteins, or glutathione and nucleic acids, via the reaction of α,β‑unsaturated carbonyl structures with free thiol (illustrated) or amine groups via the Michael addition reaction. Unsaturated hydroperoxides and the carbonyl groups of the other cleavage product 12‑oxo‑phytodienoic acid may react in the same way. In this manner, they may have signalling functions by reacting with redox-regulated proteins, but they can act as damage/signalling agents in many different physiological situations, including root injury, programmed cell death, senescence, stomata response to abscisic acid and root response to auxin.

Reaction of 2-alkenals with free thiol groups

8.   Esterified Oxylipins

The phytoprostanes and other non-enzymatically produced oxylipins are formed when esterified to galactolipids and phospholipids, and it has long been known that plant oxylipins synthesised by enzymatic means are often present in esterified form. For example, ester-linked (13S)‑hydroxy-(9Z,11E)-octadecadienoic acid is formed during germination of cucumber cotyledons, suggesting a role in plant development. New lipidomic methodologies have now simplified the analysis of intact lipids containing oxylipins and stimulated research into the topic, although knowledge of the biological properties of such lipids remains sparse.

The best known of these new complex lipids are the arabidopsides. Thus, a number of different mono- and digalactosyldiacylglycerols containing 12‑oxo‑phytodienoic acid and/or dinor-oxo-phytodienoic acids in positions sn-1 and/or sn-2 (and even linked to the carbohydrate moiety) and termed 'arabidopsides A to G' have been isolated from stressed plants of A. thaliana after leaf wounding, during the hypersensitive response to bacterial pathogens, or under cold stress. In plants challenged by a bacterial pathogen, a monogalactosyldiacylglycerol containing two 12‑oxo‑phytodienoate and one dinor-oxo-phytodienoate acyl chain (arabidopside E) was found to accumulate in amounts up to 8% of the total lipids, up to 150 times more than in the free state, and it was shown to have anti-bacterial properties in vitro. Not only do they accumulate in stressed leaves of A. thaliana but, to a limited extent, in distal non-wounded leaves of the same plant, as well as at trace levels in normal plants. It is now apparent that fatty acids esterified to galactolipids, as well as the free acids, may be substrates for biosynthesis of OPDA, and both lipoxygenase 2 and the allene oxide synthase are able to oxidize lipid-bound fatty acids.

Formulae for arabidopsides A and C

Arabidopsides are produced in the chloroplasts and are found mainly in the thylakoid membrane, although it is possible that they could be trafficked to the plasma membrane. It seems probable that they function as a storage pool, which upon stimulation could release OPDA for direct signalling or as a substrate for production of jasmonic acid, although there are suggestions that the intact lipids have antibacterial and antifungal properties and may have a role in senescence.

Although these intriguing lipids do not appear to have a widespread distribution in the Brassica family, more lipids of this kind are being reported from other species, and other oxylipins are known to be present in esterified form in plants subject to damage by pathogens or freeze/thawing. Oxylipins including 12‑oxo‑phytodienoate linked to phosphatidylinositol and phosphatidylglycerol have been detected in stressed Arabidopsis, and lipoxygenase products have been found esterified to various phospholipids in other plant species. In addition, it has been established that acylation of the head-group of monogalactosyldiacylglycerol is a common stress response in plants, with the nature of the added acyl group, which can include OPDA, varying with the plant species and the stress applied.

Colneleic and colnelenic acids, produced by the action of 9-LOX, occur esterified to the sn-2 position of phospholipids in potato, suggesting the presence of a preformed pool that, like the arabidopsides, would be immediately available in response to challenge by pathogens, for example to inhibit germination of fungal spores. Mono- and digalactosyldiacylglycerols containing divinyl ether fatty acids and termed 'linolipins' occur in leaves of flax plants (Linum usitatissimum L.) subjected to damage by freezing-thawing and in those inoculated with cells of the phytopathogenic bacterium Pectobacterium atrosepticum, and related lipids have been found in damaged leaves of the meadow buttercup (Ranunculus acris).

Plant N-acylethanolamides, in which the fatty acid moiety is a lipoxygenase product of linoleic acid, have been detected in seeds of A. thaliana, where they act synergistically with abscisic acid to modulate the transition from embryo to seedling.

