Lipoxygenase Transcripts Research Articles

RNAseq analysis of treatment-dependent signaling changes during inflammation in a mouse cutaneous wound healing model

BackgroundDespite proven therapeutic effects in inflammatory conditions, the specific mechanisms of phytochemical therapies are not well understood. The transcriptome effects of Traumeel (Tr14), a multicomponent natural product, and diclofenac, a non-selective cyclooxygenase (COX) inhibitor, were compared in a mouse cutaneous wound healing model to identify both known and novel pathways for the anti-inflammatory effect of plant-derived natural products.MethodsSkin samples from abraded mice were analyzed by single-molecule, amplification-free RNAseq transcript profiling at 7 points between 12 and 192 h after injury. Immediately after injury, the wounds were treated with either diclofenac, Tr14, or placebo control (n = 7 per group/time). RNAseq levels were compared between treatment and control at each time point using a systems biology approach.ResultsAt early time points (12–36 h), both control and Tr14-treated wounds showed marked increase in the inducible COX2 enzyme mRNA, while diclofenac-treated wounds did not. Tr14, in contrast, modulated lipoxygenase transcripts, especially ALOX12/15, and phospholipases involved in arachidonate metabolism. Notably, Tr14 modulated a group of cell-type specific markers, including the T cell receptor, that could be explained by an overarching effect on the type of cells that were recruited into the wound tissue.ConclusionsTr14 and diclofenac had very different effects on the COX/LOX synthetic pathway after cutaneous wounding. Tr14 allowed normal autoinduction of COX2 mRNA, but suppressed mRNA levels for key enzymes in the leukotriene synthetic pathway. Tr14 appeared to have a broad ‘phytocellular’ effect on the wound transcriptome by altering the balance of cell types present in the wound.

The Rice GLYCINE-RICH PROTEIN 3 Confers Drought Tolerance by Regulating mRNA Stability of ROS Scavenging-Related Genes

BackgroundPlant glycine-rich proteins are categorized into several classes based on their protein structures. The glycine-rich RNA binding proteins (GRPs) are members of class IV subfamily possessing N-terminus RNA-recognition motifs (RRMs) and proposed to be involved in post-transcriptional regulation of its target transcripts. GRPs are involved in developmental process and cellular stress responses, but the molecular mechanisms underlying these regulations are still elusive.ResultsHere, we report the functional characterization of rice GLYCINE-RICH PROTEIN 3 (OsGRP3) and its physiological roles in drought stress response. Both drought stress and ABA induce the expression of OsGRP3. Transgenic plants overexpressing OsGRP3 (OsGRP3OE) exhibited tolerance while knock-down plants (OsGRP3KD) were susceptible to drought compared to the non-transgenic control. In vivo, subcellular localization analysis revealed that OsGRP3-GFP was transported from cytoplasm/nucleus into cytoplasmic foci following exposure to ABA and mannitol treatments. Comparative transcriptomic analysis between OsGRP3OE and OsGRP3KD plants suggests that OsGRP3 is involved in the regulation of the ROS related genes. RNA-immunoprecipitation analysis revealed the associations of OsGRP3 with PATHOGENESIS RELATED GENE 5 (PR5), METALLOTHIONEIN 1d (MT1d), 4,5-DOPA-DIOXYGENASE (DOPA), and LIPOXYGENASE (LOX) transcripts. The half-life analysis showed that PR5 transcripts decayed slower in OsGRP3OE but faster in OsGRP3KD, while MT1d and LOX transcripts decayed faster in OsGRP3OE but slower in OsGRP3KD plants. H2O2 accumulation was reduced in OsGRP3OE and increased in OsGRP3KD plants compared to non-transgenic plants (NT) under drought stress.ConclusionOsGRP3 plays a positive regulator in rice drought tolerance and modulates the transcript level and mRNA stability of stress-responsive genes, including ROS-related genes. Moreover, OsGRP3 contributes to the reduction of ROS accumulation during drought stress. Our results suggested that OsGRP3 alleviates ROS accumulation by regulating ROS-related genes’ mRNA stability under drought stress, which confers drought tolerance.

