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 .