Oxylipins in Breast Implant-Associated Systemic Symptoms.

BACKGROUND: A subset of females with breast implants have reported a myriad of nonspecific systemic symptoms collectively termed systemic symptoms associated with breast implants (SSBI). SSBI symptoms are similar to manifestations associated with autoimmune and connective tissue disorders. Breast tissue is rich in adipose cells, comprised of lipids. Insertion of an implant creates an oxidative environment leading to lipid oxidation. Oxylipins can influence immune responses and inflammatory processes. OBJECTIVES: In this study we explored the abundance of a spectrum of oxylipins in the periprosthetic tissue surrounding the breast implant. Because oxylipins are immunogenic, we sought to determine if they were associated with the SSBI patients. We have also attempted to determine if the common manifestations exhibited by such patients have any association with oxylipin abundance. METHODS: The study included 120 patients divided into 3 cohorts. We analyzed 46 patients with breast implants exhibiting manifestations associated with SSBI; 29 patients with breast implants not exhibiting manifestations associated with SSBI (control cohort I, non-SSBI); and 45 patients without implants (control cohort II, no-implant tissue). Lipid extraction and oxylipin quantification were performed with liquid chromatography mass spectrometry (LC-MS/MS). LC-MS/MS targeted analysis of the breast adipose tissue was performed. RESULTS: Of the 15 oxylipins analyzed, 5 exhibited increased abundance in the SSBI cohort when compared to the non-SSBI and no-implant cohorts. CONCLUSIONS: The study documents the association of the oxylipins with each manifestation reported by the patient. This study provides an objective assessment of the subjective questionnaire, highlighting which symptoms may be more relevant than the others.

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.

[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.

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.

Quantitation of Oxylipins in Fish and Algae Oil Supplements Using Optimized Hydrolysis Procedures and Ultra-High Performance Liquid Chromatography Coupled to Tandem Mass-Spectrometry.

Fish and algae oil supplements are enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are precursors to oxidized fatty acids, known as oxylipins. Here, we optimized a base hydrolysis method for measuring oxylipins in oil with ultrahigh-performance liquid chromatography coupled to tandem mass-spectrometry (UPLC-MS/MS) and quantified them in fish and algae oil supplements. Hydrolysis of 2 µL of oil with sodium carbonate resulted in greater oxylipin concentrations and minimal matrix effects, compared to higher oil volumes (10, 20, and 30 µL). Oxylipin yield was higher when oil was hydrolyzed in methanol containing 0.1% acetic acid and 0.1% butylated hydroxytoluene, compared to no methanol, and using sodium hydroxide versus sodium carbonate. Oxylipins extracted from 2 µL of oil using sodium hydroxide in solvent showed that EPA-derived oxylipins were most abundant in fish oil (84-87%), whereas DHA-oxylipins were abundant in algae oil (83%). This study shows that fish and algae oils are direct sources of EPA- and DHA-derived oxylipins.

High Dietary Protein Does Not Alter Renal Prostanoids and Other Oxylipins in Normal Mice or in Those with Inherited Kidney Disease.

