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

Indole-3-acetic acid (IAA) is a crucial auxin governing plant development and environmental responses. While the indole-3-pyruvic acid (IPyA) pathway is the predominant IAA biosynthesis route, other pathways, like the indole-3-acetaldoxime (IAOx) pathway, have been proposed. 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 supporting evidence for individual steps, conclusive physiological relevance of the IAOx pathway remains unproven. Using a comprehensive genetic approach combined with metabolic and phenotypic profiling, we demonstrate that mutating gene families proposed to function in the IAOx pathway does not result in prominent auxin-deficient phenotypes, nor are these genes required for high-auxin production in the sur2 mutant. Our findings also challenge the previously postulated linear IAOx pathway. While exogenously provided IAOx, IAN, and IAM can be converted to IAA in vivo, 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 auxin biosynthetic route, likely involving IAOx as an intermediate, for the production of IAA in the sur2 mutant. Future identification of the metabolic steps and the corresponding genes in this new pathway may uncover the previously unknown way of synthesizing IAA in plants.