Accumulation of free tryptophan in azuki bean ( Vigna angularis) induced by expression of a gene ( OASA1D) for a modified α-subunit of rice anthranilate synthase

Abstract

Anthranilate synthase (AS) is a key metabolic enzyme of the tryptophan (Trp) biosynthetic pathway in plants and microbes, and its activity is regulated by feedback inhibition by Trp. A rice gene ( OASA1D) for a modified α-subunit of anthranilate synthase that is resistant to such feedback inhibition was introduced into azuki bean [ Vigna angularis (Willd.) Ohwi & Ohashi] by Agrobacterium-mediated transformation followed by selection with hygromycin B. Five independent azuki bean lines expressing OASA1D in which the transgene was fixed in the T 2 generation were obtained. The free Trp level in seeds of these lines (1.3–3.3 mg/g of dry weight) was increased compared with that in wild-type seeds (0.2 mg/g). In contrast, the amount of protein-bound (fixed) Trp in seeds was not affected by expression of the transgene. The AS activity in the transgenic seeds showed a reduced sensitivity to feedback inhibition by Trp or the Trp analog 5-methyltryptophan, indicating that the rice OASA1D subunit was able to interact functionally with the β-subunit of azuki bean AS. These results reveal that transformation with OASA1D is a feasible approach to increase Trp synthesis and accumulation in mature seeds of grain legumes.