Lipid hydroperoxides in plants

Hydroperoxides are the primary oxygenated products of polyunsaturated fatty acids and were determined spectrophotometrically based on their reaction with an excess of Fe2+ 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 a range of plant tissues including Phaseolus hypocotyls (26 +/- 5 nmol.g of fresh weight(-1); mean +/- S.D.), Alstroemeria floral tissues (sepals, 66+/-13 nmol.g of fresh weight(-1); petals, 49+/-6 nmol.g of fresh weight(-1)), potato leaves (334+/-75 nmol.g of fresh weight(-1)), broccoli florets (568+/-68 nmol.g of fresh weight(-1)) and Chlamydomonas cells (602+/-40 nmol.g of wet weight(-1)). Relative to the total fatty acid content of the tissues, the percentage hydroperoxide content 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. Leaves of transgenic potato with the fatty acid hydroperoxide lyase enzyme expressed in the antisense orientation were elevated by 38%, indicating a role for this enzyme in the maintenance of cellular levels of lipid hydroperoxides.     

Root uptake, transport, and metabolism of externally applied glutathione in Phaseolus vulgaris seedlings

The most abundant thiol in beans (Phaseolus vulgaris L. cv. Saxa) is the tripeptide homoglutathione (hGSH) rather than glutathione (GSH). At the whole-plant level the GSH content is less than 0.5% of the hGSH content. In the present study GSH was supplied to the roots of bean seedlings to test whether GSH can be taken up by roots and transported to the shoot. Therefore, 12-day-old plants were exposed to 1 mmol/L GSH for 4, 8 and 24 h prior to harvest. In response to this GSH exposure, elevated GSH contents were found in all tissues. After 4 h the GSH content increased in the roots from 1 +/- 1 to 22 +/- 2 nmol GSH g(-1) fresh weight (FW), in the leaves from 2 +/- 1 to 9 +/- 4 nmol GSH g(-1) FW, and in the apex from 30 +/- 5 to 75 +/- 4 nmol GSH g(-1) FW. These data indicate that GSH is taken up by bean roots and is transported to above above-ground parts of the plants. Roots exposed to GSH for 24 h contained 2-fold higher cysteine (Cys) and hGSH contents than the controls. Apparently, GSH taken up by the roots is not only loaded into the xylem but also partially degraded and used for hGSH synthesis.     

Lipoxygenase H1 gene silencing reveals a specific role in supplying fatty acid hydroperoxides for aliphatic aldehyde production.

Lipoxygenases catalyze the formation of fatty acid hydroperoxide precursors of an array of compounds involved in the regulation of plant development and responses to stress. To elucidate the function of the potato 13-lipoxygenase H1 (LOX H1), we have generated transgenic potato plants with reduced expression of the LOX H1 gene as a consequence of co-suppression-mediated gene silencing. Three independent LOX H1-silenced transgenic lines were obtained, having less than 1% of the LOX H1 protein present in wild-type plants. This depletion of LOX H1 has no effect on the basal or wound-induced levels of jasmonates derived from 13-hydroperoxylinolenic acid. However, LOX H1 depletion results in a marked reduction in the production of volatile aliphatic C6 aldehydes. These compounds are involved in plant defense responses, acting as either signaling molecules for wound-induced gene expression or as antimicrobial substances. LOX H1 protein was localized to the chloroplast and the protein, expressed in Escherichia coli, showed activity toward unesterified linoleic and linolenic acids and plastidic phosphatidylglycerol. The results demonstrate that LOX H1 is a specific isoform involved in the generation of volatile defense and signaling compounds through the HPL branch of the octadecanoid pathway.     

