Differential induction of oxylipin pathway in potato and tobacco cells by bacterial and oomycete elicitors

Key message

Potato and tobacco cells are differentially suited to study oxylipin pathway and elicitor-induced responses.

Abstract

Synthesis of oxylipins via the lipoxygenase (LOX) pathway provides plant cells with an important class of signaling molecules, related to plant stress responses and innate immunity. The aim of this study was to evaluate the induction of LOX pathway in tobacco and potato cells induced by a concentrated culture filtrate (CCF) from Phytophthora infestans and lipopolysaccharide (LPS) from Pectobacterium atrosepticum. Oxylipin activation was evaluated by the measurement of LOX activity and metabolite quantification. The basal levels of oxylipins and fatty acids showed that potato cells contained higher amounts of linoleic (LA), linolenic (LnA) and stearic acids than tobacco cells. The major oxylipin in potato cells, 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid (9,10,11-THOD), was not detected in tobacco cells. CCF induced a sharp increase of LA and LnA at 8 h in tobacco cells. In contrast they decreased in potato cells. In CCF-treated tobacco cells, colneleic acid increased up to 24 h, colnelenic acid and 9(S)-hydroxyoctadecatrienoic acid (9(S)-HOT) increased up to 16 h. In potato cells, only colneleic acid increased slightly until 16 h. A differential induction of LOX activity was measured in both cells treated by CCF. With LPS treatment, only 9,10,11-THOD accumulation was significantly induced at 16 h in potato cells. Fatty acids were constant in tobacco but decreased in potato cells over the studied time period. These results showed that the two elicitors were differently perceived by the two Solanaceae and that oxylipin pathway is strongly induced in tobacco with the CCF. They also revealed that elicitor-induced responses depended on both cell culture and elicitor.     

Oxylipin profiling in pathogen-infected potato leaves

Plants respond to pathogen attack with a multicomponent defense response. Synthesis of oxylipins via the lipoxygenase (LOX) pathway appears to be an important factor for establishment of resistance in a number of pathosystems. In potato cells, pathogen-derived elicitors preferentially stimulate the 9-LOX-dependent metabolism of polyunsaturated fatty acids (PUFAs). Here we show by oxylipin profiling that potato plants react to pathogen infection with increases in the amounts of the 9-LOX-derived 9,10,11- and 9,12,13-trihydroxy derivatives of linolenic acid (LnA), the divinyl ethers colnelenic acid (CnA) and colneleic acid (CA) as well as 9-hydroxy linolenic acid. Accumulation of these compounds is faster and more pronounced during the interaction of potato with the phytopathogenic bacterium Pseudomonas syringae pv. maculicola, which does not lead to disease, compared to the infection of potato with Phytophthora infestans, the causal agent of late blight disease. Jasmonic acid (JA), a 13-LOX-derived oxylipin, accumulates in potato leaves after infiltration with P. syringae pv. maculicola, but not after infection with P. infestans.     

Overexpression of hydroperoxide lyase,peroxygenase and epoxide hydrolase in tobacco for the biotechnological production of flavours and polymer precursors

Plants produce short‐chain aldehydes and hydroxy fatty acids, which are important industrial materials, through the lipoxygenase pathway. Based on the information that lipoxygenase activity is up‐regulated in tobacco leaves upon infection with tobacco mosaic virus (TMV), we introduced a melon hydroperoxide lyase (CmHPL) gene, a tomato peroxygenase (SlPXG) gene and a potato epoxide hydrolase (StEH) into tobacco leaves using a TMV‐based viral vector system to afford aldehyde and hydroxy fatty acid production. Ten days after infiltration, tobacco leaves infiltrated with CmHPL displayed high enzyme activities of 9‐LOX and 9‐HPL, which could efficiently transform linoleic acid into C₉ aldehydes. Protein extracts prepared from 1 g of CmHPL‐infiltrated tobacco leaves (fresh weight) in combination with protein extracts prepared from 1 g of control vector‐infiltrated tobacco leaves (as an additional 9‐LOX source) produced 758 ± 75 μg total C₉ aldehydes in 30 min. The yield of C₉ aldehydes from linoleic acid was 60%. Besides, leaves infiltrated with SlPXG and StEH showed considerable enzyme activities of 9‐LOX/PXG and 9‐LOX/EH, respectively, enabling the production of 9,12,13‐trihydroxy‐10(E)‐octadecenoic acid from linoleic acid. Protein extracts prepared from 1 g of SlPXG‐infiltrated tobacco leaves (fresh weight) in combination with protein extracts prepared from 1 g of StEH‐infiltrated tobacco leaves produced 1738 ± 27 μg total 9,12,13‐trihydroxy‐10(E)‐octadecenoic acid isomers in 30 min. The yield of trihydroxyoctadecenoic acids from linoleic acid was 58%. C₉ aldehydes and trihydroxy fatty acids could likely be produced on a larger scale using this expression system with many advantages including easy handling, time‐saving and low production cost.     