BACKGROUND: Ex vivo studies suggest that increased renal prostanoids can mediate effects of high-protein (HP) compared with low-protein (LP) diets on normal and diseased kidneys. However, a short-term HP feeding study in normal male rats failed to demonstrate higher renal prostanoids in vivo. OBJECTIVES: The aim of the present study was to investigate whether long-term HP feeding alters renal prostanoids in male and female mice, with and without kidney disease. METHODS: Weanling normal mice (CD1) and mice with kidney disease (CD1-pcy/pcy mice) were fed standard diets with normal protein [NP, 20% of energy (%E)] or HP (35%E) for 13 wk. Renal disease was assessed by histomorphometric analysis of cysts and fibrosis, and measurement of serum urea nitrogen (SUN) and creatinine concentrations. Targeted analysis of renal oxylipins was performed by HPLC-MS/MS. RESULTS: The HP diet increased kidney size and water content of normal kidneys, and worsened disease in CD1-pcy/pcy mice as indicated by higher (P < 0.05) kidney weights (8-31%), water content (8-10%), cyst volume (36-60%), fibrous volume (44-53%), and SUN (47-55%). Diseased compared with normal kidneys had higher (P < 0.05) concentrations of 6 of 11 prostanoids and lower (P < 0.05) concentrations of 33 of 54 other oxylipins. This is consistent with previously known effects of dietary HP and disease effects on the kidney. However, the HP diet did not alter renal prostanoids and other renal oxylipins in either normal or diseased kidneys (P < 0.05), despite having the expected physiological effects on normal and diseased kidneys. This study also showed that females have higher concentrations of renal prostanoids [9 of 11 prostanoids higher (P < 0.05) in females], but lower concentrations of other oxylipins [28 of 54 other oxylipins lower (P < 0.05) in females]. CONCLUSIONS: The effects of HP diets on normal and diseased kidneys in CD1 and CD1-pcy/pcy mice are independent of renal oxylipin alterations.

Agrobacterium rhizogenes mediated transformation of Ficus carica L. for the efficient production of secondary metabolites.

BACKGROUND: Ficus carica L., an ancient source of food and medicines, is rich in valuable nutritional and secondary compounds with antioxidant, antimicrobial, and anticancer effects. The present study is the first attempt to examine hairy root (HR) induction of F. carica (Sabz and Siah) by inoculating the 3-week-old shoots and leaves with different strains of Agrobacterium rhizogenes and also to investigate methyl jasmonate (MeJA) elicitation of HRs to produce a fast and high-yield production method for secondary metabolites. RESULTS: The maximum transformation rate (100%) was achieved by inoculating the shoots with Agrobacterium rhizogenes strain A7. Siah HRs elicited with 100 and 200 µmol L-1 MeJA and Sabz HRs with 100 µmol L-1 MeJA showed the highest total phenolic content. The highest flavonoid content was 3.935 mg QE g-1 DW in Siah HRs treated with 200 µmol L-1 MeJA and 2.762 mg QE g-1 DW in Sabz HRs treated with 300 µmol L-1 MeJA. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity and ferric reducing antioxidant power (FRAP) value of HRs were affected by MeJA treatments. Methyl jasmonate elicitation also significantly enhanced the content of six phenolic acids (gallic acid, caffeic acid, chlorogenic acid, coumaric acid, rosmarinic acid, and cinnamic acid) and three flavonoids (rutin, quercetin, and apigenin). Thymol, a monoterpene phenol, was the main HR compound detected in gas chromatography mass spectrometry (GC-MS) analysis of the essential oils. CONCLUSION: Induction of HRs and elicitation of F. carica HRs by MeJA resulted in a significant increase in the production of important phenolic compounds and a significant increase in antioxidant capacity. © 2020 Society of Chemical Industry.

In†Vivo Solid-Phase Microextraction for Sampling of Oxylipins in Brain of Awake, Moving Rats.

Oxylipins are key lipid mediators of important brain processes, including pain, sleep, oxidative stress, and inflammation. For the first time, an in-depth profile of up to 52 oxylipins can be obtained from the brains of awake moving animals using in vivo solid-phase microextraction (SPME) chemical biopsy tool in combination with liquid chromatography-high resolution mass spectrometry. Among these, 23 oxylipins are detectable in the majority of healthy wildtype samples. This new approach successfully eliminates the changes in oxylipin concentrations routinely observed during the analysis of post-mortem samples, allows time-course monitoring of their concentrations with high spatial resolution in specific brain regions of interest, and can be performed using the same experimental set-up as in vivo microdialysis (MD) thus providing a new and exciting tool in neuroscience and drug discovery.

Oxylipin concentrations in bovine corpora lutea during maternal recognition of pregnancy.

