From MS/MS library implementation to molecular networks: Exploring oxylipin diversity with NEO-MSMS.

Oxylipins, small polar molecules derived from the peroxidation of polyunsaturated fatty acids (PUFAs), serve as biomarkers for many diseases and play crucial roles in human physiology and inflammation. Despite their significance, many non-enzymatic oxygenated metabolites of PUFAs (NEO-PUFAs) remain poorly reported, resulting in a lack of public datasets of experimental data and limiting their dereplication in further studies. To overcome this limitation, we constructed a high-resolution tandem mass spectrometry (MS/MS) dataset comprising pure NEO-PUFAs (both commercial and self-synthesized) and in vitro free radical-induced oxidation of diverse PUFAs. By employing molecular networking techniques with this dataset and the existent ones in public repositories, we successfully mapped a wide range of NEO-PUFAs, expanding the strategies for annotating oxylipins, and NEO-PUFAs and offering a novel workflow for profiling these molecules in biological samples.

Do meals contain a relevant amount of oxylipins? LC-MS-based analysis of oxidized fatty acids in food.

Several oxylipins are potent lipid mediators and are discussed to be absorbed after oral intake. However, information about their concentrations in oils and processed foods are scarce. Here, we analyzed the concentrations of mono-, di- and multihydroxy- as well as epoxy-PUFA in virgin and refined oils as well as in different foods/meals. Oil refining causes hydrolysis of epoxy-PUFA and thus high dihydroxy-PUFA concentrations (e.g. 15,16-DiHODE 290 µg/g in refined vs. 15 µg/g in virgin rapeseed oil), making the epoxy-to-diol ratio a potential marker for refined oils. Low oxylipin levels were found in foods with high amounts of saturated fatty acids such as Hamburger patties (around 30 µg/g). High concentrations (up to 1200 µg/g, 80 mg per serving) and high oxylipin/precursor-PUFA ratios were found in fried falafel and processed foods such as vegetarian sausage/fish fingers. Our study provides first insights in the oxylipin concentrations of our daily food, indicating a relevant intake.

Analytical Strategy for Oxylipin Annotation by Combining Chemical Derivatization-Based Retention Index Algorithm and Feature Tandem Mass Spectrometric Fragmentation as a Biomarker Discovery Tool.

Accurate oxylipin annotation is crucial for advancing our understanding of physiological processes in health and disease and identifying biomarkers. However, a full view of oxylipins for early diagnosis needs further attention due to the lack of proper analytical methods, which may be attributed to the wide dynamic range, poor sensitivity, extreme molecular complexity, and limited commercially available standards of oxylipins. Here, we devised a novel method by combining a chemical derivatization (CD)-based retention index (RI) algorithm and feature tandem mass spectrometric fragmentation annotation (CD-RI-LC-MS/MS) for identification and quantification of oxylipins. To this end, N,N-diethyl-1,3-diaminopropane (DEPA) was used for fast labeling of oxylipin (within 0.5 min at room temperature) to improve separation resolution and detection sensitivity. The RI algorithm was established to calibrate the retention time variances and assist the identification of oxylipins during liquid chromatography-tandem mass spectrometry (LC-MS) analysis. MS/MS analysis of in total 58 DEPA derivatives of authentic oxylipin standards was subsequently employed to obtain the tandem mass spectrometric feature fragmentation rules for further structure elucidation of the unknown regio-isomers. Finally, a method based upon CD-RI-LC-MS/MS was established for profiling oxylipins from Standard Reference Material (SRM) 1950 human plasma and nonalcoholic fatty liver disease (NAFLD) mouse liver tissue samples. A total of 87 and 96 potential oxylipins including 12 and 14 unreported oxylipins were detected and identified from human plasma and mouse liver tissues, respectively. The results showed that compared to the control group, in the liver samples of the NAFLD mouse, the content levels of prostaglandin (PG) E2, PGF2a, 8-hydroxy-eicosatrienoic acid (8-HETrE), and the newly discovered 2-hydroxy-octadecatrienoic acid (2-HOTrE) were remarkably increased, while the oxidation product of n-3 PUFA (p < 0.05) and all hydroperoxy oxylipins significantly decreased. On balance, this method contributes to future studies on oxylipin screening and application in other biological samples for facilitating the understanding of oxylipin roles in metabolic regulation of numerous diseases.

