Vacuolar H+-ATPase Is Required for Efficient Vacuolar Phosphate Storage and Systemic Pi Homeostasis in Arabidopsis.

The vacuolar H+-ATPase (V-ATPase) plays a crucial role in facilitating nutrient ions storage in vacuoles, whereas its direct impact on vacuolar phosphate (Pi) accumulation has not been fully elucidated. Previous research revealed that the absence of VPT1 and VPT3, two major vacuolar Pi influx transporters, significantly affected vacuolar Pi storage. This study shows that disrupting V-ATPase function could mimic the vpt1 vpt3 mutant phenotypes. The vha-a2 a3 mutant, lacking V-ATPase activity, had lower vacuolar Pi levels, higher cytoplasmic Pi and increased resistance to As(V) toxicity under sufficient Pi conditions. Complementation assays in Pi transport-deficient yeast confirmed that high pH suppressed VPT1 activity, while overexpressing VPT1 couldn't overload Pi in vacuoles of vha-a2 a3 mutants. These data illustrate the reliance of VPT1's activity on V-ATPase-generated proton gradients. Furthermore, we find V-ATPase activity correlates positively with Pi availability, and varying across developmental stages. During flowering, V-ATPase activity decreases to enhance Pi allocation in xylem sap for long-distance transport when external Pi is replete, akin to the vpt1 vpt3 mutant. Thus, V-ATPase could cooperate with VPT proteins to regulate Pi homeostasis at both subcellular and systemic levels.

Mechanisms of vacuolar phosphate efflux supporting soybean root hair growth in response to phosphate deficiency.

Phosphorus is an essential macronutrient for plant growth and development. In response to phosphate (Pi) deficiency, plants rapidly produce a substitutive amount of root hairs; however, the mechanisms underlying Pi supply for root hair growth remain unclear. Here, we observed that soybean (Glycine max) plants maintain a consistent level of Pi within root hairs even under external Pi deficiency. We therefore investigated the role of vacuole-stored Pi, a major Pi reservoir in plant cells, in supporting root hair growth under Pi-deficient conditions. Our findings indicated that two vacuolar Pi efflux (VPE) transporters, GmVPE1 and GmVPE2, remobilize vacuolar stored Pi to sustain cytosolic Pi content in root hair cells. Genetic analysis showed that double mutants of GmVPE1 and GmVPE2 exhibited reduced root hair growth under low Pi conditions. Moreover, GmVPE1 and GmVPE2 were highly expressed in root hairs, with their expression levels significantly upregulated by low Pi treatment. Further analysis revealed that GmRSL2 (ROOT HAIR DEFECTIVE 6-like 2), a transcription factor involved in root hair morphogenesis, directly binds to the promoter regions of GmVPE1 and GmVPE2, and promotes their expressions under low Pi conditions. Additionally, mutants lacking both GmRSL2 and its homolog GmRSL3 exhibited impaired root hair growth under low Pi stress, which was rescued by overexpressing either GmVPE1 or GmVPE2. Taken together, our study has identified a module comprising vacuolar Pi exporters and transcription factors responsible for remobilizing vacuolar Pi to support root hair growth in response to Pi deficiency in soybean.

Genetically controlling VACUOLAR PHOSPHATE TRANSPORTER 1 contributes to low-phosphorus seeds in Arabidopsis.

Phosphorus (P) is an indispensable nutrient for seed germination, but the seeds always store excessive P over demand. High-P seeds of feeding crops lead to environmental and nutrition issues, because phytic acid (PA), the major form of P in seeds, cannot be digested by mono-gastric animals. Therefore, reduction of P level in seeds has become an imperative task in agriculture. Our study here suggested that both VPT1 and VPT3, two vacuolar phosphate transporters responsible for vacuolar Pi sequestration, were downregulated in leaves during the flowering stage, which led to less Pi accumulated in leaves and more Pi allocated to reproductive organs, and thus high-P containing seeds. To reduce the total P content in seeds, we genetically regulated VPT1 during the flowering stage and found that overexpression of VPT1 in leaves could reduce P content in seeds without affecting the production and seed vigor. Therefore, our finding provides a potential strategy to reduce the P level of the seeds to prevent the nutrition over-accumulation pollution.

A SPX domain vacuolar transporter links phosphate sensing to homeostasis in Arabidopsis.