Wounding stimulates the accumulation of glycerolipids containing oxophytodienoic acid and dinor-oxophytodienoic acid in Arabidopsis leaves

Although oxylipins can be synthesized from free fatty acids, recent evidence suggests that oxylipins are components of plastid-localized polar complex lipids in Arabidopsis (Arabidopsis thaliana). Using a combination of electrospray ionization (ESI) collisionally induced dissociation time-of-flight mass spectrometry (MS) to identify acyl chains, ESI triple-quadrupole (Q) MS in the precursor mode to identify the nominal masses of complex polar lipids containing each acyl chain, and ESI Q-time-of-flight MS to confirm the identifications of the complex polar lipid species, 17 species of oxylipin-containing phosphatidylglycerols, monogalactosyldiacylglycerols (MGDG), and digalactosyldiacylglycerols (DGDG) were identified. The oxylipins of these polar complex lipid species include oxophytodienoic acid (OPDA), dinor-OPDA (dnOPDA), 18-carbon ketol acids, and 16-carbon ketol acids. Using ESI triple-Q MS in the precursor mode, the accumulation of five OPDA- and/or dnOPDA-containing MGDG and two OPDA-containing DGDG species were monitored as a function of time in mechanically wounded leaves. In unwounded leaves, the levels of these oxylipin-containing complex lipid species were low, between 0.001 and 0.023 nmol/mg dry weight. However, within the first 15 min after wounding, the levels of OPDA-dnOPDA MGDG, OPDA-OPDA MGDG, and OPDA-OPDA DGDG, each containing two oxylipin chains, increased 200- to 1,000-fold. In contrast, levels of OPDA-hexadecatrienoic acid MGDG, linolenic acid (18:3)-dnOPDA MGDG, OPDA-18:3 MGDG, and OPDA-18:3 DGDG, each containing a single oxylipin chain, rose 2- to 9-fold. The rapid accumulation of high levels of galactolipid species containing OPDA-OPDA and OPDA-dnOPDA in wounded leaves is consistent with these lipids being the primary products of plastidic oxylipin biosynthesis.     

Divinyl ether fatty acid synthesis in late blight-diseased potato leaves

We conducted a study of the patterns and dynamics of oxidized fatty acid derivatives (oxylipins) in potato leaves infected with the late-blight pathogen Phytophthora infestans. Two 18-carbon divinyl ether fatty acids, colneleic acid and colnelenic acid, accumulated during disease development. To date, there are no reports that such compounds have been detected in higher plants. The divinyl ether fatty acids accumulate more rapidly in potato cultivar Matilda (a cultivar with increased resistance to late blight) than in cultivar Bintje, a susceptible cultivar. Colnelenic acid reached levels of up to approximately 24 nmol (7 microgram) per g fresh weight of tissue in infected leaves. By contrast, levels of members of the jasmonic acid family did not change significantly during pathogenesis. The divinyl ethers also accumulated during the incompatible interaction of tobacco with tobacco mosaic virus. Colneleic and colnelenic acids were found to be inhibitory to P. infestans, suggesting a function in plant defense for divinyl ethers, which are unstable compounds rarely encountered in biological systems.     