Determination of oxylipins and their precursors in breast milk by solid phase extraction-ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry.

Oxylipins and their precursors (long-chain polyunsaturated fatty acids, LCPUFAs) are key intercellular signaling molecules influencing the inflammatory response. Each oxylipin has pro- and/or anti-inflammatory effects, and the relative abundance of different oxylipins can alter the inflammatory balance, making it important to clarify the oxylipin profile of breast milk for optimal infant health. The extraction, identification, and simultaneous quantification of oxylipins in breast milk are challenging due to the structural similarity, limited stability, and the low endogenous concentration of oxylipins and the complex matrix of breast milk. This study aimed to develop a solid phase extraction-ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry (SPE-UPLC-MS/MS) method for the comprehensive and specific quantification of oxylipins and their precursors in breast milk. The LC conditions (including column, mobile phase, and gradient conditions) and SPE procedure (including SPE cartridges, elution solvent, and elution volume) were optimized to achieve accurate quantification and better analyte recovery. A single 18-minute chromatographic run allows for the quantification of 20 oxylipins and 5 PUFAs. The results showed good linearity (R2 > 0.99) over the concentration range of 2 to 100 ng/mL, with the instrument detection limits ranging from 0.01 to 0.90 ng/mL for oxylipins and 0.02 to 0.59 ng/mL for PUFAs. The method is rapid, sensitive, and reproducible (RSD ≤ 10%) and is suitable for the quantitative analysis of oxylipins and their precursors in infant formula samples.

Multiplexed targeted analysis of polyunsaturated fatty acids and oxylipins using liquid chromatography-tandem mass spectrometry.

Polyunsaturated fatty acids (PUFAs) and their oxidized products (oxylipins) are important mediators in intra- and extra-cellular signaling. We describe here the simultaneous quantification of 163 PUFAs and oxylipins using liquid chromatography-mass spectrometry (LC-MS). The protocol details steps for PUFA purification from various biological materials, the conditions for LC-MS analysis, as well as quantitative approaches for data evaluation. We provide an example of PUFA quantification in animal tissue along with the bioinformatic protocol, enabling efficient inter-sample comparison and statistical analysis. For complete details on the use and execution of this protocol, please refer to Vila et al.,1 Costanza et al.,2 Blomme et al.,3 and Blomme et al.4.

Development of a quantitative proteomics approach for cyclooxygenases and lipoxygenases in parallel to quantitative oxylipin analysis allowing the comprehensive investigation of the arachidonic acid cascade.

Oxylipins derived from the cyclooxygenase (COX) and lipoxygenase (LOX) pathways of the arachidonic acid (ARA) cascade are essential for the regulation of the inflammatory response and many other physiological functions. Comprehensive analytical methods comprised of oxylipin and protein abundance analysis are required to fully understand mechanisms leading to changes within these pathways. Here, we describe the development of a quantitative multi-omics approach combining liquid chromatography tandem mass spectrometry-based targeted oxylipin metabolomics and proteomics. As the first targeted proteomics method to cover these pathways, it enables the quantitative analysis of all human COX (COX-1 and COX-2) and relevant LOX pathway enzymes (5-LOX, 12-LOX, 15-LOX, 15-LOX-2, and FLAP) in parallel to the analysis of 239 oxylipins with our targeted oxylipin metabolomics method from a single sample. The detailed comparison between MRM3 and classical MRM-based detection in proteomics showed increased selectivity for MRM3, while MRM performed better in terms of sensitivity (LLOQ, 16-122 pM vs. 75-840 pM for the same peptides), linear range (up to 1.5-7.4 µM vs. 4-368 nM), and multiplexing capacities. Thus, the MRM mode was more favorable for this pathway analysis. With this sensitive multi-omics approach, we comprehensively characterized oxylipin and protein patterns in the human monocytic cell line THP-1 and differently polarized primary macrophages. Finally, the quantification of changes in protein and oxylipin levels induced by lipopolysaccharide stimulation and pharmaceutical treatment demonstrates its usefulness to study molecular modes of action involved in the modulation of the ARA cascade.