Potential of zeolite to control Sclerotinia sclerotiorum and Rhizoctonia solani in lettuce and the induction of defence‐related genes

AbstractThe use of zeolite in agriculture as a soil additive has strongly increased in the last decade. However, little experimental evidence exists about the plant protective potential of zeolite and its mode of action. In the present study, we aimed to elucidate the activity of zeolite to control the soil‐borne pathogens Sclerotinia sclerotiorum and Rhizoctonia solani in lettuce. Incorporation of zeolite at a rate of 10% into S. sclerotiorum‐infested soil resulted in significant plant protection, accompanied by increased growth compared with the control plants, whereas zeolite was not effective against R. solani. The plant protective activity of zeolite was studied in association with its potential to induce systemic resistance. For this purpose, the expression of pathogenesis‐related (PR) gene PR1, LIPOXYGENASE (LOX) and ETHYLENE RESPONSE FACTOR 1 (ERF1), marker genes of the salicylic acid (SA), jasmonate (JA) and ethylene (ET) dependent defence, respectively, was assessed. Upon infection of lettuce with S. sclerotiorum, zeolite induced the accumulation of LOX and ERF1 transcripts with time. Therefore, it is suggested that zeolite primes the ET/JA‐dependent defence against S. sclerotiorum. In contrast, zeolite‐treated plants showed decreasing levels of LOX and ERF1 transcripts in plants infected with R. solani with time. This discrepancy in the regulation of plant defence mechanisms may explain the contrasting plant protective activity of zeolite against S. sclerotiorum and R. solani.

Volatile compounds released by maize grains and silks in response to infection by Fusarium verticillioides and its association with pathogen resistance

Many plants respond to fungal infections by producing and/or emitting a specific blend of oxylipins, some of them through the lipoxygenase (LOX) pathway. In vitro bioassays revealed that volatiles from silks and grains of a set of six maize genotypes with variable resistance to Fusarium verticillioides affected fungal growth in different ways. Analyses by solid phase microextraction and GC‐MS showed different volatile profiles in silks and grains of each genotype. Susceptible genotypes with higher concentrations of precursor polyunsaturated fatty acids produced more volatiles, mainly C9 compounds, in comparison to moderately resistant ones. Real‐time PCR from several lipoxygenase transcripts revealed that levels of gene expression depended on the genotype and the inoculation level and suggested that F. verticillioides could use compounds from the 9‐LOX pathway to promote infection in grains. On the other hand, volatiles produced by the 13‐LOX pathway were more important in moderately resistant genotypes and could be associated to field resistance. Results from this set of genotypes indicate that LOX‐regulated volatile compounds might be important in F. verticillioides infection and should be targeted in further research to understand disease resistance.

Cyclic LIPopeptides from Bacillus subtilis ABS-S14 elicit defense-related gene expression in citrus fruit.

Effects of cyclic lipopeptides (CLPs) obtained from Bacillus subtilis ABS-S14 on eliciting defense-related gene transcription and activity of defense-related enzymes; glucanase (GLU), chitinase (CHI), peroxidase (POX) and lipoxygenase (LOX) in Citrus sinensis cv. Valencia fruit were determined. The maximum level of GLU transcripts induced in fruit treated with fengycin was significantly greatest among treatments at 48 h. Surfactin enhanced the LOX and POX transcripts. In parallel, corresponding enzyme activities were correlated with changes in gene expression observed in fruit inoculated with Penicillium digitatum following treatment with individual CLPs. Synergistic effects of fengycin and iturin A, fengycin and surfactin were shown in gene transcript of GLU and CHI, respectively, and surfactin induced POX and LOX gene expression of citrus flavedo without pathogen infection. These results suggest that fengycin and surfactin act as elicitors of defense-related gene expression in “Valencia” fruit following infection.

Transcriptional response of sugarcane roots to methyl jasmonate

The effect of methyl jasmonate treatment on gene expression in sugarcane roots signalling between roots and shoots was studied. A collection of 829 ESTs were obtained from sugarcane roots treated with the defence-regulator methyl jasmonate (MJ) treatment. A subset of 747 of these were combined with 4793 sugarcane ESTs obtained from stem tissues in a cDNA microarray and experiments undertaken to identify genes that were induced in roots 24–120 h following treatment with MJ. Two data analysis systems ( t-statistic and tRMA) were used to analyse the microarray results and these methods identified a common set of 21 ESTs corresponding to transcripts significantly induced by MJ in roots and 23 that were reduced in expression following MJ treatment. The induction of six transcripts identified in the microarray analysis was tested and confirmed using northern blotting. Homologues of genes encoding lipoxygenase and PR-10 proteins were induced 8–24 h after MJ treatment while the other four selected transcripts were induced at later time points. Following treatment of roots with MJ, the lipoxygenase homologue, but not the PR-10 homologue, was induced in untreated stem and leaf tissues. The PR-10 homologue and a PR-1 homologue, but not the lipoxygenase homologue, were induced in untreated tissues after the application of SA to roots. Repeated foliar application of MJ had no apparent effects on plant growth and was demonstrated to increase lipoxygenase transcripts in roots, but did not increase transcript levels of other genes tested. These results lay a foundation for further studies of induced pest and disease resistance in sugarcane roots.

