In recent years, substantial progress has been made in exploring auxin conjugation and metabolism, primarily aiming at indole-3-acetic acid (IAA). However, the metabolic regulation of another key auxin, phenylacetic acid (PAA), remains largely uncharacterized. Here, we provide a comprehensive exploration of PAA metabolism in land plants. Through LC-MS screening across multiple plant species and their organs, we identified four previously unreported endogenous PAA metabolites: phenylacetyl-leucine, phenylacetyl-phenylalanine, phenylacetyl-valine, and phenylacetyl-glucose. Enzyme assays, genetic evidence, crystal structures, and docking studies demonstrate that PAA and IAA share core metabolic machinery, revealing a complex regulatory network that maintains auxin homeostasis. Furthermore, our study of PAA conjugation with amino acids and glucose suggests limited compensatory mechanisms within known conjugation pathways, pointing to the existence of alternative metabolic routes in land plants. These insights advance our knowledge of auxin-specific metabolic networks and highlight the unique complexity within plant hormone regulation.

• Klíčová slova
• Auxin, Gretchen Hagen 3, HPLC-MS/MS, conjugation, glucosyl ester, indole-3-acetic acid, metabolism, phenylacetic acid, plant,
• MeSH
• fenylacetáty * metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• metabolické sítě a dráhy * MeSH
• regulátory růstu rostlin * metabolismus   MeSH
• vyšší rostliny * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fenylacetáty * MeSH
• kyseliny indoloctové MeSH
• phenylacetic acid MeSH Prohlížeč
• regulátory růstu rostlin * MeSH

Root hairs (RHs) are mixed tip- and non-tip-growing protrusions derived from root epidermal cells that play essential roles in nutrient and water uptake, root anchorage, and interactions with soil microorganisms. Nutrient availability and temperature are critical and interconnected factors for sustained plant growth, but the molecular mechanisms that underlie their perception and downstream signaling pathways remain unclear. Here, we show that moderately low temperature (10°C) induces a strong RH elongation response mediated by several molecular components of the auxin pathway. Specifically, auxin biosynthesis mediated by TAA1/YUCCAs, auxin transport via PIN2, PIN4, and AUX1, and auxin signaling regulated by TIR1/AFB2 in conjunction with specific ARFs (ARF6/ARF8 and ARF7, but not ARF19) contribute to the RH response under moderately low temperature. These findings establish the auxin biosynthesis and signaling pathway as a central regulatory process driving RH growth under moderate low-temperature conditions in roots. Our work underscores the importance of moderately low temperature as a stimulus that interacts with complex nutritional signaling originating from the growth medium and the plant nutritional status; this process has the potential to be fine-tuned for future biotechnological applications to enhance nutrient uptake.

• Klíčová slova
• Arabidopsis, auxin, moderately low temperature, root hairs,
• MeSH
• Arabidopsis * růst a vývoj   metabolismus   genetika   fyziologie   MeSH
• kořeny rostlin * růst a vývoj   metabolismus   genetika   MeSH
• kyseliny indoloctové * metabolismus   MeSH
• nízká teplota MeSH
• proteiny huseníčku metabolismus   genetika   MeSH
• regulace genové exprese u rostlin MeSH
• signální transdukce * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kyseliny indoloctové * MeSH
• proteiny huseníčku MeSH

Auxin glycosylation plays a fundamental role in the regulation of auxin homeostasis, activity, and transport, contributing to the dynamic control of plant growth and development. Glycosylation enhances auxin stability, solubility, and storage capacity, serving as a key mechanism for both temporary inactivation and long-term storage of auxin molecules. Specific glycosyltransferases are critical for this process, catalyzing glycosylation at either the carboxyl group or the nitrogen atom of the indole ring. The storage roles of glycosylated auxins, such as IAA-N-Glc, have been shown to be essential during embryogenesis and seed germination, while irreversible conjugation into catabolic products helps to maintain auxin homeostasis in vegetative tissues. This review highlights the diversity, enzymatic specificity, and physiological relevance of auxin glycosylation pathways, including a frequently overlooked N-glycosylation, underscoring its importance in the complex network of auxin metabolism.

