Lipoxygenase Gene Research Articles

Structural and Functional Biology of Mammalian ALOX Isoforms with Particular Emphasis on Enzyme Dimerization and Their Allosteric Properties

The human genome involves six functional arachidonic acid (AA) lipoxygenase (ALOX) genes, and the corresponding enzymes (ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5) have been implicated in cell differentiations and in the pathogenesis of inflammatory, hyperproliferative, metabolic, and neurological disorders. Humans express two different AA 15-lipoxygenating ALOX isoforms, and these enzymes are called ALOX15 (15-LOX1) and ALOX15B (15-LOX2). Chromosomal localization, sequence alignments, and comparison of the enzyme properties suggest that pig and mouse ALOX15 orthologs (leukocyte-type 12-LOX) on the one hand and rabbit and human ALOX15 orthologs on the other (reticulocyte-type 15-LOX1) belong to the same enzyme family despite their different reaction specificities with AA as a substrate. In contrast, human ALOX12 (platelet-type 12-LOX), as well as pig and mouse ALOX15 (leukocyte-type 12-LOX), belong to different enzyme families, although they exhibit a similar reaction specificity with AA as a substrate. The complex multiplicity of mammalian ALOX isoforms and the controversial enzyme nomenclatures are highly confusing and prompted us to summarize the current knowledge on the biological functions, enzymatic properties, and allosteric regulation mechanisms of mammalian ALOX15, ALOX15B, and ALOX12 orthologs that belong to three different enzyme sub-families.

Evolution of the lipoxygenase gene family in Pyropia haitanensis and its response to biotic and abiotic stresses

Macroalgae contain a chemically diverse and unique set of oxylipins that derived from lipid peroxidation play pivotal roles in response to various environmental stresses in the intertidal zone. Specific enzymes involved in biosynthesizing these oxylipins remain largely unknown. Lipoxygenase (LOX) is a key enzyme in the oxylipin biosynthetic pathway. Here, six homologous LOX genes were identified in Pyropia haitanensis. To investigate the evolutionary status of the LOX gene family in red algae and its role in the adaptation of P. haitanensis to the intertidal environment, this study analyzed the evolution, structure, and enzymatic activities of these genes. Phylogenetic analysis and homologous sequence alignment indicated that the P. haitanensis LOX genes had two evolutionary origins. PhLOX and PhLOX2 possess an SRPBCC domain at the N-terminus that may have been acquired from marine bacteria through horizontal gene transfer, while PhLOX3–6 may have been acquired from cyanobacteria. Cis-Regulatory analysis revealed numerous elements upstream of the PhLOX genes associated with abiotic stress and hormone regulation, as well as binding sites for defense-related transcription factors. The in-vitro enzymatic activity of PhLOXs was detected, revealing that PhLOX and PhLOX2 exhibited the highest activities. PhLOX3–6 showed a substrate preference for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), but it had lower activity. The transcriptional expression levels of the six PhLOX genes varied under different biotic and abiotic stresses. For example, all PhLOX genes were significantly upregulated after mechanical damage, while PhLOX5 responded to low temperatures. In summary, P. haitanensis acquired a series of LOX genes from marine bacteria and cyanobacteria during its evolutionary history, among which PhLOX and PhLOX2 were selected as the likely primary functional genes. Its functional diversity to cope with various environmental stresses is achieved through the complex transcriptional regulation of PhLOX genes.

Genome-Wide Identification of LOX Gene Family and Its Expression Analysis under Abiotic Stress in Potato (Solanum tuberosum L.).

The lipoxygenases (LOXs) are non-heme iron-containing dioxygenases that play an important role in plant growth and defense responses. There is scarce knowledge regarding the LOX gene family members and their involvement in biotic and abiotic stresses in potato. In this study, a total of 17 gene family members (StLOXs) in potato were identified and clustered into three subfamilies: 9-LOX type I, 13-LOX type I, and 13-LOX type II, with eleven, one, and five members in each subfamily based on phylogenetic analysis. By exploiting the RNA-seq data in the Potato Genome Sequencing Consortium (PGSC) database, the tissue-specific expressed and stress-responsive StLOX genes in double-monoploid (DM) potato were obtained. Furthermore, six candidate StLOX genes that might participate in drought and salt response were determined via qPCR analysis in tetraploid potato cultivars under NaCl and PEG treatment. Finally, the involvement in salt stress response of two StLOX genes, which were significantly up-regulated in both DM and tetraploid potato under NaCl and PEG treatment, was confirmed via heterologous expression in yeast under salt treatment. Our comprehensive analysis of the StLOX family provides a theoretical basis for the potential biological functions of StLOXs in the adaptation mechanisms of potato to stress conditions.

