Gaetan Glauser

Spatial and Temporal Dynamics of Jasmonate Synthesis and Accumulation in Arabidopsis in Response to Wounding

A new metabolite profiling approach combined with an ultrarapid sample preparation procedure was used to study the temporal and spatial dynamics of the wound-induced accumulation of jasmonic acid (JA) and its oxygenated derivatives in Arabidopsis thaliana. In addition to well known jasmonates, including hydroxyjasmonates (HOJAs), jasmonoyl-isoleucine (JA-Ile), and its 12-hydroxy derivative (12-HOJA-Ile), a new wound-induced dicarboxyjasmonate, 12-carboxyjasmonoyl-l-isoleucine (12-HOOCJA-Ile) was discovered. HOJAs and 12-HOOCJA-Ile were enriched in the midveins of wounded leaves, strongly differentiating them from the other jasmonate metabolites studied. The polarity of these oxylipins at physiological pH correlated with their appearance in midveins. When the time points of accumulation of different jasmonates were determined, JA levels were found to increase within 2–5 min of wounding. Remarkably, these changes occurred throughout the plant and were not restricted to wounded leaves. The speed of the stimulus leading to JA accumulation in leaves distal to a wound is at least 3 cm/min. The data give new insights into the spatial and temporal accumulation of jasmonates and have implications in the understanding of long-distance wound signaling in plants.

Velocity Estimates for Signal Propagation Leading to Systemic Jasmonic Acid Accumulation in Wounded Arabidopsis

The wound response prohormone jasmonic acid (JA) accumulates rapidly in tissues both proximal and distal to injury sites in plants. Using quantitative liquid chromatography-mass spectrometry after flash freezing of tissues, we found that JA accumulated within 30 s of injury in wounded Arabidopsis leaves (p = 3.5 e−7). JA augmentation distal to wounds was strongest in unwounded leaves with direct vascular connections to wounded leaves wherein JA levels increased significantly within 120 s of wounding (p = 0.00027). This gave conservative and statistically robust temporal boundaries for the average velocity of the long distance signal leading to distal JA accumulation in unwounded leaves of 3.4–4.5 cm min−1. Like JA, transcripts of the JA synthesis gene LIPOXYGENASE2 (LOX2) and the jasmonate response gene JAZ10.3 also accumulated to higher levels in directly interconnected leaves than in indirectly connected leaves. JA accumulation in a lox2-1 mutant plant was initiated rapidly after wounding then slowed progressively compared with the wild type (WT). Despite this, JAZ10.3 expression in the two genotypes was similar. Free cyclopentenone jasmonate levels were similar in both resting WT and lox2-1. In contrast, bound cyclopentenone jasmonates (arabidopsides) were far lower in lox2-1 than in the WT. The major roles of LOX2 are to generate arabidopsides and the large levels of JA that accumulate proximal to the wound. LOX2 is not essential for some of the most rapid events elicited by wounding.

UPLC–TOF-MS for plant metabolomics: A sequential approach for wound marker analysis in Arabidopsis thaliana ☆

The model plant Arabidopsis thaliana was studied for the search of new metabolites involved in wound signalling. Diverse LC approaches were considered in terms of efficiency and analysis time and a 7-min gradient on a UPLC-TOF-MS system with a short column was chosen for metabolite fingerprinting. This screening step was designed to allow the comparison of a high number of samples over a wide range of time points after stress induction in positive and negative ionisation modes. Thanks to data treatment, clear discrimination was obtained, providing lists of potential stress-induced ions. In a second step, the fingerprinting conditions were transferred to longer column, providing a higher peak capacity able to demonstrate the presence of isomers among the highlighted compounds.

Optimized liquid chromatography-mass spectrometry approach for the isolation of minor stress biomarkers in plant extracts and their identification by capillary nuclear magnetic resonance.

A LC-MS approach is presented for the isolation of minor key plant biomarkers, in view of their characterization by NMR at the microgram scale. Due to the complexity of plant extracts, the purification of metabolites present in low concentrations is critical. The strategy used relies on the optimization of the chromatographic analysis using ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOF-MS), thanks to modelling software. The optimized method is then transferred to semi-preparative LC conditions with MS detection. The approach is illustrated by the isolation of wound-induced jasmonate derivatives revealed by a metabolomic study in Arabidopsis thaliana leaves and their subsequent characterization by capillary NMR (CapNMR).