Subcellular localisation of <Emphasis Type="Italic">Medicago truncatula</Emphasis>9/13-hydroperoxide lyase reveals a new localisation pattern and activation mechanism for CYP74C enzymes

Background  

Hydroperoxide lyase (HPL) is a key enzyme in plant oxylipin metabolism that catalyses the cleavage of polyunsaturated fatty acid hydroperoxides produced by the action of lipoxygenase (LOX) to volatile aldehydes and oxo acids. The synthesis of these volatile aldehydes is rapidly induced in plant tissues upon mechanical wounding and insect or pathogen attack. Together with their direct defence role towards different pathogens, these compounds are believed to play an important role in signalling within and between plants, and in the molecular cross-talk between plants and other organisms surrounding them. We have recently described the targeting of a seed 9-HPL to microsomes and putative lipid bodies and were interested to compare the localisation patterns of both a 13-HPL and a 9/13-HPL from Medicago truncatula, which were known to be expressed in leaves and roots, respectively.     

Differential distribution of the lipoxygenase pathway enzymes within potato chloroplasts

The lipoxygenase pathway is responsible for the production of oxylipins, which are important compounds for plant defence responses. Jasmonic acid, the final product of the allene oxide synthase/allene oxide cyclase branch of the pathway, regulates wound-induced gene expression. In contrast, C6 aliphatic aldehydes produced via an alternative branch catalysed by hydroperoxide lyase, are themselves toxic to pests and pathogens. Current evidence on the subcellular localization of the lipoxygenase pathway is conflicting, and the regulation of metabolic channelling between the two branches of the pathway is largely unknown. It is shown here that while a 13-lipoxygenase (LOX H3), allene oxide synthase and allene oxide cyclase proteins accumulate upon wounding in potato, a second 13-lipoxygenase (LOX H1) and hydroperoxide lyase are present at constant levels in both non-wounded and wounded tissues. Wound-induced accumulation of the jasmonic acid biosynthetic enzymes may thus commit the lipoxygenase pathway to jasmonic acid production in damaged plants. It is shown that all enzymes of the lipoxygenase pathway differentially localize within chloroplasts, and are largely found associated to thylakoid membranes. This differential localization is consistently observed using confocal microscopy of GFP-tagged proteins, chloroplast fractionation, and western blotting, and immunodetection by electron microscopy. While LOX H1 and LOX H3 are localized both in stroma and thylakoids, both allene oxide synthase and hydroperoxide lyase protein localize almost exclusively to thylakoids and are strongly bound to membranes. Allene oxide cyclase is weakly associated with the thylakoid membrane and is also detected in the stroma. Moreover, allene oxide synthase and hydroperoxide lyase are differentially distributed in thylakoids, with hydroperoxide lyase localized almost exclusively to the stromal part, thus closely resembling the localization pattern of LOX H1. It is suggested that, in addition to their differential expression pattern, this segregation underlies the regulation of metabolic fluxes through the alternative branches of the lipoxygenase pathway.     

Peroxidation stimulated by lipid hydroperoxides on bovine retinal pigment epithelium mitochondria: effect of cellular retinol-binding protein

This study analyzes the effect of cellular retinol-binding protein (CRBP), partially purified from retinal pigment epithelium (RPE) cytosol, on the non-enzymatic lipid peroxidation induced by fatty acid hydroperoxides of mitochondrial membranes isolated from bovine RPE. The effect of different amounts (50, 75 and 100 nmol) of linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) on the lipid peroxidation of RPE mitochondria was studied; RPE mitochondria deprived of exogenously added hydroperoxide was utilized as control. The process was measured simultaneously by determining chemiluminescence as well as polyunsaturated fatty acid (PUFA) degradation of total lipids isolated from RPE mitochondria. The addition of hydroperoxides to RPE mitochondria produces a marked increase in light emission that was hydroperoxide concentration dependent. The highest value of activation was produced by LHP. The major difference in the fatty acid composition of total lipids isolated from native and peroxidized RPE mitochondria incubated with and without hydroperoxides was found in the docosahexaenoic acid content, this decreased 40.90+/-3.01% in the peroxidized group compared to native RPE mitochondria. The decrease was significantly high: 86.32+/-2.57% when the lipid peroxidation was stimulated by 100 nmol of LHP. Inhibition of lipid peroxidation (decrease of chemiluminescence) was observed with the addition of increasing amounts (100-600 microg) of CRBP to RPE mitochondria. The inhibitory effect reaches the highest values in the presence of LHP.     

