Lipid Body Lipoxygenase Research Articles

(168) Phospholipase Dα and Lipoxygenase Gene Expression in Fruit, Floral, and Vegetative Tissues of `Honey Brew' Hybrid Honeydew Melon

Increases in phospholipase D (PLD) and lipoxygenase (LOX) activities are thought to play a key role in senescence of mesocarp tissues in muskmelon fruit. We have cloned and characterized two full-length cDNAs, CmPLDα and CmLOX1, encoding PLDα and LOX proteins in honeydew melon (Cucumis melo L. Inodorus Group). Levels of expression of the corresponding genes were determined by semi-quantitative RT-PCR in developing and mature fruit mesocarp tissues (20–60 d after pollination; DAP), and in roots, leaves, and stems from 4-week-old and flowers from 6-week-old plants. The coding regions of CmPLDα1 and CmLOX1 cDNAs are, respectively, 2427 and 2634 nucleotides long, encoding proteins 808 and 877 amino acids in length. CmPLDα1 is most similar to PLDα genes in castor bean, cowpea, strawberry, and tomato (77% nucleotide identity), and is the first cucurbit PLD gene cloned. CmLOX1 has 94% nucleotide identity to a cucumber LOX gene expressed in roots and 80% identity to cucumber cotyledon lipid body LOX. Transcript of CmPLDα1 was much more abundant than that of CmLOX1, but relative levels of transcript in the various organs and tissues were similar for the two genes. Expression was highest in roots, flowers, and fruit mesocarp tissues. CmPLDα1 expression in fruit was high throughout development, although maximum levels occurred at 50 and 55 DAP, respectively, in middle and hypodermal mesocarp. CmLOX1 expression was generally higher in middle than in hypodermal mesocarp with maximum transcript levels at 55 and 50 DAP, respectively. Overall, the patterns of expression of CmPLDα1 and CmLOX1 are consistent with a model in which their encoded enzymes act in tandem to promote or accelerate senescence in fruit mesocarp tissues.

Lipoxygenase-mediated metabolism of storage lipids in germinating sunflower cotyledons and ?-oxidation of (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid by the cotyledonary glyoxysomes

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.

Metabolic profiling of oxylipins in germinating cucumber seedlings--lipoxygenase-dependent degradation of triacylglycerols and biosynthesis of volatile aldehydes.

A particular isoform of lipoxygenase (LOX) localized on lipid bodies was shown by earlier investigations to play a role in initiating the mobilization of triacylglycerols during seed germination. Here, further physiological functions of LOXs within whole cotyledons of cucumber (Cucumis sativus L.) were analyzed by measuring the endogenous amounts of LOX-derived products. The lipid-body LOX-derived esterified (13 S)-hydroperoxy linoleic acid was the dominant metabolite of the LOX pathway in this tissue. It accumulated to about 14 micromol/g fresh weight, which represented about 6% of the total amount of linoleic acid in cotyledons. This LOX product was not only reduced to its hydroxy derivative, leading to degradation by beta-oxidation, but alternatively it was metabolized by fatty acid hydroperoxide lyase leading to formation of hexanal as well. Furthermore, the activities of LOX forms metabolizing linolenic acid were detected by measuring the accumulation of volatile aldehydes and the allene oxide synthase-derived metabolite jasmonic acid. The first evidence is presented for an involvement of a lipid-body LOX form in the production of volatile aldehydes.

The N-terminal beta-barrel structure of lipid body lipoxygenase mediates its binding to liposomes and lipid bodies.

Phospholipase A2 and a particular isoform of lipoxygenase are synthesized and transferred to lipid bodies during the stage of triacylglycerol mobilization in germinating cucumber seedlings. Lipid body lipoxygenase (LBLOX) is post-translationally transported to lipid bodies without proteolytic modification. Fractionation of homogenates from cucumber cotyledons or transgenic tobacco leaves expressing LBLOX showed that a small but significant amount was detectable in the microsomal fraction. A beta-barrel-forming N-terminal domain in the structure of LBLOX, as deduced from sequence data, was shown to be crucial for selective intracellular transport from the cytosol to lipid bodies. Although a specific signal sequence for targeting protein domains to the lipid bodies could not be established, it was evident that the beta-barrel represents a membrane-binding domain that is functionally comparable with the C2 domains of mammalian phospholipases. The intact beta-barrel of LBLOX was demonstrated to be sufficient to target in vitro a fusion protein of LBLOX beta-barrel with glutathione S-transferase (GST) to lipid bodies. In addition, binding experiments on liposomes using lipoxygenase isoforms, LBLOX deletions and the GST-fusion protein confirmed the role of the beta-barrel as the membrane-targeting domain. In this respect, the cucumber LBLOX differs from cytosolic isoforms in cucumber and from the soybean LOX-1. When the beta-barrel of LBLOX was destroyed by insertion of an additional peptide sequence, its ability to target proteins to membranes was abolished.

Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis.

Multiple lipoxygenase sequence alignments and structural modeling of the enzyme/substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L+H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and/or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.

All three acyl moieties of trilinolein are efficiently oxygenated by recombinant His-tagged lipid body lipoxygenase in vitro

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 His-tagged 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.

Structural Elucidation of Oxygenated Storage Lipids in Cucumber Cotyledons: IMPLICATION OF LIPID BODY LIPOXYGENASE IN LIPID MOBILIZATION DURING GERMINATION

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.

Lipid body lipoxygenase characterized by protein fragmentation, cDNA sequence and very early expression of the enzyme during germination of cucumber seeds.

