A new 9-lipoxygenase cDNA from developing rice seeds

We isolated a novel C9 position specific lipoxygenase (r9-LOX1) cDNA from developing rice seeds. The enzymatic features of r9-LOX1 resembled those of rice LOX-L3 known to be contained in rice germ and to have C9-specific LOX activity. However, the expression level of the r9-LOX1 gene was higher in imbibed seeds rather than developing seeds. A homology search against the rice nucleotide database revealed the r9-LOX1 gene to be on rice chromosome 3 (accession number AC093017). The restriction enzyme map of the reported genomic sequence agreed with the result of the Southern blot analysis for the r9-LOX1. The enzyme could be useful for in vitro synthesis of 9,10-ketol-octadecadienoic acid.     

9-lipoxygenase metabolism is involved in the almond/Aspergillus carbonarius interaction

Phyto-oxylipins are a group of biologically active molecules that play an important role in plant defence. Their production begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases (LOX) to form 9- or 13-hydroperoxides that are substrates for several enzymes involved in the synthesis of final oxylipins, which can act as signal molecules and/or direct antimicrobials. In the present work, the response of the 9-LOX pathway in the almond/Aspergillus carbonarius (producer of ochratoxin A) interaction was studied. Both LOX gene expression and activity are up-regulated over the course of fungal infection in immature and mature almonds. The biochemical characterization of major LOX and hydroperoxide lyase (HPL) isoforms indicated that 9-LOX metabolism is specifically induced by A. carbonarius. Lipid peroxidation profiling showed that, in infected immature almonds, enzymatically produced 9-hydro(peroxy) fatty acids (HFAs) were the main HFAs and are further metabolized by HPL into C9-aldehydes. Both HPL gene expression and C9-aldehydes increased over the course of fungal infection. In mature almonds infected with A. carbonarius, levels of LOX expression and activity were lower than those found in immature seeds, and 9-HFA represented the minority of total HFA, which consisted of mostly 13- and non-enzymatically produced HFA. In these experimental conditions, no volatile aldehydes were recorded from these samples, even though HPL was up-regulated in infected mature almonds. The effects on the growth of A. carbonarius of the aldehydes produced by these enzymes were also tested in vitro. Results reported here led to the proposal that, in almond seed, the association of 9-LOX and HPL has an important role in seed defence mechanism against pathogen infection.     

Biochemical and molecular characterization of hazelnut (Corylus avellana) seed lipoxygenases.

Plant lipoxygenases (LOXs) are a class of dioxygenases which display diverse functions in several physiological processes such as growth, development and response to biotic and abiotic stresses. Even though LOXs have been characterized from several plant species, the physiological role of seed LOXs is still unclear. With the aim to better clarify the occurrence of LOXs and their influence on hazelnut seed quality, we carried out the biochemical and molecular characterization of the main LOX isoforms expressed during seed development. A genomic clone containing a complete LOX gene was isolated and fully characterized. The 9887 bp sequence reported contains an open reading frame of 5334 bp encoding a putative polypeptide of 99 kDa. Semiquantitative RT-PCR carried out from RNAs extracted from seeds at different maturation stages showed that LOXs are mainly expressed at early developmental stages. These results were confirmed by LOX activity assays. Biochemical characterization of the reaction products of the hazelnut LOX indicated that it is a 9-LOX. Two cDNAs were isolated by RT-PCR carried out on total RNA from immature hazelnut seeds. Sequence analysis indicated that the two cDNAs are highly homologous (91.9% degree of identity) and one of these corresponded exactly to the genomic clone. The deduced amino acid sequences of the hazelnut LOXs showed that they are closely related to a previously reported almond LOX (79.5% identity) and, to a lesser extent, to some LOXs involved in plant responses to pathogens (cotton and tobacco LOXs, 75.5 and 74.6% identity, respectively). The physiological role of hazelnut LOXs and their role in influencing seed quality are also discussed.     

