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

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.

• Keywords
• Arabidopsis, auxin, moderately low temperature, root hairs,
• MeSH
• Arabidopsis * growth & development   metabolism   genetics   physiology   MeSH
• Plant Roots * growth & development   metabolism   genetics   MeSH
• Indoleacetic Acids * metabolism   MeSH
• Cold Temperature MeSH
• Arabidopsis Proteins metabolism   genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Signal Transduction * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Indoleacetic Acids * MeSH
• Arabidopsis Proteins 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

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

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

Photosynthesis is among the first processes negatively affected by environmental cues and its performance directly determines plant cell fitness and ultimately crop yield. Primarily sites of photosynthesis, chloroplasts are unique sites also for the biosynthesis of precursors of the growth regulator auxin and for sensing environmental stress, but their role in intracellular auxin homeostasis, vital for plant growth and survival in changing environments, remains poorly understood. Here, we identified two ATP-binding cassette (ABC) subfamily B transporters, ABCB28 and ABCB29, which export auxin across the chloroplast envelope to the cytosol in a concerted action in vivo. Moreover, we provide evidence for an auxin biosynthesis pathway in Arabidopsis thaliana chloroplasts. The overexpression of ABCB28 and ABCB29 influenced stomatal regulation and resulted in significantly improved water use efficiency and survival rates during salt and drought stresses. Our results suggest that chloroplast auxin production and transport contribute to stomata regulation for conserving water upon salt stress. ABCB28 and ABCB29 integrate photosynthesis and auxin signals and as such hold great potential to improve the adaptation potential of crops to environmental cues.

• Keywords
• ABC transporter, auxin, drought, hormone transport, photosynthesis, salinity, stress,
• 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

Cytokinin and auxin are plant hormones that coordinate many aspects of plant development. Their interactions in plant underground growth are well established, occurring at the levels of metabolism, signaling, and transport. Unlike many plant hormone classes, cytokinins are represented by more than one active molecule. Multiple mutant lines, blocking specific parts of cytokinin biosynthetic pathways, have enabled research in plants with deficiencies in specific cytokinin-types. While most of these mutants have confirmed the impeding effect of cytokinin on root growth, the ipt29 double mutant instead surprisingly exhibits reduced primary root length compared to the wild type. This mutant is impaired in cis-zeatin (cZ) production, a cytokinin-type that had been considered inactive in the past. Here we have further investigated the intriguing ipt29 root phenotype, opposite to known cytokinin functions, and the (bio)activity of cZ. Our data suggest that despite the ipt29 short-root phenotype, cZ application has a negative impact on primary root growth and can activate a cytokinin response in the stele. Grafting experiments revealed that the root phenotype of ipt29 depends mainly on local signaling which does not relate directly to cytokinin levels. Notably, ipt29 displayed increased auxin levels in the root tissue. Moreover, analyses of the differential contributions of ipt2 and ipt9 to the ipt29 short-root phenotype demonstrated that, despite its deficiency on cZ levels, ipt2 does not show any root phenotype or auxin homeostasis variation, while ipt9 mutants were indistinguishable from ipt29. We conclude that IPT9 functions may go beyond cZ biosynthesis, directly or indirectly, implicating effects on auxin homeostasis and therefore influencing plant growth.

• Keywords
• auxin, cytokinin, metabolism, plant hormones, root growth,
• 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