Quantification of Free Plus Conjugated Indoleacetic Acid in Arabidopsis Requires Correction for the Nonenzymatic Conversion of Indolic Nitriles

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Abstract
The genetic advantages to the use of Arabidopsis thaliana mutants for the study of auxin metabolism previously have been partially offset by the complexity of indolic metabolism in this plant and by the lack of proper methods. To address some of these problems, we developed isotopic labeling methods to determine amounts and examine the metabolism of indolic compounds in Arabidopsis. Isolation and identification of endogenous indole-3-acetonitrile (IAN; a possible precursor of the auxin indole-3-acetic acid [IAA]) was carried out under mild conditions, thus proving its natural occurrence. We describe here the synthesis of 13C1-labeled IAN and its utility in the gas chromatography-mass spectrometry quantification of endogenous IAN levels. We also quantified the nonenzymatic conversion of IAN to IAA under conditions used to hydrolyze IAA conjugates. 13C1-Labeled IAN was used to assess the contribution of IAN to measured IAA following hydrolysis of IAA conjugates. We studied the stability and breakdown of the indolic glucosinolate glucobrassicin, which is known to be present in Arabidopsis. This is potentially an important concern when using Arabidopsis for studies of indolic biochemistry, since the levels of indolic auxins and auxin precursors are well below the levels of the indolic glucosinolates. We found that under conditions of extraction and base hydrolysis, formation of IAA from glucobrassicin was negligible.