Lipoxygenase Gene Research Articles

Functional characterization of genetic enzyme variations in human lipoxygenases

Mammalian lipoxygenases play a role in normal cell development and differentiation but they have also been implicated in the pathogenesis of cardiovascular, hyperproliferative and neurodegenerative diseases. As lipid peroxidizing enzymes they are involved in the regulation of cellular redox homeostasis since they produce lipid hydroperoxides, which serve as an efficient source for free radicals. There are various epidemiological correlation studies relating naturally occurring variations in the six human lipoxygenase genes (SNPs or rare mutations) to the frequency for various diseases in these individuals, but for most of the described variations no functional data are available. Employing a combined bioinformatical and enzymological strategy, which included structural modeling and experimental site-directed mutagenesis, we systematically explored the structural and functional consequences of non-synonymous genetic variations in four different human lipoxygenase genes (ALOX5, ALOX12, ALOX15, and ALOX15B) that have been identified in the human 1000 genome project. Due to a lack of a functional expression system we resigned to analyze the functionality of genetic variations in the hALOX12B and hALOXE3 gene. We found that most of the frequent non-synonymous coding SNPs are located at the enzyme surface and hardly alter the enzyme functionality. In contrast, genetic variations which affect functional important amino acid residues or lead to truncated enzyme variations (nonsense mutations) are usually rare with a global allele frequency<0.1%. This data suggest that there appears to be an evolutionary pressure on the coding regions of the lipoxygenase genes preventing the accumulation of loss-of-function variations in the human population.

Expression of defence-related genes against Phytophthora cinnamomi in five avocado rootstocks

Avocado (Persea americana) – a major fruit crop worldwide – is threatened by root rot caused by Phytophthora cinnamomi. This pathogen is known to infect the plant via the feeder roots leading to branch dieback, and eventually tree mortality. While it is known that different avocado rootstocks have varying degrees of susceptibility to Phytophthora root rot, little research has been done on the avocado–Phytophthora interaction. In this study, transcript abundance levels of defence-related genes coding for phenylalanine ammonia-lyase, lipoxygenase, pathogenesis-related protein 5, endochitinase, gluthathionine S-transferase and metallothionein were characterised and compared among five rootstocks with varying susceptibility to root rot, after exposure to P. cinnamomi. Root samples were collected at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h post-infection and transcript abundance of the defence-related genes was determined using quantitative real-time reverse transcription PCR. The results indicated the involvement of PR-5 and endochitinase in the defence response of all avocado rootstocks to P. cinnamomi but these genes could not be directly linked to the observed phenotypic resistance. PR-5 and endochitinase were highly upregulated at 72 h post-infection. Differences in transcript abundance of phenylalanine ammonia-lyase and lipoxygenase genes were seen when comparing tolerant and less tolerant rootstocks, which may suggest that transcripts of these genes contribute to resistance. These data provide important insights into plant defence and into how different avocado rootstocks may exhibit increased resistance to infection by P. cinnamomi.

Aloxe3 Knockout Mice Reveal a Function of Epidermal Lipoxygenase-3 as Hepoxilin Synthase and Its Pivotal Role in Barrier Formation

Loss-of-function mutations in the lipoxygenase (LOX) genes ALOX12B and ALOXE3 are the second most common cause of autosomal recessive congenital ichthyosis. The encoded proteins, 12R-LOX and epidermal LOX-3 (eLOX-3), act in sequence to convert fatty acid substrates via R-hydroperoxides to specific epoxyalcohol derivatives and have been proposed to operate in the same metabolic pathway during epidermal barrier formation. Here, we show that eLOX-3 deficiency in mice results in early postnatal death, associated with similar but somewhat less severe barrier defects and morphological changes than reported earlier for the 12R-LOX-knockout mice. Skin lipid analysis demonstrated that the severity of barrier failure is related to the loss of covalently bound ceramides in both 12R-LOX- and eLOX-3-null mice, confirming a proposed functional linkage of the LOX pathway to ceramide processing and formation of the corneocyte lipid envelope. Furthermore, analysis of free oxygenated fatty acid metabolites revealed strongly reduced levels of hepoxilin metabolites in eLOX-3-deficient epidermis, indicating an additional function of eLOX-3 in mammalian skin as a hepoxilin synthase linked to the 12S-LOX pathway.

Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration

Leukotriene (LT)A₄ and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA₄ is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide configuration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities sufficient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog 14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, confirming the proposed structure of eoxin A₄. Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA₄ and converts 5R-HPETE to the cis epoxide 5-epi-LTA₄, establishing substrate chirality as a determinant of the cis or trans epoxide configuration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations.

The omega-6 arachidonic fatty acid, but not the omega-3 fatty acids, inhibits osteoblastogenesis and induces adipogenesis of human mesenchymal stem cells: potential implication in osteoporosis

Arachidonic fatty acid (AA) induces adipogenesis in human mesenchymal stem cells cultures, and high concentrations inhibit osteoblastogenesis; whereas eicosapentaenoic and docosahexaenoic fatty acids do not induce adipogenesis and do not inhibit osteoblastogenesis. In mesenchymal stem cells, omega-6 arachidonic polyunsaturated fatty acid promotes the differentiation of adipocytes and inhibits the osteoblast differentiation. While omega-3 fatty acids do not affect the adipogenic differentiation their effects on osteoblastogenesis are less relevant. An increased ratio of omega-3/omega-6 fatty acid consumption can prevent bone mass loss. Consumption of omega-3 may protect against osteoporosis since they may inhibit osteoclastogenesis. However, with aging, MSC in bone marrow are increasingly differentiated into adipocytes, reducing the number of osteoblasts. Products derived from omega-6 and omega-3 metabolism may affect MSC differentiation into osteoblasts and adipocytes. Human MSC have been differentiated into osteoblasts or adipocytes in the presence of omega-6 (AA), or omega-3 (DHA and EPA), and osteoblastic and adipocytic markers have been analyzed. AA decreases the expression of osteogenic markers and the osteoprotegerin/receptor activator of nuclear factor kappa β ligand gene expression ratio (opg/rankl). High concentrations of AA inhibit the mineralization and cause the appearance of adipocytes in MSC differentiating into osteoblasts to a higher extent than DHA or EPA. In MSC differentiated into adipocytes, AA increases adipogenesis, while DHA and EPA do not affect it. AA caused the appearance of adipocytes in undifferentiated MSC. The lipoxygenase gene (alox15b) is induced by omega-3 in MSC induced to osteoblasts, and by omega-6 in MSC induced to adipocytes. An increase in the intake of omega-3 respect to omega-6 may provide protection against the loss of bone mass, since omega-6 favors the osteoclastic activity by diminishing the opg/rankl gene expression in osteoblasts and promotes MSC differentiation into adipocytes, thus diminishing the production of osteoblasts.

Transcriptomic analysis of oxylipin biosynthesis genes and chemical profiling reveal an early induction of jasmonates in chickpea roots under drought stress

Drought is one of the major constraints in subtropical agriculture. Therefore improving water stress tolerance is of great importance to breed for drought tolerance in future. The first plant organ sensing dehydration is the root. Aim of the present work was to clarify the potential impact of the phyto-oxylipins pathway on drought tolerance of chickpea (Cicer arietinum), the third important legume crop worldwide. Therefore, we measured the expression of key genes involved in oxylipins metabolism by qPCR on samples from stressed and non-stressed roots of a drought-tolerant and a drought-sensitive chickpea variety using commercially available TaqMan assays. We demonstrate that the drought tolerant variety reacts to drought with sustained and earlier activation of a specific lipoxygenase (Mt-LOX 1) gene, two hydroperoxide lyases (Mt-HPL 1 and Mt-HPL 2), an allene oxide synthase (Mt-AOS), and an oxo-phytodienoate reductase (Mt-OPR). We further show that gene over-expression positively correlates with the levels of major oxylipin metabolites from the AOS branch of the pathway, which finally leads to the synthesis of jasmonates. Higher levels of jasmonic acid (JA), its precursor 12-oxophytodienoic acid (OPDA) and the active form JA-isoleucine (JA-Ile) were especially detected in the root tissues of the tolerant variety, prompting us to assume a role of jasmonates in the early signalling of drought stress in chickpea and its involvement in the tolerance mechanism of the drought-tolerant variety.