The Role of MaWRKY70 in Regulating Lipoxygenase Gene Transcription during Chilling Injury Development in Banana Fruit.

Banana is a typical cold-sensitive fruit; it is prone to chilling injury (CI), resulting in a quality deterioration and commodity reduction. However, the molecular mechanism underlying CI development is unclear. In this study, cold storage (7 °C for 5 days) was used to induce CI symptoms in bananas. As compared with the control storage (22 °C for 5 days), cold storage increased the CI index and cell membrane permeability. Moreover, we found that the expression levels of the WRKY transcription factor MaWRKY70 were increased consistently with the progression of CI development. A subcellular localization assay revealed that MaWRKY70 was localized in the nucleus. Transcriptional activation analyses showed that MaWRKY70 processed a transactivation ability. Further, an electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter (DLR) assays showed that MaWRKY70 was directly bound to the W-box motifs in the promoters of four lipoxygenase (LOX) genes associated with membrane lipid degradation and activated their transcription. Collectively, these findings demonstrate that MaWRKY70 activates the transcription of MaLOXs, thereby acting as a possible positive modulator of postharvest CI development in banana fruit.

A genome-wide study of the lipoxygenase gene families in Medicago truncatula and Medicago sativa reveals that MtLOX24 participates in the methyl jasmonate response

BackgroundLipoxygenase (LOX) is a multifunctional enzyme that is primarily related to plant organ growth and development, biotic and abiotic stress responses, and production of flavor-associated metabolites. In higher plants, the LOX family encompasses several isozymes with varying expression patterns between tissues and developmental stages. These affect processes including seed germination, seed storage, seedling growth, fruit ripening, and leaf senescence. LOX family genes have multiple functions in response to hormones such as methyl jasmonate (MeJA) and salicylic acid.ResultsIn this study, we identified 30 and 95 LOX homologs in Medicago truncatula and Medicago sativa, respectively. These genes were characterized with analyses of their basic physical and chemical properties, structures, chromosomal distributions, and phylogenetic relationships to understand structural variations and their physical locations. Phylogenetic analysis was conducted for members of the three LOX subfamilies (9-LOX, type I 13-LOX, and type II 13-LOX) in Arabidopsis thaliana, Glycine max, M. truncatula, and M. sativa. Analysis of predicted promoter elements revealed several relevant cis-acting elements in MtLOX and MsLOX genes, including abscisic acid (ABA) response elements (ABREs), MeJA response elements (CGTCA-motifs), and antioxidant response elements (AREs). Cis-element data combined with transcriptomic data demonstrated that LOX gene family members in these species were most likely related to abiotic stress responses, hormone responses, and plant development. Gene expression patterns were confirmed via quantitative reverse transcription PCR. Several MtLOX genes (namely MtLOX15, MtLOX16, MtLOX20, and MtLOX24) belonging to the type I 13-LOX subfamily and other LOX genes (MtLOX7, MtLOX11, MsLOX23, MsLOX87, MsLOX90, and MsLOX94) showed significantly different expression levels in the flower tissue, suggesting roles in reproductive growth. Type I 13-LOXs (MtLOX16, MtLOX20, MtLOX21, MtLOX24, MsLOX57, MsLOX84, MsLOX85, and MsLOX94) and type II 13-LOXs (MtLOX5, MtLOX6, MtLOX9, MtLOX10, MsLOX18, MsLOX23, and MsLOX30) were MeJA-inducible and were predicted to function in the jasmonic acid signaling pathway. Furthermore, exogenous MtLOX24 expression in Arabidopsis verified that MtLOX24 was involved in MeJA responses, which may be related to insect-induced abiotic stress.ConclusionsWe identified six and four LOX genes specifically expressed in the flowers of M. truncatula and M. sativa, respectively. Eight and seven LOX genes were induced by MeJA in M. truncatula and M. sativa, and the LOX genes identified were mainly distributed in the type I and type II 13-LOX subfamilies. MtLOX24 was up-regulated at 8 h after MeJA induction, and exogenous expression in Arabidopsis demonstrated that MtLOX24 promoted resistance to MeJA-induced stress. This study provides valuable new information regarding the evolutionary history and functions of LOX genes in the genus Medicago.