Steric analysis of 8-hydroxy- and 10-hydroxyoctadecadienoic acids and dihydroxyoctadecadienoic acids formed from 8R-hydroperoxyoctadecadienoic acid by hydroperoxide isomerases

8-Hydroxyoctadeca-9Z,12Z-dienoic acid (8-HODE) and 10-hydroxyoctadeca-8E,12Z-octadecadienoic acid (10-HODE) are produced by fungi, e.g., 8R-HODE by Gaeumannomyces graminis (take-all of wheat) and Aspergillus nidulans, 10S-HODE by Lentinula edodes, and 10R-HODE by Epichloe typhina. Racemic [8-(2)H]8-HODE and [10-(2)H]10-HODE were prepared by oxidation of 8- and 10-HODE to keto fatty acids by Dess-Martin periodinane followed by reduction to hydroxy fatty acids with NaB(2)H(4). The hydroxy fatty acids were analyzed by chiral phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with 8R-HODE and 10S-HODE as standards. 8R-HODE eluted after 8S-HODE on silica with cellulose tribenzoate (Chiralcel OB-H), and 10S-HODE eluted before 10R-HODE on silica with an aromatic chiral selector (Reprosil Chiral-NR). 5S,8R-Dihydroxyoctadeca-9Z,12Z-dienoic acid (5S,8R-DiHODE) is formed from 18:2n-6 by A. nidulans and 8R,11S-dihydroxyoctadeca-9Z,12Z-dienoic acid (8R,11S-DiHODE) by Agaricus bisporus. 8R-Hydroperoxylinoleic acid (8R-HPODE) can be transformed to 5S,8R-DiHODE and 8R,11-DiHODE by Aspergillus spp., and 8R,13-dihydroxy-9Z,11E-dienoic acid (8R,13-DiHODE) can also be detected. We prepared racemic [5,8-(2)H(2)]5,8- and [8,11-(2)H(2)]8,11-DiHODE by oxidation and reduction as above and 8R,13S- and 8R,13R-DiHODE by oxidation of 8R-HODE by S and R lipoxygenases. The diastereoisomers were separated and identified by normal phase HPLC-MS/MS analysis. We used the methods for steric analysis of fungal oxylipins. Aspergillus spp. produced 8R-HODE (>95% R), 10R-HODE (>70% R), and 5S,8R- and 8R,11S-DiHODE with high stereoselectivity (>95%), whereas 8R,13-DiHODE was likely formed by nonenzymatic hydrolysis of 8R,11S-DiHODE.     

alpha-oxidation of fatty acids in higher plants. Identification of a pathogen-inducible oxygenase (piox) as an alpha-dioxygenase and biosynthesis of 2-hydroperoxylinolenic acid.

A pathogen-inducible oxygenase in tobacco leaves and a homologous enzyme from Arabidopsis were recently characterized (Sanz, A., Moreno, J. I., and Castresana, C. (1998) Plant Cell 10, 1523-1537). Linolenic acid incubated at 23 degrees C with preparations containing the recombinant enzymes underwent alpha-oxidation with the formation of a chain-shortened aldehyde, i.e., 8(Z),11(Z), 14(Z)-heptadecatrienal (83%), an alpha-hydroxy acid, 2(R)-hydroxy-9(Z),12(Z),15(Z)-octadecatrienoic acid (15%), and a chain-shortened fatty acid, 8(Z),11(Z),14(Z)-heptadecatrienoic acid (2%). When incubations were performed at 0 degrees C, 2(R)-hydroperoxy-9(Z),12(Z),15(Z)-octadecatrienoic acid was obtained as the main product. An intermediary role of 2(R)-hydroperoxy-9(Z), 12(Z),15(Z)-octadecatrienoic acid in alpha-oxidation was demonstrated by re-incubation experiments, in which the hydroperoxide was converted into the same alpha-oxidation products as those formed from linolenic acid. 2(R)-Hydroperoxy-9(Z),12(Z), 15(Z)-octadecatrienoic acid was chemically unstable and had a half-life time in buffer of about 30 min at 23 degrees C. Extracts of cells expressing the recombinant oxygenases accelerated breakdown of the hydroperoxide (half-life time, about 3 min at 23 degrees C), however, this was not attributable to the recombinant enzymes since the same rate of hydroperoxide degradation was observed in the presence of control cells not expressing the enzymes. No significant discrimination between enantiomers was observed in the degradation of 2(R,S)-hydroperoxy-9(Z)-octadecenoic acid in the presence of recombinant oxygenases. A previously studied system for alpha-oxidation in cucumber was re-examined using the newly developed techniques and was found to catalyze the same conversions as those observed with the recombinant enzymes, i.e. enzymatic alpha-dioxygenation of fatty acids into 2(R)-hydroperoxides and a first order, non-stereoselective degradation of hydroperoxides into alpha-oxidation products. It was concluded that the recombinant enzymes from tobacco and Arabidopsis were both alpha-dioxygenases, and that members of this new class of enzymes catalyze the first step of alpha-oxidation in plant tissue.     

