The diphenylurea-type GH3 enzyme inhibitor Auxurea A selectively inhibits the auxin inactivation pathway in dicots

Abstract Indole-3-acetic acid (IAA), the predominant natural auxin, plays a crucial role in plant growth and development. However, the molecular mechanism underlying the precise regulation of IAA homeostasis is not fully understood. The GH3 family of IAA–amino acid-conjugating enzymes is crucial for maintaining auxin homeostasis by converting IAA into inactive IAA–amino acid conjugates. Here, we report on Auxurea A (AuR), a diphenylurea compound derived from the structural optimization of a GH3 inhibitor identified through a chemical library screen. AuR specifically inhibited GH3-mediated IAA inactivation both in vivo and in vitro. AuR increased endogenous IAA levels in Arabidopsis (Arabidopsis thaliana) plants, phenocopying the IAA-overaccumulating gh3-septuple mutants. Kinetic analyses revealed that AuR acted as a selective competitive inhibitor of GH3 enzymes. The high-auxin phenotypes induced by AuR were significantly suppressed in the aux1 mutant, which is defective in auxin influx. The AuR-induced phenotypes were also suppressed by AUX1 inhibitors. In contrast, auxin efflux inhibitors enhanced the AuR-induced high-auxin phenotypes. These findings, together with IAA inactivation, underscore the pivotal role of IAA transport between the apoplast and cytosol in maintaining intracellular auxin homeostasis. Notably, AuR exhibited selective activity toward dicotyledonous plants, including tobacco, lettuce, and carrot, but was not effective in monocotyledonous plants, such as rice and barley. These results demonstrate that AuR is a valuable chemical tool for investigating auxin homeostasis and a promising compound for the development of agrochemicals.