Fine mapping of ClLOX, a QTL for powdery mildew resistance in watermelon (Citrullus lanatus L.).

ClLOX, is located on chromosome 2 and encodes a lipoxygenase gene, which induced watermelon powdery mildew resistance by inhibiting pathogen spread. Powdery mildew is one of the most severe fungal diseases reducing yield and quality of watermelon (Citrullus lanatus L.) and other cucurbit crops. Genes responsible for powdery mildew resistance in watermelon are highly valuable. In this study, we first identified the QTL pm-lox for powdery mildew resistance in watermelon, located within a 0.93Mb interval of chromosome 2, via XP-GWAS method using two F2 populations. The F2:3 families from one of the F2 populations were then used for fine-mapping the pm-lox locus into a 9,883bp physical region between 29,581,906 and 29,591,789, containing only two annotated genes. Of these, only ClG42_02g0161300 showed a significant differential expression between the resistant and susceptible lines after powdery mildew inoculation based on RNA sequencing (RNA-seq) and qRT-PCR analysis, and is designated ClLOX. Derived Cleaved Amplified Polymorphic Sequence (dCAPs) markers were developed and validated. In addition, our tests showed that the resistance was anti-spread rather than anti-infection of the pathogen. This study identified a new resistance gene (ClLOX), provided insights into the mechanism of powdery mildew resistance, and developed a molecular marker for watermelon breeding.

Comprehensive phytohormone metabolomic and transcriptomic analysis of tobacco (Nicotiana tabacum) infected by tomato spotted wilt virus (TSWV)

Tomato spotted wilt virus (TSWV) is ranked among the top 10 most destructive viruses globally. It results in abnormal leaf growth, stunting, and even death, significantly affecting crop yield and quality. Phytohormones play a crucial role in regulating plant-virus interactions. However, there is still limited research on the effect of TSWV on phytohormone levels, particularly growth hormones and genes involved in the phytohormone pathway. In our study, we combined phytohormone metabolomics and transcriptomics to examine the impact of TSWV infection on phytohormone content and gene expression profile. Metabolomic results showed that 41 metabolites, including major phytohormones and their precursors and derivatives were significantly altered after 14 days of TSWV inoculation tobacco plants cvK326, with 31 being significantly increased and 10 significantly reduced. Specifically, the levels of abscisic acid (ABA) and jasmonoyl-isoleucine (JA-Ile) were significantly reduced. The levels of indole-3-acetic acid (IAA) have remained unchanged. However, the levels of cytokinin isopentenyladenine (iP) and salicylic acid (SA) significantly increased. The transcriptome analysis revealed 2,746 genes with significant changes in expression. Out of these, 1,072 genes were significantly downregulated, while 1,674 genes were significantly upregulated. Among them, genes involved in ABA synthesis and signaling pathways, such as 9-cis-epoxycarotenoid dioxygenase (NCED), protein phosphatase 2C (PP2C), serine/threonine-protein kinase (SnRK2), and abscisic acid responsive element binding factor (ABF), exhibited significant downregulation. Additionally, expression of the lipoxygenase gene LOX, Jasmonate ZIM domain-containing protein gene JAZ, and transcription factor gene MYC were significantly down-regulated. In the cytokinin pathway, while there were no significant changes in the expression of the cytokinin synthesis genes, a significant downregulation of transcriptionally active factor type-B response regulators (type-B RRs) was observed. In terms of SA synthesis and signaling pathways, the isochorismate synthase gene ICS1 and the pathogenesis-related gene PR1 were significantly upregulated. These results can strengthen the theoretical foundation for understanding the interaction between TSWV and tobacco and provide new insights for the future prevention and control of TSWV.