Synthesis, structural identification and biological activity of 11,12-dihydroxyeicosatetraenoic acids formed in human platelets

An enantiospecific route for the synthesis of 11,12-dihydroxyeicosatetraenoic acids was developed and used to synthesize 11,12-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acids. The 11,12-DHETEs were synthesized with the stereochemistry of the hydroxyl group being 11(R),12(S) and 11(S),12(S). The synthetic compounds were used to elucidate the structure of 11,12-DHETEs formed in human platelets by comparison of the chromatographic retention time in HPLC and GC as well as their ion fragmentation pattern in GC-MS. The major 11,12-DHETE formed in human platelets was found to be identical with 11(R),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid. Two more compounds were tentatively identified as 11(S),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid and 11,12-dihydroxy-5(E),7(E),9(E),14(Z)-eicosatetraenoic acid. Furthermore, the 11(S),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid was found to possess biological activity on neutrophil functional responses. However, the major compound, 11(R),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid, formed in platelets lacks biological activity in the test systems used. The present data further support that 11,12-dihydroxy-eicosatetraenoic acids are formed in human platelets via a leukotriene like mechanism presumably by the 12-lipoxygenase. Furthermore, the biological effects of one of the compounds showed a unique activity profile compared to other lipoxygenase products.     

Lipid hydroperoxide levels in plant tissues

Hydroperoxides are the primary oxygenated products of polyunsaturated fatty acids and are key intermediates in the octadecanoid signalling pathway in plants. Lipid hydroperoxides (LHPO) were determined spectrophotometrically based on their reaction with an excess of Fe(2+)at low pH in the presence of the dye xylenol orange. Triphenylphosphine-mediated hydroxide formation was used to authenticate the signal generated by the hydroperoxides. The method readily detected lipid peroxidation in Phaseolus: microsomes, senescing potato leaves and in a range of other plant tissues including Phaseolus hypocotyls (26+/-5 nmol g(-1) FW), Alstroemeria floral tissues (sepals 66+/-13 nmol g(-1) FW petals 49+/-6 nmol g(-1) FW), potato leaves (334+/-75 nmol g(-1) FW), broccoli florets (568+/-68 nmol g(-1) FW) and Chlamydomonas cells (602+/-40 nmol g(-1) FW). Relative to the total fatty acid content of the tissues, the % LHPO was within the range of 0.6-1.7% for all tissue types (photosynthetic and non-photosynthetic) and represents the basal oxidation level of membrane fatty acids in plant cells. In order to relate the levels of LHPO to specific signalling pathways, transgenic potato plant lines were used in which lipoxygenase (LOX) (responsible for hydroperoxide biosynthesis) and hydroperoxide lyase (a route of hydroperoxide degradation) activities were largely reduced by an antisense-mediated approach. While the LHPO levels were similar to wild type in the individual LOX antisensed plants, basal LHPO levels, by contrast, were elevated by 38% in transgenic potato leaves antisensed in hydroperoxide lyase, indicating a role for this enzyme in the maintenance of cellular levels of LHPOs.     

Organ-dependent oxylipin signature in leaves and roots of salinized tomato plants (Solanum lycopersicum)

