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

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

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.

• Keywords
• 1-aminocyclopropane-1-carboxylic acid, ACC, Abscisic acid, Arabidopsis, Auxin, Cytokinin, Ethylene, Jasmonic acid, Liquid chromatography, Mass spectrometry, Plant hormones, Salicylic acid,
• Publication type
• Journal Article MeSH

BACKGROUND: Acidic phytohormones are small molecules controlling many physiological functions in plants. A comprehensive picture of their profiles including the active forms, precursors and metabolites provides an important insight into ongoing physiological processes and is essential for many biological studies performed on plants. RESULTS: A high-throughput sample preparation method for liquid chromatography-tandem mass spectrometry determination of 25 acidic phytohormones classed as auxins, jasmonates, abscisates and salicylic acid was optimised. The method uses a small amount of plant tissue (less than 10 mg fresh weight) and acidic extraction in 1 mol/L formic acid in 10% aqueous methanol followed by miniaturised purification on reverse phase sorbent accommodated in pipette tips organised in a 3D printed 96-place interface, capable of processing 192 samples in one run. The method was evaluated in terms of process efficiency, recovery and matrix effects as well as establishing validation parameters such as accuracy and precision. The applicability of the method in relation to the amounts of sample collected from distantly related plant species was evaluated and the results for phytohormone profiles are discussed in the context of literature reports. CONCLUSION: The method developed enables high-throughput profiling of acidic phytohormones with minute amounts of plant material, and it is suitable for large scale interspecies studies.

• Keywords
• 3D printing, Evolutionarily distant plant species, High-throughput, In-tip microSPE, Liquid chromatography, Mass spectrometry, Miniaturisation, Plant hormones,
• Publication type
• Journal Article MeSH

Indole-3-acetic acid (IAA) controls a plethora of developmental processes. Thus, regulation of its concentration is of great relevance for plant performance. Cellular IAA concentration depends on its transport, biosynthesis and the various pathways for IAA inactivation, including oxidation and conjugation. Group II members of the GRETCHEN HAGEN 3 (GH3) gene family code for acyl acid amido synthetases catalysing the conjugation of IAA to amino acids. However, the high degree of functional redundancy among them has hampered thorough analysis of their roles in plant development. In this work, we generated an Arabidopsis gh3.1,2,3,4,5,6,9,17 (gh3oct) mutant to knock out the group II GH3 pathway. The gh3oct plants had an elaborated root architecture, showed an increased tolerance to different osmotic stresses, including an IAA-dependent tolerance to salinity, and were more tolerant to water deficit. Indole-3-acetic acid metabolite quantification in gh3oct plants suggested the existence of additional GH3-like enzymes in IAA metabolism. Moreover, our data suggested that 2-oxindole-3-acetic acid production depends, at least in part, on the GH3 pathway. Targeted stress-hormone analysis further suggested involvement of abscisic acid in the differential response to salinity of gh3oct plants. Taken together, our data provide new insights into the roles of group II GH3s in IAA metabolism and hormone-regulated plant development.

• Keywords
• Arabidopsis, GH3, auxin, drought, salinity, stress tolerance,
• MeSH
• Arabidopsis * metabolism   MeSH
• Hormones metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators metabolism   MeSH
• Salinity MeSH
• Water metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hormones MeSH
• Indoleacetic Acids MeSH
• Plant Growth Regulators MeSH
• Water MeSH

Together with auxin transport, auxin metabolism is a key determinant of auxin signaling output by plant cells. Enzymatic machinery involved in auxin metabolism is subject to regulation based on numerous inputs, including the concentration of auxin itself. Therefore, experiments characterizing altered auxin availability and subsequent changes in auxin metabolism could elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. Here, we studied auxin metabolism in auxin-dependent tobacco BY-2 cells. We revealed that the concentration of N-(2-oxindole-3-acetyl)-l-aspartic acid (oxIAA-Asp), the most abundant auxin metabolite produced in the control culture, dramatically decreased in auxin-starved BY-2 cells. Analysis of the transcriptome and proteome in auxin-starved cells uncovered significant downregulation of all tobacco (Nicotiana tabacum) homologs of Arabidopsis (Arabidopsis thaliana) DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), at both transcript and protein levels. Auxin metabolism profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis plants, showed not only the expected lower levels of oxIAA, but also significantly lower abundance of oxIAA-Asp. Finally, ability of DAO1 to oxidize IAA-Asp was confirmed by an enzyme assay in AtDAO1-producing bacterial culture. Our results thus represent direct evidence of DAO1 activity on IAA amino acid conjugates.

• MeSH
• Amino Acids metabolism   MeSH
• Dioxygenases metabolism   MeSH
• Oxidation-Reduction MeSH
• Plant Proteins metabolism   MeSH
• Nicotiana enzymology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Amino Acids MeSH
• Dioxygenases MeSH
• Plant Proteins MeSH