Lipidomics, transcription analysis, and hormone profiling unveil the role of CsLOX6 in MeJA biosynthesis during black tea processing.

Jasmonates, such as jasmonic acid (JA) and methyl jasmonate (MeJA), are crucial aspect of black tea quality. However, lipids species, hormones, and genes regulated mechanism in the jasmonate biosynthesis during black tea processing are lacking. In this study, we employed lipidomics, hormone metabolism analysis, and transcriptome profiling of genes associated with the MeJA biosynthesis pathway to investigate these factors. The contents of lipids GLs, PLs, and TAG are decreased, accompanied by the main lipids species reduced during black tea processing. Galactolipids, primarily 34:3/36:6/36:3 DGDG and 36:6/36:5/36:4 MGDG, are transformed into massive MeJA and JA in black tea processing, accompanied by the decreased SA, MeSA, IAA, and BA and increased zeatin. Additionally, the transcriptional activity of the primary genes in MeJA biosynthesis pathway exhibited downregulated trends except for AOS and OPR and non-primary genes tend to be a little high or have fluctuation of expression. Coordinated expression of main CsHPL (TEA008699), CsAOS (TEA001041), and CsJMT (TEA015791) control the flow of lipids degradation and MeJA production. A strong infected reduction of a key lipoxygenase gene, CsLOX6 (TEA009423), in tea buds significantly reduced the level of jasmonates and expression of downstream genes, accompanied by SA, MeSA level rising, and ABA declining. We have identified a key CsLOX6, as well as established galactolipids, mainly 34:3/36:6/36:3 DGDG and 36:6/36:5/36:4 MGDG, sources for MeJA biosynthesis regulated by dynamics hormone and controlled by coordinated expressed CsHPL (TEA008699), CsAOS (TEA001041), and CsJMT (TEA015791). Our findings provide a theoretical basis for breeding high-quality black tea and offer valuable insights for improving processing methods.

Phylogenetic Analysis and Lipoxygenase (LOX) Gene Family Variation in The Pistachio

Lipoxygenases (LOX) gene family is a type of nonheme iron-containing dioxygenases, which has a very important aspect in plant development and fruit quality. LOX gene, which is responsible for lipid oxidation, the main role for the biosynthesis pathway of unsaturated fatty acids. Although some studies have investigated the LOX gene family in several species including arabidopsis, soybean, peanut and apple, there is no information from Pistachio; and the phylogeny of this gene family in the Pistacia genus is still not determined. In this study, Arabidopsis thaliana LOX1 gene (NCBI Reference Sequence: NM_104376.3) was selected and used as a query sequence for performing a BLASTN search. Among all sequence query which was found by NCBI platform, 9 sequences were selected for further analysis. Phylogenetic tree of full-length LOX gene sequences from the Pistacia genus was constructed using the Maximum Likelihood method with MEGA software. By using phylogenic analysis, we identified variations in gene structure and revealed the phylogenetic evolutionary relationship of this gene family. Additionally, this may serve as a reference value for assessing the genetic relationships among various LOX genes in Pistacia genus species. This variations provides us the possibility the design the primer to achieve us to find the exact LOX gene in Pistacia genus and future research on the evolutionary history and transgenic research on LOX genes.

Identification, characterization, and expression of lipoxygenase genes in sweet cherry (Prunus avium L.) cv. Regina and their relationship with the development of an herbaceous off-flavor during fruit ripening

