Vacuolar Pi influx is reduced in BnPHT5;1b double mutants
It can be argued that vacuolar Pi concentrations would be decreased inBnPHT5;1b loss-of-function mutants if they mediate vacuolar Pi influx. To avoid the effects of functional redundancy of homologous genes, we constructed BnPHT5;1b double mutants (DM) using a CRISPR-Cas9 based gene-editing system (Figure 5a). We then used Nuclear Magnetic Resonance (NMR) spectroscopy to compare the root vacuolar Pi concentrations in the DM plants and the wild-type W10 plants. Plants grown in 100 μM Pi conditions were transferred to 1,000 μM Pi supply for 2 hours before NMR analysis. The vacuolar Pi concentration in W10 was 17.85 ± 2.3 mmol Pi g-1 FW, while it was significantly less in the vacuoles of DM plants (8.41 ± 1.7 mmol Pi g-1 FW) (Figure 5b, c). This result demonstrates that BnPHT5;1b proteins mediate vacuolar Pi influx.