Introduction
Phosphorus (P) is an essential component of many macromolecules such as nucleic acid, phospholipid and ATP and plays vital roles in numerous metabolism pathways and signal transduction (White and Hammond 2008). Although high amounts of P exist in soil, the low availability of phosphate (Pi) is a key challenge for crop production due to its easily high fixation in soils. Phosphorus (P) deficiency unbalances plant cellular Pi homeostasis, a primary determinant of plant growth (Veneklaas et al., 2012). To adapt to P limitation, plants have evolved several mechanisms to fine-tune cellular Pi homeostasis. For example, under Pi-limited conditions, organic acids and acid phosphatase exuded by plants can improve Pi availability (Joan et al. 2017; Wang et al. 2018; Yang et al. 2019, 2021; Deng et al., 2022). In addition, the core transcription factor PHOSPHATE STARVATION RESPONSE 1 (PHR1) is activated rapidly to up-regulate the high-affinity Pi transporters PHOSPHATE TRANSPORTER 1 (PHT1) (Chiou and Lin 2011). Subsequently, dephosphorylation of endoplasmic reticulum (ER)-localized PHT1 proteins facilitate their trafficking to the plasma membrane (González et al., 2005; Bayle et al., 2011; Chen et al., 2011). These processes are required to facilitate greater Pi acquisition from the environment.
Excess P supply disrupts cellular Pi homeostasis. The formation of SPX-PHR complexes down-regulates PHT1 expression by preventing the PHRs from entering the nucleus and inhibiting promoter binding capability, leading to reduction of Pi influx from the environment (Lv et al., 2014; Zhong et al., 2018; Osorio et al., 2019; Ried et al., 2021). In parallel, the degradation of PHT1 protein ubiquitinated by NLA1 also contributes to cellular Pi homeostasis under excess P conditions (Lin et al., 2013; Park et al., 2014). AtPHO1 and its homologous gene PHO1;H1 function in loading Pi into the root xylem for transport to the shoot (Hamburger et al. 2002; Stefanovic et al. 2007). Excess Pi prevents Pi transportation from roots to shoots by initiating the PHO2/NLA1 mediated degradation of PHO1 (Lin et al., 2013; Huang et al., 2013; Yue et al., 2017). The PHO1 family members are SPX-EXS (SYG1/PHO81/XPR1- ERD1/XPR1/SYG1) proteins and NLA1 is an SPX-RING (Really Interesting New Gene) protein (Liu et al., 2018).
Besides the regulation of cellular Pi homeostasis through Pi acquisition from the environment and translocation from the roots to the shoots, the vacuole-localized SPX-MFS (Major Facility Superfamily) proteins (also known as PHT5 proteins), which mediate Pi transport into the vacuole, can also contribute to cellular Pi homeostasis. In higher plants, the vacuole is the major Pi reservoir (Veneklaas et al., 2012) and provides strong buffering capacity for cellular Pi dynamics because the ratio of vacuolar Pi/total Pi can be controlled over a large range of concentrations (Dietz et al., 1986). In rice, the SPX-MFS3 protein was initially proposed to be a Pi efflux transporter, as evidenced by the lower vacuolar Pi content in an overexpression line of SPX-MFS3 compared to wild-type rice (Wang et al., 2015), but this interpretation was recently challenged by another group (Xu et al., 2019), who found that rice overexpressing SPX-MFS3 had higher vacuolar Pi content and mutants lacking SPX-MFS3 had lower vacuolar Pi concentrations than that wild-type rice, suggesting that SPX-MFS3 encoded a vacuolar Pi influx transporter. The rice SPX-MFS1 was found to mediate vacuolar Pi influx when expressed in yeast (Wang et al., 2012). In Arabidopsis, PHT5;1/VPT1 was identified as a vacuolar Pi influx transporter (Liu et al., 2015; Liu et al., 2016). VPT1 has the highest expression in root and young leaves, then mature leaves followed by old leaves and is strongly induced by high Pi conditions in roots, mature leaves and old leaves (Liu et al., 2015). Disruption of VPT1 rendered plants phenotypically hypersensitive to variations in Pi supply accompanied with reduced cellular Pi contents and vacuolar Pi contents (Liu et al., 2015; Liu et al., 2016), suggesting that VPT1 functions in Pi storage in vacuole under conditions of Pi sufficiency and excess. Two vacuolar Pi efflux transporters VPE1 and VPE2 were recently identified in rice and the vpe1 vpe2 double mutants (DM) had higher vacuolar Pi concentrations than the wild-type rice (Xu et al., 2019). In particular, the adaptability of vpe1 vpe2 double mutants to changing Pi availability was significantly reduced (Xu et al., 2019). These results indicate that both vacuolar Pi influx and efflux transporters are essential for cellular Pi homeostasis and plant growth.
Oilseed rape (Brassica napus L .) is a major oil crop with a complex genome. Brassica napus has high P demand and is sensitive to Pi fluctuations in the environment. Its vacuolar Pi influx/efflux transporters have not yet been identified and the mechanisms underling cellular Pi homeostasis by vacuole remain elusive. In this study, we identified eight PHT5 (SPX-MFS) genes in B. napus and further investigated the role of two BnPHT5;1b genes in cellular P dynamics. The predicted BnPHT5;1b proteins located in the tonoplast and expression of BnPHT5;1bs complemented the Pi adaptability of Arabidopsis pht5/vpt1 mutant. We further performed the Pi transport activity assay of BnPHT5;1b proteins in yeast and NMR spectroscopy analysis of vacuolar Pi concentrations between wild typeB. napus cultivar Westar 10 (W10) and BnPHT5;1b double mutants (DM). The results indicated that BnPHT5;1b proteins function as Pi influx transporters for vacuolar Pi storage. Genetic disruption of two BnPHT5;1b genes significantly reduced the adaptability ofB. napus seedlings to Pi supply accompanied by high shoot cellular Pi concentrations, which is different from the pht5/vpt1mutant seedlings. At the reproductive stage, disruption ofBnPHT5;1b genes caused Pi toxicity in floral organs and reduced seed yields under Pi-sufficient and high Pi conditions. In addition,BnPHT5;1b DM had abnormal seed traits and higher seed P concentrations compared to W10. Our results highlight the importance of BnPHT5;1b proteins as vacuolar Pi influx transporters in cellular Pi homeostasis in B. napus.