Flavor is an essential characteristic of fruit quality and is significant for consumers. Off-flavors have been reported in several fruits, including sweet cherry. This fruit has been reported to show an herbaceous/grassy-like flavor. The herbaceous off-flavor in sweet cherries detected in cultivar Regina has been related to the differential development of aroma compounds such as short-chain aldehydes and esters. One of the main biosynthesis pathways for these compounds is the fatty acid oxidation mediated by lipoxygenases (LOX). In order to have a better understanding of the biological basis of the differences in the volatile profile, the LOX gene expression profile was characterized during fruit development with and without herbaceous off-flavor. A genome-wide analysis of LOX in sweet cherry was carried out and compared to other species such as Arabidopsis, tomato, apple, prunus and strawberry. The structural features of 9-LOX and 13-LOX genes, encoded protein domains and their synteny were examined. Moreover, we analyzed the LOX expression at four developmental stages along ripening by RT-qPCR. Thirteen LOX gene candidates (six 9-LOX and seven 13-LOX) were identified. The 13-LOXs, PaLOX10, PaLOX11, and PaLOX12 were differentially expressed in herbaceous sweet cherries. Furthermore, their expression profile positively correlated with key volatile compounds linked to the herbaceous off-flavor. Overall, this study involves the genome-wide characterization of the LOX family in Prunus avium cv. Regina and provides information that can aid in studying LOX-related fruit deterioration in sweet cherries and associated species.

Molecular, in silico and expression studies on lipoxygenases (LOXs) in potato (Solanum tuberosum L.).

Lipoxygenases (LOXs) namely 9-LOXs and 13-LOXs catalyse the oxygenation of polyunsaturated fatty acids to produce fatty acid hydroperoxides which are crucial in growth, development and stress responses in plants. Here, we isolated and characterized a 2723-bp cDNA encoding a distinct 861-aa 9-LOX form, designated StKCLX-1, using tuber total RNA from an Indian potato cultivar, Kufri Chipsona-1 through RT-PCR. A total of 17 LOX genes distributed in different chromosomes were identified and characterized in the potato genome. Multiple sequence alignment revealed highly conserved amino acids in the crucial domains, motifs and variable N-terminal regions between the LOX classes. A total of 36 LOXs from potato, tomato and Arabidopsis were used in phylogenetic analysis. A 3-D structure of StKCLX-1 was predicted by AlphaFold tool, validated through the predicted local-distance difference test (pLDDT) and Ramachandran Plot. Molecular docking predicted the nature of receptor-ligand interactions. STRING database was used to predict the protein-protein interactions. Expression patterns of the LOXs in the potato organs were examined by Expression Atlas and semi-quantitative RT-PCR. 9-LOX activity was noticed at early stages of tuberization, and significantly increased in the freshly-harvested mature tubers. This report would be useful in gaining insights into the structure-function relationships of the LOXs and corresponding multigene family-prerequisites for understanding tuber development in potato.

DNA methylation and lipid metabolism are involved in GA-induced maize aleurone layers PCD as revealed by transcriptome analysis

BackgroundThe aleurone layer is a part of many plant seeds, and during seed germination, aleurone cells undergo PCD, which is promoted by GA from the embryo. However, the numerous components of the GA signaling pathway that mediate PCD of the aleurone layers remain to be identified. Few genes and transcriptomes have been studied thus far in aleurone layers to improve our understanding of how PCD occurs and how the regulatory mechanism functions during PCD. Our previous studies have shown that histone deacetylases (HDACs) are required in GA-induced PCD of aleurone layer. To further explore the molecular mechanisms by which epigenetic modifications regulate aleurone PCD, we performed a global comparative transcriptome analysis of embryoless aleurones treated with GA or histone acetylase (HAT) inhibitors.ResultsIn this study, a total of 7,919 differentially expressed genes (DEGs) were analyzed, 2,554 DEGs of which were found to be common under two treatments. These identified DEGs were involved in various biological processes, including DNA methylation, lipid metabolism and ROS signaling. Further investigations revealed that inhibition of DNA methyltransferases prevented aleurone PCD, suggesting that active DNA methylation plays a role in regulating aleurone PCD. GA or HAT inhibitor induced lipoxygenase gene expression, leading to lipid degradation, but this process was not affected by DNA methylation. However, DNA methylation inhibitor could regulate ROS-related gene expression and inhibit GA-induced production of hydrogen peroxide (H2O2).ConclusionOverall, linking of lipoxygenase, DNA methylation, and H2O2 may indicate that GA-induced higher HDAC activity in aleurones causes breakdown of lipids via regulating lipoxygenase gene expression, and increased DNA methylation positively mediates H2O2 production; thus, DNA methylation and lipid metabolism pathways may represent an important and complex signaling network in maize aleurone PCD.

