Hormonal dynamics after Plasmodiophora brassicae infection were compared in two Brassica napus cultivars-more resistant SY Alister and more sensitive Hornet, in order to elucidate responses associated with efficient defense. Both cultivars responded to infection by the early transient elevation of active cytokinins (predominantly cis-zeatin) and auxin indole-3-acetic acid (IAA) in leaves and roots, which was longer in Hornet. Moderate IAA levels in Hornet roots coincided with a high expression of biosynthetic gene nitrilaseNIT1 (contrary to TAA1, YUC8, YUC9). Alister had a higher basal level of salicylic acid (SA), and it stimulated its production (via the expression of isochorismate synthase (ICS1)) in roots earlier than Hornet. Gall formation stimulated cytokinin, auxin, and SA levels-with a maximum 22 days after inoculation (dai). SA marker gene PR1 expression was the most profound at the time point where gall formation began, in leaves, roots, and especially in galls. Jasmonic acid (JA) was higher in Hornet than in Alister during the whole experiment. To investigate SA and JA function, SA was applied before infection, and twice (before infection and 15 dai), and JA at 15 dai. Double SA application diminished gall formation in Alister, and JA promoted gall formation in both cultivars. Activation of SA/JA pathways reflects the main differences in clubroot resistance.
- MeSH
- Aminohydrolases genetics MeSH
- Brassica napus growth & development metabolism parasitology MeSH
- Cyclopentanes analysis MeSH
- Cytokinins analysis MeSH
- Intramolecular Transferases genetics MeSH
- Plant Roots growth & development metabolism parasitology MeSH
- Indoleacetic Acids analysis MeSH
- Plant Leaves growth & development metabolism parasitology MeSH
- Plant Diseases parasitology MeSH
- Disease Resistance MeSH
- Oxylipins analysis MeSH
- Plasmodiophorida pathogenicity MeSH
- Gene Expression Regulation, Plant MeSH
- Plant Growth Regulators analysis MeSH
- Plant Proteins genetics MeSH
- Gene Expression Regulation, Developmental MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
The methodology described here represents an improved strategy for analysis of a broad range of stress-related plant hormones including jasmonates, salicylic acid, abscisic acid, and auxin metabolites. The method conditions are optimized in order to reduce the background effect of complicated plant matrix, allow effective preconcentration and thus perform highly sensitive profiling of multiple plant hormones by ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).
- MeSH
- Cyclopentanes analysis MeSH
- Abscisic Acid analysis MeSH
- Salicylic Acid analysis MeSH
- Indoleacetic Acids analysis MeSH
- Oxylipins analysis MeSH
- Plant Growth Regulators analysis MeSH
- Tandem Mass Spectrometry methods MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cyclopentenediones (CPDs) are compounds with a variety of applications ranging from the preparation of functional polymers to the development of antimicrobial agents, suggesting the potential use of CPDs as novel bioactive compounds or drugs. For this reason, a detailed characterization of CPDs and the development of robust analytical methods for their trace analysis are being sought. Here we focused on the design and synthesis of a library of novelized benzylidene CPD derivatives that were consequently characterized by ultra-high performance liquid chromatography (UHPLC) on-line connected with tandem mass spectrometry (MS/MS). The library design was based on a 2-benzylidene-4-cyclopentene-1,3-dione skeleton substituted with a variety of hydroxy, methoxy, halogen, linear aliphatic, heterocyclic and saccharide moieties, primarily modulating the skeleton's hydrophobicity. The prepared CPDs were effectively ionized by positive/negative atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI). After careful optimization of the dopant composition and flow rate, positive-mode APPI proved to be more sensitive than APCI. In negative mode, both ionization techniques gave similar results. Further, a detailed MS fragmentation study was performed, confirming the structure of the compounds and enabling positional isomers of CPDs to be differentiated on the basis of their collision spectra analysis. Finally, an optimization of the composition of the mobile phase and reversed-phased separation mode were done, followed by a selection of the most suitable UHPLC stationary phases, i.e. C18, C8 and phenyl. The applicability of the method was evaluated by the inclusion of the other two substances in the study, i.e. monomeric and dimeric bioactive CPDs, compound TX-1123 and nostotrebin 6 with cytostatic and antimicrobial activities, respectively. The results presented here could be used in further investigations of the chromatographic retention and MS behavior of CPDs, which could be utilized for their isolation, detailed characterization and analysis in biological systems.