The objective was to determine the effects of pregnancy status on oxylipin profiles and eicosanoid metabolizing enzymes and in corpora lutea (CL) or endometrial (caruncle; CAR and intercaruncle; IC) tissues. Angus crossed cattle were synchronized with the CO-Synch protocol and artificially inseminated (AI). Sixteen days after AI, cattle were euthanized, and reproductive tracts collected from 6 non-pregnant and 6 pregnant cows. Oxylipin profiles and concentrations of progesterone (P4) were obtained from CL tissues. The activity of cytochrome P450 1A (CYP1A) and UDP-glucuronosyltransferase (UGT) enzymes were determined using specific luminogenic substrates. Data were analyzed using the MIXED procedure of SAS, and the model included pregnancy status. Corpora lutea of pregnant cattle contained greater (P < 0.05) concentrations of 9,10-DiHODE, 15,16-DiHODE, and 9,10-DiHOME. These oxylipins have been observed to increase cellular proliferation and vasodilation. Activity of CYP1A in the CL and UGT in CAR and IC was not different (P > 0.05) between pregnant and non-pregnant cattle. In the CL, activity of UGT was decreased (P < 0.05) in pregnant vs. non-pregnant cattle. The decrease in CL UGT activity during pregnancy indicates alterations in local hormone metabolism, while no differences in CL weight nor amount of P4 in CL were different between pregnant and non-pregnant cattle. Moreover, the increase in specific concentrations of oxylipins in the CL may indicate a novel pathway of steroid and eicosanoid metabolism during maternal recognition of pregnancy.

Phenolic, oxylipin and fatty acid profiles of the Chilean hazelnut (Gevuina avellana): Antioxidant activity and inhibition of pro-inflammatory and metabolic syndrome-associated enzymes.

Roasted cotyledons of the Chilean hazelnut (Gevuina avellana) are appreciated as snacks. The aim of our work was to assess the fatty acid, oxylipin and phenolic composition using gas chromatography (GC) coupled to mass spectrometry (MS), ultra- high performance liquid chromatography (UHPLC) coupled to MS and HPLC coupled to diode array detector (HPLC-DAD). Additionally, various antioxidant activities were assessed. The inhibition of α-glucosidase, α-amylase, lipase, cyclooxygenases-1 and -2 (COX-1/COX-2), and lipoxygenase was determined. The main fatty acids were oleic and 7-hexadecenoic acids. Eight phytoprostanes and three phytofurans were identified and quantified. Hydroxybenzoic and hydroxycinnamic acids were the main phenolic compounds. Oils showed antioxidant activity determined by EPR, and inhibition of COX-1/COX-2. The statistical analysis showed that the roasting does not affect the composition of the samples. The occurrence of oxylipins in this species is reported for the first time. Chilean hazelnuts can be considered a source of health promoting compounds.

Effect of Major Tea Insect Attack on Formation of Quality-Related Nonvolatile Specialized Metabolites in Tea ( Camellia sinensis) Leaves.

Insect attack is known to induce a high accumulation of volatile metabolites in tea ( Camellia sinensis). However, little information is available concerning the effect of insect attack on tea quality-related nonvolatile specialized metabolites. This study aimed to investigate the formation of characteristic nonvolatile specialized metabolites in tea leaves in response to attack by major tea insects, namely, tea green leafhoppers and tea geometrids, and determine the possible involvement of phytohormones in metabolite formation resulting from insect attack. Both tea green leafhopper and tea geometrid attacks increased the jasmonic acid and salicylic acid contents. The abscisic acid content was only increased under tea green leafhopper attack, perhaps due to special continuous piercing-sucking wounding. Tea green leafhopper attack induced the formation of theaflavins from catechins under the action of polyphenol oxidase, while tea geometrid attack increased the l-theanine content. Exogenous phytohormone treatments can affect the caffeine and catechin contents. These results will help to determine the influence of major tea pest insects on important tea quality-related metabolites and enhance understanding of the relationship of phytohormones and quality-related nonvolatile metabolite formation in tea exposed to tea pest insect attacks.

