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

The accumulation of conjugates of indole-3-acetic acid (IAA) in Arabinopsis thaliana was studied by incubating tissues with high concentrations of exo genous IAA, followed by reverse phase HPLC analysis of the extracts, Using fluorescence detection, indole-3-acetyl-aspartate, indole-3-acetyl-glutamat e, and indole-3-acetyl-glucose were observed and quantitated in extracts of tissue? after 24 h incubation,sith 500 mu M IAA. In addition, a new metabo lite was detected and positively identified as indole-3-acetyl-glutamine by fast atom bombardment mass spectrometry, exact mass measurement, and tande m mass spectrometry in comparison with a synthetic standard. The amounts of individual conjugates formed differed between lea ces, shoot axes and root s. In all three tissues, indole-3-acetyl-aspartate was the most abundant co njugate, the highest lec el being observed in roots, Highest levels of indo le-3-acetyl-glutamine were observed in leaves, where it was the second most abundant conjugate and comprised approximately 12% of the fluorescent meta bolites. Accumulation of the three amide conjugates was dramatically inhibi ted by cycloheximide, whereas accumulation of indole-3-acetyl-glucose was l ittle affected. Based on these data, a screen for Arabidopsis mutants alter ed in the IAA-inducible system for auxin conjugate formation was initiated. The first mutant to be isolated and characterized produces more indole-3-a cetyl-glutamine and less indole-3-acetyl-aspartate than wild-type, and is a llelic to an existing class of photorespiration mutants (gluS) deficient in chloroplastic glutamate synthase.