31P NMR analysis of vacuolar Pi.
The method was slightly modified from that of Xu et al. (2019).
Approximately 0.05 g B. napus roots were used for NMR analysis.
The roots were packed into a 5-mm-diameter NMR tube equipped with a
perfusion system (5 mM glucose, 10 mM KNO3, 0.5 mM
Ca(NO3)2, 1 mM KCl, 0.5 mM
MgSO4, 100 μM KH2PO4, 2
mM MES, pH 5.5) connected to a peristaltic pump.31P-NMR spectra were recorded using a standard
broadband 5-mm probe on a Bruker Ascend 500 spectrometer with TopSpin
software v.3.0. The 31P-NMR spectra were recorded at
242.9 MHz lock with deuteroxide in two 0.5-mm glass capillaries. The31P-NMR acquisition conditions were set as follows:
30° radiofrequency pulses (70 μs) at 2 s relaxation delay; 24 KHz
spectral width; Waltz-based broadband proton decoupling (44 W during
acquisition time, 0.6 W during delay) with a probe temperature of 25 °C.
Free induction decays (FIDs) were collected as 16 K data points,
zero-filled to 32 K, and processed with a 30-Hz exponential line
broadening. Each spectrum was acquired over 0.34 s and was the sum of
3,600 scans. Other conditions followed the description by Xu et al.
(2019).