Metabolism of exogenous auxin by Arabidopsis thaliana: Identification of the conjugate N‐(indol‐3‐ylacetyl)‐glutamine and initiation of a mutant screen

Abstract

The accumulation of conjugates of indole‐3‐acetic acid (IAA) in Arabidopsis thaliana was studied by incubating tissues with high concentrations of exogenous IAA, followed by reverse phase HPLC analysis of the extracts. Using fluorescence detection, indole‐3‐acetyl‐aspartate, indole‐3‐acetyl‐glutamate, and indole‐3‐acetyl‐glucose were observed and quantitated in extracts of tissue after 24 h incubation with 500 μM IAA. In addition, a new metabolite was detected and positively identified as indole‐3‐acetyl‐glutamine by fast atom bombardment mass spectrometry, exact mass measurement, and tandem mass spectrometry in comparison with a synthetic standard. The amounts of individual conjugates formed differed between leaves, shoot axes and roots. In all three tissues, indole‐3‐acetyl‐aspartate was the most abundant conjugate, the highest level being observed in roots. Highest levels of indole‐3‐acetyl‐glutamine were observed in leaves, where it was the second most abundant conjugate and comprised approximately 12% of the fluorescent metabolites. Accumulation of the three amide conjugates was dramatically inhibited by cycloheximide, whereas accumulation of indole‐3‐acetyl‐glucose was little affected. Based on these data, a screen for Arabidopsis mutants altered in the IAA‐inducible system for auxin conjugate formation was initiated. The first mutant to be isolated and characterized produces more indole‐3‐acetyl‐glutamine and less indole‐3‐acetyl‐aspartate than wild‐type, and is allelic to an existing class of photorespiration mutants (gluS) deficient in chloroplastic glutamate synthase.