PHT5;1-VPTs play distinct regulatory mechanisms of cellular Pi homeostasis between B. napus seedlings and Arabidopsisseedlings
In Arabidopsis thaliana , the PHT5 family includes 3 members (PHT5;1 , PHT5;2 and PHT5;3 ) andPHT5;1 plays a predominant role in cellular Pi homeostasis (Liu et al., 2015, 2016). PHT5;1 is expressed mainly in roots and young leaves and its expression is increased by increasing Pi supply in roots, mature and old leaves (Liu et al., 2015). Consistent with the response of AtPHT5;1 to Pi supply, both BnA09PHT5;1b andBnCnPHT5;1b were strongly induced by high Pi supply in roots and older leaves when plants were transferred from 5 μM Pi to 100 μM Pi supply and in all tissues when transferred to 1,000 μM Pi supply (Figure 2). Slower growth than wild-type plants was observed in the roots and shoots of Atpht5;1 mutant at Pi concentrations from 13 μM to 6.5 mM (Liu et al., 2015), while only in the shoots of BnPHT5;1b DM plants at Pi concentrations 100 and 1,000 μM (Figure 6). These results suggested that AtPHT5;1 functions in both roots and shoots, but BnPHT5;1b affect primarily shoot growth.
Differences in fresh weights of shoots were always greater than differences in fresh weights of roots between W10 and BnPHT5;1bDM plants when grown under both Pi-sufficient conditions (100 μM) and high Pi conditions (1,000 μM) (Figure 6 and Figure 7). Consistently,BnPH5;1b DM plants had increased root-to-shoot ratios compared with W10 (Figure 6c). Notably, the BnPHT5;1b DM plants had greater Pi concentrations in shoots than W10 when grown under both Pi-sufficient and high Pi conditions (Figure 6d). Given thatBnPHT5;1bs had strong expression in root vascular tissue (Figure 2b, c), dead cells of xylem are not the target of BnPHT5;1b . Disruption of BnPHT5;1bs function might not have interference on the long-distance transport of Pi from roots to shoots. Because vacuolar Pi was lower in BnPHT5;1b DM plants than in W10, thus the higher Pi concentrations in the shoot of BnPHT5;1b DM plants can only be the consequence of cytoplasmic Pi accumulation. The elevated cytoplasmic Pi level is harmful to shoot growth. In contrast, Atpht5;1/vpt1mutant has lower shoot Pi content and cytosolic Pi level than the wild-type (Liu et al., 2016, Luan et al., 2019). ProAtPHT5;1:GUS showed strong expression of non-vascular cells, which may provide an interpretation that loss-of-function in AtPHT5;1 severely hinders root growth (Liu et al., 2015).
VPE1 and VPE2 are the vacuolar Pi efflux transporters and direct vacuole-to-cytosol Pi movement for Pi remobilization under Pi-deprived conditions (Xu et al., 2019). We proposed that reduced vacuole storage of Pi in BnPHT5;1b DM plants would render plants sensitive to Pi-deficient conditions. Brassica napus seedlings grown for 5 days with 100 μM, 1,000 μM and 2,000 μM Pi supplies, were subjected to 14 days of Pi deprivation. The slower growth of BnPHT5;1 DM plants than W10 plants (Figure 7a, b) hardly be attributed to the decreased ability of vacuolar Pi remobilization because the Pi concentrations constantly higher in the shoots of BnPHT5;1b DM plants than the W10 plants (Figure 7c), indicating that BnPHT5;1DM plants had lower Pi reuse efficiency than W10 under Pi deprivation condition. Nonetheless, these results suggest that PHT5;1s play distinct regulatory mechanisms of cellular Pi homeostasis between B. napusseedlings and Arabidopsis seedlings.
PHT5;1-VPTs play a conserved role in floral Pi homeostasis between B. napus and Arabidopsis .
In the Arabidopsis pht5;1/vpt1 mutant, the absence ofPHT5;3 further reduced seedling Pi concentration, and vacuolar and cytosolic Pi concentrations when grown under Pi-sufficient conditions (Luan et al., 2019). In particular, the pht5;1 pht5;3(vpt1 vpt3 ) double mutant rather than pht5;1 /vpt1single mutant showed Pi toxicity in floral organs and reproductive defects (Luan et al., 2019). This result agrees with our findings thatBnPHT5;1b DM plants had larger floral Pi concentrations than W10 plants when grown under Pi-sufficient conditions (Figure 8c) and also displayed reproductive defects when grown under both Pi-sufficient and high Pi conditions in pots (Figure 8f). It was previously shown that Pi-toxicity has little effect on the morphology of floral organs but inhibits pollen tube growth as a consequence of excessive Pi concentration in the pistil (Luan et al., 2019). The BnPHT5;1bgenes had strong expression in leaves and flowers (Figure 8a). Loss-of-function of BnPHT5;1b reduced Pi storage in leaves and lead to excessive Pi concentrations in flowers of plants grown under Pi-sufficient conditions (Figure S6c, d). Therefore, the excessive Pi concentration in the pistil of BnPHT5;1b DM plants was correlated with the reduction in seed yield. These results suggest that PHT5;1s play a conserved role in floral Pi homeostasis between B. napusand Arabidopsis.
In addition, the BnPHT5;1b DM seeds had abnormal phenotypes, particularly a larger P concentration than W10 seeds (Figure 9), suggesting that BnPHT5;1b genes might also play a crucial role in seed development. Although the role of BnPHT5;1b genes in seed development and the interrelationship between BnA09PHT5;1b andBnCnPHT5;1b in cellular Pi homeostasis needed further investigation, our findings demonstrate that BnPHT5;1b proteins play an essential role in cellular Pi homeostasis throughout the plant.