9.   Biological Activity of Plant Oxylipins

When plants are attacked by bacterial or 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, such as the jasmonates and their precursors the oxo-phytodienoic acids, are potent regulators of defence mechanisms by stimulating proteinase inhibitors or by promoting the accumulation of antimicrobial secondary metabolites (phytoalexins). Oxylipins also function in plant adaptation to abiotic stresses, including wounding, suboptimal light and temperature, dehydration and osmotic stress, and the effects of heavy metals. They take part in the complex interactive networks of phytohormones, including salicylic acid, ethylene, auxin, brassinosteroids, gibberellic acid and abscisic acid, that control all aspects of plant growth and development and how plants adapt to the environment. This 'crosstalk' is too complex a topic to be discussed here (but see the reading list below).

Simplistically, hydroperoxide lyase and allene oxide synthase actively compete for substrate within the lipoxygenase pathway. It is believed that the latter mediates plant defence responses directly by forming jasmonic acid, whereas the volatile products of the hydroperoxide lyase pathway initiate defence responses indirectly by attracting the natural enemies of plant invaders. Most organelles in plants have the capacity to produce oxylipins, but it is increasingly being recognized that lipid droplets in which triacylglycerols are stored have a role in mediating stress responses as well as supplying substrates and sequestering potentially toxic products. These are formed in the endoplasmic reticulum and thylakoid membranes (plastoglobules) and are associated with a range of biosynthetic enzymes (see our web page on triacylglycerol biosynthesis).

Jasmonates: (9S,13S)-12-Oxo-phytodienoic (OPDA) acid and jasmonic acid metabolites like (+)-7-jasmonoyl-L-isoleucine (JA-Ile), are phytohormones that are involved in growth and developmental processes, secondary metabolism, defence against attack by insects and pathogens, and protection from many other abiotic stresses, including those induced by salt, drought, heavy metals, freezing and light. In response to changes in the environment and transient endogenous signalling events, jasmonates are part of an intricate web of interactions involving gene networks, regulatory proteins and signalling intermediates. They can induce stomatal opening, inhibit photosynthetic CO2 assimilation, and affect the uptake of nitrogen and phosphorus and the transport of organic nutrients such as glucose. By integrating and coordinating primary and secondary metabolism, jasmonates orchestrate the balance between plant growth and defence. Long-distance transmission of jasmonate signals within the plant occurs via the vascular bundle.

Jasmonoyl-isoleucine is the primary jasmonate known to be active at the molecular level at the nucleus via a receptor complex that contains a 'Coronatine Insensitive 1 or COI1' protein. At low JA-Ile levels, the transcription of JA-responsive genes is repressed because of the accumulation of transcriptional repressor proteins (so-called ‘JAZ’ proteins). This repressed state is then de-repressed by elevated JA-Ile levels in response to environmental and developmental cues, which promote the binding of JA-Ile with JAZ in the COI1 coreceptor and initiate ubiquitination and the subsequent degradation of JAZ by proteasomes. It is believed that a co-receptor complex is formed between COI1 and a JAZ protein in the presence of JA-Ile with inositol pentakisphosphate (IP5) as a cofactor. A specific transporter (JAT1) acts as a JA-Ile carrier into the nucleus and a jasmonic acid carrier out of the cytoplasm and across the plasma membrane. 12‑Hydroxy-JA-Ile synthesised in the endoplasmic reticulum has biological activity by differentially activating a subset of the JA‑Ile co-receptors to control and/or modulate some jasmonate-dependent responses that improve plant resilience.

In relation to fertility, jasmonates have a role in pollen maturation, and in such varied processes as seed germination, fruit ripening, root growth, flower (including sex determination) development, flowering time, senescence and tendril coiling; plants defective in jasmonate biosynthesis or signalling are male-sterile. They are believed to act by mechanisms that are as yet poorly understood to activate signalling pathways both intra- and inter-cellularly that modulate the expression of a number of genes and thence the synthesis of many key proteins often by interacting with abscisic acid, gibberellin and light. With many aspects awaiting clarification, the picture emerging is a highly complex one, and there appear to be functional differences among plant species that have yet to be explained.