Some solutions to obtain very efficient separations in isocratic and gradient modes using small particles size and ultra-high pressure

The UHPLC strategy which combines sub-2 microm porous particles and ultra-high pressure (>1000 bar) was investigated considering very high resolution criteria in both isocratic and gradient modes, with mobile phase temperatures between 30 and 90 degrees C. In isocratic mode, experimental conditions to reach the maximal efficiency were determined using the kinetic plot representation for DeltaP(max)=1000 bar. It has been first confirmed that the molecular weight of the compounds (MW) was a critical parameter which should be considered in the construction of such curves. With a MW around 1000 g mol(-1), efficiencies as high as 300,000 plates could be theoretically attained using UHPLC at 30 degrees C. By limiting the column length to 450 mm, the maximal plate count was around 100,000. In gradient mode, the longest column does not provide the maximal peak capacity for a given analysis time in UHPLC. This was attributed to the fact that peak capacity is not only related to the plate number but also to column dead time. Therefore, a compromise should be found and a 150 mm column should be preferentially selected for gradient lengths up to 60 min at 30 degrees C, while the columns coupled in series (3x 150 mm) were attractive only for t(grad)>250 min. Compared to 30 degrees C, peak capacities were increased by about 20-30% for a constant gradient length at 90 degrees C and gradient time decreased by 2-fold for an identical peak capacity.

Metabolite profiling of plant extracts by ultra-high-pressure liquid chromatography at elevated temperature coupled to time-of-flight mass spectrometry

Detailed metabolite profiling of crude plant extracts, mandatory for both quality control and metabolomics purposes, requires high-resolution separation and sensitive detection with a reasonable sample throughput. In this respect, the use of ultra-high-pressure liquid chromatography (UHPLC) working at high temperature (HT) and coupled to time-of-flight mass spectrometry (TOF-MS) was evaluated in the present study in terms of achievable peak capacities for given analysis times. Prior to the analysis of complex mixtures, the effects of TOF-MS detection on peak capacity were evaluated, and a loss of 15-30% compared to UV was observed due to the additional band broadening generated by this detector. Extracts from a model plant Arabidopsis thaliana and from a widely used phytochemical preparation Ginkgo biloba, as well as a standard mixture of representative natural products (NPs), have been analyzed. As expected from the theory, the increase in mobile phase temperature of up to 90 degrees C for the profiling of extracts containing metabolites spread over a large polarity range (e.g., Arabidopsis thaliana) generated similar peak capacities to those obtained at room temperature, but with a 2- to 3-fold reduction in analysis time, demonstrating the power of this approach for such applications. On the other hand, for the analysis of more polar extracts (e.g., Ginkgo biloba), the use of higher temperature was not beneficial, as it induced a significant decrease in retention, and thus resolving power, because of the increase in elution strength. The use of HT-UHPLC-TOF-MS raised the question of NP stability under high temperature conditions. This work demonstrated that no apparent degradation was evidenced at high temperature for a representative mixture of NPs and also for the different metabolites detected in the selected plant extracts.

Differential Analysis of Mycoalexins in Confrontation Zones of Grapevine Fungal Pathogens by Ultrahigh Pressure Liquid Chromatography/Time-of-Flight Mass Spectrometry and Capillary Nuclear Magnetic Resonance

An original approach was developed for the chemical and biological investigation of zone lines formed by the confrontation of fungi growing in confined spaces. Two wood-decaying fungi involved in esca disease, Eutypa lata and Botryosphaeria obtusa, were grown in Petri dishes. Metabolic profiles of pure fungal strains and confrontation zones were differentially analyzed by ultrahigh pressure liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC/TOFMS). Selected metabolites induced by the confrontation were isolated and characterized by capillary NMR (CapNMR) at the submilligram level. Fungitoxic and phytotoxic assays were applied to the crude extracts and isolated molecules. While the extracts of pure strains were inactive, the extract from confrontation zones exhibited significant activities. A very strongly induced compound, O-methylmellein, may explain these toxic properties. The developed approach demonstrates the use of fungal confrontations as an original source of bioactive molecules and gives new insights into the study of esca disease.