Influence of Dietary n-3 Long Chain Polyunsaturated Fatty Acid Intake on Oxylipins in Erythrocytes of Women with Rheumatoid Arthritis.

Oxylipins derived from n-3 fatty acids are suggested as the link between these fatty acids and reduced inflammation. The aim of the present study was to explore the effect of a randomized controlled cross-over intervention on oxylipin patterns in erythrocytes. Twenty-three women with rheumatoid arthritis completed 2 × 11-weeks exchanging one cooked meal per day, 5 days a week, for a meal including 75 g blue mussels (source for n-3 fatty acids) or 75 g meat. Erythrocyte oxylipins were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results were analyzed with multivariate data analysis. Orthogonal projections to latent structures (OPLS) with effect projections and with discriminant analysis were performed to compare the two diets' effects on oxylipins. Wilcoxon signed rank test was used to test pre and post values for each dietary period as well as post blue-mussel vs. post meat. The blue-mussel diet led to significant changes in a few oxylipins from the precursor fatty acids arachidonic acid and dihomo-É£-linolenic acid. Despite significant changes in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and free EPA in erythrocytes in the mussel group, no concurrent changes in their oxylipins were seen. Further research is needed to study the link between n-3 fatty-acid intake, blood oxylipins, and inflammation.

Early detection of lipid oxidation in infant milk formula by measuring free oxylipins-Comparison with hydroperoxide value and thiobarbituric acid reactive substance methods.

Infant milk formula was used as a model food to compare the sensitivity of thiobarbituric acid reactive substances (TBARS) and hydroperoxide methods to UPLC-MS/MS oxylipin analysis for detecting early lipid oxidation. Two different infant milk formulas were tested during 21 days of storage at 4°C. Formulas 1 and 2 contained canola oil and canola oil + 1% docosahexaenoic acid ethyl ester, respectively. Formulas were sampled up to 21 days of storage. Formula 2 had higher peroxide values than Formula 1 across all time points. However, no significant differences over time in TBARS and peroxide values in either formula were observed. Several oxylipins increased in both formulas starting on day 7 (linoleic acid and alpha-linolenic acid-derived oxylipins in Formula 1 and DHA-derived oxylipins in Formula 2). These results indicate that free oxylipins are effective in detecting early lipid oxidation and distinguishing between formulations containing different fatty acids. PRACTICAL APPLICATION: We have recently shown that primary oxidation products known as oxylipins can be measured in their free form by ultra-high pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to detect early lipid oxidation. However, a head-to-head comparison of the sensitivity of this approach to conventional spectrophotometric methods has not been evaluated. Our results indicate that free oxylipin measurements are better than conventional methods in detecting early lipid oxidation in milk infant formula  distinguishing between different formulations.

The Plasma Oxylipin Signature Provides a Deep Phenotyping of Metabolic Syndrome Complementary to the Clinical Criteria.

Metabolic syndrome (MetS) is a complex condition encompassing a constellation of cardiometabolic abnormalities. Oxylipins are a superfamily of lipid mediators regulating many cardiometabolic functions. Plasma oxylipin signature could provide a new clinical tool to enhance the phenotyping of MetS pathophysiology. A high-throughput validated mass spectrometry method, allowing for the quantitative profiling of over 130 oxylipins, was applied to identify and validate the oxylipin signature of MetS in two independent nested case/control studies involving 476 participants. We identified an oxylipin signature of MetS (coined OxyScore), including 23 oxylipins and having high performances in classification and replicability (cross-validated AUCROC of 89%, 95% CI: 85-93% and 78%, 95% CI: 72-85% in the Discovery and Replication studies, respectively). Correlation analysis and comparison with a classification model incorporating the MetS criteria showed that the oxylipin signature brings consistent and complementary information to the clinical criteria. Being linked with the regulation of various biological processes, the candidate oxylipins provide an integrative phenotyping of MetS regarding the activation and/or negative feedback regulation of crucial molecular pathways. This may help identify patients at higher risk of cardiometabolic diseases. The oxylipin signature of patients with metabolic syndrome enhances MetS phenotyping and may ultimately help to better stratify the risk of cardiometabolic diseases.

Evidence of increased sequestration of pro-resolving lipid mediators within brain esterified lipid pools of multiple sclerosis patients.