Scottish thistleEach of the various jasmonate derivatives, i.e., the free acid, methyl ester and conjugates with amino acids, has distinct biological effects, often in defence mechanisms. Wound response is one of the most-studied pathways of jasmonates in signal transduction with the tomato often as the experimental model. Jasmonic acid levels in undamaged leaves of mature plants are barely detectable, but wounding activates a rapid synthesis of jasmonic acid and jasmonoyl-isoleucine in both local and systemic tissues. In brief, local wounding results in breakdown of cells and release of fatty acids, and at the same time, cleavage of the octadecapeptide systemin is initiated from prosystemin to stimulate jasmonic acid biosynthesis and the formation of various jasmonic acid conjugates. This is believed to act as a signal that leads to systemic expression of genes encoding proteinase inhibitors and anti-feedant and poisonous compounds, which deter insect herbivores by inhibiting their digestive capabilities and 'immunizing' the plant against further herbivore attacks. At the same time, biosynthesis of surface waxes is promoted. Jasmonates are part of the defence system against viruses carried by insects, and they participate in many other abiotic stress responses, such as to excessive cold and heat. The relationship with other defence mechanisms including those based on phytohormones are under active study.

Volatile jasmonate metabolites, such as cis-jasmone (a decarboxylation product) and methyl jasmonate, may regulate the behaviour of some insects by deterring herbivorous species or attracting their predators. Methyl jasmonate can influence other plants at a distance by activating defensive genes encoding proteins and secondary compounds such as anthocyanins and alkaloids. In a study of the mechanism of this interaction with the model bryophyte Marchantia polymorpha, it has been demonstrated that airborne methyl jasmonate is metabolized to jasmonic acid and 12‑hydroxyjasmonic acid, while increased expression of biosynthetic genes for jasmonic acid is induced.

Similarly, jasmonates are involved in defence against bacterial pathogens such as those that feed on necrotic tissue or cells undergoing apoptosis. The bacterial phytotoxin coronatine exerts is toxic effects by binding to the receptor for jasmonoyl-isoleucine and by interfering with the response to salicylic acid. While certain soil-borne microorganisms have beneficial effects by enhancing the defensive capacity of plants, with jasmonic acid as a regulator of the process, others can hijack jasmonate synthesis to cause hormonal signalling imbalances and enhance infection. Jasmonates promote the beneficial interactions between certain mycorrhizal fungi and nitrogen-fixing bacteria, and they may even enable communication between plants.

Jasmonates have pivotal roles in root regeneration by signalling to induce cell proliferation and restore the root meristem by stimulating specific transcription factors to promote callus formation and shoot regeneration. They may down-regulate genes involved in the core metabolism of the plant simultaneously by interacting with gibberellins to in effect decide whether defence or growth should have priority. As well as its tuber-inducing properties, tuberonic acid is a leaf-closing factor that induces leaf movement in motor cells of certain plants.

12-Oxo-phytodienoic acid (cis-OPDA), a biosynthetic precursor of jasmonic acid, controls a set of genes that are independent of the latter in the regulation of seed germination, the development of the embryo and plant defence. Working together with salicylic acid, it recognizes the bacterial quorum-sensing lipoamino acid N‑3‑oxo-tetradecanoyl-L-homoserine lactone and primes the plant for an enhanced defence by a mechanism that is independent of the jasmonic acid signalling cascade. There is also believed to be crosstalk between OPDA and the phytohormone abscisic acid. While no receptor for OPDA has yet been discovered, it does bind to cyclophilin in plastids and thence induces a signalling pathway. OPDA and dinor-OPDA are present in nonvascular land plants (e.g., mosses and liverworts), but jasmonoyl-isoleucine is not.

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 and appear to have therapeutic potential for this and other human diseases. Many other plant oxylipins, including aldehydes, oxy-, epoxy- and hydroxy-derivatives of fatty acids, phytoprostanes and phytofurans are reported to have beneficial properties for human health.

Other oxylipins: Aside from their function as precursors of jasmonates, oxylipins derived from the lipoxygenase pathway have characteristic biological activities of their own. Even the primary 9(S)- and 13(S)‑hydroperoxides and hydroxides have antifungal and anti-microbial properties, and for example, in Arabidopsis, LOX1-derived fatty acid hydroxides regulate stomatal closure in an abscisic acid–independent way and are believed to be involved in defence reactions against bacterial pathogens. 2-Hydroxy-linolenic acid produced in leaf oil bodies and the epoxy and hydroxy derivatives of linoleic acid resulting from the peroxygenase pathway are toxic to fungal pathogens, while 9-keto-10(E),12(Z),15(Z)-octadecatrienoic acid, produced from linoleic acid by the action of 9-LOX, is highly active against the plant pathogen Pseudomonas syringae pv. tomato. They may be part of a short-term local response, while the jasmonates operate over a longer time scale.

