BnPHT5;1b genes are required for the adaptability ofB. napus to fluctuations in Pi supply
To investigate the role of BnPHT5;1b genes in the cellular Pi homeostasis in B. napus , the P response for BnPHT5;1b DM plants and W10 plants were compared phenotypically. 10-day-old plants grown with 100 μM Pi supply in a hydroponic system were transferred to 100 μM or 1,000 μM Pi supply for 7 days.BnPHT5;1b DM plants had lower shoot fresh weight than W10 at both Pi supplies and the fresh weight of the root was slightly less in BnPHT5;1b DM plants compared to W10 at both Pi supplies (Figure 6a, b), leading to increased root-shoot ratios of BnPHT5;1b DM plants, especially at high Pi supply (Figure 6c). Notably, the difference in shoot fresh weight between W10 and BnPHT5;1b DM plants was greater in 1,000 μM Pi supply (fresh weight 64% - 71% relative to W10) than in 100 μM Pi supply (fresh weight 59% - 66% relative to W10) (Figure 6b). Interestingly, the Pi concentrations were greater in shoots and lower in roots of BnPHT5;1b DM plants than in W10 at both Pi supplies (Figure 6d), while the percentage of Pi increase after 1,000 μM Pi treatment was lower in all leaves but rather in the roots ofBnPHT5;1b DM plants compared to W10 (Figure 6e). This result suggests that loss-of-function of BnPHT5;1b genes reduce the Pi storage capacity in leaves of BnPHT5;1b DM plants grown with a sufficient Pi supply.
It is reasonable to assume that reduced vacuolar Pi storage capacity rendered weak buffering capacity to Pi deficiency stress. To test this, five -day-old plants (W10 and DM) grown with 100 μM, 1,000 μM and 2,000 μM Pi supply were transferred to a solution lacking Pi for 14 days of growth. As expected, Pi deprivation dramatically inhibited plant growth, of which the biggest difference in biomass between DM plants and W10 plants was observed in plants grown initially with 2,000 μM Pi supply (40.3%-55.2%) compared with plants grown initially with 1,000 μM Pi supply (29.3%-37.5%) and 100 μM Pi supply (29.0%-37.4%) (Figure 7a, b). Interestingly, leaves of BnPHT5;1b DM plants still had significantly greater Pi concentrations than W10 plants grown initially with 1,000 μM and 2,000 μM Pi supply (Figure 7c). Consistently, the leaves of the BnPHT5;1b DM plants had significantly higher anthocyanin contents than W10 plants grown initially with 1,000 μM and 2,000 μM Pi supplies, suggesting that the larger Pi concentrations caused more acute Pi stress in leaves of BnPHT5;1b DM plants than in W10 plants (Figure 7d). Nevertheless, loss-of-function inBnPHT5;1b genes disrupted cellular Pi homeostasis, which reduced the adaptability of B.napus seedlings to fluctuations in Pi supply.