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.

In addition to a previously characterized 13-lipoxygenase of 100 kDa encoded by LOX2:Hv:1 [Vörös et al., Eur. J. Biochem. 251 (1998), 36-44], two full-length cDNAs (LOX2:Hv:2, LOX2:Hv:3) were isolated from barley leaves (Hordeum vulgare cv. Salome) and characterized. Both of them encode 13-lipoxygenases with putative target sequences for chloroplast import. Immunogold labeling revealed preferential, if not exclusive, localization of lipoxygenase proteins in the stroma. The ultrastructure of the chloroplast was dramatically altered following methyl jasmonate treatment, indicated by a loss of thylakoid membranes, decreased number of stacks and appearance of numerous osmiophilic globuli. The three 13-lipoxygenases are differentially expressed during treatment with jasmonate, salicylate, glucose or sorbitol. Metabolite profiling of free linolenic acid and free linoleic acid, the substrates of lipoxygenases, in water floated or jasmonate-treated leaves revealed preferential accumulation of linolenic acid. Remarkable amounts of free 9- as well as 13-hydroperoxy linolenic acid were found. In addition, metabolites of these hydroperoxides, such as the hydroxy derivatives and the respective aldehydes, appeared following methyl jasmonate treatment. These findings were substantiated by metabolite profiling of isolated chloroplasts, and subfractions including the envelope, the stroma and the thylakoids, indicating a preferential occurrence of lipoxygenase-derived products in the stroma and in the envelope. These data revealed jasmonate-induced activation of the hydroperoxide lyase and reductase branch within the lipoxygenase pathway and suggest differential activity of the three 13-lipoxygenases under different stress conditions.

No abstract

Recently, we found a 13-lipoxygenase in germinating cucumber cotyledons, which was located at the lipid body membrane. Based on its products formed mobilization of storage lipids seems to be initiated by this 13-lipoxygenase. For biochemical characterization its cDNA was expressed as Histagged protein. Active recombinant enzyme was obtained from low temperature cultivation of E. coli after affinity purification. It (i) exhibited an unchanged region specificity, and (ii) showed a pH optimum of 7.2 against trilinolein as substrate. We compared its ability to oxygenate trilinolein with the one of another 13-lipoxygenase, soybean lipoxygenase-1. At the pH optimum of soybean lipoxygenase-1 (9.0), trilinolein was oxygenated only to 28% of the amount converted by the lipid body lipoxygenase. Moreover, trilinolein oxygenation by soybean lipoxygenase-1 leads mainly to monohydroperoxy derivatives, whereas oxygenation by lipid body LOX leads to a trihydroperoxy derivative.z 1998 Federation of European Biochemical Societies.

Olive oil is one of the oldest known vegetable oils, and it is almost unique in that it can be consumed without any refining treatment. One of its most important quality problems is oxidative rancidity due to the oxygenation of polyenoic fatty acids and formation of compounds that derive from these fatty acid hydroperoxides. Beside autoxidation, lipoxygenases (LOXs) were suggested to be involved in this process. Here we show, that approximately 1.6% of all linoleic acid (LA) molecules within olive oil samples had been converted into LOX‐derived (13S,9Z,11E)‐13‐hydroperoxy‐9,11‐octadecadienoic acid (13‐HPODE) as determined by 1H NMR‐ and HPLC analysis. LOX activity tests indicated the occurrence of an active 13‐LOX exhibiting a pH optimum between pH 5.5 and 6.0. Furthermore, this enzyme preferentially metabolized free fatty acids. In order to elucidate the origin of this LOX, we analyzed olive endosperms for LOX forms. Chromatography of total protein extracts of the tissue showed LOX activity almost exclusively associated with a high molecular mass fraction. Light microscopic inspection, as well as the calculated phosphate, neutral lipid, and protein content of this fraction, suggested that this fraction may contain oil bodies and that LOX activity was associated with their membrane. This LOX activity had a pH optimum of 6.0. Activity assays at various temperatures indicated a significant catalytic efficiency of the enzyme up to 55°C. HPLC analysis of LA oxygenation products within the lipid fraction and of activity tests of isolated oil bodies showed that the LOX present in mature olive endosperm oil bodies was, as the enzyme from olive oil, a linoleate 13‐LOX preferentially active on free LA. We suggest, that this oil body LOX from olive endosperm, is the one detected originally in olive oil and may survive at least in part olive oil production.