Oxylipins have been extensively studied in plant defense mechanisms or as signal molecules. Depending on the stress origin (e.g. wounding, insect, pathogen), and also on the plant species or organ, a specific oxylipin signature can be generated. Salt stress is frequently associated with secondary stress such as oxidative damage. Little is known about the damage caused to lipids under salt stress conditions, especially with respect to oxylipins. In order to determine if an organ-specific oxylipin signature could be observed during salt stress, tomato (Solanum lycopersicum cv. Money Maker) plants were submitted to salt stress (100 mM of NaCl) for a 30-d period. A complete oxylipin profiling and LOX related-gene expression measurement were achieved in leaves and roots. As expected, salt stress provoked premature senescence in leaves, as revealed by a decrease in photosystem II efficiency (F(v)/F(m) ratio) and sodium accumulation in leaves. In roots, a significant decrease in several oxylipins (9- and 13-hydro(pero)xy linole(n)ic acids, keto and divinyl ether derivatives) was initiated at day 5 and intensified at day 21 after salt treatment, whereas jasmonic acid content increased. In leaves, the main changes in oxylipins were observed later (at day 30), with an increase in some 9- and 13-hydro(pero)xy linole(n)ic acids and a decrease in some keto-derivatives and in jasmonic acid. Oxylipin enantiomeric characterization revealed that almost all compounds were formed enzymatically, and therefore a massive auto-oxidation of lipids that can be encountered in abscission processes can be excluded here.     

Analysis of the subcellular localisation of lipoxygenase in legume and actinorhizal nodules

Plant lipoxygenases (LOXs; EC 1.13.11.12) catalyse the oxygenation of polyunsaturated fatty acids, linoleic (18:2) and α-linolenic acid (18:3(n-3)) and are involved in processes such as stress responses and development. Depending on the regio-specificity of a LOX, the incorporation of molecular oxygen leads to formation of 9- or 13-fatty acid hydroperoxides, which are used by LOX itself as well as by members of at least six different enzyme families to form a series of biologically active molecules, collectively called oxylipins. The best characterised oxylipins are the jasmonates: jasmonic acid (JA) and its isoleucine conjugate that are signalling compounds in vegetative and propagative plant development. In several types of nitrogen-fixing root nodules, LOX expression and/or activity is induced during nodule development. Allene oxide cyclase (AOC), a committed enzyme of the JA biosynthetic pathway, has been shown to localise to plastids of nodules of one legume and two actinorhizal plants, Medicago truncatula, Datisca glomerata and Casuarina glauca, respectively. Using an antibody that recognises several types of LOX interspecifically, LOX protein levels were compared in roots and nodules of these plants, showing no significant differences and no obvious nodule-specific isoforms. A comparison of the cell-specific localisation of LOXs and AOC led to the conclusion that (i) only cytosolic LOXs were detected although it is generally assumed that the (13S)-hydroperoxy α-linolenic acid for JA biosynthesis is produced in the plastids, and (ii) in cells of the nodule vascular tissue that contain AOC, no LOX protein could be detected.     

A multifunctional lipoxygenase with fatty acid hydroperoxide cleaving activity from the moss Physcomitrella patens

A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C(18)-C(22) fatty acids giving rise to even more LOX-derived products. In contrast to other lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.     

A 49-kDa mini-lipoxygenase from Anabaena sp. PCC 7120 retains catalytically complete functionality

Anabaena sp. PCC 7120 is one of the few prokaryotes harboring a lipoxygenase (LOX) gene. The sequence resides in an open reading frame encoding a fusion protein of a catalase-like hemoprotein with an unusually short LOX (approximately 49 kDa) at the C terminus. The recombinant mini-LOX contains a non-heme iron in the active site and is highly active with linoleic and alpha-linolenic acids (which occur naturally in Anabaena) giving the respective 9R-hydroperoxides, the mirror image of the 9S-LOX products of plants. Using stereospecifically labeled [11-(3)H]linoleic acids we show that reaction is catalyzed via a typical antarafacial relationship of initial hydrogen abstraction and oxygenation. The mini-LOX oxygenated C16/C18:2-phosphatidylcholine with 9R specificity, suggesting a "tail first" mode of fatty acid binding. Site-directed mutagenesis of an active site Ala (Ala215), typically conserved as Gly in R-LOX, revealed that substitution with Gly retained 9R specificity, whereas the larger Val substitution switched oxygenation to 13S, implying that Ala215 represents the functional equivalent of the Gly in other R-LOX. Metabolism studies using a synthetic fatty acid with extended double bond conjugation, 9E,11Z,14Z-20:3omega6, showed that the mini-LOX can control oxygenation two positions further along the fatty acid carbon chain. We conclude that the mini-LOX, despite lacking the beta-barrel domain and much additional sequence, is catalytically complete. Interestingly, animal and plant LOX, which undoubtedly share a common ancestor, are related in sequence only in the catalytic domain; it is possible that the prokaryotic LOX represents a common link and that the beta-barrel domain was then acquired independently in the animal and plant kingdoms.     