Activation of the fatty acid alpha-dioxygenase pathway during bacterial infection of tobacco leaves. Formation of oxylipins protecting against cell death

A pathogen-induced oxygenase showing homology to prostaglandin endoperoxide synthases-1 and -2 was recently characterized by in vitro experiments as a fatty acid alpha-dioxygenase catalyzing formation of unstable 2(R)-hydroperoxy fatty acids. To study the activity of this enzyme under in vivo conditions and to elucidate the fate of enzymatically produced 2-hydroperoxides, leaves of tobacco were analyzed for the presence of alpha-dioxygenase-generated compounds as well as for lipoxygenase (LOX) products and free fatty acids. Low basal levels of 2-hydroxylinolenic acid (0.4 nmol/g leaves fresh weight) and 8,11,14-heptadecatrienoic acid (0.1 nmol/g) could be demonstrated. These levels increased strongly upon infection with the bacterium Pseudomonas syringae pv syringae (548 and 47 nmol/g, respectively). Transgenic tobacco plants overexpressing alpha-dioxygenase were developed, and incompatible infection of such plants led to a dramatic elevation of 2-hydroxylinolenic acid (1778 nmol/g) and 8,11,14-heptadecatrienoic acid (86 nmol/g), whereas the levels of LOX products were strongly decreased. Further analysis of oxylipins in infected leaves revealed the presence of a number of 2-hydroxy fatty acids differing with respect to chain length and degree of unsaturation as well as two new doubly oxygenated oxylipins identified as 2(R),9(S)-dihydroxy-10(E),12(Z),15(Z)-octadecatrienoic acid and 2(R),9(S)-dihydroxy-10(E),12(Z)-octadecadienoic acid. alpha-Dioxygenase-generated 2-hydroxylinolenic acid, and to a lesser extent lipoxygenase-generated 9-hydroxyoctadecatrienoic acid, exerted a tissue-protective effect in bacterially infected tobacco leaves.     

Exposure to nitric oxide protects against oxidative damage but increases the labile iron pool in sorghum embryonic axes

Sodium nitroprusside (SNP) and diethylenetriamine NONOate (DETA NONOate), were used as the source of exogenous NO to study the effect of NO upon germination of sorghum (Sorghum bicolor (L.) Moench) seeds through its possible interaction with iron. Modulation of cellular Fe status could be an important factor for the establishment of oxidative stress and the regulation of plant physiology. Fresh and dry weights of the embryonic axes were significantly increased in the presence of 0.1 mM SNP, as compared to control. Spin trapping EPR was used to assess the NO content in axes from control seeds after 24 h of imbibition (2.4+/-0.2 nmol NO g(-1) FW) and seeds exposed to 0.01, 0.1, and 1 mM SNP (3.1+/-0.3, 4.6+/-0.2, and 6.0+/-0.9 nmol NO g(-1) FW, respectively) and 1 mM DETA NONOate (6.2+/-0.6 nmol NO g(-1) FW). Incubation of seeds with 1 mM SNP protected against oxidative damage to lipids and maintained membrane integrity. The content of the deferoxamine-Fe (III) complex significantly increased in homogenates of axes excised from seeds incubated in the presence of 1 mM SNP or 1 mM DETA NONOate as compared to the control (19+/-2 nmol Fe g(-1) FW, 15.2+/-0.5 nmol Fe g(-1) FW, and 8+/-1 nmol Fe g(-1) FW, respectively), whereas total Fe content in the axes was not affected by the NO donor exposure. Data presented here provide experimental evidence to support the hypothesis that increased availability of NO drives not only protective effects to biomacromolecules, but to increasing the Fe availability for promoting cellular development as well.     