Oxidative stress and abnormal bioactive lipids in early cystic fibrosis lung disease.

BACKGROUND: Clinical data indicate that airway inflammation in children with cystic fibrosis (CF) arises early, is associated with structural lung damage, and predicts progression. In bronchoalveolar lavage fluid (BALF) from CFTR mutant mice, several aspects of lipid metabolism are abnormal that contributes to lung disease. We aimed to determine whether lipid pathway dysregulation is also observed in BALF from children with CF, to identify biomarkers of early lung disease and potential therapeutic targets. METHODS: A comprehensive panel of lipids that included Sphingolipids, oxylipins, isoprostanes and lysolipids, all bioactive lipid species known to be involved in inflammation and tissue remodeling, were measured in BALF from children with CF (1-6 years, N = 33) and age-matched non-CF patients with unexplained inflammatory disease (N = 16) by HPLC-MS/MS. Lipid data were correlated with chest CT scores and BALF inflammation biomarkers. RESULTS: The ratio of long chain to very long chain ceramide species (LCC/VLCC) and lysolipid levels were enhanced in CF compared to non-CF patients, despite comparable neutrophil counts and bacterial load. In CF patients both LCC/VLCC and lysolipid levels correlated with inflammation and chest CT scores. The ceramide precursors Sphingosine, Sphinganine, Sphingomyelin, correlated with inflammation, whilst the oxidative stress marker isoprostane correlated with inflammation and chest CT scores. No correlation between lipids and current bacterial infection in CF (N = 5) was observed. CONCLUSIONS: Several lipid biomarkers of early CF lung disease were identified, which point toward potential disease monitoring and therapeutic approaches that can be used to complement CFTR modulators.

Transcriptome and metabolome analysis reveal candidate genes and biochemicals involved in tea geometrid defense in Camellia sinensis.

Tea plant (Camellia sinensis (L) O. Kuntze) respond to herbivore attack through large changes in defense related metabolism and gene expression. Ectropis oblique (Prout) is one of the most devastating insects that feed on tea leaves and tender buds, which can cause severe production loss and deteriorate the quality of tea. To elucidate the biochemicals and molecular mechanism of defense against tea geometrid (TG), transcriptome and metabolome of TG interaction with susceptible (SG) and resistance (RG) tea genotypes were analyzed by using UPLC-Q-TOF-MS, GC-MS, and RNA-seq technologies. This revealed that jasmonic acid was highly induced in RG, following a plethora of secondary metabolites involved in defense against TG could be induced by jasmonic acid signaling pathway. However, the constitutively present of salicylic acid in SG might be a suppressor of jasmonate signaling and thus misdirect tea plants against TG. Furthermore, flavonoids and terpenoids biosynthesis pathways were highly activated in RG to constitute the chemical barrier on TG feeding behavior. In contrast, fructose and theanine, which can act as feeding stimulants were observed to highly accumulate in SG. Being present in the major hub, 39 transcription factors or protein kinases among putative candidates were identified as master regulators from protein-protein interaction network analysis. Together, the current study provides a comprehensive gene expression and metabolite profiles, which can shed new insights into the molecular mechanism of tea defense against TG. The candidate genes and specific metabolites identified in the present study can serve as a valuable resource for unraveling the possible defense mechanism of plants against various biotic stresses.

A Maize Inbred Exhibits Resistance Against Western Corn Rootwoorm, Diabrotica virgifera virgifera.

