Brassinosteroids (BRs) are plant steroidal hormones that play crucial roles in plant growth and development. Accurate quantification of BRs in plant tissues is essential for understanding their biological functions. This study presents a comprehensive overview of the latest methods used for the quantification of BRs in plants. We discuss the principles, advantages and limitations of various analytical techniques, including immunoassays, gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry that are used for the detection and quantification of BRs from complex plant matrixes. We also explore the use of isotopically labeled internal standards to improve the accuracy and reliability of BR quantification.
- Klíčová slova
- Brassinosteroids, Chemical synthesis, Chromatography, Immunoassays, Mass spectrometry, Quantification,
- MeSH
- brassinosteroidy * metabolismus analýza MeSH
- chromatografie kapalinová metody MeSH
- imunoanalýza metody MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí metody MeSH
- regulátory růstu rostlin metabolismus analýza MeSH
- rostliny metabolismus chemie MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- brassinosteroidy * MeSH
- regulátory růstu rostlin MeSH
We have developed and validated a novel LC-MS/MS method for simultaneously analyzing amino acids, biogenic amines, and their acetylated and methylated derivatives in plants. This method involves a one-step extraction of 2-5 mg of lyophilized plant material followed by fractionation of different biogenic amine forms, and exploits an efficient combination of hydrophilic interaction liquid chromatography (HILIC), reversed phase (RP) chromatography with pre-column derivatization, and tandem mass spectrometry (MS). This approach enables high-throughput processing of plant samples, significantly reducing the time needed for analysis and its cost. We also present a new synthetic route for deuterium-labeled polyamines. The LC-MS/MS method was rigorously validated by quantifying levels of nitrogen-related metabolites in seedlings of seven plant species, including Arabidopsis, maize, and barley, all of which are commonly used model organisms in plant science research. Our results revealed substantial variations in the abundance of these metabolites between species, developmental stages, and growth conditions, particularly for the acetylated and methylated derivatives and the various polyamine fractions. However, the biological relevance of these plant metabolites is currently unclear. Overall, this work contributes significantly to plant science by providing a powerful analytical tool and setting the stage for future investigations into the functions of these nitrogen-related metabolites in plants.
- Klíčová slova
- Acetylated amino acids, LC-MS/MS, acetylated biogenic amines, amino acids, biogenic amines, methylated amino acids, plant metabolism,
- MeSH
- Arabidopsis metabolismus růst a vývoj MeSH
- chromatografie kapalinová MeSH
- dusík * metabolismus MeSH
- ječmen (rod) metabolismus růst a vývoj MeSH
- kapalinová chromatografie-hmotnostní spektrometrie MeSH
- kukuřice setá metabolismus růst a vývoj MeSH
- polyaminy metabolismus analýza MeSH
- rostliny metabolismus MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- dusík * MeSH
- polyaminy MeSH
BACKGROUND AND AIMS: Turions are vegetative, dormant overwintering organs formed in aquatic plants in response to unfavourable ecological conditions. Contents of cytokinin (CK), auxin metabolites and abscisic acid (ABA) as main growth and development regulators were compared in innately dormant autumnal turions of 22 aquatic plant species of different functional ecological or taxonomic groups with those in non-dormant winter apices in three aquatic species and with those in spring turions of four species after their overwintering. METHODS: The hormones were analysed in miniature turion samples using ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry. KEY RESULTS: In innately dormant turions, the total contents of each of the four main CK types, biologically active forms and total CKs differed by two to three orders of magnitude across 22 species; the proportion of active CK forms was 0.18-67 %. Similarly, the content of four auxin forms was extremely variable and the IAA proportion as the active form was 0.014-99 %. The ABA content varied from almost zero to 54 µmol kg-1 dry weight and after overwintering it usually significantly decreased. Of all functional traits studied, hormone profiles depended most on the place of turion sprouting (surface vs bottom) and we suggest that this trait is crucial for turion ecophysiology. CONCLUSIONS: The key role of ABA in regulating turion dormancy was confirmed. However, the highly variable pattern of the ABA content in innately dormant and in overwintered turions indicates that the hormonal mechanism regulating the innate dormancy and its breaking in turions is not uniform within aquatic plants.