The aroma biogenesis-related Olea europaea ALCOHOL DEHYDROGENASE gene is developmentally regulated in the fruits of two O. europaea L. cultivars

In plants, the aroma imprint is expressed through well-established and constant ratios between different compounds, which mainly develop through the Lipoxygenase pathway. Such imprint strongly relies on plant genetic traits but can be modulated by exogenous environmental factors. In order to enlarge knowledge on the genetic control underlying aroma biogenesis in olive (Olea europaea L.) drupes, in the present study we characterized one member of ALCOHOL DEHYDROGENASE gene family. The ALCOHOL DEHYDROGENASE activities act along Lipoxygenase enzymatic cascade by interconverting aldehydes into alcohols. The temporal expression pattern of O. europaea ALCOHOL DEHYDROGENASE was monitored by quantitative Real Time-Polymerase Chain Reaction in drupes at different developmental stages (i.e. from light green phase to ripening stage) of two olive cultivars widely used for olive and oil production. The expression of a known LIPOXYGENASE gene (ACG56281.1) was jointly monitored as marker of drupe development. Results were analysed in relation to the spectrum of volatile compounds developed from crushed drupes estimated by Solid-Phase Micro-Extraction method in-line with a Gas Chromatograph/ion trap Mass Spectrometer. The obtained results showed that both cultivar and maturation stage impact on the expression of O. europaea ALCOHOL DEHYDROGENASE gene which in the pathway is co-ordinately modulated with LIPOXYGENASE expression. The transcriptional differences between cultivars appeared to be related to variation of metabolite contents at early developmental stages. The joint role of genes acting in the Lipoxygenase pathway as molecular markers for flavour characteristic of olive drupes is discussed.

Expression patterns of defense genes in resistance of the panicles exserted from the caulis and from the tillers to neck blast in rice

The rice variety Xiushui227 is resistant to neck blast in the panicles exserted from the caulis but susceptible in the panicles from the tillers, however, the other variety Xiushui09 is susceptible to neck blast in the panicles from the caulis but resistant in the panicles from the tillers. These two varieties were used to analyze the expression patterns of defense genes in the panicles from the caulis and the first first-class tiller at the preliminary heading stage, after inoculating the necks invitro with Magnaporthe oryzae, respectively. All defense genes (pathogenesis-related genes PR1a, Gns1 (1,3; 1,4-β-glucanase), Cht-1 (chitinase), PR4, PR5, and PR10a, secondary metabolite pathway genes PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), and LOX (lipoxygenase), and oxidative stress-related protein genes POX22.3 (peroxidase), and PPO (polyphenol oxidase)) used in this experiment except Cht-1 and PR5 could participate in defending Xiushui227 against neck blast in the panicles from the caulis. All defense genes used in this study except Cht-1, PR10a, and PPO may play roles in defending Xiushui09 against neck blast in the panicles from the tillers.

OsNPR1 negatively regulates herbivore‐induced JA and ethylene signaling and plant resistance to a chewing herbivore in rice

NPR1 (a non-expressor of pathogenesis-related genes1) has been reported to play an important role in plant defense by regulating signaling pathways. However, little to nothing is known about its function in herbivore-induced defense in monocot plants. Here, using suppressive substrate hybridization, we identified a NPR1 gene from rice, OsNPR1, and found that its expression levels were upregulated in response to infestation by the rice striped stem borer (SSB) Chilo suppressalis and rice leaf folder (LF) Cnaphalocrocis medinalis, and to mechanical wounding and treatment with jasmonic acid (JA) and salicylic acid (SA). Moreover, mechanical wounding induced the expression of OsNPR1 quickly, whereas herbivore infestation induced the gene more slowly. The antisense expression of OsNPR1 (as-npr1), which reduced the expression of the gene by 50%, increased elicited levels of JA and ethylene (ET) as well as of expression of a lipoxygenase gene OsHI-LOX and an ACC synthase gene OsACS2. The enhanced JA and ET signaling in as-npr1 plants increased the levels of herbivore-induced trypsin proteinase inhibitors (TrypPIs) and volatiles, and reduced the performance of SSB. Our results suggest that OsNPR1 is an early responding gene in herbivore-induced defense and that plants can use it to activate a specific and appropriate defense response against invaders by modulating signaling pathways.

