Figure 3.

Transgene expression and metabolite levels in TT‐engineered polished rice. In the different panels, the pyrimidine branch (THIC and HMP) is indicated in blue, the thiazole branch (THI1 and HET) in orange/red and thiamin in green. (a) Relative transgene expression of AtTHIC and AtTHI1 in rice seeds of homozygous T2 plants originating from independent plant transformation events revealed by real‐time quantitative PCR. Values are means of two independent biological repeats (± SE). Data analysis and normalization were performed using the qBASE software, based on the 2^–ΔΔCt method (Hellemans et al., 2007; Livak and Schmittgen, 2001) using OsActin (LOC_Os03g50885.1), LOC_Os11g43900 and LOC_Os07g02340 as reference genes. Specific information regarding primers and conditions can be retrieved from Table S1. Level (mean ± SE) of 4‐amino‐2‐methyl‐5‐hydroxymethylpyrimidine (HMP) (b), 4‐methyl‐5‐β‐hydroxyethylthiazole (HET) and total HET (sum of non‐phophorylated and phosphorylated HET) (c) and thiamin (d) as measured by quantitative LC‐MS/MS analysis. Metabolites were measured in thoroughly polished white rice (60s M + 16h SP, see Figure S2). AnalysIs of metabolites was performed on at least three biological repeats (transgenic lines) and 15 biological repeats (WT). Statistical difference (see Methods S2) in mean metabolite level compared to WT is indicated to be significant (single asterisk, P < 0.05) or very significant (double asterisks, P < 0.01).