Putative NAD(P)-Binding Rossmann Fold Protein Is Involved in Chitosan-Induced Peroxidase Activity and Lipoxygenase Expression in Physcomitrium patens.

Oxidative burst, the rapid production of high levels of reactive oxygen species in response to external stimuli, is an early defense reaction against pathogens. The fungal elicitor chitosan causes an oxidative burst in the moss Physcomitrium patens (formerly Physcomitrella patens), mainly due to the peroxidase enzyme Prx34. To better understand the chitosan responses in P. patens, we conducted a screen of part of a P. patens mutant collection to isolate plants with less peroxidase activity than wild-type (WT) plants after chitosan treatment. We isolated a P.patens mutant that affected the gene encoding NAD(P)-binding Rossmann fold protein (hereafter, Rossmann fold protein). Three Rossmann fold protein-knockout (KO) plants (named Rossmann fold KO lines) were generated and used to assess extracellular peroxidase activity and expression of defense-responsive genes, including alternative oxidase, lipoxygenase (LOX), NADPH oxidase, and peroxidase (Prx34) in response to chitosan treatment. Extracellular (apoplastic) peroxidase activity was significantly lower in Rossmann fold KO lines than in WT plants after chitosan treatments. Expression of the LOX gene in Rossmann fold KO plants was significantly lower before and after chitosan treatment when compared with WT. Peroxidase activity assays together with gene expression analyses suggest that the Rossmann fold protein might be an important component of the signaling pathway leading to oxidative burst and basal expression of the LOX gene in P. patens. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome-Wide Identification and Expression Analysis of Lipoxygenase Genes in Rose (Rosa chinensis).

Lipoxygenases (LOX) play pivotal roles in plant resistance to stresses. However, no study has been conducted on LOX gene identification at the whole genome scale in rose (Rosa chinensis). In this study, a total of 17 RcLOX members were identified in the rose genome. The members could be classified into three groups: 9-LOX, Type I 13-LOX, and Type II 13-LOX. Similar gene structures and protein domains can be found in RcLOX members. The RcLOX genes were spread among all seven chromosomes, with unbalanced distributions, and several tandem and proximal duplication events were found among RcLOX members. Expressions of the RcLOX genes were tissue-specific, while every RcLOX gene could be detected in at least one tissue. The expression levels of most RcLOX genes could be up-regulated by aphid infestation, suggesting potential roles in aphid resistance. Our study offers a systematic analysis of the RcLOX genes in rose, providing useful information not only for further gene cloning and functional exploration but also for the study of aphid resistance.

Abscisic acid- and ethylene-induced abscission of yellow lupine flowers is mediated by jasmonates

The appropriate timing of organ abscission determines plant growth, development, reproductive success, and yield in relation to crop species. Among these, yellow lupine is an example of a crop species that loses many fully developed flowers, which limits the formation of pods with high-protein seeds and affects its economic value. Lupine flower abscission, similarly to the separation of other organs, depends on a complex regulatory network functioning in the cells of the abscission zone (AZ). In the present study, genetic, biochemical, and cellular methods were used to highlight the complexity of the interactions among strong hormonal stimulators of abscission, including abscisic acid (ABA), ethylene, and jasmonates (JAs) precisely in the AZ cells, with all results supporting that the JA-related pathway has an important role in the phytohormonal cross-talk leading to flower abscission in yellow lupine. Based on obtained results, we conclude that ABA and ET have positive influence on JAs biosynthesis and signaling pathway in time-dependent manner. Both phytohormones changes lipoxygenase (LOX) gene expression, affects LOX protein abundance, and JA accumulation in AZ cells. We have also shown that the signaling pathway of JA is highly sensitive to ABA and ET, given the accumulation of COI1 receptor and MYC2 transcription factor in response to these phytohormones. The results presented provide novel information about the JAs-dependent separation of organs and provide insight and details about the phytohormone-related mechanisms of lupine flower abscission.

Identification and characterization of lipoxygenase (LOX) genes involved in abiotic stresses in yellow horn.