Expression of defense genes and antioxidant defense responses in rice resistance to neck blast at the preliminary heading stage and full heading stage

The rice variety Xiushui227 is resistant to neck blast at three crucial panicle stages (the booting stage, the preliminary heading stage (PHS), and the full heading stage (FHS)) that controlling neck blast. The other rice variety Liangyou6326 is susceptible to neck blast at these three panicle stages. These two varieties were used to analyze the expression patterns of defense genes and antioxidant defense responses at the PHS and FHS, after inoculating the necks in vitro with Magnaporthe oryzae, respectively. All defense genes (pathogenesis-related genes PR1a, Gns1 (1,3; 1,4-β-glucanase), Cht-1 (chitinase), PR4, PR5, and PR10a, secondary metabolite pathway genes PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), and LOX (lipoxygenase), and oxidative stress-related protein genes POX22.3 (peroxidase), and PPO (polyphenol oxidase)) used in this experiment except Cht-1, CHS and PPO could mainly play important roles in the resistance to neck blast at the PHS in Xiushui227, and CHS and PPO may primarily participate in fighting back against to neck blast at the FHS. Conversely, PR1a, Cht-1, PR4, PR10a, CHS, LOX-RLL, and PPO could chiefly play participate in defending Liangyou6326 against neck blast at the FHS, and PR5, PAL, and POX22.3 may be primarily involved in fighting back against to neck blast at the PHS. Furthermore, the antioxidant enzymes superoxide dismutase, peroxidase, and catalase may mainly participate in defending Xiushui227 against neck blast at the PHS and defending Liangyou6326 against neck blast at the FHS, respectively. Therefore oxidative damage is less at the PHS in Xiushui227 and at the FHS in Liangyou6326, respectively.

12/15‐Lipoxygenase gene knockout severely impairs ischemia‐induced angiogenesis due to lack of Rac1 farnesylation

Previously, we have reported that 15(S)‐hydroxyeicosatetraenoic acid (15(S)‐HETE)‐induced migration and tube formation of endothelial cells require Rac1 activation. To understand the mechanisms by which 15(S)‐HETE activates Rac1, here, we have studied the role of HMG‐CoA reductase and RhoGFP, α‐Pix. 15(S)‐HETE by inducing HMG‐CoA reductase expression caused increased farnesylation and membrane translocation of Rac1, where it became activated by Src‐dependent α‐Pix stimulation in human dermal microvascular endothelial cells (HDMVECs) and these events enhanced the migration and tube formation of these cells. Mevalonate rescued 15(S)‐HETE‐induced Rac1 farnesylation in HDMVECs and the migration and tube formation of these cells from inhibition by simvastatin. Hind limb ischemia induced Rac1 farnesylation and activation leading to increased angiogenesis and these effects were blocked by simvastatin and rescued by mevalonate in C57BL/6 mice. In contrast, hind limb ischemia failed to induce Rac1 farnesylation and activation as well as angiogenic response in 12/15‐Lox−/− mice. Activation of Src and α‐Pix were also compromised at least to some extent in 12/15‐Lox−/− mice. Together, these findings demonstrate that HMG‐CoA reductase‐dependent farnesylation and α‐Pix‐dependent GDP/GTP exchange of Rac1 is essential for 12/15‐Lox‐12/15 (S)‐HETE‐induced angiogenesis.

Methods for identifying lipoxygenase producing microorganisms on agar plates

Plate assays for lipoxygenase producing microorganisms on agar plates have been developed. Both potassium iodide-starch and indamine dye formation methods were effective for detecting soybean lipoxygenase activity on agar plates. A positive result was also achieved using the β-carotene bleaching method, but the sensitivity of this method was lower than the other two methods. The potassium iodide-starch and indamine dye formation methods were also applied for detecting lipoxygenase production by Trichoderma reesei and Pichia pastoris transformants expressing the lipoxygenase gene of the fungus Gaeumannomyces graminis. In both cases lipoxygenase production in the transformants could be identified. For detection of the G. graminis lipoxygenase produced by Aspergillus nidulans the potassium iodide-starch method was successful. When Escherichia coli was grown on agar and soybean lipoxygenase was applied on the culture lipoxygenase activity could clearly be detected by the indamine dye formation method. This suggests that the method has potential for screening of metagenomic libraries in E. coli for lipoxygenase activity.