BACKGROUND: Unresolved inflammation in multiple sclerosis (MS) is associated with progressive demyelination and symptom worsening. In the brain, both inflammation and resolution pathways are mediated by free lipid mediators (i.e., oxylipins) that can be derived from the enzymatic hydrolysis of esterified oxylipins . It is not known whether disturbances in the turnover of free lipid mediators from esterified pools exist in postmortem brain of MS patients. We hypothesized that resolution pathways are impaired in MS patients because of disturbances in the turnover of free pro-resolving lipid mediators from esterified lipids. The objective was to characterize free and esterified oxylipins in postmortem prefrontal cortex of MS and unaffected control participants. METHODS: Oxylipins in free, neutral lipid and phospholipid pools were extracted from prefrontal cortex of 10 MS participants and 5 unaffected controls, separated by solid phase extraction columns, and quantified by ultra-high-pressure liquid chromatography-tandem mass spectrometry. Significant differences between the control and MS groups were determined by an unpaired t-test with Benjamini and Hochberg False Discovery Rate correction (10%) applied to oxylipins within each lipid pool. RESULTS: The concentration of 7 esterified pro-resolving fatty acid epoxides within neutral lipids were significantly higher by 126%-285% in postmortem prefrontal cortex of MS compared to control participants. The concentration of esterified linoleic acid-derived 9(10)-epoxy-octadecenoic acid, a pro-inflammatory epoxide, was higher by 206% in MS compared to controls. No significant changes were observed in free or phospholipid-bound oxylipins. CONCLUSION: In MS, several pro-resolving lipid mediators are trapped within prefrontal cortex neutral lipids, potentially limiting their supply and availability in the free bioactive form. This may explain why inflammation resolution is impaired in MS patients.

Feasibility and reliability of measures of bioactive lipids in human plasma and nasal mucosa.

Analysis of bioactive lipids is increasingly useful in clinical studies, and there is a need for non-invasive and easy-to-use sampling methods that meet the demands of reliability. Samples that can be taken by a non-professional and that can be taken repeatedly so as to provide more detailed information about the inflammatory process are often desired. In this study, the feasibility of non-invasive sampling of nasal mucosa and saliva for the analysis of bioactive lipid mediators (e.g. oxylipins and endocannabinoids) was evaluated in a pilot study (n = 10). In a second study, the reliability (relative and absolute) of sampling of these lipid mediators derived from nasal mucosa and from plasma was assessed by calculation of the intraclass correlation coefficient and Bland-Altman's limit of agreement. Samples were taken at the same time of day on two occasions from a cohort of individuals with and without building-related intolerance (n = 37). Nasal mucosa proved to be a suitable matrix for the analysis of bioactive lipids and was therefore included in the study on reliability together with the plasma samples. Relative reliability varied among the identified oxylipins and endocannabinoids. Arachidonic acid derivatives showed generally better reliability. Absolute reliability measures also varied indicating that only a subset of the oxylipins and endocannabinoids were suitable as biomarkers in either nasal mucosa or plasma and should therefore be used with caution for that purpose.

Quantitative profiling of differentially expressed oxylipins in ADSCs under proinflammatory cytokine stimulation.

Mesenchymal stem cells (MSCs) have been proved to have anti-inflammatory capabilities, but the mechanisms are still under investigation. Recently, oxylipins have been identified as being related to the immuno-regulation function of MSCs, but the MSC-derived oxylipins, especially under the stimulation of versatile pro-inflammatory cytokines, have never been comprehensively analyzed. In the present research, a UPLC-MS/MS method was employed to identify and quantify the oxylipin profiles of adipose-derived mesenchymal stem cells (ADSCs) under cytokine stimulation (IL-1ß, TNF-α, IFN-𝛾 and TNF-α + IFN-𝛾). The differentially produced oxylipins between experimental groups were analyzed and compared. The elevated level of lipoxygenase-15 (LOX-15) mRNA was further verified by qRT-PCR analysis. From the targeted 71 oxylipins, we detected and quantified 57 oxylipins, while 14 were not detected. Distinctive from other cytokines, ADSCs activated by the combination of IFN-𝛾 and TNF-α up-regulated LOX-15 products 7-HDHA and 15-HEPE, which were metabolized from docosahexaenoic acid (DHA) and eicosapentaenoic acid, respectively, and involved in the pro-resolution phase of inflammation. The results reported here make a first step towards a comprehensive characterization of MSC-derived oxylipins under differential proinflammatory cytokine stimulation. The findings may lay a fundamental foundation for MSC-based therapies and further determine ways to optimize the therapeutic potential of MSCs.