Aspergillus infection inhibits the expression of peanut 13S-HPODE-forming seed lipoxygenases

Oxylipins recently have been implicated as signaling molecules for cross-kingdom communication in plant-pathogen interactions. Linoleic acid and its two plant lipoxygenase (LOX) oxylipin products 9- and 13-hydroperoxy fatty acids (9S- and 13S-HPODE) have been shown to have a significant effect on differentiation processes in the mycotoxigenic seed pathogens Aspergillus spp. Whereas both fatty acids promote sporulation, 9S-HPODE stimulates and 13S-HPODE inhibits mycotoxin production. Additionally, Aspergillus flavus infection of seed promotes linoleate 9-LOX expression and 9S-HPODE accumulation. Here, we describe the characterization of two peanut seed lipoxygenase alleles (PnLOX2 and PnLOX3) highly expressed in mature seed. PnLOX2 and PnLOX3 both are 13S-HPODE producers (linoleate 13-LOX) and, in contrast to previously characterized 9-LOX or mixed function LOX genes, are repressed between 5-fold and 250-fold over the course of A. flavus infection. The results of these studies suggest that 9S-HPODE and 13S-HPODE molecules act as putative susceptibility and resistance factors respectively, in Aspergillus seed-aflatoxin interactions.     

Characterization of a membrane-associated apoplastic lipoxygenase in Phaseolus vulgaris L.

An extracytoplasmic 86.7 kDa protein was isolated from intercellular washing fluids (IWF) of Phaseolus vulgaris etiolated hypocotyls. Micro sequencing of tryptic peptides of the 86.7 kDa protein revealed 100% identity with a bean lipoxygenase (LOX) protein fragment. Purified P87-LOX exhibited LOX activity characterized by an optimal pH of 6.0 and linolenic acid as an optimal substrate, and was classified as a 13-LOX with respect to its positional specificity of linoleic acid oxygenation. A protein identical to P87-LOX, as determined by MALDI-TOF analysis and biochemical characterization, was purified from hypocotyl microsomes. Immunoblot analysis showed that P87-LOX is present in plasma membrane-enriched fractions, from which it was solubilized using high ionic strength buffers. These observations suggest that P87-LOX is a peripheral protein associated to the apoplastic face of the plasma membrane.     

Characterization of a membrane-associated apoplastic lipoxygenase in Phaseolus vulgaris L

An extracytoplasmic 86.7 kDa protein was isolated from intercellular washing fluids (IWF) of Phaseolus vulgaris etiolated hypocotyls. Micro sequencing of tryptic peptides of the 86.7 kDa protein revealed 100% identity with a bean lipoxygenase (LOX) protein fragment. Purified P87-LOX exhibited LOX activity characterized by an optimal pH of 6.0 and linolenic acid as an optimal substrate, and was classified as a 13-LOX with respect to its positional specificity of linoleic acid oxygenation. A protein identical to P87-LOX, as determined by MALDI-TOF analysis and biochemical characterization, was purified from hypocotyl microsomes. Immunoblot analysis showed that P87-LOX is present in plasma membrane-enriched fractions, from which it was solubilized using high ionic strength buffers. These observations suggest that P87-LOX is a peripheral protein associated to the apoplastic face of the plasma membrane.     

A lipoxygenase with dual positional specificity is expressed in olives (Olea europaea L.) during ripening

Plant lipoxygenases (LOXs) are a class of widespread dioxygenases catalysing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological roles remain to be clarified. With the aim to clarify the occurrence of LOXs in olives and their contribution to the elaboration of the olive oil aroma, we cloned and characterized the first cDNA of the LOX isoform which is expressed during olive development. The open reading frame encodes a polypeptide of 864 amino acids. This olive LOX is a type-1 LOX which shows a high degree of identity at the peptide level towards hazelnut (77.3%), tobacco (76.3%) and almond (75.5%) LOXs. The recombinant enzyme shows a dual positional specificity, as it forms both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio, and would be defined as 9/13-LOX. Although a LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest that there are at least two Lox genes expressed in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity and its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered.     

Disruption of a maize 9-lipoxygenase results in increased resistance to fungal pathogens and reduced levels of contamination with mycotoxin fumonisin

Plant oxylipins, produced via the lipoxygenase (LOX) pathway, function as signals in defense and development. In fungi, oxylipins are potent regulators of mycotoxin biosynthesis and sporogenesis. Previous studies showed that plant 9-LOX-derived fatty acid hydroperoxides induce conidiation and mycotoxin production. Here, we tested the hypothesis that oxylipins produced by the maize 9-LOX pathway are required by pathogens to produce spores and mycotoxins and to successfully colonize the host. Maize mutants were generated in which the function of a 9-LOX gene, ZmLOX3, was abolished by an insertion of a Mutator transposon in its coding sequence, which resulted in reduced levels of several 9-LOX-derived hydroperoxides. Supporting our hypothesis, conidiation and production of the mycotoxin fumonisin B1 by Fusarium verticillioides were drastically reduced in kernels of the lox3 mutants compared with near-isogenic wild types. Similarly, conidia production and disease severity of anthracnose leaf blight caused by Colletotrichum graminicola were significantly reduced in the lox3 mutants. Moreover, lox3 mutants displayed increased resistance to southern leaf blight caused by Cochliobolus heterostrophus and stalk rots caused by both F. verticillioides and C. graminicola. These data strongly suggest that oxylipin metabolism mediated by a specific plant 9-LOX isoform is required for fungal pathogenesis, including disease development and production of spores and mycotoxins.     