Cloning and expression of three lipoxygenase genes from liverwort, Marchantia polymorpha L., in Escherichia coli

Three genes homologous to plant lipoxygenase genes were identified from the EST libraries of Marchantia polymorpha, in order to clarify the function of LOXs in bryophytes. Full-length genes were isolated using 5′- and 3′-RACE methods and named MpLOX1, MpLOX2, and MpLOX3, respectively. To investigate the enzymatic activities of liverwort LOXs, recombinant MpLOX1, MpLOX2, and MpLOX3 proteins were prepared from Escherichia coli cells expressing the corresponding gene. LC–MS/MS analyses and chiral column chromatography of their reaction products showed that MpLOX1 codes for 11S/15S-lipoxygenase against eicosapentaenoic acid and for 15S-lipoxygenase against arachidonic acid, and that MpLOX2 and MpLOX3 code for 15S-lipoxygenase against eicosapentaenoic and arachidonic acids. Phylogenetic analysis showed that the liverwort lipoxygenase genes separated from the ancestor of higher plants in the early stages of plant evolution. Quantification analyses suggested that arachidonic acid and eicosapentaenoic acid were preferred substrates. Furthermore, each liverwort lipoxygenase exhibited highest activity at pH 7.0 and dependency on Ca2+ ion in the oxygenation reaction.

An ex vivo model of severe asthma using reconstituted human bronchial epithelium

Structural changes to the airways are features of severe asthma. The bronchial epithelium facilitates this remodeling process. Learning about the changes that develop in the airway epithelium could improve our understanding of asthma pathogenesis and lead to new therapeutic approaches. We sought to determine the feasibility and relevance of air-liquid interface cultures of bronchial epithelium derived from endobronchial biopsy specimens of patients with different severities of asthma for studying the airway epithelium. Human bronchial epithelial cells derived from endobronchial biopsy specimens of patients with mild and severe asthma were maintained in culture for 21 days in an air-liquid interface to reproduce a fully differentiated airway epithelium. Initially, features of remodeling that included epithelial and subepithelial layers, as well as mucus production, were assessed in paraffin-embedded endobronchial biopsy specimens to evaluate morphologic characteristics of asthmatic patients' epithelia. Ex vivo differentiated epithelia were then analyzed for morphology and function based on ultrastructural analysis, IL-8 release, lipoxin A(4) generation, mucin production, and lipoxygenase gene expression. Morphologic and inflammatory imbalances initially observed in endobronchial biopsy specimens obtained from patients with severe or mild asthma persisted in the air-liquid interface reconstituted epithelium throughout the differentiation process to 21 days. Epithelium from patients with severe asthma produced greater levels of mucin, released more IL-8, and produced lower levels of lipoxin A(4) than that from patients with mild asthma. Expression of 15-lipoxygenase 2 was increased in epithelium from patients with severe asthma, whereas expression levels of MUC5AC, MUC5B, 5-lipoxygenase, and 15-lipoxygeanse 1 were similar to those of patients with mild asthma. Ex vivo cultures of fully differentiated bronchial epithelium from endobronchial biopsy specimens maintain inherent phenotypic differences specifically related to the severity of asthma.

Development of Functional Markers for a Lipoxygenase Gene TaLox‐B1 on Chromosome 4BS in Common Wheat

