The CYP71A, NIT, AMI, and IAMH gene families are dispensable for indole-3-acetaldoxime-mediated auxin biosynthesis in Arabidopsis.

The auxin indole-3-acetic acid (IAA) governs plant development and environmental responses. Although the indole-3-pyruvic acid (IPyA) pathway is the predominant route for IAA biosynthesis, other pathways have been proposed, such as the indole-3-acetaldoxime (IAOx) pathway. The IAOx pathway has garnered attention due to its supposed activation in auxin-overproducing mutants (e.g. sur1, sur2, ugt74b1) and the auxin-like responses triggered by exogenous application of its proposed intermediates IAOx, indole-3-acetonitrile (IAN), and indole-3-acetamide (IAM). However, despite the supporting evidence for individual steps of the IAOx pathway, its overall physiological relevance remains inconclusive. Here, using a comprehensive genetic approach combined with metabolic and phenotypic profiling, we demonstrate that mutating gene families proposed to function in the IAOx pathway in Arabidopsis (Arabidopsis thaliana) does not result in prominent auxin-deficient phenotypes, nor are these genes required for the high auxin production in the sur2 mutant. Our findings also challenge the previously postulated linear IAOx pathway. Exogenously provided IAOx, IAN, and IAM can be converted to IAA in vivo, but they do not act as precursors for each other. Finally, our findings question the physiological relevance of IAM and IAN as IAA precursors in plants and suggest the existence of a yet-uncharacterized route for IAA production in the sur2 mutant, likely involving IAOx as an intermediate. The identification of the metabolic steps and the corresponding genes in this pathway may uncover another IAA biosynthesis route in plants.