砂梨脂氧合酶cDNA片段克隆与RNAi载体构建

以砂梨‘若光’的成熟果实为材料,根据脂氧合酶氨基酸保守区设计1对简并引物,采用RT-PCR克隆到1段长827 bp的序列,经Blast比对和DNAstar软件聚类分析,结果表明由该序列推导出的氨基酸序列含有脂氧合酶氨基酸保守结构域,与马铃薯脂氧合酶基因的氨基酸序列相似性达到77.5%,判断其为砂梨脂氧合酶基因片段,命名为LOX1,并将序列登录到GenBank,登录号为EF215448。根据RNAi载体的构建原则,选择LOX1一开放阅读框设计携带酶切位点的特异引物,通过PCR扩增正、反向基因片段,再与YYT间隔区串连,插入植物表达载体pYF7713中的相应位置,成功地构建了干扰LOX基因表达的RNAi的植物双元表达载体pYL028,为深入研究该基因在砂梨果实成熟过程中的功能及耐贮藏基因工程育种奠定了基础。     

Accumulation of chloroplast-targeted lipoxygenase in passion fruit leaves in response to methyl jasmonate

Wounding caused local and systemic induction of lipoxygenase (LOX) activity in passion fruit (Passiflora edulis f. flavicarpa) leaves, while exposing intact plants to methyl jasmonate (MJ) vapor provoked a much stronger response. Western blot analysis of these leaf protein extracts using polyclonal antibodies against cucumber LOX, revealed an accumulation of a 90 kDa protein, consistent with LOX enzymatic assays. The inducible LOX was purified to apparent homogeneity, and in vitro analysis of LOXactivity using linoleic acid as substrate showed that it possesses C-13 specificity. Immunocytochemical localization studies using leaf tissue from MJ-treated plants demonstrated that the inducible LOX was compartmented in large quantities in the chloroplasts of mesophyll cells, associated with the stroma. The results suggest that the wound response in passion fruit plants may be mediated by a chloroplast 13-LOX, a key enzyme of the octadecanoid defense-signaling pathway.     

The LOX1 Gene of Arabidopsis Is Temporally and Spatially Regulated in Germinating Seedlings

We examined the temporal and spatial expression patterns of the LOX1 gene during the development of Arabidopsis thaliana seedlings. Measurements of steady-state LOX1 mRNA levels indicated that this gene is transiently expressed during germination. LOX1 mRNA was not detected in seed that had imbibed (T0) but reached a maximum level by 1 d in both light- and dark-grown seedlings. The induction of the LOX1 gene was not light dependent; however, mRNA levels were 4-fold greater in light-grown seedlings. Immunoblot analysis of lipoxygenase protein levels and measurements of enzyme activity suggested that the induction of the LOX1 gene resulted in the production of functional lipoxygenase enzyme. Lipoxygenase protein was not present in dry seed or seed that had imbibed, but was first detected by immunoblot analysis after 1 and 2 d of growth in the light and dark, respectively. In both cases, lipoxygenase protein levels remained high for 2 d and then declined. Lipoxygenase activity paralleled the changes in protein levels. In situ hybridization studies revealed that the LOX1 gene is transiently expressed in the epidermis and the aleurone layer during germination. LOX1 mRNA levels were particularly high in the epidermis of the radicle and the adaxial side of the cotyledons. These results suggest that the LOX1 gene product is produced specifically during early germination and plays a role in the functioning of the epidermis.     

COX-2/12-LOX dual pathway, a novel strategy for treatment of multiple myeloma?