ABSTRACTLipoxygenase (LOX) activity in grain influences the color and processing quality of wheat (Triticum spp.)‐based products. Characterization of LOX genes and development of functional markers are of importance for marker‐assisted selection in common wheat (Triticum aestivum L.) breeding. In the present study, the full‐length genomic DNA (gDNA) sequence of a LOX gene (designated TaLox‐B1) located on chromosome 4BS was characterized by in silico cloning and experimental validation. Two complementary dominant sequence tagged site (STS) markers, LOX16 and LOX18, were developed based on the single nucleotide polymorphism (SNP) of two alleles at the TaLox‐B1 locus, amplifying 489‐ and 791‐bp fragments in cultivars with higher and lower LOX activities, respectively. The two markers were mapped on chromosome 4BS using a doubled haploid (DH) population derived from Zhongyou 9507 × CA9632, a set of Chinese Spring nullisomic–tetrasomic lines, ditelosomic line 4BS, and Langdon (LDN) 4D(4B) chromosome substitution line. Quantitative trait loci (QTL) analysis indicated that TaLox‐B1 co‐segregated with the two functional markers and was closely linked to simple sequence repeat (SSR) locus Xgwm251 on chromosome 4BS with a genetic distance of 1.8 cM. LOX16 and LOX18 were validated on 198 Chinese wheat cultivars and advanced lines and showed highly significant (p &lt; 0.01) associations with LOX activity. These results suggested that LOX16 and LOX18, co‐segregating with TaLox‐B1, could be used for the improvement of color attributes of noodles and other wheat‐based products in wheat breeding programs.

The Expression Profiling of the Lipoxygenase (LOX) Family Genes During Fruit Development, Abiotic Stress and Hormonal Treatments in Cucumber (Cucumis sativus L.)

Lipoxygenases (LOXs) are non-haem iron-containing dioxygenases that catalyse oxygenation of polyunsaturated fatty acids and lipids to initiate the formation of a group of biologically active compounds called oxylipins. Plant oxylipins play important and diverse functions in the cells. In the current study, expression analysis during cucumber development using semi-quantitative RT-PCR revealed that 13 of 23 CsLOX genes were detectable, and were tissue specific or preferential accumulation. In total, 12 genes were found to be differentially expressed during fruit development and have different patterns of expression in exocarp, endocarp and pulp at day 5 after anthesis. The expression analysis of these 12 cucumber LOX genes in response to abiotic stresses and plant growth regulator treatments revealed their differential transcript in response to more than one treatment, indicating their diverse functions in abiotic stress and hormone responses. Results suggest that in cucumber the expanded LOX genes may play more diverse roles in life cycle and comprehensive data generated will be helpful in conducting functional genomic studies to understand their precise roles in cucumber fruit development and stress responses.

Extracellular production of lipoxygenase from Anabaena sp. PCC 7120 in Bacillus subtilis and its effect on wheat protein

In this study, the lipoxygenase (ana-LOX) gene from Anabaena sp. PCC 7120 was successful expressed and secreted in Bacillus subtilis. Under the control of the P43 promoter, with a signal peptide from the B. subtilis 168 nprB gene, and facilitated by the molecular chaperone PrsA, the production of the recombinant ana-LOX (ana-rLOX)reached 76 U/mL (171.9 μg/ml) in the supernatant. The purified ana-rLOX was investigated for its effect on dough protein. Ana-rLOX treatment decreased free sulfhydryl groups, increased glutenin macropolymer content, promoted the formation of covalent bonds between gluten protein, and affected protein crosslinking. The results indicated that large aggregates involving gliadin and glutenin were formed. The glutenin macropolymer played a role in the formation of the dough network structure through the exchange of thiol disulfide bonds and the formation of hydrogen or hydrophobic bonds with other proteins.

Molecular sequence variations of the lipoxygenase-2 gene in soybean

Soybean lipoxygenase genes comprise a multi-gene family, with the seed lipoxygenase isozymes LOX1, LOX2, and LOX3 present in soybean seeds. Among these, the LOX2 isozyme is primarily responsible for the "beany" flavor of most soybean seeds. The variety, Jinpumkong 2, having null alleles (lx1, lx2, and lx3) lacks the three seed lipoxygenases; so, sequence variations between the lipoxygenase-2 genes of Pureunkong (Lx2) and Jinpumkong 2 (lx2) cultivars were examined. One indel, four single nucleotide polymorphisms (SNPs), a 175-bp fragment in the 5'-flanking sequence, and a missense mutation within the coding region were found in Jinpumkong 2. The distribution of the sequence variations was investigated among 90 recombinant inbred lines (RILs) derived from a cross of Pureunkong × Jinpumkong 2 and in 480 germplasm accessions with various origins and maturity groups. Evidence for a genetic bottleneck was observed: the 175-bp fragment was rare in Glycine max, but present in the majority of the G. soja accessions. Furthermore, the 175-bp fragment was not detected in the 5' upstream region of the Lx2 gene on chromosome (Chr) 13 in Williams 82; instead, a similar 175-bp fragment was positioned in the homeologous region on Chr 15. The findings indicated that the novel fragment identified was originally present in the Lx2 region prior to the recent genome duplication in soybean, but became rare in the G. max gene pool. The missense mutation of the conserved histidine residue of the lx2 allele was developed into a single nucleotide-amplified polymorphism (SNAP) marker. The missense mutation showed a perfect correlation with the LOX2-lacking phenotype, so the SNAP marker is expected to facilitate breeding of soybean cultivars which lack the LOX2 isozyme.