Lipoxygenase (LOX) gene plays an essential role in plant growth, development, and stress response. 15 LOX genes were identified, which were unevenly distributed on chromosomes and divided into three subclasses in this study. In promoter region analysis, many cis-elements were identified in growth and development, abiotic stress response, hormonal response, and light response. qRT-PCR showed that the LOX gene showed tissue specificity in seven tissues, especially XsLOX1, 3, and 7 were relatively highly expressed in roots, stems, and axillary buds. The different expression patterns of LOX genes in response to abiotic stress and hormone treatment indicate that different XsLOX genes have different reactions to these stresses and play diversified roles. This study improves our understanding of the mechanism of LOX regulation in plant growth, development, and stress and lays a foundation for further analysis of biological functions.

Polymorphisms in Cyclooxygenase, Lipoxygenase and TP53 genes predict colorectal polyp risk reduction by aspirin in the seAFOod polyp prevention trial.

Aspirin and eicosapentaenoic acid (EPA) reduce colorectal adenomatous polyp risk and affect synthesis of oxylipins including prostaglandin E2. We investigated whether 35 single nucleotide polymorphisms (SNPs) in oxylipin metabolism genes such as cyclooxygenase [PTGS] and lipoxygenase [ALOX], as well as 7 SNPs already associated with colorectal cancer (CRC) risk reduction by aspirin (eg. TP53; rs104522), modified the effects of aspirin and EPA on colorectal polyp recurrence in the randomised 2x2 factorial seAFOod trial. Treatment effects were reported as the incidence rate ratio (IRR) and 95% confidence interval (CI) by stratifying negative binomial and Poisson regression analyses of colorectal polyp risk on SNP genotype. Statistical significance was reported with adjustment for the false discovery rate as the P and q value. Five hundred and forty-two (of 707) trial participants had both genotype and colonoscopy outcome data. Reduction in colorectal polyp risk in aspirin users compared with non-aspirin users was restricted to rs4837960 (PTGS1) common homozygotes (IRR 0.69 [95%CI 0.53,0.90]; q=0.06), rs2745557 (PTGS2) compound heterozygote-rare homozygotes (IRR 0.60 [0.41,0.88]; q=0.06), rs7090328 (ALOX5) rare homozygotes (IRR 0.27 [0.11,0.64]; q=0.05), rs2073438 (ALOX12) common homozygotes (IRR 0.57 [0.41,0.80]; q=0.05), and rs104522 (TP53) rare homozygotes (IRR 0.37 [0.17,0.79]; q=0.06). No modification of colorectal polyp risk in EPA users was observed. In conclusion, genetic variants relevant to the proposed mechanism of action on oxylipins are associated with differential colorectal polyp risk reduction by aspirin in individuals who develop multiple colorectal polyps. SNP genotypes should be considered during development of personalised, predictive models of CRC chemoprevention by aspirin.

CRISPR/Cas9-mediated lipoxygenase gene-editing in yellow pea leads to major changes in fatty acid and flavor profiles.

Although pulses are nutritious foods containing high amounts of protein, fiber and phytochemicals, their consumption and use in the food industry have been limited due to the formation of unappealing flavors/aromas described as beany, green, and grassy. Lipoxygenase (LOX) enzymes are prevalent among pulse seeds, and their activity can lead to the formation of specific volatile organic compounds (VOCs) from certain polyunsaturated fatty acids (PUFAs). As a widespread issue in legumes, including soybean, these VOCs have been linked to certain unappealing taste perception of foods containing processed pulse seeds. To address this problem in pea and as proof of principle to promote the wider use of pulses, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) construct was designed to create null alleles (knockouts) of PsLOX2 which had been implicated in the generation of VOCs in peas. Successful CRISPR/Cas9-mediated LOX gene editing of stable transgenic pea lines (TGP) was confirmed by DNA sequencing of the wild type (WT) and TGP pslox2 mutant lines. These lines were also assessed for LOX activity, PUFA levels, and VOCs. Compared to WT peas, the TGP lines showed a significant reduction (p < 0.05) in LOX activity and in the concentration of key VOCs, including hexanal, 2-hexenal, heptanal, (E)-2-heptenal, (E,E)-2,4-heptadienal, 1-octen-3-ol, octanal, (E)-2-octenal (E,E)-2,4-nonadienal and furan-2-pentyl. The content of two essential PUFAs, linoleic and α-linolenic acids, the known substrates of LOX in plants, was higher in TGP flours, indicating the efficacy of the CRISPR-mediated gene editing in minimizing their oxidation and the further modification of PUFAs and their products. The collection of VOCs from the headspace of ground pea seeds, using a portable eNose also distinguished the TGP and WT lines. Multiple regression analysis showed that LOX activity correlated with the two VOCs, heptanal and (E,E)-2,4-heptadienal in pea flours. Partial Least Squares Regression (PLS-R) plot for selected PUFAs, VOCs, and sensor responses in WT and TGP lines showed distinct clusters for WT and TGP lines. Together this data demonstrates the utility of CRISPR mediated mutagenesis of PsLOX2 to quickly improve aroma and fatty acid (FA) profiles of pea seeds of an elite Canadian variety.

