A 5-methyltryptophan resistant mutant of rice has an altered regulation of anthranilate synthase gene expression Yuichi Ishikawa a , Jin-Heui Park a , Hiroaki Kisaka a , Hyo-Yeon Lee b , Akira Kanno a , Toshiaki Kameya a, * a Graduate School of Life Sciences, Tohoku University, Katahira 2-1-1, Aoba-Ku, Sendai 980-8577, Japan b School of Plant Science and Production, Suncheon National University, 315 Maegok-Dong, Sunchon, Jeonnam 540-742, South Korea Received 5 July 2002; received in revised form 24 February 2003; accepted 24 February 2003 Abstract The rice mutant, TR-1, was previously selected for resistance to growth inhibition by the tryptophan (Trp) analog 5- methyltryptophan (5MT). This mutant has an elevated Trp level. Since anthranilate synthase (AS) catalyzes the first step of Trp biosynthesis and is normally subject to feedback regulation by Trp, alteration of AS causes elevated Trp levels. In this study, we performed a physiological, biochemical and molecular characterization of the 5MT resistant mutant TR-68, a derivative of TR-1. The extracts of TR-68 exhibited 6-fold higher AS activity than that of the wild-type under standard conditions. However, the AS activity of TR-68 was as sensitive to feedback inhibition by Trp as that of the wild-type. We determined the nucleotide sequences of a part of rice AS gene (OASA1 ) corresponding to the site of mutation in 5MT resistant mutant in Arabidopsis and to the site of naturally feedback insensitive AS in Nicotiana tabacum and Ruta graveolens , however, there were no differences in these regions between TR-68 and wild-type. In the wild-type, the expression of two AS genes (OASA1 and OASA2 ) was inhibited simultaneously by 5MT and the addition of Trp partly suppressed this inhibition. In contrast, 5MT did not affect the expression of AS genes in TR- 68. These data indicate that the 5MT resistance of TR-68 may be caused by mutation of a regulator of the expression of the AS genes. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Anthranilate synthase (AS); Feedback regulation; Rice; 5-Methyltryptophan (5MT) 1. Introduction In plants, the tryptophan (Trp) biosynthetic pathway provides not only the amino acid Trp for protein synthesis, but also a wide array of important secondary metabolites, including the growth regulator IAA [1,2], antimicrobial phytoalexins [3], and other indolic mole- cules that influence plant /microbe and plant  /animal interactions. Since these secondary metabolites are not produced in microorganisms, the control of Trp gene expression in plants is likely to be more complex than that in microorganisms. Anthranilate synthase (AS) catalyzes the conversion of chorismate into anthranilate, the first reaction leading from the common aromatic amino acid (shikimic acid) pathway towards the biosynthesis of Trp in both microorganisms and plants, and is feedback inhibited by the end product, Trp (Fig. 1). As a branch point enzyme in the synthesis of aromatic amino acids, AS plays a key role in the diversion of chorismate into Trp and indolic secondary compound biosynthesis. AS is composed of two catalytic alpha and beta subunits [4 / 10]. The alpha subunit can convert chorismate to anthranilate in the presence of high levels of ammonia (ammonia-dependent AS activity), whereas the beta subunit is responsible for the use of glutamine as the amino donor [7]. Both AS reactions require Mg 2 as a cofactor. Since specific amino acid residues in the AS alpha-subunit of bacteria and plants are import- ant for feedback inhibition of the enzyme by Trp, AS plays a key role in the regulation of Trp biosynthesis [6 / 12]. * Corresponding author. Tel.:  /81-22-217-5681; fax:  /81-22-217- 5725. E-mail address: kameya@ige.tohoku.ac.jp (T. Kameya). Plant Science 164 (2003) 1037  /1045 www.elsevier.com/locate/plantsci 0168-9452/03/$ - see front matter # 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0168-9452(03)00107-9