Metabolism of indole-3-acetic acid in Arabidopsis

Abstract

The metabolism of indole-3-acetic acid (IAA) was investigated in 14-d-old Arabidopsis plants grown in liquid culture. After ruling out metabolites formed as an effect of nonsterile conditions, high-level feeding, and spontaneous interconversions, a simple metabolic pattern emerged. Oxindole-3-acetic acid (OxIAA), OxIAA conjugated to a hexose moiety via the carboxyl group, and the conjugates indole-3-acetyl aspartic acid (IAAsp) and indole-3-acetyl glutamate (IAGlu) were identified by mass spectrometry as primary products of IAA fed to the plants. Refeeding experiments demonstrated that none of these conjugates could be hydrolyzed back to IAA to any measurable extent at this developmental stage. IAAsp was further oxidized, especially when high levels of IAA were fed into the system, yielding OxIAAsp and OH-IAAsp. This contrasted with the metabolic fate of IAGlu, since that conjugate was not further metabolized. At IAA concentrations below 0.5 microM, most of the supplied IAA was metabolized via the OxIAA pathway, whereas only a minor portion was conjugated. However, increasing the IAA concentrations to 5 microM drastically altered the metabolic pattern, with marked induction of conjugation to IAAsp and IAGlu. This investigation used concentrations for feeding experiments that were near endogenous levels, showing that the metabolic pathways controlling the IAA pool size in Arabidopsis are limited and, therefore, make good targets for mutant screens provided that precautions are taken to avoid inducing artificial metabolism.

Figure 1

Gradient-elution reversed-phase HPLC-radioactivity chromatogram of partially purified extracts of Arabidopsis plants fed [1-¹⁴C]IAA. All incubations were done in darkness.

Figure 2

Frit-FAB-MS spectra of metabolite 1 (A); and GC-MS spectrum of silylated metabolite 1 (B) and of the silylated hydrolysis product of metabolite 1 (C).

Figure 3

Time-course study of a [1-¹⁴C]IAA feed to Arabidopsis plants grown in liquid culture for 2 to 120 h and of partially purified extracts spiked with [¹⁴C]OxIAA and [¹⁴C]IAAsp.

Figure 4

Time-course study of [1-¹⁴C]IAAsp (A), [1-¹⁴C]IAGlu (B), and [1-¹⁴C]OxIAA (C) feeds to Arabidopsis plants grown in liquid culture for 6, 12, and 48 h.

Figure 5

Gradient-elution reversed-phase HPLC-radioactivity chromatogram of aliquots of partially purified extracts of sterile-grown plants fed [5-³H]IAA in liquid culture in darkness for 6 to 48 h.

Figure 6

Proposed metabolic pathway for IAA in Arabidopsis plants. The solid arrows represent the steps that have been demonstrated by in planta conversions under physiologically relevant conditions.