Measurement of Pi concentrations and total P concentration
The Pi concentration was measured using the method described previously by Lu et al. (2016). 50 mg of fresh tissue was harvested and mixed with 50 µL of 5 M H2SO4, then 1.5 mL pure water was added to each sample after grinding. The mixture was centrifuged at 12,000 g for 10 min at 4°C. The supernatant was collected and diluted to an appropriate concentration. The diluted supernatant was reacted with a malachite green reagent (19.4 mM H3BO3, 27.64 mM (NH4)6MO7O24·4H2O, 2.38 M H2SO4, 627.5 μM malachite green, and 0.1% polyvinyl alcohol) in a 3:1 ratio for 30 min. 200 µl reaction mixture was taken to measure the absorption values at 650 nm by an ELIASA (Spark, TECAN). A standard curve was established using varying concentrations of KH2PO4 for the calculation of sample Pi concentrations. For measurement of total P concentration, 150 mg of dried plant tissue was pre-digested overnight in glass tubes with concentrated sulfuric acid. The tubes were then heated to 120 °C with 4–5 drops of 30% H2O2 every 30 min until the solution turned colorless. After 30 min extension of heat digestion, the total P concentration was determined by molybdenum blue colorimetry at 700 nm by an ELIASA (Spark, TECAN).