Insect resistance against root herbivores like the western corn rootworm (WCR, Diabrotica virgifera virgifera) is not well understood in non-transgenic maize. We studied the responses of two American maize inbreds, Mp708 and Tx601, to WCR infestation using biomechanical, molecular, biochemical analyses, and laser ablation tomography. Previous studies performed on several inbreds indicated that these two maize genotypes differed in resistance to pests including fall armyworm (Spodoptera frugiperda) and WCR. Our data confirmed that Mp708 shows resistance against WCR, and demonstrates that the resistance mechanism is based in a multi-trait phenotype that includes increased resistance to cutting in nodal roots, stable root growth during insect infestation, constitutive and induced expression of known herbivore-defense genes, including ribosomal inhibitor protein 2 (rip2), terpene synthase 23 (tps23) and maize insect resistance cysteine protease-1 (mir1), as well high constitutive levels of jasmonic acid and production of (E)-ß-caryophyllene. In contrast, Tx601 is susceptible to WCR. These findings will facilitate the use of Mp708 as a model to explore the wide variety of mechanisms and traits involved in plant defense responses and resistance to herbivory by insects with several different feeding habits.

Quantification of phytoprostanes - bioactive oxylipins - and phenolic compounds of Passiflora edulis Sims shell using UHPLC-QqQ-MS/MS and LC-IT-DAD-MS/MS.

The genus Passiflora, comprising about 500 species, is the largest in the Passion flower family. Passiflora edulis Sims f. edulis (gulupa) is one of the most important fruits cultivated in Colombia. In recent years and due to its organoleptic and bioactive properties, its exports have significantly increased. In this work, six new bioactive oxylipins -phytoprostanes - were detected in gulupa shell by a UHPLC-QqQ-MS/MS method: F1t-phytoprostanes and D1t-phytoprostanes were the predominant and minor classes, respectively. Moreover, the polyphenol profile of the shell was investigated and we were able to detect and quantify phenolic compounds that have not been described previously, like luteolin-8-C-(2-O-rhamnosyl)hexoside and quercetin-3-O-(6″-acetyl)glucosyl-2″-sinapic acid. Consequently, this study provides new insights into the importance of gulupa shell as a valuable option in the design of new beverages rich in antioxidant phytochemicals, as part of a well-balanced diet, and in the process and quality control of such products.

Separating and Identifying the Four Stereoisomers of Methyl Jasmonate by RP-HPLC and using Cyclodextrins in a Novel Way.

INTRODUCTION: Several authors have reported on the different bioactivities of methyl jasmonate (MeJA) stereoisomers. However, no simple, precise and cheap method for separating and identifying them using reversed-phase high performance liquid chromatography (RP-HPLC) has been developed. OBJECTIVE: (1) To create a simple, precise and cheap method for separating and identifying the four stereoisomers present in commercial racemic mixtures of MeJA and (2) to identify the four stereoisomers using molecular docking techniques and coinjection. Materials and Methods - RP-HPLC using a 250 mm C18 column and different proportions of cyclodextrins (CDs) and organic solvents was applied to a commercial sample of racemic MeJA. RESULTS: The results show that the best conditions for separating the MeJA stereoisomers are: 20% methanol in the mobile phase, a temperature of 45 °C and a 16 mM concentration of methyl-ß-cyclodextrin (M-ß-CD). A simple C18 250 mm column and a flow rate of 1.25 mL/min were used. The reduction in the retention time of MeJA observed when M-ß-CD is added to the mobile phases was used to determine the complexation constants of the guest/CD complex and compared with the obtained when other CDs were used. The KF for M-ß-CD (117.49 ± 5.9 1/M) was obtained with a 1:1 stoichiometry. The four stereoisomers were identified by molecular docking techniques and coinjection of a commercially available rosemary essential oil. CONCLUSION: The new method identified and classified the four stereoisomers of MeJA in the following ordination: (-)epiMeJA, (-)MeJA; (+)MeJA and (+)epiMeJA. These results could be used to improve the elicitation of cell cultures with only the best isomer. Copyright © 2016 John Wiley & Sons, Ltd.

Phytohormone profile in Lactuca sativa and Brassica oleracea plants grown under Zn deficiency.