10. Oxylipins from Fungi, Mosses and Algae

Mosses, fungi and yeasts produce a variety of oxylipins from saturated and unsaturated fatty acids that include oleate through to arachidonic and eicosapentaenoic acids, which can occur naturally in these organisms. In addition, fungi generate many endogenous oxylipins from the fatty acids of host plants and animals during infections by the action of linoleate diol synthases, cytochrome P450 dioxygenases and lipoxygenases, and enzymes with a LOX domain fused to a functional cytochrome P450 at the C-terminal end are known. Many filamentous fungi pathogens contain dioxygenases with homology to human cyclooxygenases, and the prostaglandin metabolites PGF2 and PGF2-lactone, derived from arachidonic acid, have been detected in yeasts of the Lipomycetaceae family, while PGE2 is produce by Candida albicans and other species. The latter organism is a pathogen that utilizes host arachidonic acid for this purpose and as a first step in β‑oxidation to generate 3-hydroxy-eicosanoids, which stimulate its growth and virulence. On the other hand, octadecanoids are more common, most with an 8R‑hydroxyl group though some 5S-,9R- or 10R-substituents occur, and dihydroxy- and hydroxy,epoxy-metabolites are known. 8R‑Hydroxy-octadeca-9,12-dienoic and 5S,8R-dihydroxy-octadec-9-enoic acids are produced by Aspergillus species, and comparable oxylipins can be formed from oleate. In ascomycetes, the linoleic acid-derived oxylipin 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) is produced for the transcriptional reprogramming required for asexual development.

Fungal oxylipins

While fungal pathogens can synthesise jasmonates by essentially the same pathway as in plants and then exploit host oxylipins to increase their own virulence, oxylipins such as the jasmonates in plant hosts function in the opposite sense to resist the attack of fungal pathogens. Volatile C8 oxylipins, 1-octen-3-ol, 3‑octanone, 3-octanol and others are synthesised in fungi from the longer-chain oxylipins, and they are believed to participate in the regulation of the germination of fungal spores and the subsequent developmental processes. They have antibacterial and antifungal properties and act as signalling molecules during the interactions with higher plants, nematodes and insects. They are responsible for the characteristic smell of mushrooms.

The mechanisms of fungal-plant interactions are now being unravelled, but it appears that C20 polyenoic fatty acids, which do not occur in higher plants and are released during fungal infection, engage plant signalling networks to induce resistance to the pathogens. They elicit a cascade of responses, including an oxidative burst and the transcriptional activation of genes involved in the hypersensitive response.

Mosses are of interest in that they are intermediate between unicellular algae and flowering plants in evolutionary terms. As the complete genome of the moss Physcomitrella patens is available, it makes an ideal model organism for oxylipin study. It produces a range of C18 and C20 oxylipins, including 12‑hydroperoxy-eicosatetraenoic acid (12-HPETE) and further metabolites from arachidonate, which makes up 30% of the total fatty acids in the organism, but it does not synthesise jasmonic acid.

In unicellular algae, aldehydes derived from C20 fatty acids are formed as a defence response on wounding, while multicellular algae produce oxylipins from C18 and C20 polyunsaturated fatty acids. Thus, 12- and 5-HPETEs and a range of further lipoxygenase metabolites are produced by red algae. Marine diatoms produce many distinctive oxylipins, and some have even been found in prokaryotes. The functions of most of these compounds have yet to be determined, although in fungi at least, they are known to act as hormone-like signals that regulate such processes as asexual and sexual spore development and toxin production.

Some bacteria have lipoxygenase enzymes that can oxidize host unsaturated fatty acids during infections of plants and animals to produce oxylipins with signalling functions that influence virulence, and for example, the opportunistic bacterial pathogen Pseudomonas aeruginosa uses a diol synthase to catalyse the stereospecific oxygenation of oleic acid to 10-HOME and 7,10-diHOME.

11. Analysis

As with the eicosanoids, methods involving gas chromatography-mass spectrometry or high-performance liquid chromatography allied to electrospray tandem mass spectrometry are preferred for the analysis of the plant oxylipins, with HPLC and UV detection as a useful complementary technique. Internal standards labelled with stable isotopes, such as O18, are essential for quantification.

Recommended Reading

Lipid listings © Author: William W. Christie LipidWeb icon
Contact/credits/disclaimer Updated: February 14th, 2024