Measuring Stress-Induced Changes in Defense Phytohormones and Related Compounds.

Measuring quantitative changes in plant hormones and derivatives is crucial to understand how reactive oxygen species trigger signaling cascades to regulate stress responses. In this chapter, we describe the liquid chromatography-mass spectrometry procedure that we use to extract and quantify salicylic acid (SA), jasmonic acid (JA), and related compounds in common extracts of Arabidopsis tissue. The method can provide quantitative data on SA, SA glucosides, and JA, as well as information on oxidized and conjugated forms of these compounds and related derivatives of benzoic acid.

[Effects of simulated insect herbivory on defense traits of Galinsoga parviflora.] / 模拟昆虫取食对牛膝菊防御特征的影响.

To understand mechanisms underlying Galinsoga parviflora invasion and its responses to simulated insect herbivory, individuals of Galinsoga parviflora were treated with different concentrations of methyl jasmonate (MeJA) before blooming. We measued plant height, abundance of leaves and inflorescences, biomass, specific leaf area, trichome density, condensed tannins, total polyphenols, and flavonoids in leaves and inflorescences. The growth and reproduction parameters of G. parviflora treated with 5 mmol·L-1 MeJA were not significantly different from those of control, higher than those of control when treated with 10 mmol·L-1 MeJA, with significant difference except plant height, and declined when treated with 20 mmol·L-1 MeJA. The trichome density of leaf upper epidermis increased and specific leaf area decreased with increasing MeJA concentration, with both being significantly different from that of control. The contents of flavonoids, total polyphenols, and condensed tannins in leaves treated with 5 mmol·L-1MeJA were not significantly different from those of control. These defensive substances in leaves and inflorescences were highest under 10 mmol·L-1MeJA treatment. The contents of flavonoids and total polyphenols in inflorescences being higher than those of leaves, while condensed tannins was opposite. The defensive substances in leaves declined under 20 mmol·L-1MeJA treatment. The results suggested that G. parviflora could use tolerance and resistance strategies comprehensively, and adopted a variety of defense strategies such as compensatory growth, physical defense, and chemical defense, which was conducive to its success in invasion.

Antioxidant Potential and Enhancement of Bioactive Metabolite Production in In Vitro Cultures of Scutellaria lateriflora L. by Biotechnological Methods.

Studies carried out using three different in vitro assays and a biological setting (Escherichia coil) demonstrated the antioxidant activity of Scutellaria lateriflora microshoot extract. Moreover, the extract exhibited no toxicity in a brine shrimp lethality bioassay. These results indicated that microshoots are a rich, safe source of antioxidants, which encouraged us to enhance their production in vitro. In agar and agitated cultures, two biotechnological strategies were applied: feeding the cultures with the biogenetic precursors of the phenolics-phenylalanine and tyrosine, and eliciting them with methyl jasmonate. Specific Scutellaria flavonoids and verbascoside were analysed by HPLC. Feeding with precursors (1 g/L) in agar cultures decreased the production of the metabolites. In agitated cultures, different concentrations of precursors (1.0-2.5 g/L) and the elicitor (10; 50; 100 µM) were tested. Additionally, parallel feeding with the precursor and elicitor in a concentration of 50 µM were applied. The best strategy for total flavonoid and verbascoside production was phenylalanine feeding (1.5 g/L), max. 3765 and 475 mg/100 g DW, respectively, after 7 days. This is the first report documenting the high antioxidant production in S. lateriflora microshoots after feeding with phenylalanine. Moreover, for the first time, bioreactor cultures were successfully maintained, obtaining attractive results (max. total flavonoid content 2348 and verbascoside 485 mg/100 g DW).

Omega-6 and Omega-3 Fatty Acid-Derived Oxylipins from the Lipoxygenase Pathway in Maternal and Umbilical Cord Plasma at Delivery and Their Relationship with Infant Growth.

