Jasmonates are a family of oxylipin phytohormones regulating plant development and growth and mediating "defense versus growth" responses. The upstream JA biosynthetic precursor cis-(+)-12-oxo-phytodienoic acid (cis-OPDA) acts independently of CORONATIVE INSENSITIVE 1-mediated JA signaling in several stress-induced and developmental processes. However, its perception and metabolism are only partially understood. An isoleucine analog of the biologically active JA-Ile, OPDA-Ile, was detected years ago in wounded leaves of flowering plants, opening up the possibility that conjugation of cis-OPDA to amino acids might be a relevant mechanism for cis-OPDA regulation. Here, we extended the analysis of amino acid conjugates of cis-OPDA and identified naturally occurring OPDA-Val, OPDA-Phe, OPDA-Ala, OPDA-Glu, and OPDA-Asp accumulating in response to biotic and abiotic stress in Arabidopsis (Arabidopsis thaliana). The OPDA amino acid conjugates displayed cis-OPDA-related plant responses in a JA-Ile-dependent manner. We also showed that the synthesis and hydrolysis of cis-OPDA amino acid conjugates are mediated by members of the amidosynthetase GRETCHEN HAGEN 3 and the amidohydrolase INDOLE-3-ACETYL-LEUCINE RESISTANT 1/ILR1-like families. Thus, OPDA amino acid conjugates function in the catabolism or temporary storage of cis-OPDA in stress responses instead of acting as chemical signals per se.

• MeSH
• amidy metabolismus   MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• cyklopentany * metabolismus   MeSH
• fyziologický stres * MeSH
• homeostáza * MeSH
• isoleucin analogy a deriváty   metabolismus   MeSH
• nenasycené mastné kyseliny * metabolismus   MeSH
• oxylipiny * metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• regulátory růstu rostlin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 12-oxophytodienoic acid MeSH Prohlížeč
• amidy MeSH
• cyklopentany * MeSH
• isoleucin MeSH
• jasmonic acid MeSH Prohlížeč
• nenasycené mastné kyseliny * MeSH
• oxylipiny * MeSH
• regulátory růstu rostlin MeSH

Formation of the apical hook in etiolated dicot seedlings results from differential growth in the hypocotyl apex and is tightly controlled by environmental cues and hormones, among which auxin and gibberellins (GAs) play an important role. Cell expansion is tightly regulated by the cell wall, but whether and how feedback from this structure contributes to hook development are still unclear. Here, we show that etiolated seedlings of the Arabidopsis (Arabidopsis thaliana) quasimodo2-1 (qua2) mutant, defective in pectin biosynthesis, display severe defects in apical hook formation and maintenance, accompanied by loss of asymmetric auxin maxima and differential cell expansion. Moreover, qua2 seedlings show reduced expression of HOOKLESS1 (HLS1) and PHYTOCHROME INTERACTING FACTOR4 (PIF4), which are positive regulators of hook formation. Treatment of wild-type seedlings with the cellulose inhibitor isoxaben (isx) also prevents hook development and represses HLS1 and PIF4 expression. Exogenous GAs, loss of DELLA proteins, or HLS1 overexpression partially restore hook development in qua2 and isx-treated seedlings. Interestingly, increased agar concentration in the medium restores, both in qua2 and isx-treated seedlings, hook formation, asymmetric auxin maxima, and PIF4 and HLS1 expression. Analyses of plants expressing a Förster resonance energy transfer-based GA sensor indicate that isx reduces accumulation of GAs in the apical hook region in a turgor-dependent manner. Lack of the cell wall integrity sensor THESEUS 1, which modulates turgor loss point, restores hook formation in qua2 and isx-treated seedlings. We propose that turgor-dependent signals link changes in cell wall integrity to the PIF4-HLS1 signaling module to control differential cell elongation during hook formation.

• MeSH
• Arabidopsis * genetika   růst a vývoj   metabolismus   MeSH
• benzamidy MeSH
• buněčná stěna * metabolismus   MeSH
• gibereliny metabolismus   MeSH
• hypokotyl růst a vývoj   genetika   metabolismus   MeSH
• kyseliny indoloctové * metabolismus   MeSH
• mutace genetika   MeSH
• pektiny metabolismus   MeSH
• proteiny huseníčku * metabolismus   genetika   MeSH
• regulace genové exprese u rostlin * MeSH
• semenáček * genetika   růst a vývoj   metabolismus   MeSH
• transkripční faktory bHLH * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• benzamidy MeSH
• gibereliny MeSH
• isoxaben MeSH Prohlížeč
• kyseliny indoloctové * MeSH
• pektiny MeSH
• PIF4 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku * MeSH
• transkripční faktory bHLH * 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