• MeSH
• glykosylace MeSH
• homeostáza * MeSH
• kyseliny indoloctové * metabolismus   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• kyseliny indoloctové * MeSH
• regulátory růstu rostlin MeSH

Indole-3-acetic acid (IAA), the most common form of auxin, is involved in a great range of plant physiological processes. IAA is synthesized from the amino acid tryptophan and can be transported and inactivated in a myriad of ways. Despite intense research efforts, there are still dark corners in our comprehension of IAA metabolism and its interplays with other pathways. Genetic screens are a powerful tool for unbiasedly looking for new players in a given biological process. However, pleiotropism of auxin-related phenotypes and indirect effects make it necessary to incorporate additional screening steps to specifically find mutants affected in IAA homeostasis. We previously developed and validated a high-throughput methodology to simultaneously quantify IAA, key precursors, and inactive forms from as little as 10 mg of fresh tissue. We have carried out a genetic screening to identify mutants involved in IAA metabolism. Auxin reporters DR5pro:VENUS and 35Spro:DII-VENUS were EMS-mutagenized and subjected to a parallel morphological and reporter-signal pre-screen. We then obtained the auxin metabolite profile of 325 M3 selected lines and used multivariate data analysis to identify potential IAA-metabolism mutants. To test the screening design, we identified the causal mutations in three of the candidate lines by mapping-by-sequencing: dii365.3, dii571.1 and dr693. These carry new alleles of CYP83A1, MIAO, and SUPERROOT2, respectively, all of which have been previously involved in auxin homeostasis. Our results support the suitability of this approach to find new genes involved in IAA metabolism.

• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• fenotyp MeSH
• kyseliny indoloctové * metabolismus   MeSH
• mutace * genetika   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• regulátory růstu rostlin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• indoleacetic acid MeSH Prohlížeč
• kyseliny indoloctové * MeSH
• proteiny huseníčku MeSH
• regulátory růstu rostlin MeSH

BACKGROUND: Hormonal homeostasis plays a critical role in the regulation of microspore embryogenesis (ME). The balance between endogenous phytohormones must be altered to induce microspore reprogramming from the classical pollen-formation pathway to embryogenic development, but too extensive changes may be detrimental. In the present study, the levels of auxins, cytokinins and abscisic acid were monitored in the anthers of two Polish winter wheat F1 lines and the spring cultivar Pavon highly differentiated in terms of ME effectiveness. Analyses were carried out at subsequent steps of the ME induction procedure that combined low temperature, sodium selenate and mannitol tiller pre-treatment. RESULTS: Of all the factors tested, mannitol induced the most profound effect on phytohormones and their homeostasis in wheat anthers. It significantly increased the accumulation of all auxins and decreased the levels of most cytokinins, while the change in ABA content was limited to cv. Pavon. In an attempt to alleviate this hormonal shock, we tested several modifications of the induction medium hormonal composition and found thidiazuron to be the most promising in stimulating the embryogenic development of wheat microspores. CONCLUSIONS: The lack of ABA-driven stress defence responses may be one of the reasons for the low effectiveness of ME induction in winter wheat microspore cultures. Low cytokinin level and a disturbed auxin/cytokinin balance may then be responsible for the morphological abnormalities observed during the next phases of embryogenic microspore development. One possible solution is to modify the hormonal composition of the induction medium with thidiazuron identified as the most promising component.

• Klíčová slova
• Triticum aestivum, Abscisic acid, Auxins, Cytokinins, Hormonal homeostasis, Microspore embryogenesis,
• MeSH
• cytokininy metabolismus   MeSH
• kyselina abscisová metabolismus   MeSH
• kyseliny indoloctové * metabolismus   MeSH
• mannitol * farmakologie   MeSH
• pšenice * embryologie   účinky léků   metabolismus   MeSH
• pyl * embryologie   účinky léků   metabolismus   MeSH
• regulátory růstu rostlin * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokininy MeSH
• kyselina abscisová MeSH
• kyseliny indoloctové * MeSH
• mannitol * MeSH
• regulátory růstu rostlin * MeSH

N-Sulfonated IAA was discovered as a novel auxin metabolite in Urtica where it is biosynthesized de novo utilizing inorganic sulfate. It showed no auxin activity in DR5::GUS assay, implying possible inactivation/storage mechanism. A novel auxin derivative, N-sulfoindole-3-acetic acid (IAA-N-SO3H, SIAA), was discovered in stinging nettle (Urtica dioica) among 116 sulfonated metabolites putatively identified by a semi-targeted UHPLC-QqTOF-MS analysis of 23 plant/algae/fungi species. These sulfometabolites were detected based on the presence of a neutral loss of sulfur trioxide, as indicated by the m/z difference of 79.9568 Da in the MS2 spectra. The structure of newly discovered SIAA was confirmed by synthesizing its standard and comparing retention time, m/z and MS2 spectrum with those of SIAA found in Urtica. To study its natural occurrence, 73 species in total were further analyzed by UHPLC-QqTOF-MS or targeted UHPLC-MS/MS method with a limit of detection of 244 fmol/g dry weight. However, SIAA was only detected in Urtica at a concentration of 13.906 ± 9.603 nmol/g dry weight. Its concentration was > 30 times higher than that of indole-3-acetic acid (IAA), and the SIAA/IAA ratio was further increased under different light conditions, especially in continuous blue light. In addition to SIAA, structurally similar metabolites, N-sulfoindole-3-lactic acid, 4-(sulfooxy)phenyllactic acid and 4-(sulfooxy)phenylacetic acid, were detected in Urtica for the first time. SIAA was biosynthesized from inorganic sulfate in seedlings, as confirmed by the incorporation of exogenous 34S-ammonium sulfate (1 mM and 10 mM). SIAA exhibited no auxin activity, as demonstrated by both the Arabidopsis DR5::GUS assay and the Arabidopsis phenotype analysis. Sulfonation of IAA may therefore be a mechanism for IAA deactivation and/or storage in Urtica, similar to sulfonation of the jasmonates in Arabidopsis.