Analysis of lipoxygenase mRNA accumulation in the common bean (Phaseolus vulgaris L.) during development and under stress conditions.

Plant lipoxygenases (LOX, EC 1.13.11.12) have been involved in processes such as stress responses and development. The levels of these enzymes and their corresponding mRNAs are modulated during these processes as well as by different effectors such as jasmonic acid (JA), its methyl ester (MeJA) or abscisic acid (ABA). A new lipoxygenase (LOX) cDNA clone, PvLOX2, was isolated from a Phaseolus vulgaris nodule library and used to study the LOX mRNA accumulation pattern in some developmental stages and in plants subjected to hormone and stress treatments. In nodules, LOX mRNA reaches a maximum level around day 14 to 16 after Rhizobium tropici inoculation, as compared with LOX mRNA present in uninoculated and inoculated roots at the same days. LOX antigen is detected in the nodule parenchyma and in the uninfected cells. During germination, bean LOX transcripts start to accumulate 48 h after imbibition, remains at the same level until 72 h after imbibition and then declines. In hypocotyl, LOX mRNA is abundant in the growing region and almost absent in the mature region. After water stress or ABA treatment, this mRNA increases in the mature region and decreases in the growing region. In bean seedlings, LOX mRNA is accumulated in response to some types of stresses such as cold and desiccation. Wounding, MeJA or ABA treatment of mature leaves also induces LOX mRNA accumulation. These results indicate that in common bean plants LOX is required during development and stress conditions.

Germination-associated lipoxygenase transcripts persist in maturing soybean plants and are induced by jasmonate

Full-length and nearly full-length cDNA clones for the two major lipoxygenase (LOX) transcripts in the axis of germinating soybean ( Glycine max [L.] Merr.) represent transcripts distinct from those of the three seed LOX isozymes. Gene-specific probes revealed that the seedling axis LOX transcripts were absent in developing embryos, while seed LOX-3 transcripts did not accumulate in seedlings or in vegetative tissues. Seedling transcripts appeared in axes between 8 and 16 h after the start of imbihition (seeds germinated 24 h after the start of imbibition) and reached maximal levels at 32 h. One seedling transcript (pTK18) was found in all non-embryonic tissues examined: axes and roots and leaves of two-week plants; the second transcript (pTK11) was also found in vegetative tissues but in reduced levels when compared to pTK18, especially in the leaf. Both transcripts were elevated by methyl jasmonate (MeJA) in the seedling. Thus, gene-specific probes allowed us to monitor the accumulation of two specific LOX transcripts under a variety of developmental and environmental conditions. Their accumulation in the seedling, persistence in vegetative tissues and their deduced consensus signal peptide sequences make them candidates for the MeJA-induced vacuolar vegetative storage protein LOXs. This identification awaits employing gene-specific LOX probes on wounded tissues and those from plants grown under nitrogen excess/deficiency and under water stress.

Complex spatial and temporal expression of lipoxygenase genes during Phaseolus vulgaris (L.) development

A new member (Lox1) of the lipoxygenase (LOX) gene family in French bean was isolated and its specific expression compared to the general expression pattern of related LOX genes. LOX transcripts detected by a French bean cDNA LOX probe were generally abundant in young, developing tissues, and LOX protein, detected on immunoblots showed a similar distribution. Tissue prints showed that cell-specific, locally high amounts of LOX protein were observed in some tissues, for example in parts of the starch sheath in the hypocotyl and a cell layer in the pericarp. The Lox1 gene from Phaseolus vulgaris was shown in genomic Southerns to be present as a single copy per haploid genome. RNase protection studies showed that the gene is active and the transcription start site was mapped by cDNA primer extension. RNase protection studies showed that Lox1 mRNA was present only in flowers and embryonic primary leaves, in stems and most strongly, in young secondary leaves but not in any of the other organs tested (i.e. not in roots, nodules, hypocotyls, cotyledons, older secondary leaves, pericarp or seeds). Lox1 gene transcripts did not accumulate in leaves in response to methyl jasmonate or pathogen inoculation, even though these treatments caused the accumulation of other LOX transcripts. Thus, the various lipoxygenase genes are regulated differentially during French bean development and in response to different stress stimuli, and possible functions are discussed in view of the specific expression patterns observed.

Spatial and temporal accumulation of defense gene transcripts in bean (Phaseolus vulgaris) leaves in relation to bacteria-induced hypersensitive cell death.

