4.5. Inorganic Phosphate Assay

WT, vip1-1/vip2-1, vip1-1/vip2-2, vip1-2/vip2-2, and ipk1 seedlings were grown on 0.5× MS media at 21 °C under an 8 h photoperiod for 18 days. Separately, a replicate panel of seedlings were transplanted to soil and grown to maturity for an additional 28 days. Shoot tissues from ~50 seedlings/genotype/bioreplicate were harvested into liquid nitrogen and ground to a fine powder. For assays of mature plants, 2 leaves were collected from each of 8 plants/genotype/bioreplicate and ground in a similar manner. Inorganic phosphate was extracted by incubating in ten volumes 1% v/v acetic acid. Extracted fractions were analyzed for Pi concentration by a modified micro-titer assay [54]. Briefly, 50 µL sample extract was diluted to a volume of 150 µL, mixed 1:1 with an ammonium molybdate working reagent (5% w/v FeH₁₄O₁₁S·7H₂O, 1% w/v (NH₄)₂MoO₄, 1N H₂SO₄ aq.) and incubated for 1 h. Absorbance at 660 nm was measured on a plate spectrophotometer and phosphate concentrations were calculated using a standard curve of known phosphate amounts in the form of KH₂PO_4.