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Plant and Cell Physiology Advance Access published online on February 2, 2005
Plant and Cell Physiology, doi:10.1093/pcp/pci051
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Plant and Cell Physiology 2005 © The Japanese Society of Plant Physiologists (JSPP); all rights reserved.
Received August 18, 2004
Revised January 3, 2005
Accepted January 7, 2005

Regular Paper

Metabolic Profiling of Tryptophan-Overproducing Rice Calli that Express a Feedback-Insensitive {alpha} Subunit of Anthranilate Synthase

Keiko Morino 1, Fumio Matsuda 2, Haruna Miyazawa 1, Akiko Sukegawa 3, Hisashi Miyagawa 1, and Kyo Wakasa 4*

1 Division of Applied Life Sciences, Department of Agriculture, Kyoto University, Kyoto 606-8502, Japan; CREST, Japan Science and Technology Agency, Tokyo 103-0027, Japan
2 CREST, Japan Science and Technology Agency, Tokyo 103-0027, Japan
3 Biotechnology Laboratory, National Institute of Crop Science, Ibaraki 305-8518, Japan
4 CREST, Japan Science and Technology Agency, Tokyo 103-0027, Japan; Biotechnology Laboratory, National Institute of Crop Science, Ibaraki 305-8518, Japan

* To whom correspondence should be addressed.
Kyo Wakasa, E-mail: kwakasa{at}naro.affrc.go.jp


  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 ~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.

Keywords: anthranilate synthase; indole-3-acetic acid; metabolic profiling; Oryza sativa; rice; tryptophan.
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