Excess phosphate (Pi) is stored into the vacuole through Pi transporters so that cytoplasmic Pi levels remain stable in plant cells. We hypothesized that the vacuolar Pi transporters may harbor a Pi-sensing mechanism so that they are activated to deliver Pi into the vacuole only when cytosolic Pi reaches a threshold high level. We tested this hypothesis using Vacuolar Phosphate Transporter 1 (VPT1), a SPX domain-containing vacuolar Pi transporter, as a model. Recent studies have defined SPX as a Pi-sensing module that binds inositol polyphosphate signaling molecules (InsPs) produced at high cellular Pi status. We showed here that Pi-deficient conditions or mutation of the SPX domain severely impaired the transport activity of VPT1. We further identified an auto-inhibitory domain in VPT1 that suppresses its transport activity. Taking together the results from detailed structure-function analyses, our study suggests that VPT1 is in the auto-inhibitory state when Pi status is low, whereas at high cellular Pi status InsPs are produced and bind SPX domain to switch on VPT1 activity to deliver Pi into the vacuole. This thus provides an auto-regulatory mechanism for VPT1-mediated Pi sensing and homeostasis in plant cells.

Vacuolar Phosphate Transporters Contribute to Systemic Phosphate Homeostasis Vital for Reproductive Development in Arabidopsis.

Vacuolar storage of phosphate (Pi) is essential for Pi homeostasis in plants. Recent studies have identified a family of vacuolar Pi transporters, VPTs (PHT5s), responsible for vacuolar sequestration of Pi. We report here that both VPT1 and VPT3 contribute to cytosol-to-vacuole Pi partitioning. Although VPT1 plays a predominant role, VPT3 is particularly important when VPT1 is absent. Our data suggested that the vpt1 vpt3 double mutant was more defective in Pi homeostasis than the vpt1 single mutant, as indicated by Pi accumulation capacity, vacuolar Pi influx, subcellular Pi allocation, and plant adaptability to changing Pi status. The remaining member of the VPT family, VPT2 (PHT5;2), did not appear to contribute to Pi homeostasis in such assays. Particularly interesting is the finding that the vpt1 vpt3 double mutant was impaired in reproductive development with shortened siliques and impaired seed set under sufficient Pi, and this phenotype was not found in the vpt1 vpt2 and vpt2 vpt3 double mutants. Measurements of Pi contents revealed Pi over-accumulation in the floral organs of vpt1 vpt3 as compared with the wild type. Further analysis identified excess Pi in the pistil as inhibitory to pollen tube growth, and thus seed yield, in the mutant plants. Reducing the Pi levels in culture medium or mutation of PHO1, a Pi transport protein responsible for root-shoot transport, restored the seed set of vpt1 vpt3 Thus, VPTs, through their function in vacuolar Pi sequestration, control the fine-tuning of systemic Pi allocation, which is particularly important for reproductive development.

Vacuolar Phosphate Transporter 1 (VPT1) Affects Arsenate Tolerance by Regulating Phosphate Homeostasis in Arabidopsis.

Arsenate [As(V)] is toxic to nearly all organisms. Soil-borne As(V) enters plant cells mainly through the plasma membrane-localized phosphate (Pi) transporter PHT1 family proteins due to its chemical similarity to Pi. We report here that VPT1, a major vacuolar phosphate transporter which contributes to vacuolar Pi sequestration, is associated with As(V) tolerance in Arabidopsis. vpt1 mutants displayed enhanced tolerance to As(V) toxicity, whereas plants overexpressing VPT1 were more sensitive to As(V) as compared with the wild-type plants. Measurements of arsenic content indicated that vpt1 mutants accumulated less arsenic and, in contrast, up-regulating VPT1 expression contributed to higher levels of arsenic accumulation in plants. To examine further how VPT1 may modulate arsenic contents in plants, we surveyed the expression patterns of all the PHT1 family members that play roles in As(V) uptake, and found that many of the PHT1 genes were down-regulated in the vpt1 mutant as compared with the wild type under Pi-sufficient conditions, but not when Pi levels were low in the medium. Interestingly, As(V) sensitivity assays indicated that As(V) resistance in vpt1 mutants was prominent only under Pi-sufficient but not under Pi-deficient conditions. These results suggest that under Pi-sufficient conditions, loss of VPT1 leads to elevated levels of Pi in the cytosol, which in turn suppressed the expression of PHT1-type transporters and reduced accumulation of arsenic.