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).