Changes in primary and secondary metabolite levels in response to gene targeting-mediated site-directed mutagenesis of the anthranilate synthase gene in rice

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.

Figure 1

Structure of the OASA2 gene. The mutations S126F (S [TCC] to F [TTC] at amino acid 126) and L530D (L [CTT] to D [GAC] at amino acid 530) were introduced successfully into the endogenous OASA2 gene via gene targeting (GT) [4]. Molecular analysis revealed that true GT, in which the wild-type OASA2 gene was mutated as expected, had occurred successfully [4].

Figure 2

Sensitivity of non-transformant and GT plants to 5MT. Plants were grown in the presence of 250 μM 5MT for 10 days.(a) Length of leaves and roots in non-transformants (NT) and plants homozygous (GT) and heterozygous (H) for the modified OASA2 gene in the T₂ generation. Values are average ± SD (NT n = 9, H n = 14, GT n = 8).(b) Photographs of 10-day-old NT and GT plants grown under 250 μM 5MT conditions. Bars = 5 cm.

Figure 3

Changes in primary and secondary metabolite levels in mature seeds and leaves. (a) Venn diagram of metabolites with >1.5-fold higher intensity in mature seeds and seedlings in GT plants compared to non-transformants. (b) Comparison of the fold change in metabolites in mature seeds and leaves. Fold change is presented as the ratio of the intensity of each metabolite in GT plants homozygous for mutated OASA2 compared to non-transformants. There is a weak positive relationship between the fold change in metabolites in mature seeds and that in leaves (R = 0.18). Metabolites are listed in Supplementary Table S2 and Supplementary Table S3.

Figure 4

Changes in metabolite levels derived from amino acids in mature seeds. Mature seeds of non-transformant (NT) and homozygous GT plants (GT) were subjected to metabolome analysis using CE-MS (see Tables S1–S3). Representative metabolites are shown in this pathway map, and the fold change in metabolite levels in mature seeds of GT plants to that in NT is shown using a color scale, as indicated.

Figure 5

Principal component analysis (PCA) of untargeted negatively charged metabolic profile. PCA was performed using Simca-P11. Black squares and red circles represent samples obtained from mature seeds of NT and GT, respectively.

Figure 6

An indole alkaloid present in increased amount in mature seeds of GT plants. (a) Structure of the indole alkaloid, 2-[2-hydroxy-3-β-D-glucopyranosyloxy-1-(1H-indol-3-yl)propyl]-tryptophan. (b) MS/MS spectra of peak #4673. Red arrowheads show the fragments corresponding to the fragments shown in a previous report [12].