In barley leaves, the application of jasmonates leads to dramatic alterations of gene expression. Among the up-regulated gene products lipoxygenases occur abundantly. Here, at least four of them were identified as 13-lipoxygenases exhibiting acidic pH optima between pH 5.0 and 6.5. (13S,9Z,11E,15Z)-13-hydroxy-9,11,15-octadecatrienoic acid was found to be the main endogenous lipoxygenase-derived polyenoic fatty acid derivative indicating 13-lipoxygenase activity in vivo. Moreover, upon methyl jasmonate treatment . 78% of the fatty acid hydroperoxides are metabolized by hydroperoxide lyase activity resulting in the endogenous occurrence of volatile aldehydes. (2E)-4-Hydroxy-2-hexenal, hexanal and (3Z)-plus (2E)-hexenal were identified as 2,4-dinitrophenylhydrazones using HPLC and identification was confirmed by GC/MS analysis. This is the first proof that (2E)-4-hydroxy-2-hexenal is formed in plants under physiological conditions. Quantification of (2E)-4-hydroxy-2-hexenal, hexanal and hexenals upon methyl jasmonate treatment of barley leaf segments revealed that hexenals were the major aldehydes peaking at 24 h after methyl jasmonate treatment. Their endogenous content increased from 1.6 nmol´g 21 fresh weight to 45 nmol´g 21 fresh weight in methyl-jasmonate-treated leaf segments, whereas (2E)-4-hydroxy-2-hexenal, peaking at 48 h of methyl jasmonate treatment increased from 9 to 15 nmol´g 21 fresh weight. Similar to the hexenals, hexanal reached its maximal amount 24 h after methyl jasmonate treatment, but increased from 0.6 to 3.0 nmol´g 21 fresh weight. In addition to the classical leaf aldehydes, (2E)-4-hydroxy-2-hexenal was detected, thereby raising the question of whether it functions in the degradation of chloroplast membrane constituents, which takes place after methyl jasmonate treatment.

A glycopeptide elicitor prepared from germ tubes of the rust fungus Puccinia graminis Pers. f. sp. tritici Erikss. & Henn (Pgt), as well as chitin oligosaccharides, chitosan, and methyl jasmonate (MJ) stimulated lipoxygenase (LOX) activity (E.C. 1.1 3.1 1.1 2) in wheat (Triticum aestivum) leaves. lmmunoblot analysis using anti-LOX antibodies revealed the induction of 92-and 103-kD LOX species after Pgt elicitor treatment. In contrast, MJ treatment led to a significant increase of a 100-kD LOX species, which was also detected at lower levels in control plants. The effects of chitin oligomers and chitosan resembled those caused by MJ. In conjunction with other observations the results suggest that separate reaction cascades exist, and that jasmonates may not be involved in Pgt elicitor action. LOX-92 appears to be mainly responsible for the increase in LOX activity after Pgt elicitor treatment because its appearance on western blots coincided with high LOX activity in distinct anion-exchange chromatography fractions. It is most active at pH 5.5 to 6.0, and product formation from linoleic and a-linolenic acid is clearly in favor of the 9-LOOHs. It is interesting that a 92-kD LOX species, which seems to correspond to the Pgt elicitor-induced LOX species, was also detected in rust-inoculated leaves.The ubiquitous LOXs (E.C. 1.13.11.12) identified in animais and plants are non-heme-iron-containing enzymes, which catalyze the oxidation of polyunsaturated fatty acids containing a cis,cis-1,4-pentadiene site. Linoleic (182) and linolenic (18:3) acid, components of plant lipids, represent potential substrates for these enzymes, which in plants are predominantly present as soluble proteins in the cytosol but are also found in membranes and in different organelles (for review, see Siedow [1991]). Generally, free fatty acids have been regarded as the natural LOX substrate; however, in vitro reactions of plant lipoxygenases with membrane components have been shown (Maccarrone et al., 1994;Regdel et al., 1994). A precise physiological role for LOX in plants has not been defined so far, but the diversity of isozymes and the subcellular distribution suggest multiple functions (Siedow, 1991;Rosahl, 1996). LOX is required for the syn-