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.     

On the role of molecular oxygen in lipoxygenase activation: comparison and contrast of epidermal lipoxygenase-3 with soybean lipoxygenase-1

The oxygenation of polyunsaturated fatty acids by lipoxygenases (LOX) is associated with a lag phase during which the resting ferrous enzyme is converted to the active ferric form by reaction with fatty acid hydroperoxide. Epidermal lipoxygenase-3 (eLOX3) is atypical in displaying hydroperoxide isomerase activity with fatty acid hydroperoxides through cycling of the ferrous enzyme. Yet eLOX3 is capable of dioxygenase activity, albeit with a long lag phase and need for high concentrations of hydroperoxide activator. Here, we show that higher O(2) concentration shortens the lag phase in eLOX3, although it reduces the rate of hydroperoxide consumption, effects also associated with an A451G mutation known to affect the disposition of molecular oxygen in the LOX active site. These observations are consistent with a role of O(2) in interrupting hydroperoxide isomerase cycling. Activation of eLOX3, A451G eLOX3, and soybean LOX-1 with 13-hydroperoxy-linoleic acid forms oxygenated end products, which we identified as 9R- and 9S-hydroperoxy-12S,13S-trans-epoxyoctadec-10E-enoic acids. We deduce that activation partly depends on reaction of O(2) with the intermediate of hydroperoxide cleavage, the epoxyallylic radical, giving an epoxyallylic peroxyl radical that does not further react with Fe(III)-OH; instead, it dissociates and leaves the enzyme in the activated free ferric state. eLOX3 differs from soybean LOX-1 in more tightly binding the epoxyallylic radical and having limited access to O(2) within the active site, leading to a deficiency in activation and a dominant hydroperoxide isomerase activity.     

Regulation of oxylipin synthesis

Two very common groups of oxylipins formed in plants involve the conversion of fatty acid hydroperoxides, such as hydroperoxy-octadecatrienoic acid, into further metabolites by allene oxide synthase and hydroperoxide lyase. Both of these oxylipin branch pathways appear to be ubiquitous or nearly so in plants, but the relative activities of these two branches vary among plant species. In most plants examined, including Arabidopsis, product formation from either of these pathways is minimal until elicited by wounding or some other means, upon which products from both pathways, such as jasmonic acid and C(6) aldehydes and alcohols, can increase by orders of magnitude. In some plant species such as Artemisia and Jasminum spp. oxylipin product formation is heavily skewed towards allene oxide synthase products. Others such as watermelon (Citrullus lanatus) produce 10-fold higher amounts or more of hydroperoxide lyase than allene oxide synthase products. Arabidopsis and tobacco are intermediate between these extremes. Artemisia and Jasminum are also unusual in that they do not require wounding or other types of induction for high oxylipin product formation. Release of non-esterified fatty acids appears to be correlated with oxylipin formation, but phospholipase A(2) appears not to be involved with oxylipin production, at least in the case of Artemisia leaves.     

Formation of a new class of oxylipins from N-acyl(ethanol)amines by the lipoxygenase pathway.