Mass spectrometry‐based metabolomics oriented by correlation analysis for wound‐induced molecule discovery: identification of a novel jasmonate glucoside

INTRODUCTION Jasmonates are members of the oxylipin family involved in various plant regulatory processes, particularly in the response to biotic and abiotic stresses. They are present in very low amounts in wounded plants, which complicates their detection within complex plant extracts. Therefore, advanced analytical methods are needed for the profiling and characterisation of novel jasmonate derivatives. OBJECTIVE To use metabolomics to search for original wound-induced metabolites belonging to the jasmonate family. METHODOLOGY Numerous Arabidopsis specimens harvested at various time points after wounding were analysed by ultra-high pressure liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC/TOFMS). A correlation analysis based on kinetic profile similarities with known jasmonates was applied to find wound-induced molecules having a potential role in defence signalling. Their characterisation was performed by tandem mass spectrometry and capillary nuclear magnetic resonance spectroscopy. RESULTS The statistical data treatment highlighted several previously reported jasmonates as well as a new glucoside derivative of the jasmonate 3-oxo-2-(2Z-pentenyl) cyclopentane-1-butyric acid (OPC-4). The monitoring of its kinetics in response to wounding revealed a delayed accumulation compared to the profile of OPC-4. This suggests an inactivation or elimination of OPC-4 through the formation of a polar glucosylated metabolite. CONCLUSION The metabolomic approach developed has proved useful in the discovery of original jasmonates synthesised in response to plant wounding.

High-resolution profiling of oxylipin-containing galactolipids in Arabidopsis extracts by ultra-performance liquid chromatography/time-of-flight mass spectrometry.

A high-resolution ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOFMS) method using in-source collision-induced dissociation (CID) was developed for globally profiling oxylipin-containing galactolipids in Arabidopsis wounded leaves. MS and pseudo-MS/MS spectra were obtained during a single analytical run by switching a lens of the TOFMS transfer optics from low to high voltage. Numerous known galactolipids were observed, and four novel mono- or di-galactosyl monoacylglycerides (MGMGs or DGMGs) containing oxophytodienoic acid (OPDA) or dinor-oxophytodienoic acid (dn-OPDA), esterified respectively at the sn1 and the sn2 positions, were identified. Rapid microisolation of the galactolipids followed by alkaline and enzymatic hydrolyses enabled the release of the esterified oxylipins, which allowed for the unambiguous characterization of the oxylipin-containing monoacylglycerides. Their strong induction in response to wounding indicates that these compounds are probably lysogalactolipids formed from galactosyldiglycerides in the injured tissues.

Isomeric tropane alkaloids from the aerial parts of Schizanthus tricolor.

Investigation of the aerial parts of Schizanthus tricolor yielded seven isomeric tropane alkaloids: 3alpha-(1-methylitaconyl)-6beta-senecioyloxytropane (1), 3alpha-(1-methylitaconyl)-6beta-angeloyloxytropane (2), 3alpha-(1-methylmesaconyl)-6beta-senecioyloxytropane (3), 3alpha-(1-methylmesaconyl)-6beta-angeloyloxytropane (4), 3alpha-(1-methylmesaconyl)-6beta-tigloyloxytropane (5), 3alpha-(1-methylcitraconyl)-6beta-senecioyloxytropane (6), and 3alpha-(1-methylcitraconyl)-6beta-angeloyloxytropane (7). Their structures were established by NMR including (1)H, (13)C NMR, HSQC, HMBC, COSY, and NOESY experiments, UV, IR, and mass spectrometry. Compounds 1, 6, and 7 are new to the literature. Alkaloids 1, 3, 4, and 5 and a mixture of 3, 4, and 5 were evaluated for in vitro antiplasmodial and cytotoxic activity. Compounds 1, 4, and 5 showed marginal inhibition of Plasmodium falciparum strain K1 with IC(50) values of 22.8, 24.8, and 36.0 microM and displayed no cytotoxicity on MRC-65 cells (CC(50) > 64 microM). Alkaloid 3 was inactive (IC(50) 63.5 microM). The alkaloid mixture exhibited slightly higher activity (IC(50) 17.0 microM) than the pure compounds, indicating some synergy between the different isomers.