Omega-3 and omega-6 fatty acids are important for neonatal development and health. One mechanism by which omega-3 and omega-6 fatty acids exert their effects is through their metabolism into oxylipins and specialized pro-resolving mediators. However, the influence of oxylipins on fetal growth is not well understood. Therefore, the objective of this study was to identify oxylipins present in maternal and umbilical cord plasma and investigate their relationship with infant growth. Liquid chromatography-tandem mass spectrometry was used to quantify oxylipin levels in plasma collected at the time of delivery. Spearman's correlations highlighted significant correlations between metabolite levels and infant growth. They were then adjusted for maternal obesity (normal body mass index (BMI: ≤30 kg/m2) vs. obese BMI (>30 kg/m2) and smoking status (never vs. current/former smoker) using linear regression modeling. A p-value < 0.05 was considered statistically significant. Our study demonstrated a diverse panel of oxylipins from the lipoxygenase pathway present at the time of delivery. In addition, both omega-3 and omega-6 oxylipins demonstrated potential influences on the birth length and weight percentiles. The oxylipins present during pregnancy may influence fetal growth and development, suggesting potential metabolites to be used as biomarkers for infant outcomes.

One-anastomosis gastric bypass modulates the serum levels of pro- and anti-inflammatory oxylipins, which may contribute to the resolution of inflammation.

BACKGROUND/OBJECTIVES: Oxylipins are polyunsaturated fatty acid derivatives involved in the regulation of various processes, including chronic inflammation, insulin resistance and hepatic steatosis. They can be synthesized in various tissues, including adipose tissue. There is some evidence that obesity is associated with the deregulation of serum oxylipin levels. The aim of this study was to evaluate the effect of bariatric surgery (one-anastomosis gastric bypass) on the serum levels of selected oxylipins and their fatty acid precursors and to verify the hypothesis that their changes after surgery can contribute to the resolution of inflammation. Moreover, we compared the oxylipin levels (prostaglandin E2, 13-HODE, maresin 1 and resolvin E1), fatty acids and the expression of enzymes that synthesize oxylipins in adipose tissue of lean controls and subjects with severe obesity. SUBJECTS/METHODS: The study included 50 patients with severe obesity that underwent bariatric surgery and 41 subjects in lean, control group. Fatty acid content was analyzed by GC-MS, oxylipin concentrations were measured with immunoenzymatic assay kits and real-time PCR analysis was used to assess mRNA levels in adipose tissue. RESULTS: Our results show increased expression of some enzymes that synthesize oxylipins in adipose tissue and alterations in the levels of oxylipins in both adipose tissue and serum of subjects with obesity. After bariatric surgery, the levels of anti-inflammatory oxylipins increased, whereas pro-inflammatory oxylipins decreased. CONCLUSIONS: In patients with obesity, the metabolism of oxylipins is deregulated in adipose tissue, and their concentrations in serum are altered. Bariatric surgery modulates the serum levels of pro- and anti-inflammatory oxylipins, which may contribute to the resolution of inflammation.

Distribution analysis of jasmonic acid-related compounds in developing Glycine max L. (soybean) seeds using mass spectrometry imaging and liquid chromatography-mass spectrometry.

INTRODUCTION: Jasmonic acid (JA) and its precursors are oxylipins derived from α-linolenic acid (αLA) and hexadecatrienoic acid, and regulate seed development. However, their spatial distribution in the developing Glycine max L. (soybean) seeds has not been elucidated. OBJECTIVE: To investigate the distribution of JA-related compounds in the developing soybean seeds using desorption electrospray ionisation-mass spectrometry imaging (DESI-MSI) and liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) analyses. METHODS: Cryosections of developing seeds were prepared using adhesive films, and subjected to DESI-MSI analysis. Verification of the DESI-MSI ion images were performed using DESI-tandem MSI (MS/MSI), LC-ESI-MS and tandem MS (MS/MS). RESULTS: In the DESI-MSI mass spectrum, peaks matching the chemical formulae of αLA, 12-oxo-phytodienoic acid (OPDA), and 3-oxo-2-(2-(Z)-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0) were detected. These compounds were mainly distributed in the seed coat, especially near the hilum. This was consistent with the quantitative results obtained by LC-ESI-MS. While, DESI-MS/MSI and LC-ESI-MS/MS suggested the presence of isomers for OPDA and OPC-8:0. The effect of isomers on the DESI-MSI ion images was small for OPDA, and considerable for OPC-8:0. CONCLUSION: These results demonstrated that free αLA, OPDA, and OPC-8:0 were the abundant JA-related compounds mainly distributed in the seed coat of the developing soybeans. OPDA and OPC-8:0 might exert a biological role in the seed coat. To the best of my knowledge, this is the first report on the accumulation of OPDA and OPC-8:0 in the seed coat. The combination of DESI-MSI and LC-ESI-MS is a useful tool for distribution analysis of JA-related compounds in the developing seeds.