N-Acylethanolamines (NAEs) constitute a new class of plant lipids and are thought to play a role in plant defense strategies against pathogens. In plant defense systems, oxylipins generated by the lipoxygenase pathway are important actors. To date, it is not known whether plants also use endogeneous oxylipins derived from NAEs in their defense reactions. We tested whether members of the NAE class can be converted by enzymes constituting this pathway, such as (soybean) lipoxygenase-1, (alfalfa) hydroperoxide lyase and (flax seed) allene oxide synthase. We found that both alpha-N-linolenoylethanolamine and gamma-N-linolenoylethanolamine (18:3), as well as alpha-N-linolenoylamine and gamma-N-linolenoylamine were converted into their (13S)-hydroperoxide derivatives by lipoxygenase. Interestingly, only the hydroperoxides of alpha-N-linolenoyl(ethanol)amines and their linoleic acid analogs (18:2) were suitable substrates for hydroperoxide lyase. Hexanal and (3Z)-hexenal were identified as volatile products of the 18:2 and 18:3 fatty acid (ethanol)amides, respectively. 12-Oxo-N-(9Z)-dodecenoyl(ethanol)amine was the nonvolatile hydrolysis product. Kinetic studies with lipoxygenase and hydroperoxide lyase revealed that the fatty acid ethanolamides were converted as readily or even better than the corresponding free fatty acids. Allene oxide synthase utilized all substrates, but was most active on (13S)-hydroperoxy-alpha-N-linolenoylethanolamine and the (13S)-hydroperoxide of linoleic acid and its ethanolamine derivative. alpha-Ketols and gamma-ketols were characterized as products. In addition, cyclized products, i.e. 12-oxo-N-phytodienoylamines, derived from (13S)-hydroperoxy-alpha-N-linolenoylamines were found. The results presented here show that, in principle, hydroperoxide NAEs can be formed in plants and subsequently converted into novel phytooxylipins.     

Formation of lipoxygenase-pathway-derived aldehydes in barley leaves upon methyl jasmonate treatment.

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-dinitro-phenylhydrazones 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-1 fresh weight to 45 nmol.g-1 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-1 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-1 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.     

Jasmonate-induced lipid peroxidation in barley leaves initiated by distinct 13-LOX forms of chloroplasts

In addition to a previously characterized 13-lipoxygenase of 100 kDa encoded by LOX2:Hv:1 [V?r?s 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.     

Expression of a Flax Allene Oxide Synthase cDNA Leads to Increased Endogenous Jasmonic Acid (JA) Levels in Transgenic Potato Plants but Not to a Corresponding Activation of JA-Responding Genes

Both jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA), are thought to be significant components of the signaling pathway regulating the expression of plant defense genes in response to various stresses. JA and MeJA are plant lipid derivatives synthesized from [alpha]-linolenic acid by a lipoxygenase-mediated oxygenation leading to 13-hydroperoxylinolenic acid, which is subsequently transformed by the action of allene oxide synthase (AOS) and additional modification steps. AOS converts lipoxygenase-derived fatty acid hydroperoxide to allene epoxide, which is the precursor for JA formation. Overexpression of flax AOS cDNA under the regulation of the cauliflower mosaic virus 35S promoter in transgenic potato plants led to an increase in the endogenous level of JA. Transgenic plants had six- to 12-fold higher levels of JA than the nontransformed plants. Increased levels of JA have been observed when potato and tomato plants are mechanically wounded. Under these conditions, the proteinase inhibitor II (pin2) genes are expressed in the leaves. Despite the fact that the transgenic plants had levels of JA similar to those found in nontransgenic wounded plants, pin2 genes were not constitutively expressed in the leaves of these plants. Transgenic plants with increased levels of JA did not show changes in water state or in the expression of water stress-responsive genes. Furthermore, the transgenic plants overexpressing the flax AOS gene, and containing elevated levels of JA, responded to wounding or water stress by a further increase in JA and by activating the expression of either wound- or water stress-inducible genes. Protein gel blot analysis demonstrated that the flax-derived AOS protein accumulated in the chloroplasts of the transgenic plants.     

Involvement of phospholipase D in wound-induced accumulation of jasmonic acid in arabidopsis

Multiple forms of phospholipase D (PLD) were activated in response to wounding, and the expressions of PLDalpha, PLDbeta, and PLDgamma differed in wounded Arabidopsis leaves. Antisense abrogation of the common plant PLD, PLDalpha, decreased the wound induction of phosphatidic acid, jasmonic acid (JA), and a JA-regulated gene for vegetative storage protein. Examination of the genes involved in the initial steps of oxylipin synthesis revealed that abrogation of the PLDalpha attenuated the wound-induced expression of lipoxygenase 2 (LOX2) but had no effect on allene oxide synthase (AOS) or hydroperoxide lyase in wounded leaves. The systemic induction of LOX2, AOS, and vegetative storage protein was lower in the PLDalpha-suppressed plants than in wild-type plants, with AOS exhibiting a distinct pattern. These results indicate that activation of PLD mediates wound induction of JA and that LOX2 is probably a downstream target through which PLD promotes the production of JA.     