Identification of oxylipins with antifungal activity by LC–MS/MS from the supernatant of Pseudomonas 42A2

In microorganisms hydroxy fatty acids are produced from the biotransformation of unsaturated fatty acids. Such compounds belong to a class of oxylipins which are reported to perform a variety of biological functions such as anti-inflammatory or cytotoxic activity. These compounds have been found in rice and timothy plants after being infected by specific fungus. When grown in submerged culture with linoleic acid, Pseudomonas 42A2 accumulated in the supernatant several hydroxy fatty acids. In this work LC–MS/MS has been used to elucidate the structure of the components form the organic extract: 9-hydroxy-10,12-octadecadienoic acid; 13-hydroxy-9,11-octadecadienoic acid; 7,10-dihydroxy-8E-octadecenoic acid; 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid. Antimicrobial activity against several pathogenic fungal strains is presented: MIC (μg/mL) Verticillium dhaliae, 32; Macrophonia phaesolina, 32; Arthroderma uncinatum, 32; Trycophyton mentagrophytes, 64.     

Oxylipin profiling of the hypersensitive response in Arabidopsis thaliana. Formation of a novel oxo-phytodienoic acid-containing galactolipid, arabidopside E

Oxidation products of unsaturated fatty acids, collectively known as oxylipins, function as signaling molecules in plants during development, wounding, and insect and pathogen attack. Certain oxylipins are also known to have direct cytotoxic effects on pathogens. We used inducible expression of bacterial avirulence proteins in planta to study the involvement of oxylipins in race-specific defense against bacterial pathogens. We demonstrate that recognition of the Pseudomonas syringae avirulence protein AvrRpm1 induces 9- and 13-lipoxygenase-dependent oxylipin synthesis in Arabidopsis thaliana. The major oxylipins accumulated were jasmonic acid, 12-oxo-phytodienoic acid, and dinor-oxo-phytodienoic acid. The majority of the newly formed oxylipins (>90%) was found to be esterified to glycerolipids, whereby 12-oxo-phytodienoic acid and dinor-oxo-phytodienoic acid were found to be esterified to a novel galactolipid. The structure of the substance was determined as a monogalactosyldiacylglycerol containing two 12-oxo-phytodienoic acids and one dinor-oxo-phytodienoic acid acyl chain and was given the trivial name arabidopside E. This substance accumulated to surprisingly high levels, 7-8% of total lipid content, and was shown to inhibit growth of a bacterial pathogen in vitro. Arabidopside E was formed also after recognition of the avirulence protein AvrRpt2, suggesting that this could be a conserved feature of defense reactions against bacterial pathogens. In conclusion, the data presented suggest a role of enzymatically formed oxylipins, especially the octadecanoids and arabidopside E in race-specific resistance against bacterial pathogens.     

灵芝液态深层发酵产物中10种不饱和脂肪酸类化合物的鉴定

通过规模化液态深层发酵获得灵芝发酵产物,采用多种硅胶色谱柱层析及重结晶的方式,从中分离得到10个化合物.通过核磁、质谱等波谱分析,鉴定出这些化合物均属于含羟基或酮基的不饱和脂肪酸类化合物,分别为(9S,10R,11E,13R)-9,10,13-trihydroxyoctadec-11-enoic acid(1)和(9S...     

Peroxygenase-Catalyzed Fatty Acid Epoxidation in Cereal Seeds (Sequential Oxidation of Linoleic Acid into 9(S),12(S),13(S)-Trihydroxy-10(E)-Octadecenoic Acid)