• Klíčová slova
• N-Sulfoindole-3-acetic acid, Indole-3-acetic acid, Mass spectrometry, Metabolomics, Phytohormone, Sulfonated,
• MeSH
• Arabidopsis metabolismus   MeSH
• kyseliny indoloctové * metabolismus   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Urtica dioica metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• indoleacetic acid MeSH Prohlížeč
• kyseliny indoloctové * MeSH
• regulátory růstu rostlin MeSH

Smoke-water (SW) and Karrikinolide1 (KAR1) release dormancy and improve seed germination in many plant species. Therefore, we tested SW (1:2500 v/v) and KAR1 (10-7 M) to break the morphological dormancy of celery cultivar (Apium graveolens L.). In the first trial, seeds were subjected to a 21-day incubation period at 20 °C with SW and KAR1 applied as single treatments. KAR1 showed significantly improved germination (30.7%) as compared to SW (17.2%) and a water control (14.7%). In seed soaking experiments, SW, KAR1, and gibberellic acid (GA3) treatments showed higher germination percentages than the water control after 3 and 6 h of soaking. However, prolonged soaking (12 h) reduced germination percentages for all treatments, indicating a detrimental effect. Analysis of KAR1 content dynamics in 7-day- and 21-day-old celery seeds indicated its prolonged effects on germination and dormancy alleviation. Phytohormones, including auxins in 7-day-old and cytokinins in 7-day- and 21-day-old celery seedlings, along with their precursors and metabolites, were analyzed using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) after treatment with KAR1 and SW. The analysis of auxin levels in 7-day-old seeds revealed a negative correlation between seed germination and auxin (indole-3-acetic acid, IAA) content. Notably, it was found that KAR1-treated seeds significantly reduced IAA levels in all treatments. SW and KAR1 did not significantly affect cytokinin levels during celery germination except for N6-Isopentenyladenine. Hence, further research is needed to understand their precise role in celery seed germination. This work will improve our understanding of the role of bioactive compounds from plant-derived smoke and how they regulate hormonal responses and improve germination efficiency in celery.

• Klíčová slova
• Karrikinolide, biostimulants, celery, phytohormones, smoke-water,
• Publikační typ
• časopisecké články 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

Auxin amino acid conjugates are considered to be storage forms of auxins. Previous research has shown that indole-3-acetyl-L-alanine (IAA-Ala), indole-3-propionyl-L-alanine (IPA-Ala) and indole-3-butyryl-L-alanine (IBA-Ala) affect the root growth of Brassica rapa seedlings. To elucidate the potential mechanism of action of the conjugates, we treated B. rapa seedlings with 0.01 mM IAA-, IPA- and IBA-Ala and investigated their effects on the auxin metabolome and transcriptome. IBA-Ala and IPA-Ala caused a significant inhibition of root growth and a decrease in free IAA compared to the control and IAA-Ala treatments. The identification of free auxins IBA and IPA after feeding experiments with IBA-Ala and IPA-Ala, respectively, confirms their hydrolysis in vivo and indicates active auxins responsible for a stronger inhibition of root growth. IBA-Ala caused the induction of most DEGs (807) compared to IPA-Ala (417) and IAA-Ala (371). All treatments caused similar trends in transcription profile changes when compared to control treatments. The majority of auxin-related DEGs were found after IBA-Ala treatment, followed by IPA-Ala and IAA-Ala, which is consistent with the apparent root morphology. In addition to most YUC genes, which showed a tendency to be downregulated, transcripts of auxin-related DEGs that were identified (UGT74E2, GH3.2, SAUR, IAA2, etc.) were more highly expressed after all treatments. Our results are consistent with the hypothesis that the hydrolysis of conjugates and the release of free auxins are responsible for the effects of conjugate treatments. In conclusion, free auxins released by the hydrolysis of all auxin conjugates applied affect gene regulation, auxin homeostasis and ultimately root growth inhibition.

• Klíčová slova
• Brassica rapa, amino acid auxin conjugates, auxin metabolome, indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, root growth inhibition, transcriptome,
• MeSH
• alanin MeSH
• Brassica rapa * genetika   MeSH
• indoly MeSH
• kyseliny indoloctové farmakologie   MeSH
• plži * MeSH
• semenáček genetika   MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alanin MeSH
• indoly MeSH
• kyseliny indoloctové MeSH