Lipid bodies are cellular compartments containing triacylglycerols. They are encompassed by a phospholipid monolayer and decorated with characteristic proteins. In plants, lipid bodies are synthesized during seed formation but acquire new proteins during seed germination. In germinating cucumber (Cucumis sativus) seeds, the set of newly synthesized proteins appearing in the lipid bodies at the early stage of triacylglycerol mobilization comprises a special form of lipoxygenase. We isolated the lipid body lipoxygenase and characterized fragments prepared by limited proteolysis and cleavage with cyanogen bromide. A very early expression of lipid body lipoxygenase was found by studying the rate of de novo synthesis of lipoxygenase forms during germination. This allowed a clear distinction of this enzyme from other lipoxygenase isoforms. Hence, for determining the molecular structure of lipid body lipoxygenase we analyzed a cDNA prepared from mRNA of cotyledons at day 1 of germination. From the cDNA sequence, oligonucleotides were derived that specifically detected lipid body lipoxygenase mRNA on northern blots. The very early expression of lipid body lipoxygenase was corroborated by this approach. Good agreement was observed between the amino acid sequence deduced from the cDNA sequence and the peptide structures analyzed biochemically. In particular, the cleavage products of cyanogen bromide treatment indicated that we had isolated the lipid body lipoxygenase cDNA. The sequence data show a lipoxygenase form characterized by a molecular mass of 99655 Da, which is significantly higher than the molecular masses of the cytosolic forms. Compared to the cytosolic forms that exhibit a molecular mass of 95 kDa, the lipid body form has an N-terminal extension of 34 amino acid residues. No evidence for a cotranslational or post-translational proteolytic processing was obtained by the size comparison of the in vitro-translated lipoxygenase and the lipid body form.

Lipid-body lipoxygenase is expressed in cotyledons during germination prior to other lipoxygenase forms

Lipid bodies are degraded during germination. Whereas some proteins, e.g. oleosins, are synthesized during the formation of lipid bodies of maturating seeds, a new set of proteins, including a specific form of lipoxygenase (LOX; EC 1.13.11.12), is detectable in lipid bodies during the stage of fat degradation in seed germination. In cotyledons of cucumber (Cucumis sativus L.) seedlings at day 4 of germination, the most conspicuous staining with anti-LOX antibodies was observed in the cytosol. At very early stages of germination, however, the LOX form present in large amounts and synthesized preferentially was the lipid-body LOX. This was demonstrated by immunocytochemical staining of cotyledons from 1-h and 24-h-old seedlings: the immunodecoration of sections of 24-h-old seedlings with anti-LOX antiserum showed label exclusively correlated with lipid bodies of around 3 μm in diameter. In accordance, the profile of LOX protein isolated from lipid bodies during various stages of germination showed a maximum at day 1. By measuring biosynthesis of the protein in vivo we demonstrated that the highest rates of synthesis of lipid-body LOX occurred at day 1 of germination. The early and selective appearance of a LOX form associated with lipid bodies at this stage of development is discussed.

The lipid body lipoxygenase from cucumber seedlings exhibits unusual reaction specificity

The lipid body lipoxygenase of cucumber seedlings is at a high level expressed during the germinating process which is the stage of triglyceride mobilisation. This enzyme exhibits an unusual positional specificity which has not been described so far for any plant and animal lipoxygenase. The purified enzyme converts arachidonic acid to 15-S-hydroperoxy-5Z,8Z,11Z, 13E-eicosatetraenoic acid (15S-HPETE), 12-S-hydroperoxy-5Z, 8Z, 10E, 14Z-eicosatetraenoic acid (12S-HPETE), and 8-S-hydroperoxy-5Z,9E,11Z,14Z-eicosatetraenoic acid (8S-HPETE) in a ratio of 76:4:20 with the corresponding R-isomers being only minor contaminants. Binding to the lipid bodies enhances the arachidonic acid dioxygenase activity more than 4-times and alters positional specificity of the enzyme in favour of 8-S-hydroperoxy-5Z,9E,11Z,14Z-eicosatetraenoic acid (8S-HPETE) formation.

Particulate and soluble lipoxygenase isoenzymes

Cucumber (Cucumis sativus L.) cotyledons, a plant model system for studying changes in metabolic compartmentation, contain at least six forms of lipoxygenase. The intracellular location and organellar topology of lipoxygenase forms in lipid bodies, microsomes, and cytosol were investigated. A protocol was worked out to solubilize and prepare lipid-body lipoxygenase in an enzymatically active form. The methodology required for the solubilization of the lipid-body form differed from the procedure applicable for solubilization of two lipoxygenase forms from the microsomal membranes. Three cytosolic lipoxygenases were purified and found to be distinguishable from each other in size and charge. Further characterization and differentiation of all cellular lipoxygenase isoforms was achieved by comparison of the enzymatic properties. Marked differences in pH optima of the particle-bound lipoxygenases were found: optimal pH of 8.5 for lipid-body lipoxygenase and pH 5.5 for the microsomal lipoxygenases. In addition, analysis of the products formed showed that the catalytic properties of lipidbody lipoxygenase and microsomal lipoxygenase are clearly distinguishable from each other and from the soluble forms.

A lipoxygenase is the main lipid body protein in cucumber and soybean cotyledons during the stage of triglyceride mobilization

The 90-kDa lipid body protein characterized earlier by its high expression during the stage of fat degradation was identified as a form of lipoxygenase. This organelle form was compared with lipoxygenase species purified from the cytosol. It is further shown that the antibodies raised against the lipid body membrane lipoxygenase from cucumber cotyledons cross-react with both cytosolic and lipid body lipoxygenase from soybean.