Phytoplasma protein effector SAP11 enhances insect vector reproduction by manipulating plant development and defense hormone biosynthesis

Phytoplasmas are insect-transmitted phytopathogenic bacteria that can alter plant morphology and the longevity and reproduction rates and behavior of their insect vectors. There are various examples of animal and plant parasites that alter the host phenotype to attract insect vectors, but it is unclear how these parasites accomplish this. We hypothesized that phytoplasmas produce effectors that modulate specific targets in their hosts leading to the changes in plant development and insect performance. Previously, we sequenced and mined the genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB) and identified 56 candidate effectors. Here, we report that the secreted AY-WB protein 11 (SAP11) effector modulates plant defense responses to the advantage of the AY-WB insect vector Macrosteles quadrilineatus. SAP11 binds and destabilizes Arabidopsis CINCINNATA (CIN)-related TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORS 1 and 2 (TCP) transcription factors, which control plant development and promote the expression of lipoxygenase (LOX) genes involved in jasmonate (JA) synthesis. Both the Arabidopsis SAP11 lines and AY-WB-infected plants produce less JA on wounding. Furthermore, the AY-WB insect vector produces more offspring on AY-WB-infected plants, SAP11 transgenic lines, and plants impaired in CIN-TCP and JA synthesis. Thus, SAP11-mediated destabilization of CIN-TCPs leads to the down-regulation of LOX2 expression and JA synthesis and an increase in M. quadrilineatus progeny. Phytoplasmas are obligate inhabitants of their plant host and insect vectors, in which the latter transmits AY-WB to a diverse range of plant species. This finding demonstrates that pathogen effectors can reach beyond the pathogen-host interface to modulate a third organism in the biological interaction.

Biocontrol of black scurf on potato by seed tuber treatment with Pythium oligandrum

The biological control activity of Pythium oligandrum against black scurf of potato caused by Rhizoctonia solani AG-3 was evaluated in field experiments after treatment of potato seed tubers with P. oligandrum. Seed tubers infected with black scurf sclerotia were dipped for a few seconds in a suspension of 103, 104 or 105mL−1P. oligandrum oospores and were then air-dried. Each level of P. oligandrum-treatment significantly reduced the disease rates of stolon at a level similar to that achieved by chemical control. When P. oligandrum populations adherent to the surface of seed tubers were determined, oospore counts on tubers treated with 104 or 105oosporesmL−1 were about 540/cm2 or about 22,000/cm2 just after dipping and decreased to about 170/cm2 or 2900/cm2 after a 3-week incubation, respectively. Confocal laser scanning microscopic observation with an immuno-enzymatic staining procedure showed that P. oligandrum hyphae had colonized the sclerotia and established close contact by coiling around the R. solani hyphae present on the surface of seed tubers, in a manner similar to that observed in the dual-culture test. Quantification of R. solani DNA by PCR indicated that the R. solani population was reduced on the seed tubers treated with P. oligandrum compared to untreated tubers. Furthermore, the ability of P. oligandrum to induce resistance against black scurf was determined using a potato tuber disk assay. Treatment of tuber disks with the cell wall protein fraction of P. oligandrum enhanced the expression of defense-related genes such as 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, lipoxygenase and basic PR-6 genes, and reduced disease severity upon challenge with R. solani compared with untreated controls. These results suggest that biocontrol mechanisms employed by P. oligandrum against black scurf involve both mycoparasitism and induced resistance.