Peroxygenase-catalyzed epoxidation of oleic acid in preparations of cereal seeds was investigated. The 105,000g particle fraction of oat (Avena sativa) seed homogenate showed high peroxygenase activity, i.e. 3034 [plus or minus] 288 and 2441 [plus or minus] 168 nmol (10 min)-1 mg-1 protein in two cultivars, whereas the corresponding fraction obtained from barley (Hordeum vulgare and Hordeum distichum), rye (Secale cereale), and wheat (Triticum aestivum) showed only weak activity, i.e. 13 to 138 nmol (10 min)-1 mg-1 protein. In subcellular fractions of oat seed homogenate, peroxygenase specific activity was highest in the 105,000g particle fraction, whereas lipoxygenase activity was more evenly distributed and highest in the 105,000g supernatant fraction. Incubation of [1-14C]linoleic acid with the 105,000g supernatant of oat seed homogenate led to the formation of several metabolites, i.e. in order of decreasing abundance, 9(S)-hydroxy-10(E),12(Z)-octadecadienoic acid, 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid, cis-9,10-epoxy-12(Z)-octadecenoic acid [mainly the 9(R),10(S) enantiomer], cis-12,13-epoxy-9(Z)-octadecenoic acid [mainly the 12(R),13(S) enantiomer], threo-12,13-dihydroxy-9(Z)-octadecenoic acid, and 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid. Incubation of linoleic acid with the 105,000g particle fraction gave a similar, but not identical, pattern of metabolites. Conversion of linoleic acid into 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid, a naturally occurring oxylipin with antifungal properties, took place by a pathway involving sequential catalysis by lipoxygenase, peroxygenase, and epoxide hydrolase.     

Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense against pathogens

Plant oxylipins are a large family of metabolites derived from polyunsaturated fatty acids. The characterization of mutants or transgenic plants affected in the biosynthesis or perception of oxylipins has recently emphasized the role of the so-called oxylipin pathway in plant defense against pests and pathogens. In this context, presumed functions of oxylipins include direct antimicrobial effect, stimulation of plant defense gene expression, and regulation of plant cell death. However, the precise contribution of individual oxylipins to plant defense remains essentially unknown. To get a better insight into the biological activities of oxylipins, in vitro growth inhibition assays were used to investigate the direct antimicrobial activities of 43 natural oxylipins against a set of 13 plant pathogenic microorganisms including bacteria, oomycetes, and fungi. This study showed unequivocally that most oxylipins are able to impair growth of some plant microbial pathogens, with only two out of 43 oxylipins being completely inactive against all the tested organisms, and 26 oxylipins showing inhibitory activity toward at least three different microbes. Six oxylipins strongly inhibited mycelial growth and spore germination of eukaryotic microbes, including compounds that had not previously been ascribed an antimicrobial activity, such as 13-keto-9(Z),11(E),15(Z)-octadecatrienoic acid and 12-oxo-10,15(Z)-phytodienoic acid. Interestingly, this first large-scale comparative assessment of the antimicrobial effects of oxylipins reveals that regulators of plant defense responses are also the most active oxylipins against eukaryotic microorganisms, suggesting that such oxylipins might contribute to plant defense through their effects both on the plant and on pathogens, possibly through related mechanisms.     

Hydroperoxide lyase cascade in pea seedlings: Non-volatile oxylipins and their age and stress dependent alterations

The profiles of non-volatile oxylipins of pea (Pisum sativum) seedlings were examined by gas chromatography-mass spectrometry after invitro incubation with α-linolenic acid. The 13-lipoxygenase/hydroperoxide lyase (HPL) products were predominant in the leaves, while the roots possess both 13- and 9-HPL products. Allene oxide synthase (AOS) and divinyl ether synthase (DES) products were not detected in the leaves or in the roots of any age. The HPL cascade produces a diversity of oxylipins, including the compounds (2E)-4-hydroxy-traumatic, (10E)-9,12-dihydroxy-10-dodecenoic and 9,12-dihydroxydodecanoic acids, as well as (2E)-4-hydroxy-2-nonenoic acid, which has not yet been detected in plants. Oxylipin patterns were altered by infection, water deficit, as well as by plant age. Infection caused the specific strong accumulation of azelaic (nonane-1,9-dioic) acid in the leaves. The azelaic acid content in the aged (14 and 18day-old) leaves was significantly higher than in the younger leaves. Water deficit induced the accumulation of (2E)-4-hydroxy-2-nonenoic acid and (2E)-traumatic acid in the roots. Results demonstrate that: (1) the HPL cascade is the predominant branch of the lipoxygenase pathway in pea seedlings; (2) the HPL products may have the regulatory role both in growth control and adaptation.