BACKGROUND: Bryophytes represent a very diverse group of non-vascular plants such as mosses, liverworts and hornworts and the oldest extant lineage of land plants. Determination of endogenous phytohormone profiles in bryophytes can provide substantial information about early land plant evolution. In this study, we screened thirty bryophyte species including six liverworts and twenty-four mosses for their phytohormone profiles in order to relate the hormonome with phylogeny in the plant kingdom. METHODOLOGY: Samples belonging to nine orders (Pelliales, Jungermanniales, Porellales, Sphagnales, Tetraphidales, Polytrichales, Dicranales, Bryales, Hypnales) were collected in Central and Northern Bohemia. The phytohormone content was analysed with a high performance liquid chromatography electrospray tandem-mass spectrometry (HPLC-ESI-MS/MS). PRINCIPAL FINDINGS: As revealed for growth hormones, some common traits such as weak conjugation of both cytokinins and auxins, intensive production of cisZ-type cytokinins and strong oxidative degradation of auxins with abundance of a major primary catabolite 2-oxindole-3-acetic acid were pronounced in all bryophytes. Whereas apparent dissimilarities in growth hormones profiles between liverworts and mosses were evident, no obvious trends in stress hormone levels (abscisic acid, jasmonic acid, salicylic acid) were found with respect to the phylogeny. CONCLUSION: The apparent differences in conjugation and/or degradation strategies of growth hormones between liverworts and mosses might potentially show a hidden link between vascular plants and liverworts. On the other hand, the complement of stress hormones in bryophytes probably correlate rather with prevailing environmental conditions and plant survival strategy than with plant evolution.
- MeSH
- Bryophyta classification metabolism MeSH
- Cyclopentanes analysis metabolism MeSH
- Cytokinins analysis metabolism MeSH
- Phylogeny MeSH
- Spectrometry, Mass, Electrospray Ionization MeSH
- Abscisic Acid analysis metabolism MeSH
- Salicylic Acid analysis metabolism MeSH
- Indoleacetic Acids analysis metabolism MeSH
- Oxylipins analysis metabolism MeSH
- Plant Growth Regulators analysis metabolism MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Stress-induced changes in phytohormone metabolite profiles have rapid effects on plant metabolic activity and growth. The jasmonates (JAs) are a group of fatty acid-derived stress response regulators with roles in numerous developmental processes. To elucidate their dual regulatory effects, which overlap with those of other important defence-signalling plant hormones such as salicylic acid (SA), abscisic acid (ABA) and indole-3-acetic acid (IAA), we have developed a highly efficient single-step clean-up procedure for their enrichment from complex plant matrices that enables their sensitive quantitative analysis using hyphenated mass spectrometry technique. The rapid extraction of minute quantities of plant material (less than 20mg fresh weight, FW) into cold 10% methanol followed by one-step reversed-phase polymer-based solid phase extraction significantly reduced matrix effects and increased the recovery of labile JA analytes. This extraction and purification protocol was paired with a highly sensitive and validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method and used to simultaneously profile sixteen stress-induced phytohormones in minute plant material samples, including endogenous JA, several of its biosynthetic precursors and derivatives, as well as SA, ABA and IAA.
- MeSH
- Chromatography, Liquid MeSH
- Cyclopentanes analysis chemistry MeSH
- Solid Phase Extraction MeSH
- Stress, Physiological MeSH
- Abscisic Acid analysis chemistry MeSH
- Salicylic Acid analysis chemistry MeSH
- Indoleacetic Acids analysis MeSH
- Plant Leaves chemistry MeSH
- Fatty Acids analysis chemistry MeSH
- Molecular Structure MeSH
- Oxylipins analysis chemistry MeSH
- Plant Growth Regulators chemistry isolation & purification physiology MeSH
- Tandem Mass Spectrometry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Phytohormones are key regulators in various physiological processes of plant growth and development. Their chemical analyses, together with their genomics and proteomics, are an integral part of plant development studies. The information on hormone levels is often valuable for biologists dealing with any hormone-regulated processes. Plant tissue is a complex multicomponent mixture containing phytohormones in minute quantities (pg or ng/g fresh weight) along with many other related compounds with similar structures and/or physicochemical properties. Therefore, their analysis requires rapid, sensitive and sufficiently selective analytical methods. The significance of solid-phase extraction for purification and hyphenated techniques such as GC-MS, LC-MS and CE-MS for routine analyses of phytohormones is discussed.
- MeSH
- Brassica MeSH
- Chemistry Techniques, Analytical MeSH
- Chromatography, Liquid MeSH
- Cyclopentanes analysis MeSH
- Cytokinins analysis MeSH
- Electrophoresis, Capillary MeSH
- Financing, Organized MeSH
- Plant Physiological Phenomena MeSH
- Gibberellins analysis MeSH
- Abscisic Acid analysis MeSH
- Indoleacetic Acids analysis MeSH
- Gas Chromatography-Mass Spectrometry MeSH
- Plants MeSH
- Steroids MeSH