At early stages of germination, a special lipoxygenase is expressed in cotyledons of cucumber and several other plants. This enzyme is localized at the lipid storage organelles and oxygenates their storage triacylglycerols. We have isolated this lipid body lipoxygenase from cucumber seedlings and found that it is capable of oxygenating in vitro di-and trilinolein to the corresponding mono-, di-, and trihydroperoxy derivatives. To investigate the in vivo activity of this enzyme during germination, lipid bodies were isolated from cucumber seedlings at different stages of germination, and the triacylglycerols were analyzed for oxygenated derivatives by a combination of high pressure liquid chromatography, gas chromatography/mass spectrometry, and nuclear magnetic resonance spectroscopy. We identified as major oxygenation products triacylglycerols that contained one, two, or three 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid residues. During germination, the amount of oxygenated lipids increased strongly, reaching a maximum after 72 h and declining afterward. The highly specific pattern of hydroperoxy lipids formed suggested the involvement of the lipid body lipoxygenase in their biosynthesis.These data suggest that this lipoxygenase may play an important role during the germination process of cucumber and other plants and support our previous hypothesis that the specific oxygenation of the storage lipids may initiate their mobilization as a carbon and energy source for the growing seedling.Lipoxygenases (LOXs) 1 are non-heme iron-containing dioxygenases that catalyze the regio-and stereoselective oxygenation of polyenoic fatty acids to their corresponding hydroperoxy derivatives (1). They are widely distributed in plants and animals (2, 3). In mammals, LOXs are classified according to their positional specificity of arachidonic acid oxygenation (4). Since arachidonic acid is either not present in higher plants or is a minor constituent of cellular lipids, plant LOXs may be classified into 9-and 13-LOXs with respect to their positional specificity in linoleic acid oxygenation. Recently, a more comprehensive classification of plant LOXs has been proposed based on the comparison of their primary structures (5). Although plant and mammalian lipoxygenases have been extensively characterized with respect to their protein chemical and enzymatic properties, there is no general idea of their biological importance (1).In plants, 13-LOXs have been implicated in the biosynthesis of jasmonic acid (6). This phytohormone has been shown to be an important mediator in the wound response of plants to herbivore attack (7,8). For many years, plant and animal LOXs have been considered to oxygenate mainly free polyenoic fatty acids, forming oxygenated derivatives that may exhibit biological activities (2, 4, 9). On the other hand, more recent studies have suggested that certain plant (10, 11) and mammalian lipoxygenases (12-14) are able to oxygenate not only free polyenoic fatty acids but also ester lipid substrates, such as phosphol...

During the early stages of germination, a lipid-body lipoxygenase is expressed in the cotyledons of sunflowers (Helianthus annuus L.). In order to obtain evidence for the in vivo activity of this enzyme during germination, we analyzed the lipoxygenase-dependent metabolism of polyunsaturated fatty acids esterified in the storage lipids. For this purpose, lipid bodies were isolated from etiolated sunflower cotyledons at different stages of germination, and the storage triacylglycerols were analyzed for oxygenated derivatives. During the time course of germination the amount of oxygenated storage lipids was strongly augmented, and we detected triacylglycerols containing one, two or three residues of (9Z,11E,13S)-13-hydro(pero)xy-octadeca-9,11-dienoic acid. Glyoxysomes from etiolated sunflower cotyledons converted (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid to (9Z,11E)-13-oxo-octadeca-9,11-dienoic acid via an NADH-dependent dehydrogenase reaction. Both oxygenated fatty acid derivatives were activated to the corresponding CoA esters and subsequently metabolized to compounds of shorter chain length. Cofactor requirement and formation of acetyl-CoA indicate degradation via beta-oxidation. However, beta-oxidation only proceeded for two consecutive cycles, leading to accumulation of a medium-chain metabolite carrying an oxo group at C-9, equivalent to C-13 of the parent (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid. Short-chain beta-oxidation intermediates were not detected during incubation. Similar results were obtained when 13-hydroxy octadecanoic acid was used as beta-oxidation substrate. On the other hand, the degradation of (9Z,11E)-octadeca-9,11-dienoic acid was accompanied by the appearance of short-chain beta-oxidation intermediates in the reaction mixture. The results suggest that the hydroxyl/oxo group at C-13 of lipoxygenase-derived fatty acids forms a barrier to continuous beta-oxidation by glyoxysomes.

No abstract