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

Abstract

The genetic advantages t o the use o f 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 t o determine amounts and examine the metabolism of indolic compounds in Arabidopsis. lsolation and identification of endogenous indole-3-acetonitrile (IAN; a possible precursor of the auxin indole-3-acetic acid [IAAI) was carried out under mild conditions, thus proving i t s natural occurrence. We describe here the synthesis of 13Cl-labeled I A N and i t s utility i n the gas chromatography-mass spectrometry quantification o f endogenous IAN levels. We also quantified the nonenzymatic conversion o f I A N t o IAA under conditions used t o hydrolyze IAA conjugates. 13C1-Labeled I A N was used t o assess the contribution of I A N t o measured IAA following hydrolysis of IAA conjugates. We studied the stability and breakdown of the indolic glucosinolate glucobrassicin, which is known t o be present i n 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.The study of plant hormone biogenesis and metabolism has been enhanced greatly by the application of genetic techniques, especially the use of metabolic mutants (Normanly et al., 1995). Various plants have been used for such biochemical genetic studies and these include maize, pea, tobacco, and Lemna gibba. The use of Arabidopsis for such studies has significant advantages because of the simplicity of its genome, short life cycle, and the existence of an impressive number of mutant plants and information concerning specific genes. For studies of auxin metabolism, however, the advantages associated with Arabidopsis pre-

Arabidopsis Monothiol Glutaredoxin, AtGRXS17, Is Critical for Temperature-dependent Postembryonic Growth and Development via Modulating Auxin Response

“…For anthocyanin quantitation, 0.1 g of fresh weight of wild type and atgrxs17 KO seedlings, grown on one-half strength MS medium for 2 weeks at either 22 or 28°C, homogenized in 1.6 ml of extraction buffer (0.6 ml of methanol-1% HCl, 0.4 ml of H 2 O, and 0.6 ml of chloroform), and mixed well before spinning for 2 min at 16,000 ϫ g. One ml of supernatant was used to measure the absorbance at 535 nm. The amount of anthocyanin is expressed as cyanidin 3-glucoside equivalents (mg⅐0.1 g of fresh weight Ϫ1 ) (41 (42). After 1 h on ice, 150 l of the homogenate was purified through two sequential solid phase extraction (SPE) columns, anion exchange and plastic affinity, using a Gilson SPE 215 system, methylated, dried, and redissolved in ethyl acetate exactly as described previously (43).…”

Trp-dependent auxin biosynthesis in Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3

“…IAA was purified by the method of Chen et al (1988) with slight modifications that are described in Tam and Normanly (2002). IAN was purified by the method of Ilić et al (1996). The conditions for gas chromatography selected ion monitoring mass spectrometry of IAA and IAN are described in Tam and Normanly (2002).…”

ArabidopsisCytochrome P450 Monooxygenase 71A13 Catalyzes the Conversion of Indole-3-Acetaldoxime in Camalexin Synthesis

“…Another source of IAN is the turnover of indole glucosinolates (Bones and Rossiter, 2006). A basal level of IAN is present in healthy plants (Ilic et al, 1996).…”