The LOX Pathway

Enzymatic peroxidation of lipids occurs by stereospecific dioxygenation of PUFAs such as linolenic acid (LeA) or linoleic acid (LA) catalyzed by LOXs. LeA might be released by activity of a phospholipase A2, as shown for a wound response (Narvaez-Vasquez et al., 1999). Recently, JA deficiency in the Arabidopsis mutant dadl (defective in anther dehiscence 1), which is affected in a flower-specific chloroplast-located phospholipase A1, indicates the role of this enzyme in JA biosynthesis (Ishiguro et al., 2001). Although unknown in function, a senescence-related cytosolic lipase of carnation petals was cloned recently (Hong et al., 2000). Several forms of LOX have been shown to oxygenate LA or LeA (Brash, 1999) and many of them are chloroplast-located (Feussner et al., 1995). However, only recently reverse genetic analysis revealed specific functions in development or stress-responses (Feussner and Wasternack, 2002). The S-configured LOX-derived hydroperoxides (HPOD or HPOT) are substrates for at least seven metabolic branches (Fig. 9-1): In one of these branches, allene oxides are formed by allene oxide synthase (AOS), and its products are octadecanoids and jasmonates. Important products of other branches are the leaf aldehydes and divinyl ether. Cloning and reverse genetic studies on lipases and the various LOX forms will show in molecular terms initial events of lipid peroxidation and its role in PCD and senescence. Furthermore, autoxidative processes in lipid peroxidation occur in leaf senescence (Berger et al., 2001).

B. Octadecanoid and Jasmonate Biosynthesis

The initial step in JA biosynthesis is the synthesis of an allene oxide formed from 13-HPOT by an AOS (Fig. 9-2). AOSs have been cloned from several plants, and the AOS protein resides in the chloroplast (Maucher et al., 2000), apparently in a chloroplast envelope fraction (Froehlich et al., 2001). The allene oxide is highly unstable and needs to be converted by an allene oxide cyclase (AOC). The cis-(9S,13S)-OPDA formed is the unique precursor of the naturally occurring stereoisomer of JA and, thus, the AOC has a special regulatory role in jasmonate biosynthesis.

Recently, AOC was cloned from tomato (Ziegler et al., 2000) and later from Arabidopsis (Stenzel et al., 2002b). Wounding, osmotic stress and glucose treatment or application of jasmonates and octadecanoids lead to the expression of AOC in tomato leaves (Stenzel et al., 2002a). Similar kinetics of transcriptional up-regulation were found for the tomato AOS (Howe et al., 2000). Further conversion of OPDA takes place by an NADPH-dependent reductase (OPR). Besides the unspecific OPR1 and OPR2, only the recently cloned OPR3 of Arabidopsis forms exclusively the correct enantiomer cis-(9S,13S)-OPDA (Schaller et al., 2000). OPR3 contains a peroxisomal signal sequence suggesting its peroxisomal location. Consequently, the transport has to be assumed of OPDA, from the chloroplasts into peroxisomes, where enzymes catalyzing ^-oxidation are located. The final product of JA biosynthesis, (+)-7-iso-JA, isomerizes to the more stable (-)-JA.

A highly important issue in JA biosynthesis is its regulation. Due to the fact that all LOX, AOS, AOC and OPR3 are transcriptionally up-regulated by JA or stress, a positive feedback was assumed. However, at least within some hours, a transcriptional up-regulation for LOX, AOS and AOC upon treatment with jasmonates and octadecanoids was not accompanied by an endogenous rise in jasmonates measured by GC-MS quantification and isotopic dilution analysis (Kramell et al., 2000). Recently, for Arabidopsis, indications were found that positive feedback may occur during growth (Stenzel et al., 2002b). In addition, in tomato, substrate availability may control enzyme activity in JA biosynthesis (Stenzel et al., 2002a). Interestingly, constitutively high JA levels, found upon overexpression of AOS in potato, did not lead necessarily to constitutive expression of JA-inducible genes,

Biosynthesis
Figure 9-2. The biosynthesis of jasmonates. Peculiarities are given by instability of the allene oxide and the strict stereochemistry established by the AOC and kept by the OPR-catalyzed step.

suggesting intracellular sequestration of JA (Harms et al, 1995). Recent data emphasize cell-specific AOC expression and elevation of JA levels in leaf veins (Stenzel et al., 2002a).

C. Signaling Properties of Jasmonates and Octadecanoids—the Oxylipin Signature

The signaling role of jasmonates is based on only correlative data on (i) structure-activity relationships and (ii) a link between an endogenous rise upon an external stimulus and the corresponding expression of jasmonate-responsive genes. Numerous studies on structure-activity relations of substituted, deleted or stereospecifically altered jasmonic acid derivatives revealed that (i) an intact cyclopentanone ring with a (-)-enantiomeric or (+)-7-iso-enantiomeric structure, and (ii) a pentenyl side chain and (iii) an acetic acid side chain are necessary (Blechert et al., 1999; Miersch et al., 1999). More recently, it was shown that octadecanoids but not jasmonates were responsible for resistance against insect and fungal attack (Stintzi et al., 2001). Furthermore, octadecanoids are the preferential signals in tendril coiling (Blechert et al., 1999) and in the release of diterpenoid-derived volatiles (Koch et al., 1999), whereas jasmonates were found to be preferentially active in inducing synthesis of alkaloids in cell cultures or in the release of sesquiterpene volatiles (Koch et al., 1999). The OPDA derivative dinor OPDA, which is formed from the 16:3 fatty acid, appears beside OPDA (Weber et al., 1997). A varying amount of different oxylipins, called the "oxylipin-signature", may function as a signal (Weber et al., 1997). Such a distinct ratio of jasmonates, octadecanoids and conjugates of amino acid with JA was found recently in distinct flower organs (Hause et al., 2000).

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