Phytohormones, structurally diverse compounds, are involved in multiple processes within plants, such as controlling plant growth and stress response. Zn is an essential micronutrient for plants and its deficiency causes large economic losses in crops. Therefore, the purpose of this study was to analyse the role of phytohormones in the Zn-deficiency response of two economically important species, i.e. Lactuca sativa and Brassica oleracea. For this, these two species were grown hydroponically with different Zn-application rates: 10 µM Zn as control and 0.1 µM Zn as deficiency treatment and phytohormone concentration was determined by U-HPLC-MS. Zn deficiency resulted in a substantial loss of biomass in L. sativa plants that was correlated with a decline in growth-promoting hormones such as indole-3-acetic acid (IAA), cytokinins (CKs), and gibberellins (GAs). However these hormones increased or stabilized their concentrations in B. oleracea and could help to maintain the biomass in this species. A lower concentration of stress-signaling hormones such as ethylene precursor aminocyclopropane-1-carboxylic acid (ACC), abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) and also CKs might be involved in Zn uptake in L. sativa while a rise in GA4, isopentenyl adenine (iP), and ACC and a fall in JA and SA might contribute to a better Zn-utilization efficiency (ZnUtE), as observed in B. oleracea plants.

Simultaneous determination of shikimic acid, salicylic acid and jasmonic acid in wild and transgenic Nicotiana langsdorffii plants exposed to abiotic stresses.

The presence and relative concentration of phytohormones may be regarded as a good indicator of an organism's physiological state. The integration of the rolC gene from Agrobacterium rhizogenes and of the rat glucocorticoid receptor (gr) in Nicotiana langsdorffii Weinmann plants has shown to determine various physiological and metabolic effects. The analysis of wild and transgenic N. langsdorffii plants, exposed to different abiotic stresses (high temperature, water deficit, and high chromium concentrations) was conducted, in order to investigate the metabolic effects of the inserted genes in response to the applied stresses. The development of a new analytical procedure was necessary, in order to assure the simultaneous determination of analytes and to obtain an adequately low limit of quantification. For the first time, a sensitive HPLC-HRMS quantitative method for the simultaneous determination of salicylic acid, jasmonic acid and shikimic acid was developed and validated. The method was applied to 80 plant samples, permitting the evaluation of plant stress responses and highlighting some metabolic mechanisms. Salicylic, jasmonic and shikimic acids proved to be suitable for the comprehension of plant stress responses. Chemical and heat stresses showed to induce the highest changes in plant hormonal status, differently affecting plant response. The potential of each genetic modification toward the applied stresses was marked and particularly the resistance of the gr modified plants was evidenced. This work provides new information in the study of N. langsdorffii and transgenic organisms, which could be useful for the further application of these transgenes.

Simultaneous analysis of apolar phytohormones and 1-aminocyclopropan-1-carboxylic acid by high performance liquid chromatography/electrospray negative ion tandem mass spectrometry via 9-fluorenylmethoxycarbonyl chloride derivatization.

A strategy to detect and quantify the polar ethylene precursor 1-aminocyclopropan-1-carboxylic acid (ACC) along with the more apolar phytohormones abscisic acid (ABA), indole-3-acetic acid (IAA), jasmonic acid (JA), jasmonic acid-isoleucine conjugate (JA-Ile), 12-oxo-phytodienoic acid (OPDA), trans-zeatin, and trans-zeatin 9-riboside using a single extraction is presented. Solid phase resins commonly employed for extraction of phytohormones do not allow the recovery of ACC. We circumvent this problem by attaching an apolar group to ACC via derivatization with the amino group specific reagent 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl). Derivatization in the methanolic crude extract does not modify other phytohormones. The derivatized ACC could be purified and detected together with the more apolar phytohormones using common solid phase extraction resins and reverse phase HPLC/electrospray negative ion tandem mass spectrometry. The limit of detection was in the low nanomolar range for all phytohormones, a sensitivity sufficient to accurately determine the phytohormone levels from less than 50mg (fresh weight) of Arabidopsis thaliana and Nicotiana benthamiana tissues. Comparison with previously published phytohormone levels and the reported changes in phytohormone levels after stress treatments confirmed the accuracy of the method.