Transcriptome Analysis Reveals Roles of Anthocyanin- and Jasmonic Acid-Biosynthetic Pathways in Rapeseed in Response to High Light Stress.

Rapeseed (Brassica napus) is one of the major important oil crops worldwide and is largely cultivated in the Qinghai-Tibetan plateau (QTP), where long and strong solar-radiation is well-known. However, the molecular mechanisms underlying rapeseed's response to light stress are largely unknown. In the present study, the color of rapeseed seedlings changed from green to purple under high light (HL) stress conditions. Therefore, changes in anthocyanin metabolism and the transcriptome of rapeseed seedlings cultured under normal light (NL) and HL conditions were analyzed to dissect how rapeseed responds to HL at the molecular level. Results indicated that the contents of anthocyanins, especially glucosides of cyanidin, delphinidin, and petunidin, which were determined by liquid chromatography-mass spectrometry (LC-MS), increased by 9.6-, 4.2-, and 59.7-fold in rapeseed seedlings exposed to HL conditions, respectively. Next, RNA-sequencing analysis identified 7390 differentially expressed genes (DEGs), which included 4393 up-regulated and 2997 down-regulated genes. Among the up-regulated genes, many genes related to the anthocyanin-biosynthetic pathway were enriched. For example, genes encoding dihydroflavonol reductase (BnDFR) and anthocyanin synthase (BnANS) were especially induced by HL conditions, which was also confirmed by RT-qPCR analysis. In addition, two PRODUCTION OF ANTHOCYANIN PIGMENTATION 2 (BnPAP2) and GLABRA3 (BnGL3) genes encoding MYB-type and bHLH-type transcription factors, respectively, whose expression was also up-regulated by HL stress, were found to be associated with the changes in anthocyanin biosynthesis. Many genes involved in the jasmonic acid (JA)-biosynthetic pathway were also up-regulated under HL conditions. This finding, which is in agreement with the well-known positive regulatory role of JA in anthocyanin biosynthesis, suggests that the JA may also play a key role in the responses of rapeseed seedlings to HL. Collectively, these data indicate that anthocyanin biosynthesis-related and JA biosynthesis-related pathways mediate HL responses in rapeseed. These findings collectively provide mechanistic insights into the mechanisms involved in the response of rapeseed to HL stress, and the identified key genes may potentially be used to improve HL tolerance of rapeseed cultivars through genetic engineering or breeding strategies.

Phytohormone profiles in non-transformed and AtCKX transgenic centaury (Centaurium erythraea Rafn) shoots and roots in response to salinity stress in vitro.

Plant hormones regulate numerous developmental and physiological processes. Abiotic stresses considerably affect production and distribution of phytohormones as the stress signal triggers. The homeostasis of plant hormones is controlled by their de novo synthesis and catabolism. The aim of this work was to analyse the contents of total and individual groups of endogenous cytokinins (CKs) as well as indole-3-acetic acid (IAA) in AtCKX overexpressing centaury plants grown in vitro on graded NaCl concentrations (0, 50, 100, 150, 200 mM). The levels of endogenous stress hormones including abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) were also detected. The elevated contents of total CKs were found in all analysed centaury shoots. Furthermore, increased amounts of all five CK groups, as well as enhanced total CKs were revealed on graded NaCl concentrations in non-transformed and AtCKX roots. All analysed AtCKX centaury lines exhibited decreased amounts of endogenous IAA in shoots and roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all AtCKX lines. In shoots and roots of both non-transformed and AtCKX transgenic centaury plants, salinity was associated with an increase of ABA and JA and a decrease of SA content.