Metabolic Profiling of Tryptophan-overproducing Rice Calli that Express a Feedback-insensitive α Subunit of Anthranilate Synthase

Abstract

The profile of aromatic metabolites in calli was compared between wild-type rice (Oryza sativa cv. Nipponbare) and tryptophan-overproducing transgenic rice lines that express a gene (OASA1D) for a feedback-insensitive alpha subunit of anthranilate synthase. Metabolic profiling by high-performance liquid chromatography coupled with photodiode array detection of ultraviolet absorbance revealed a total of 71 peaks in both wild-type and transgenic calli. Only a limited effect on the pattern of major aromatic compounds was observed in tryptophan-accumulating transgenic rice lines, with the exception of an approximately 80-fold increase in the amount of tryptophan. Expression of OASA1D induced relatively small changes in several minor metabolites. One of the minor metabolites whose abundance was increased by OASA1D expression was purified and identified as a previously unknown indole-alkaloid glucoside. The levels of free and conjugated forms of indole-3-acetic acid (IAA), a plant hormone derived from the tryptophan biosynthetic pathway, were determined separately by liquid chromatography and tandem mass spectrometry (LC-MS/MS). The amounts of both free IAA and its conjugates were increased in the transgenic calli, suggesting that the activity of anthranilate synthase or the concentration of tryptophan (or both) is an important determinant of IAA biosynthesis.