In situ separation and visualization of synthetic and naturally occurring isomers from heterogeneous plant tissues, especially when they share similar molecular structures, are a challenging task. In this study, we combined the ion mobility separation with desorption electrospray ionization mass spectrometry imaging (DESI-IM-MSI) to achieve a direct separation and visualization of two synthetic auxin derivatives, auxinole and its structural isomer 4pTb-MeIAA, as well as endogenous auxins from Arabidopsis samples. Distinct distribution of these synthetic isomers and endogenous auxins in Arabidopsis primary roots and hypocotyls was achieved in the same imaging analysis from both individually treated and cotreated samples. We also observed putative metabolites of synthetic auxin derivatives, i.e. auxinole amino acid conjugates and hydrolysed 4pTb-MeIAA product - 4pTb-IAA, based on their unique drifting ion intensity patterns. Furthermore, DESI-IM-MSI-revealed abundance of endogenous auxins and synthetic isomers was validated by liquid chromatography-mass spectrometry (LC-MS). Our results demonstrate that DESI-IM-MSI could be used as a robust technique for detecting endogenous and exogenous isomers and provide a spatiotemporal evaluation of hormonomics profiles in plants.

• Klíčová slova
• Auxin, Desorption electrospray ionization, Ion mobility, Isomer, Mass spectrometry imaging, Metabolite,
• MeSH
• Arabidopsis * MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací metody   MeSH
• isomerie MeSH
• kyseliny indoloctové analýza   MeSH
• molekulární struktura MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kyseliny indoloctové 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

INTRODUCTION: Chloroplast calcium homeostasis plays an important role in modulating the response of plants to abiotic and biotic stresses. One of the greatest challenges is to understand how chloroplast calcium-permeable pathways and sensors are regulated in a concerted manner to translate specific information into a calcium signature and to elucidate the downstream effects of specific chloroplast calcium dynamics. One of the six homologs of the mitochondrial calcium uniporter (MCU) was found to be located in chloroplasts in the leaves and to crucially contribute to drought- and oxidative stress-triggered uptake of calcium into this organelle. METHODS: In the present study we integrated comparative proteomic analysis with biochemical, genetic, cellular, ionomic and hormone analysis in order to gain an insight into how chloroplast calcium channels are integrated into signaling circuits under watered condition and under drought stress. RESULTS: Altogether, our results indicate for the first time a link between chloroplast calcium channels and hormone levels, showing an enhanced ABA level in the cmcu mutant already in well-watered condition. Furthermore, we show that the lack of cMCU results in an upregulation of the calcium sensor CAS and of enzymes of chlorophyll synthesis, which are also involved in retrograde signaling upon drought stress, in two independent KO lines generated in Col-0 and Col-4 ecotypes. CONCLUSIONS: These observations point to chloroplasts as important signaling hubs linked to their calcium dynamics. Our results obtained in the model plant Arabidopsis thaliana are discussed also in light of our limited knowledge regarding organellar calcium signaling in crops and raise the possibility of an involvement of such signaling in response to drought stress also in crops.

• Klíčová slova
• calcium channel, calcium sensor, chloroplast, comparative proteomics, drought stress,
• Publikační typ
• časopisecké články MeSH

Auxins are a group of phytohormones that play a key role in plant growth and development, mainly presented by the major member of the family - indole-3-acetic acid (IAA). The levels of free IAA are regulated, in addition to de novo biosynthesis, by irreversible oxidative catabolism and reversible conjugation with sugars and amino acids. These conjugates, which serve as inactive storage forms of auxin and/or degradation intermediates, can also be oxidized to form 2-oxindole-3-acetyl-1-O-ß-d-glucose (oxIAA-glc) and oxIAA-amino acids (oxIAA-AAs). Until now, only oxIAA conjugates with aspartate and glutamate have been identified in plants. However, detailed information on the endogenous levels of these and other putative oxIAA-amino acid conjugates in various plant species and their spatial distribution is still not well understood but is finally getting more attention. Herein, we identified and characterized two novel naturally occurring auxin metabolites in plants, namely oxIAA-leucine (oxIAA-Leu) and oxIAA-phenylalanine (oxIAA-Phe). Subsequently, a new liquid chromatography-tandem mass spectrometry method was developed for the determination of a wide range of IAA metabolites. Using this methodology, the quantitative determination of IAA metabolites including newly characterized oxIAA conjugates in roots, shoots and cotyledons of four selected plant models - Arabidopsis thaliana, pea (Pisum sativum L.), wheat (Triticum aestivum L.) and maize (Zea mays L.) was performed to compare auxin metabolite profiles. The distribution of various groups of auxin metabolites differed notably among the studied species as well as their sections. For example, oxIAA-AA conjugates were the major metabolites found in pea, while oxIAA-glc dominated in Arabidopsis. We further compared IAA metabolite levels in plants harvested at different growth stages to monitor the dynamics of IAA metabolite profiles during early seedling development. In general, our results show a great diversity of auxin inactivation pathways among angiosperm plants. We believe that our findings will greatly contribute to a better understanding of IAA homeostasis.

• Klíčová slova
• 2-oxindole-3-acetic acid, HPLC-MS/MS, auxin conjugates, auxin metabolism, catabolism, indole-3-acetic acid, quantitative analysis,
• Publikační typ
• časopisecké články MeSH