Cytosolic- and membrane-localized oxidized indole-3-acetic acid formation regulates developmental auxin transients

Catabolism of the auxin indole-3-acetic acid (IAA) to terminate cellular responses primarily occurs in three steps: (i) conjugation of IAA to Asp/Glu, (ii) oxidation of the indole ring by DIOXYGENASE FOR AUXIN OXIDATION (DAO), and (iii) amidohydrolase cleavage of Asp/Glu. This study examines if IAA oxidation historically associated with membranes is mediated by DAO isoforms and if the oxidized auxin product (oxIAA) retains nominal functionality. We show that Arabidopsis thaliana DAO1 exhibits both soluble and auxin-dependent plasma membrane association, and that oxIAA exhibits weak "anti-auxin" activity. Both soluble and membrane-associated DAO1 primarily oxidized IAAsp. DAO2 activity was enzymatically similar to DAO1 and occurred where IAA levels were high. DAO1 and DAO2 functioned synergistically in adventitious root formation and in temperature-dependent petal development. In vitro assays showed that oxIAA acts as a molecular glue between repressor auxin/indole-3-acetic acid (AUX/IAA) proteins IAA7 and IAA17 and Transport Inhibitor Response 1 (TIR1) auxin co-receptors but is readily outcompeted by IAA. Biolayer interferometry and yeast degradation assays indicated weak "anti-auxin" activity, as oxIAA enhances IAA7-TIR1 interactions while retarding IAA7 and IAA17 degradation. In a low auxin/quiescent interval that precedes auxin-triggered apical hook opening in etiolated seedlings, IAA7 gain-of-function and loss-of-function mutants exhibited early apical hook opening similar to dao1, and application of oxIAA to etiolated dao1 apical hooks partially rescued the phenotype. The weak "anti-auxin" activity of oxIAA during transitional growth is an important reminder of the evolutionary processes that forge adaptive plant growth responses.