Vitamin B1 and B6 Quantification

For vitamin B₁, a Saccharomyces cerevisiae thi4 mutant deficient in vitamin B₁ biosynthesis de novo was used for quantification of total vitamin B₁ from rice tissues from plants grown in experiments 1 and 2 (Raschke et al., 2007). Fifty milligrams of frozen, ground rice leaves or seeds were used for extraction of vitamin B₁ in 500 μl of 20 mM sulfuric acid in darkness at room temperature for 30 min, before heating to 100°C for 1 h. The solution was adjusted to pH 5.7 with 3 M sodium acetate and centrifuged. To convert phosphorylated B₁ vitamers to free thiamine to permit uptake by yeast, supernatants were treated with acid phosphatase (0.2 U/10 μl per 50 μl of plant extract) overnight for 12–15 h at 37°C. Total vitamin B₁ was calculated from the linear range of a standard curve prepared with 5–100 ng of thiamine hydrochloride provided to the thi4 yeast mutant in thiamine-deficient media.

Samples from candidate accessions with contrasting total vitamin B₁ contents were selected for confirmation and vitamer profiling of thiochrome derivatives by HPLC using a method first described by Moulin et al. (2013). Fifty milligrams of frozen, ground rice leaves or seeds were used for extraction of soluble vitamin B₁ in 100 μl in 1% (v/v) trichloroacetic acid by aggressive vortexing at room temperature for 30 min. Samples were centrifuged at full speed in a tabletop microcentrifuge for 10 min at room temperature. The clear supernatant was neutralized with 3 M sodium acetate to 10% of the final volume and oxidized to thiochrome derivatives using 15 μl of freshly prepared 30 mM potassium ferracyanide in 15% (w/v) NaOH, with 15 μl 1 M NaOH and 25 μl methanol according to Moulin et al. (2013). Samples were injected into an Agilent Technologies 1260 HPLC to determine vitamer profiles by separation of thiochrome derivatives on a Cosmosil π-NAP column (150 × 4.6 mm, 3 μm pore size) using a methanol gradient at 1 ml min⁻¹ detailed in Moulin et al. (2013), with a 40 min run time. Peaks of fluorescence corresponding to the retention time of the commercial standards of B₁ vitamers TDP (Sigma), thiamine monophosphate (TMP; Fluka), and thiamine (Fluka) were integrated and extrapolated against a standard curve for each vitamer. Peak area was integrated only from non-saturated peaks and in cases of peak saturation, samples were reinjected in lower volumes. Injection volumes ranged from 10 to 40 μl.

Data for leaf samples were normalized to fresh weight (FW) and seed samples to dry weight (DW).

For vitamin B₆, a Saccharomyces pastorianus American Type Culture Collection 9080 strain was used (Tambasco-Studart et al., 2005) as reported (Mangel et al., 2019). Fifty milligrams of frozen, ground rice leaves or seeds were used for extraction of vitamin B₆ in 500 μl of 20 mM sulfuric acid in darkness at room temperature for 30 min, before heating to 100°C for 1 h. The solution was adjusted to pH 5.7 with 3 M sodium acetate and centrifuged. To convert phosphorylated and glucosylated B₆ vitamers to non-phosphorylated vitamers to permit uptake by yeast, supernatants were treated with acid phosphatase and β-glucosidase (0.2 U/10 μl of each enzyme per 50 μl of plant extract) overnight for 12–15 h at 37°C. Total vitamin B₆ was calculated from the linear range of a standard curve prepared with 0.15–2.4 ng of pyridoxine hydrochloride provided to the yeast mutant in pyridoxine-deficient media.

Samples from candidate rice accessions with contrasting total vitamin B₆ contents were selected for vitamer profiling by HPLC using an established protocol (Szydlowski et al., 2013). Fifty milligrams of frozen, ground rice leaves or seeds were used for extraction of vitamin B₆ in 100 μl of 50 mM ammonium acetate pH 4.0 with aggressive vortexing for 10 min at room temperature. Samples were centrifuged at full speed in a tabletop microcentrifuge for 15 min at room temperature. The supernatant was incubated for 3 min at 99°C and again centrifuged for 15 min at room temperature before analysis. Extracts were injected into an Agilent Technologies 1200 HPLC to separate B₆ vitamers on a Sunfire C18 column (Waters), 4.6 × 150 mm, 3.5 μm particle diameter, with post-column derivatization in 0.7 M potassium phosphate buffer with 1 g L⁻¹ sodium bisulfite added freshly, flow rate 0.3 ml min⁻¹. Samples were separated on an isocratic gradient of 50 mM ammonium acetate pH 4.0, flow rate 1 ml min⁻¹ in a 40 min run time. Quantification was carried out using the linear range of a standard curve constructed with known amounts of standards (Colinas et al., 2016), with vitamin B₆ glucoside (PN–Glu) determination calculated as PN equivalents (Mangel et al., 2019). Standards were prepared and injected into the HPLC with every set of extractions. Peak area was integrated only from non-saturated peaks and in cases of peak saturation, samples were reinjected in lower volumes. Injection volumes were typically 10–30 μl.

Data for leaf samples were normalized to fresh weight (FW) and seed samples to dry weight (DW).