Primary leaves of 7- to 9-day-old Red Mexican bean plants were inoculated with virulent or avirulent isolates of Pseudomonas syringae pv. phaseolicola, or saprophytic P. fluorescens either by vacuum infiltration of the whole leaf lamina, or by syringe-inoculation of selected leaf panels. In the incompatible combination, resistance was associated with a hypersensitive response (HR). Syringe-inoculated leaves were sampled in three zones: zone 1, the inoculated leaf area; zone 2, the surrounding 0.5-0.7 cm of leaf tissue; and zone 3, the remainder of the leaf. Northern blots of RNA from zones 1, 2, and 3 were probed with bean cDNAs for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chitinase (CHT), and lipoxygenase (LOX). Accumulation of PAL, CHS, and CHT transcripts was more rapid and generally of greater magnitude in the incompatible than in the compatible interaction and, in both cases, was observed essentially only in zone 1 tissues. Similarly, antibacterial phytoalexins were only detected in zone 1 from the incompatible interaction. Young primary leaves have a background level of LOX transcripts, which declines as leaves age. This decline was accelerated over the first 12 hr postinoculation (hpi) with avirulent bacteria, whereas a weak transient induction, peaking at 5-6 hpi, was observed in the compatible interaction. A subsequent, strong accumulation of LOX transcripts was seen in both the compatible and incompatible interactions outside the inoculation site starting about 14 hpi. LOX transcripts did not accumulate at the inoculation site itself in the incompatible interaction compared to a relatively strong induction in the compatible interaction. Interestingly, inoculation of leaves with cells of the saprophyte P. fluorescens also induced the accumulation of transcripts for CHS, CHT, and LOX, but generally to a lesser degree than in the incompatible interaction. No HR occurred and no macroscopic cell damage was apparent in leaves inoculated with P. fluorescens. However, at the microscopic level individual, trypan blue-stained, necrotic plant cells were visible. In spite of this and the accumulation of CHS transcripts, no phytoalexin accumulation was found up to 48 hr after inoculation. The spatial and temporal relationship of the hypersensitive reaction to defense gene transcript and phytoalexin accumulation is discussed.

The Effects of Wounding and Mite Infestation on Soybean Leaf Lipoxygenase Levels

Numerous studies have demonstrated induction of proteins in plant tissues by wounding and infestations by various pests and pathogens. Lipoxygenase (LOX) is among the proteins that has been found to be induced by pathogens, but detailed information on the induction of LOX has not been reported. We have found a large (up to 10-fold) increase in LOX activity upon wounding of soybean (Glycine max L. Merr.) leaves and variable increases due to feeding of the twospotted spider mite. This induction in LOX activity was reflected in increases in amounts of both LOX protein and transcripts suggesting that the induction was at the transcriptional level. LOX activity was also found to be increased in unwounded leaves from plants with wounded leaves lower on the stem indicating that translocatable factors can cause remote induction of LOX activity.

Two soybean seed lipoxygenase nulls accumulate reduced levels of lipoxygenase transcripts

Soybean (Glycine max L. [Merrill]) seed lipoxygenase cDNA clones were recovered from two cDNA libraries: a size-selected library in pBR322 and an expression library in pUC8. The pUC8 library was made with total poly(A)(+) embryo RNA and was screened with antiserum to lipoxygenase-1, one of 3 seed lipoxygenase isozymes. Three lipoxygenase antigen-producing clones were identified: two with identical cDNA inserts of 977 nucleotides representing an open-reading frame and a third truncated clone bearing a 3' end common to the longer clones. A long clone, pAL-134, was chosen for further study and was used to screen the size-selected cDNA library from which sixteen clones were identified. They fall into two homology classes represented by pLX-10 (ca. 1360 bp) and pLX-65 (2047 bp).The lipoxygenase expression clone pAL-134 hybridized much more strongly to pLX-65 than to pLX-10. pAL-134 and pLX-65 share 89% nucleotide homology and 75% deduced amino acid homology along their common sequence. Their deduced amino acid sequences each show 80% homology to sequences determined for isolated peptides of the lipoxygenase-1 isozyme.pAL-134 hybridizes poorly with a 3.8 kb RNA from LOX-1 null (lx1) embryos while pLX-65 hybridizes more strongly, but still to a lesser extent than its hybridization to standard embryo RNA or to RNA from embryos lacking lipoxygenase-2 (lx2) or lipoxygenase-3 (lx3) protein.The lx3 null lacks almost all embryo 3.8 kb RNA homologous to pLX-10. This hybridization pattern suggests that pLX-10 encodes LOX-3. Thus, the lx1 and lx3 genotypes accumulate little, if any, mRNA for the lipoxygenase-1 and lipoxygenase-3 isozymes, respectively.