BnPHT5;1b proteins function as vacuolar Pi influx transporters
The phytoavailable form of P is Pi, but soil Pi concentrations often cannot satisfy crop growth requirements (Schachtman et al., 1998). The application of large amounts of Pi-containing fertilizers by farmers is a common agronomic practice to attain maximum agronomic production. It also causes large fluctuations in soil Pi concentration, requiring plants to adapt rapidly cellular Pi fluxes to maintain cellular Pi homeostasis. The importance of the regulation of Pi transporters in adjusting Pi uptake and delivery to the xylem in plant Pi homeostasishas been established (Wang et al., 2021). However, cellular Pi homeostasis cannot be achieved without utilizing the buffering capacity of the Pi major reservoirin plants, the vacuole (Veneklaas et al., 2012; Dietz and Foyer, 1986). Vacuolar Pi influx and efflux rely on the Pi influx and efflux transporters on the tonoplast, such as SPX-MFS3 in rice (Wang et al., 2015; Xu et al., 2019) and PHT5;1/VPT1 and PHT5;3/VPT3 in Arabidopsis (Liu et al., 2015; Liu et al., 2016; Luan et al., 2019).
Brassica napus is an allotetraploid oil crop with a more complex genome (AACC, 2n = 38) than the genomes of rice and Arabidopsis and is highly sensitive to Pi stress. The vacuolar Pi transporters and their roles in cellular Pi homeostasis remain elusive. Based on the protein sequences of the AtPHT5 family, we identified 8 homologs in B. napus showing more than 90% amino acid sequence identities with AtPHT5 proteins (Figure 1a, Table 1). The BnA09PHT5;1a, BnC09PHT5;1a, BnA09PHt5;1b and BnCnPHT5;1b proteins were the homolog of AtPHT5;1 inB. napus (Figure 1a), however only the two BnPHT5;1b genes showed Pi induced expression pattern (Figure 1c, f and Figure 2), similar to the expression pattern of AtPHT5;1 (Liu et al., 2015). The predicted 3-D structures of BnPHT5;1b and AtPHT5;1 had high similarity (Figure 3a, b). We thus hypothesized that BnPHT5;1b proteins may function as the vacuolar Pi influx transporters in B. napus . The BnPHT5;1b proteins were localized on the tonoplast and showed a comparable Pi transport activity to AtVPT1 when expressed in a yeast mutant (Figure 3c, d, Figure S3). Furthermore, vacuolar Pi concentration was smaller in BnPHT5;1b DM plants than wildtype W10 plants and both BnPHT5;1b genes could significantly recover the growth defects and P concentrations of the Arabidopsis Atvpt1 mutant under various Pi conditions (Figure 4). Therefore, we conclude that BnPHT5;1b proteins function as vacuolar Pi influx transporters inB. napus .