Cytochrome P450-dependent metabolism of oxylipins in tomato. Cloning and expression of allene oxide synthase and fatty acid hydroperoxide lyase

Allene oxide synthase (AOS) and fatty acid hydroperoxide lyase (HPL) are plant-specific cytochrome P450s that commit fatty acid hydroperoxides to different branches of oxylipin metabolism. Here we report the cloning and characterization of AOS (LeAOS) and HPL (LeHPL) cDNAs from tomato (Lycopersicon esculentum). Functional expression of the cDNAs in Escherichia coli showed that LeAOS and LeHPL encode enzymes that metabolize 13- but not 9-hydroperoxide derivatives of C(18) fatty acids. LeAOS was active against both 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) and 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, whereas LeHPL showed a strong preference for 13-HPOT. These results suggest a role for LeAOS and LeHPL in the metabolism of 13-HPOT to jasmonic acid and hexenal/traumatin, respectively. LeAOS expression was detected in all organs of the plant. In contrast, LeHPL expression was predominant in leaves and flowers. Damage inflicted to leaves by chewing insect larvae led to an increase in the local and systemic expression of both genes, with LeAOS showing the strongest induction. Wound-induced expression of LeAOS also occurred in the def-1 mutant that is deficient in octadecanoid-based signaling of defensive proteinase inhibitor genes. These results demonstrate that tomato uses genetically distinct signaling pathways for the regulation of different classes of wound responsive genes.     

植物脂氧合酶蛋白特性及其在果实成熟衰老和逆境胁迫中的作用

脂氧合酶（lipoxygenase,LOx）作为植物脂肪酸氧化途径的关键酶,是多基因家族,在植物生长发育中起重要作用。本文综述了脂氧合酶的蛋白结构特征、催化反应的位置特异性、分类及其代谢途径,重点介绍了LOX代谢途径中的氢过氧化物裂解酶（hydropemxide lyase,HPL）支路和丙二烯氧化物合酶（allene oxide synthase,AOS）支路,最后阐述了LOxs在植物成熟衰老和逆境胁迫中的作用,为通过基因工程手段改善果实品质以及提高植物抗逆性提供了一定的理论依据。     

Envelope Membranes from Spinach Chloroplasts Are a Site of Metabolism of Fatty Acid Hydroperoxides

Enzymes in envelope membranes from spinach (Spinacia oleracea L.) chloroplasts were found to catalyze the rapid breakdown of fatty acid hydroperoxides. In contrast, no such activities were detected in the stroma or in thylakoids. In preparations of envelope membranes, 9S-hydroperoxy-10(E),12(Z)-octadecadienoic acid, 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, or 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid were transformed at almost the same rates (1-2 [mu]mol min-1 mg-1 protein). The products formed were separated by reversed-phase high-pressure liquid chromatography and further characterized by gas chromatography-mass spectrometry. Fatty acid hydroperoxides were cleaved (a) into aldehydes and oxoacid fragments, corresponding to the functioning of a hydroperoxide lyase, (b) into ketols that were spontaneously formed from allene oxide synthesized by a hydroperoxide dehydratase, (c) into hydroxy compounds synthesized enzymatically by a system that has not yet been characterized, and (d) into oxoenes resulting from the hydroperoxidase activity of a lipoxygenase. Chloroplast envelope membranes therefore contain a whole set of enzymes that catalyze the synthesis of a variety of fatty acid derivatives, some of which may act as regulatory molecules. The results presented demonstrate a new role for the plastid envelope within the plant cell.