Metabolic manipulation of plants to improve their nutritional value is a primary goal of plant biotechnology. Essential amino acids such as Lys, Met, Thr, and Trp contribute substantially to the nutritional quality of plant-based foods for humans and domestic animals, but the amounts of such amino acids are limited in many crops. Dietary supplementation with Trp increases the growth rate of pigs and poultry (Subcommittee on Poultry Nutrition, 1994; Subcommittee on Swine Nutrition, 1998). An inadequate supply of Trp leads to a marked reduction in food intake in pigs as a result of the reduced synthesis of serotonin in the brain (Henry et al., 1992; Sève, 1999; Eder et al., 2001). Indeed, Trp has been used as a pharmaceutical agent in the treatment of depression (Massey et al., 1998). The ability to increase the level of Trp in food crops by metabolic engineering is thus desirable from both nutritional and clinical viewpoints. The achievement of an increase in the amino acid content of seeds by genetic engineering, however, has been limited to Lys (Falco et al., 1995; Mazur et al., 1999; Zhu and Galili, 2003) and Met (Movig et al., 1997; Lai and Messing, 2002).
The accumulation of free Trp in plants has been achieved by the introduction of genes encoding feedback-insensitive 
subunits of anthranilate synthase (AS), which catalyses the conversion of chorismate to anthranilate. This approach has thus yielded increased levels of Trp in the roots of the forage legume Astragalus sinicus (Cho et al., 2000) and in the leaves of tobacco (Zhang et al., 2001). A mutant OASA1 gene, OASA1D [formerly referred to as OASA1(D323N)], that encodes a feedback-insensitive subunit of rice AS (Tozawa et al., 2001) has previously been generated. Rice calli and leaves as well as potato plants and tubers that express OASA1D accumulate large amounts of free Trp (Tozawa et al., 2001; Yamada et al., 2004).
Manipulation of metabolic pathways in plants by genetic engineering thus has the potential to improve the nutritional value of crops and to allow the production of desired natural products in higher plants (DellaPenna, 2001; Morandini and Salamini, 2003). The proteins encoded by the introduced genes might affect not only the abundance of the target metabolite, however, but also, through changes in metabolic networks, that of related compounds. Moreover, such changes may influence the physiology or morphology of plants. Little is known, however, of the consequences of such manipulation for metabolite profiles and agronomic traits in plants.
Metabolism of Trp in plants is associated with the generation of a range of secondary compounds such as indole alkaloids and indole acetic acid (IAA). Manipulation of the Trp biosynthetic pathway might thus be expected to influence the synthesis of such metabolites and thereby elicit a pronounced change in metabolite profile. Indeed, overexpression of Trp decarboxylase, which catalyses the conversion of Trp to tryptamine, resulted in a marked reduction in the amount of indole glucosinolate in canola plants (Chavadej et al., 1994) and a decreased abundance of Trp, Phe, and chlorogenic acid in potato (Yao et al., 1995).
Evaluation of transgenic plants of improved nutritional value for effects of the transgene on agronomic traits and metabolite profiles is thus essential. However, the possible effects of the accumulation of Trp or of any other essential amino acid in seeds on agronomic traits and metabolite profiles have not been determined to date. Demonstration that Trp is the only major metabolite that accumulates in transgenic rice seeds, for example, would be likely to increase their acceptability for consumption by humans and farm animals.
The effects of OASA1D expression in rice both on the amounts of Trp and other amino acids in seeds and on agronomic traits of plants cultivated in isolated field trials have now been studied. The profile of phenolic compounds as well as the content of IAA in the transgenic seeds was also determined. Rice is one of the major crops in the world and its use for animal feed has recently been developed (Sakai et al., 2003a). The results of the increase of Trp in transgenic rice prove the usefulness of the gene for improving the nutrition of other crops for animal feed.
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