Cyclooxygenase (COX) and lipoxygenase (LOX) metabolic enzymes are the two main pathways for arachidonic acid (AA) metabolism. Emerging reports now indicate alterations of arachidonic acid metabolism with carcinogenesis and many COX and LOX inhibitors are being investigated as potential anticancer drugs. COX-2 is frequently expressed in many tumors, such as multiple myeloma (MM), a disorder in which malignant plasma cells accumulate, generally derived from one clone in the bone marrow, and is an independent predictor of poor outcome. 12-LOX, an important member of LOX, is proved to be expressed in MM cells. We hypothesize that COX-2 and 12-LOX represent an integrated system, COX-2/12-LOX dual pathway, which much more efficiently enhances the intracellular levels of unesterified arachidonate and regulates cell proliferative, apoptosis and pro-angiogenic potential of MM. The COX-2/12-LOX dual pathway may act as a novel potential strategy for treatment of tumors co-expressing COX-2 and 12-LOX, and the agents that can simultaneously inhibit the two enzymes of COX-2 and 12-LOX may present a novel and promising therapeutic approach to these tumors.     

An Arabidopsis thaliana lipoxygenase gene can be induced by pathogens, abscisic acid, and methyl jasmonate.

We isolated and characterized a 2.8-kb, full-length, Arabidopsis thaliana cDNA clone encoding a lipoxygenase. DNA sequence analysis showed that the deduced amino acid sequence of the Arabidopsis protein is 72 to 78% similar to that of legume seed lipoxygenases. DNA blot analysis indicated that Arabidopsis contains a single gene, LOX1, with appreciable homology to the cDNA clone. RNA blot analysis showed that the LOX1 gene is expressed in Arabidopsis leaves, roots, inflorescences, and young seedlings. LOX1 expression levels were highest in roots and young seedlings. In mature plants, LOX1 mRNA levels increased upon treatment with the stress-related hormones abscisic acid and methyl jasmonate and remained high for at least 96 h. Expression of the LOX1 gene was examined following infiltration of leaves with virulent (Psm ES4326) and avirulent (Pst MM1065) strains of Pseudomonas syringae. LOX1 mRNA levels were induced approximately 6-fold by both virulent and avirulent strains; however, the response to avirulent strains was much more rapid. Infiltration of leaves with Pst MM1065 resulted in maximal induction within 12 h, whereas maximal induction by Psm ES4326 did not occur until 48 h. When a cloned avr gene, avrRpt2, was transferred to Psm ES4326, LOX1 mRNA accumulated in a pattern similar to that observed for the avirulent strain Pst MM1065.     

A cell-based assay for screening lipoxygenase inhibitors

Lipoxygenases (LOX) form a family of lipid peroxidizing enzymes within the plant and animal kingdoms. In humans, six functional lipoxygenase isoforms have been identified. 5-LOX, “platelet-type” 12-LOX (p12-LOX) and 15-LOX type 1 (15-LOX1), originally identified in leukocytes, platelets, and reticulocytes, respectively, generate lipid mediators involved in host cellular functions and in the pathophysiology of asthma, cardiovascular diseases, and cancer. The pharmaceutical industry has reinvigorated their programs to develop novel LOX inhibitors in view of recent findings. However, high throughput LOX screening assays to test novel agents against these intracellular enzymes are limited. We describe a cell-based 96-well microplate fluorescence assay tested against several existing LOX inhibitors, and validate the assay by comparing known IC₅₀ values and HPLC analysis, which may provide a useful screen for novel LOX inhibitors.     

Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia

Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes, which catalyze specific dioxygenation of polyunsaturated fatty acids. According to their positional specificity of linoleic acid oxygenation plant LOX have been classified into linoleate 9- and linoleate 13-LOX and recent reports identified a critical valine at the active site of 9-LOX. In contrast, more bulky phenylalanine or histidine residues were found at this position in 13-LOX. We have recently cloned a LOX-isoform from Momordica charantia and multiple amino acid alignments indicated the existence of a glutamine (Gln599) at the position were 13-LOX usually carry histidine or phenylalanine residues. Analyzing the pH-dependence of the positional specificity of linoleic acid oxygenation we observed that at pH-values higher than 7.5 this enzyme constitutes a linoleate 13-LOX whereas at lower pH, 9-H(P)ODE was the major reaction product. Site-directed mutagenesis of glutamine 599 to histidine (Gln599His) converted the enzyme to a pure 13-LOX. These data confirm previous observation suggesting that reaction specificity of certain LOX-isoforms is not an absolute enzyme property but may be impacted by reaction conditions such as pH of the reaction mixture. We extended this concept by identifying glutamine 599 as sequence determinant for such pH-dependence of the reaction specificity. Although the biological relevance for this alteration switch remains to be investigated it is of particular interest that it occurs at near physiological conditions in the pH-range between 7 and 8.