- Klíčová slova
- ABA, Cytokinins, auxins, functional traits, innate and imposed dormancy, mature winter buds, overwintering, phylogenetic correction, quiescence,
- MeSH
- cytokininy * metabolismus MeSH
- kyselina abscisová metabolismus analýza MeSH
- kyseliny indoloctové metabolismus MeSH
- regulátory růstu rostlin * metabolismus MeSH
- vegetační klid fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cytokininy * MeSH
- kyselina abscisová MeSH
- kyseliny indoloctové MeSH
- regulátory růstu rostlin * MeSH
BACKGROUND: Gaseous phytohormone ethylene levels are directly influenced by the production of its immediate non-volatile precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Owing to the strongly acidic character of the ACC molecule, its quantification has been difficult to perform. Here, we present a simple and straightforward validated method for accurate quantification of not only ACC levels, but also major members of other important phytohormonal classes - auxins, cytokinins, jasmonic acid, abscisic acid and salicylic acid from the same biological sample. RESULTS: The presented technique facilitates the analysis of 15 compounds by liquid chromatography coupled with tandem mass spectrometry. It was optimized and validated for 10 mg of fresh weight plant material. The extraction procedure is composed of a minimal amount of necessary steps. Accuracy and precision were the basis for evaluating the method, together with process efficiency, recovery and matrix effects as validation parameters. The examined compounds comprise important groups of phytohormones, their active forms and some of their metabolites, including six cytokinins, four auxins, two jasmonates, abscisic acid, salicylic acid and 1-aminocyclopropane-1-carboxylic acid. The resulting method was used to examine their contents in selected Arabidopsis thaliana mutant lines. CONCLUSION: This profiling method enables a very straightforward approach for indirect ethylene study and explores how it interacts, based on content levels, with other phytohormonal groups in plants.
- Klíčová slova
- 1-aminocyclopropane-1-carboxylic acid, ACC, Abscisic acid, Arabidopsis, Auxin, Cytokinin, Ethylene, Jasmonic acid, Liquid chromatography, Mass spectrometry, Plant hormones, Salicylic acid,
- Publikační typ
- časopisecké články MeSH
Turions are vegetative, dormant, and storage overwintering organs formed in perennial aquatic plants in response to unfavorable ecological conditions and originate by extreme condensation of apical shoot segments. The contents of cytokinins, auxins, and abscisic acid were estimated in shoot apices of summer growing, rootless aquatic carnivorous plants, Aldrovanda vesiculosa and Utricularia australis, and in developing turions at three stages and full maturity to reveal hormonal patterns responsible for turion development. The hormones were analyzed in miniature turion samples using ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry. Photosynthetic measurements in young leaves also confirmed relatively high photosynthetic rates at later turion stages. The content of active cytokinin forms was almost stable in A. vesiculosa during turion development but markedly decreased in U. australis. In both species, auxin content culminated in the middle of turion development and then decreased again. The content of abscisic acid as the main inhibitory hormone was very low in growing plants in both species but rose greatly at first developmental stages and stayed very high in mature turions. The hormonal data indicate a great strength of developing turions within sink-source relationships and confirm the central role of abscisic acid in regulating the turion development.
- Klíčová slova
- Aldrovanda vesiculosa, Utricularia australis, abscisic acid, aquatic rootless plants, auxins, cytokinins, nutrient reutilization, photosynthesis, respiration, winter buds,
- Publikační typ
- časopisecké články MeSH
The field of plant hormonomics focuses on the qualitative and quantitative analysis of the hormone complement in plant samples, akin to other omics sciences. Plant hormones, alongside primary and secondary metabolites, govern vital processes throughout a plant's lifecycle. While active hormones have received significant attention, studying all related compounds provides valuable insights into internal processes. Conventional single-class plant hormone analysis employs thorough sample purification, short analysis and triple quadrupole tandem mass spectrometry. Conversely, comprehensive hormonomics analysis necessitates minimal purification, robust and efficient separation and better-performing mass spectrometry instruments. This review summarizes the current status of plant hormone analysis methods, focusing on sample preparation, advances in chromatographic separation and mass spectrometric detection, including a discussion on internal standard selection and the potential of derivatization. Moreover, current approaches for assessing the spatiotemporal distribution are evaluated. The review touches on the legitimacy of the term plant hormonomics by exploring the current status of methods and outlining possible future trends.