Bony Fish Arachidonic Acid 15-Lipoxygenases Exhibit Different Catalytic Properties than Their Mammalian Orthologs, Suggesting Functional Enzyme Evolution during Vertebrate Development.

The human genome involves six functional arachidonic acid lipoxygenase (ALOX) genes and the corresponding enzymes (ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5) have been implicated in cell differentiation and in the pathogenesis of inflammatory, hyperproliferative, metabolic, and neurological disorders. In other vertebrates, ALOX-isoforms have also been identified, but they occur less frequently. Since bony fish represent the most abundant subclass of vertebrates, we recently expressed and characterized putative ALOX15 orthologs of three different bony fish species (Nothobranchius furzeri, Pundamilia nyererei, Scleropages formosus). To explore whether these enzymes represent functional equivalents of mammalian ALOX15 orthologs, we here compared a number of structural and functional characteristics of these ALOX-isoforms with those of mammalian enzymes. We found that in contrast to mammalian ALOX15 orthologs, which exhibit a broad substrate specificity, a membrane oxygenase activity, and a special type of dual reaction specificity, the putative bony fish ALOX15 orthologs strongly prefer C20 fatty acids, lack any membrane oxygenase activity and exhibit a different type of dual reaction specificity with arachidonic acid. Moreover, mutagenesis studies indicated that the Triad Concept, which explains the reaction specificity of all mammalian ALOX15 orthologs, is not applicable for the putative bony fish enzymes. The observed functional differences between putative bony fish ALOX15 orthologs and corresponding mammalian enzymes suggest a targeted optimization of the catalytic properties of ALOX15 orthologs during vertebrate development.

Identification of Lipoxygenase gene repertoire of Cannabis sativa and functional characterization of CsLOX13 gene

Lipoxygenase (LOX) enzymes play a pivotal role in the biosynthesis of oxylipins. The phyto-oxilipins have been implicated in diverse aspects of plant biology, from regulating plant growth and development to providing tolerance against biotic and abiotic stresses. C. sativa is renowned for its bioactive secondary metabolites, namely cannabinoids. LOX route is assumed to be involved in the biosynthesis of hexanoic acid, which is one of the precursors of cannabinoids of C. sativa. For obvious reasons, the LOX gene family deserves thorough investigation in the C. sativa. Genome-wide analysis revealed the presence of 21 LOX genes in C. sativa, which can be further grouped into 13-LOX and 9-LOX depending upon their phylogeny as well as the enzyme activity. The promoter regions of the CsLOX genes were predicted to contain cis-acting elements involved in phytohormones responsiveness and stress response. The qRT-PCR-based expression analysis of 21 LOX genes revealed their differential expression in different plant parts (root, stem, young leaf, mature leaf, sugar leaf, and female flower). The majority of CsLOX genes displayed preferential expression in the female flower, which is the primary site for the biosynthesis of cannabinoids. The highest LOX activity and expression level of a jasmonate marker gene were reported in the female flowers among all the plant parts. Several CsLOX genes were found to be upregulated by MeJA treatment. Based on the transient expression in Nicotiana benthamiana and the development of stable Nicotiana tabacum transgenic lines, we demonstrate that CsLOX13 encodes functional lipoxygenase and play an important role in the biosynthesis of oxylipins.