Abstract
Gene targeting (GT) via homologous recombination allows precise modification of a target gene of interest. In a previous study, we successfully used GT to produce rice plants accumulating high levels of free tryptophan (Trp) in mature seeds and young leaves via targeted modification of a gene encoding anthranilate synthase—a key enzyme of Trp biosynthesis. Here, we performed metabolome analysis in the leaves and mature seeds of GT plants. Of 72 metabolites detected in both organs, a total of 13, including Trp, involved in amino acid metabolism, accumulated to levels >1.5-fold higher than controls in both leaves and mature seeds of GT plants. Surprisingly, the contents of certain metabolites valuable for both humans and livestock, such as γ-aminobutyric acid and vitamin B, were significantly increased in mature seeds of GT plants. Moreover, untargeted analysis using LC-MS revealed that secondary metabolites, including an indole alkaloid, 2-[2-hydroxy-3-β-D-glucopyranosyloxy-1-(1H-indol-3-yl)propyl] tryptophan, also accumulate to higher levels in GT plants. Some of these metabolite changes in plants produced via GT are similar to those observed in plants over expressing mutated genes, thus demonstrating that in vivo protein engineering via GT can be an effective approach to metabolic engineering in crops.
Highlights
Higher plants use a myriad of metabolites to adapt to environmental change
5MT-resistant mutant rice plants have been produced by somaclonal mutation, chemical mutagenesis and γ-ray irradiation [5,6,7,8]
There are no reports of rice mutants harboring OASA2 with S126F/L530D mutations having been screened successfully by conventional mutation approaches, probably because the chances of two kinds of mutations that improve enzymatic properties occurring simultaneously in a single gene are quite low
Summary
Higher plants use a myriad of metabolites to adapt to environmental change. Some of these are valuable for human and livestock as nutritional, insecticidal and pharmaceutical bioactives. Young leaves and mature seeds of transgenic rice overexpressing OASA1D—an OASA1 gene with a Trp-insensitive mutation (D323N)—under the control of a strong constitutive promoter [3,9,10]. Untargeted metabolome analysis revealed that secondary metabolites, including an indole alkaloid, accumulate to higher levels in the leaves and calli of OASA1D overexpressing plants [11,12]. The OASA2 mRNA level is higher than that of OASA1 in leaves and roots [3] This suggested that accumulation of free Trp in rice could be achieved more by modification of the enzymatic properties of OASA2, rather than OASA1. It has been shown that free Trp accumulates to high levels in transformed rice calli overexpressing a modified OASA2 gene harboring S126F/L530D mutations [14]. The results suggest that the levels of many metabolites were altered (mainly increased) and that mutations in the OASA2 gene affected various metabolic pathways
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