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 (PAA-Leu), phenylacetyl-phenylalanine (PAA-Phe), phenylacetyl-valine (PAA-Val), and phenylacetyl-glucose (PAA-glc). 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.

Department of Chemical Biology Faculty of Science Palacký University Olomouc Czech Republic

Department of Experimental Biology Faculty of Science Palacký University Olomouc Czech Republic

Laboratory of Growth Regulators Institute of Experimental Botany The Czech Academy of Sciences and Faculty of Science Palacký University Olomouc Czech Republic

Université Paris Saclay INRAE AgroParisTech Institute Jean Pierre Bourgin for Plant Sciences 78000 Versailles France

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Glycosylation pathways in auxin homeostasis