- Klíčová slova
- Hormonomics, Internal standard, Liquid chromatography, Mass spectrometry, Matrix effect, Metabolomics, Omics, Plant hormone, Solid phase extraction,
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
BACKGROUND AND AIMS: Aquatic carnivorous plants have typical rootless linear shoots bearing traps and exhibit steep physiological polarity with rapid apical growth. The aim was to analyse auxin and cytokinin metabolites in traps, leaves/shoots and shoot apices in several species of genera Aldrovanda and Utricularia to elucidate how the hormonal profiles reflect the specific organ functions and polarity. METHODS: The main auxin and cytokinin metabolites were analysed in miniature samples (>2 mg dry weight) of different organs of Aldrovanda vesiculosa and six Utricularia species using ultraperformance liquid chromatography coupled with triple quadrupole mass spectrometry. KEY RESULTS: Total contents of biologically active forms (free bases, ribosides) of all four main endogenously occurring cytokinin types were consistently higher in traps than in leaves in four Utricularia species with monomorphic shoots and/or higher than in shoots in two Utricularia species with dimorphic shoots. In Aldrovanda traps, the total content of different cytokinin forms was similar to or lower than that in shoots. In U. australis leaves, feeding on prey increased all cytokinin forms, while no consistent differences occurred in Aldrovanda. In four aquatic Utricularia species with monomorphic shoots, the content of four auxin forms was usually higher in traps than in leaves. Zero IAA content was determined in U. australis leaves from a meso-eutrophic site or when prey-fed. CONCLUSIONS: Different cytokinin and auxin profiles estimated in traps and leaves/shoots of aquatic carnivorous plants indicate an association with different dominant functions of these organs: nutrient uptake by traps versus photosynthetic function of traps. Interplay of cytokinins and auxins regulates apical dominance in these plants possessing strong polarity.
- Klíčová slova
- Aldrovanda vesiculosa, Utricularia spp, Droseraceae, Lentibulariaceae, Phytohormone profiles, apices, aquatic rootless plants, leaves, physiological polarity, traps,
- MeSH
- cytokininy metabolismus MeSH
- Droseraceae * fyziologie MeSH
- hluchavkotvaré * MeSH
- kyseliny indoloctové metabolismus MeSH
- Magnoliopsida * fyziologie MeSH
- masožravé rostliny MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cytokininy MeSH
- kyseliny indoloctové MeSH
The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.
- Klíčová slova
- auxin, auxin metabolism, flow cytometry, nucleus, subcellular fractionation,
- MeSH
- Arabidopsis účinky léků metabolismus ultrastruktura MeSH
- buněčné jádro účinky léků metabolismus ultrastruktura MeSH
- buněčné kultury MeSH
- centrifugace metody MeSH
- frakcionace buněk přístrojové vybavení metody MeSH
- hmotnostní spektrometrie MeSH
- homeostáza fyziologie MeSH
- indoly metabolismus farmakologie MeSH
- kyseliny indoloctové metabolismus MeSH
- protoplasty chemie MeSH
- průtoková cytometrie MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné buňky účinky léků metabolismus ultrastruktura MeSH
- tabák účinky léků metabolismus ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- indole MeSH Prohlížeč
- indoly MeSH
- kyseliny indoloctové MeSH
- regulátory růstu rostlin MeSH
BACKGROUND: Karrikins (KARs) are recently described group of plant growth regulators with stimulatory effects on seed germination, seedling growth and crop productivity. So far, an analytical method for the simultaneous targeted profiling of KARs in plant tissues has not been reported. RESULTS: We present a sensitive method for the determination of two highly biologically active karrikins (KAR1 and KAR2) in minute amounts of plant material (< 20 mg fresh weight). The developed protocol combines the optimized extraction and efficient single-step sample purification with ultra-high performance liquid chromatography-tandem mass spectrometry. Newly synthesized deuterium labelled KAR1 was employed as an internal standard for the validation of KAR quantification using a stable isotope dilution method. The application of the matrix-matched calibration series in combination with the internal standard method yields a high level of accuracy and precision in triplicate, on average bias 3.3% and 2.9% RSD, respectively. The applicability of this analytical approach was confirmed by the successful analysis of karrikins in Arabidopsis seedlings grown on media supplemented with different concentrations of KAR1 and KAR2 (0.1, 1.0 and 10.0 µmol/l). CONCLUSIONS: Our results demonstrate the usage of methodology for routine analyses and for monitoring KARs in complex biological matrices. The proposed method will lead to better understanding of the roles of KARs in plant growth and development.
Strigolactones (SLs) are important plant hormones that are produced via the carotenoid biosynthetic pathway and occur at extremely low concentrations in various plant species. They regulate root development, play important roles in symbioses between higher plants and mycorrhizal fungi, and stimulate germination of plant-parasitic Orobanche and Striga species. Chemical analysis is central to research on the biochemistry of SLs and their roles in developmental biology and plant physiology. Here we summarize key issues relating to the identification and quantification of SLs isolated from plant tissues and exudates. The advantages and drawbacks of different protocols used for strigolactone analysis are discussed, and guidelines for selecting a procedure that will minimize losses during isolation and purification prior to final analysis are proposed. Hyphenated techniques suitable for SL analysis such as GC-MS and LC-MS/MS are also discussed, and newer ambient techniques such as HR-DART-MS and DESI-MS are highlighted as tools with considerable potential in SL research. A key advantage of these methods is that they require only simply sample preparation.
- Klíčová slova
- DESI-MS, Determination, GC–MS, HR-DART-MS, Isolation, LC–MS/MS, Strigolactones,
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
