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.

• Keywords
• Auxin, Gretchen Hagen 3, HPLC-MS/MS, conjugation, glucosyl ester, indole-3-acetic acid, metabolism, phenylacetic acid, plant,
• MeSH
• Phenylacetates * metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Metabolic Networks and Pathways * MeSH
• Plant Growth Regulators * metabolism   MeSH
• Embryophyta * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phenylacetates * MeSH
• Indoleacetic Acids MeSH
• phenylacetic acid MeSH Browser
• Plant Growth Regulators * 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
• Glycosylation MeSH
• Homeostasis * MeSH
• Indoleacetic Acids * metabolism   MeSH
• Plant Growth Regulators metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Indoleacetic Acids * MeSH
• Plant Growth Regulators 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.

• Keywords
• Triticum aestivum, Abscisic acid, Auxins, Cytokinins, Hormonal homeostasis, Microspore embryogenesis,
• MeSH
• Cytokinins metabolism   MeSH
• Abscisic Acid metabolism   MeSH
• Indoleacetic Acids * metabolism   MeSH
• Mannitol * pharmacology   MeSH
• Triticum * embryology   drug effects   metabolism   MeSH
• Pollen * embryology   drug effects   metabolism   MeSH
• Plant Growth Regulators * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cytokinins MeSH
• Abscisic Acid MeSH
• Indoleacetic Acids * MeSH
• Mannitol * MeSH
• Plant Growth Regulators * MeSH

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
• Amides metabolism   MeSH
• Arabidopsis * genetics   metabolism   MeSH
• Cyclopentanes * metabolism   MeSH
• Stress, Physiological * MeSH
• Homeostasis * MeSH
• Isoleucine analogs & derivatives   metabolism   MeSH
• Fatty Acids, Unsaturated * metabolism   MeSH
• Oxylipins * metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 12-oxophytodienoic acid MeSH Browser
• Amides MeSH
• Cyclopentanes * MeSH
• Isoleucine MeSH
• jasmonic acid MeSH Browser
• Fatty Acids, Unsaturated * MeSH
• Oxylipins * MeSH
• Plant Growth Regulators 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.

• Keywords
• N-Sulfoindole-3-acetic acid, Indole-3-acetic acid, Mass spectrometry, Metabolomics, Phytohormone, Sulfonated,
• MeSH
• Arabidopsis metabolism   MeSH
• Indoleacetic Acids * metabolism   MeSH
• Plant Growth Regulators metabolism   MeSH
• Tandem Mass Spectrometry MeSH
• Urtica dioica metabolism   MeSH
• Chromatography, High Pressure Liquid MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• indoleacetic acid MeSH Browser
• Indoleacetic Acids * MeSH
• Plant Growth Regulators 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.

• Keywords
• Auxin, Desorption electrospray ionization, Ion mobility, Isomer, Mass spectrometry imaging, Metabolite,
• MeSH
• Arabidopsis * MeSH
• Spectrometry, Mass, Electrospray Ionization methods   MeSH
• Isomerism MeSH
• Indoleacetic Acids analysis   MeSH
• Molecular Structure MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Indoleacetic Acids 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.

• Keywords
• 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
• Alanine MeSH
• Brassica rapa * genetics   MeSH
• Indoles MeSH
• Indoleacetic Acids pharmacology   MeSH
• Gastropoda * MeSH
• Seedlings genetics   MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Alanine MeSH
• Indoles MeSH
• Indoleacetic Acids 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.

• Keywords
• 2-oxindole-3-acetic acid, HPLC-MS/MS, auxin conjugates, auxin metabolism, catabolism, indole-3-acetic acid, quantitative analysis,
• Publication type
• Journal Article MeSH