Oxylipins are biologically active compounds generated by oxidative metabolism of polyunsaturated fatty acids. Lipooxygenase (LOX), a non-heme iron dioxygenase, initiates the biosynthesis of most plant oxylipins. They are generated as a result of the coordinated action of lipases, LOX and a group of specific cytochromes P450 CYP74 (cytochrome P74) family members. The CYP74 family includes several enzymes devoted to oxylipin biosynthesis, such as allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase (DES), epoxy alcohol synthase, peroxygenase, alkyl hydroperoxide reducase, and LOX. This large family of enzymes is believed to have originated to protect plants from severe environments and from attacks by pathogens and predators.
In the June issue of Current Opinion of Plant Biology (vol. 5:230-236), Gregg A. Howe and Anthony L Schilmiller (Michigan State University-DOE Plant Research Laboratory) discuss the most recent developments in oxylipin metabolism and the involvement of the lipoxygenase (LOX) and cytochrome P450 (CYP)74 enzyme families in this process. They have also described how phospholipases regulate oxylipin biosynthesis by releasing fatty acid precursors from membranes.
Oxylipins are not pre-formed, but rather are synthesized de novo in response to mechanical injury, herbivore and pathogen attack, and other environmental and developmental cues. Two prominant and well-studied oxylipin compounds are jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA). More recently, oxylipin precursors of JA and MeJA have begun to be recognized as signaling molecules in their own right.
The authors begin the review by describing the biosynthesis of oxylipins. Most plant oxylipins are generated by addition of oxygen to the 9 or 13 position of the C18 chain of linoleic and linolenic acids, which are two important polyunsaturated fatty acids. Different isoforms of LOX perform this reaction, and the numerous LOX isoforms present in plants have different expression patterns, subcellular localizations, and substrate preferences. Enzymes such as AOS, HPL, DES, epoxy alcohol synthase, peroxygenase and alkyl hydroperoxide reductase act upon hydroperoxy products of LOX and convert them into a variety of oxylipins. Interestingly, AOL, HPL and DES all belong to a family of cytochromes P450 called CYP74. The authors point out that there are two distinct pathways for oxylipin metabolism, a 9-LOX pathway that generates 9-hydroperoxy fatty acids and a 13-LOX pathway that generates 13-hydroperoxy fatty acids. Each pathway has several CYP74-dependent sub-branches.
The authors then describe the 13-LOX pathway in more detail. Conversion of 13-hydroperoxy linolenic acid (13-HPOT) to the jasmonate family of compounds occurs when jasmonic acid (JA), methyl jasmonate (MeJA) and their metabolic precursor, 12-oxo-phytodienoic acid (12-OPDA) are generated through action of AOS on 13-hydroperoxy linolenic acid (13-HPOT), which is the product of 13-LOX action. JA and MeJA are known to be involved in regulating stress-induced gene expression, mechanical responses such as tendril coiling, and reproductive development. A gene encoding a JA carboxyl methyltransferase (JMT) has been recently reported, indicating that methyl jasmonate (MeJA) is one of the important components of an oxylipin signals that mediates defense responses. 12-ODPA has been shown to serve as an important signaling molecule itself, and a recent study indicated that the alpha,beta-unsaturated carbonyl moiety found in 12-ODPA and other cyclopentenone oxylipins possesses novel signaling activity as compared to cyclopentanone oxylipins such as JA. The authors point out that a similar situation exists in animals, in which cyclopentenone prostaglandins serve as important signaling molecules.
The authors also describe other aspects of oxylipin metabolism, such as branches of the 13-LOX pathway. The HPL branch, for example, directs formation of C-6 aldehydes and C-12 omega-keto-fatty acids. Several of these compounds have important biological roles in wound healing, defense and as major volatile constituents of fruits, vegetables and green leaves. The 9-LOX pathway is described as well. Metabolism of 9-hydroperoxy fatty acids generated by 9-LOX by isoforms of AOS, HPL and DES results in oxylipins structurally related to, but distinct from, those generated by the 13-LOX pathway.
The authors describe biological roles of JA, MeJA, and other oxylipins in regulation of plant defense and developmental processes. The synthesis and perception of jasmonates is critical to wound-induced systemic defense responses, as illustrated in studies utilizing mutants affected in JA biosynthesis and perception. Other oxylipins are critical for defense responses as well. 9-LOX-derived divinyl ether oxylipins are reported to accumulate in Phytopthera-infected potato leaves inhibiting fungal growth. Recent study also indicates that the C12 product derived from linolenic acid is the precursor of traumatin, a compound that triggers cell division, implicated in wound healing. Short-chain aldehyde products of the 13-HPL pathway also play important roles in defenses against microbial pathogens and insects. Oxylipins also play a role in developmental processes. JA has been implicated in development of the male gametophyte in Arabidopsis thaliana, but may not play a similar role in other plants such as tomato. Floral initiation in duckweed (Lemna) is regulated by jasmonates. Transgenic studies in potato have provided evidence that 10-OPDA or another 9-LOX-derived oxylipin regulates tuber development.
The authors discuss the CYP74 family of P450s in terms of evolutionary implications and the relationship of oxylipin metabolism CYP74 enzymes to diversity of oxylipin structure and function. Unlike classical cytochrome P450 monooxygenases, CYP74 P450s use a hydroperoxide group as an oxygen donor and source of reducing equivalents. Phylogenetic trees of the CYP74 family indicate that four sub-families can be grouped together on the basis of sequence similarity. Each sub-family is also distinguished by enzymatic identity and substrate specificity. Bioinformatics approaches based on sequences analyses such as these have helped to identify novel CYP74 genes in databases, such as three uncharacterized CYP74 sequences represented in a tomato EST database.
Several stress-induced and developmental cues regulate oxylipin biosynthesis; one of the major regulatory mechanisms in this event is phospholipases (PLs). These enzymes release fatty-acid precursors from membrane lipids. Purified PLA2 isoforms have been studied recently, and may have a role in production of oxylipin precursors during the hypersensitive response to pathogens. In addition to PLA2, PLD-alpha has been implicated in wound-induced accumulated of JA in Arabidopsis leaves. Recently, the dad1 (defective in anther dehiscence1) male-sterile Arabidopsis mutant has allowed identification of DAD1 as a PLA1 that liberates fatty acid precursors from of JA from the stereospecifically numbered-1 position of chloroplast lipids. The identification of this enzyme and its localization to plastids has raised interesting possibilities regarding substrates for 12-OPDA production, suggesting that initial enzymes of JA biosynthesis could use a lipid substrate rather than a fatty acid. The authors note that JA synthesis may be regulated by lipases that release 12-OPDA, rather than linolenic acid, in response to specific signals.
In conclusion, the authors state that although a lot of ground has been covered in research on jasmonates, there are still some unexplored areas. For instance, molecular genetic and biochemical analyses of non-jasmonate oxylipins have lagged behind. The authors advocate a thorough investigation of these compounds and the pathways that generate them in order to gain insights into the whole operational system of oxylipin metabolism. On the positive side, recent research on plant oxylipins in plants has demonstrated parallels to the eicosanoid group of metabolites that have broad regulatory roles in animal physiology. The identification of oxylipin biosynthetic genes, mostly in the model plant Arabidopsis, has allowed progress, but the authors suggest extending such studies to a broad spectrum of plant species. This will be necessary to determine how various oxylipins generated from diverse pathways function in response to developmental- and stress-related cues.
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