Molecular Identification And Functions Of Vacular Phosphate Transporters In Arabidopsis

Phosphate is a macroelement essential for plant kingdom, but it always limits crop growth due to the low dose of available phosphorus in the environment, and thus, large amount of phosphorous fertilizer is applied to increase crop products in the modern agriculture. Whereas, phosphate is a non-renewable resource, and current global reserves may be depleted in the near future, and what’s more, large application of phosphorus fertilizer also will cause environmental contamination, such as water eutrophication. How to improve the phosphorus ultilization efficiency of plants is an extremely urgent prolem now.In order to improving the phosphorus ultilization efficiency of plants, we need to know the mechanisms of uptake, tanlocation and storage of inorganic phosphate (Pi) in plant kingdom sufficiently. So far, we have known that plasma membrane located proteins of PHT1 family are mainly responsible for Pi uptake, and the Golgi located transporter PHO1 has a important role in Pi translocation, and several phosphate transporters located on mitochondria and chloroplasts also have been identified. The most of Pi is stored in vacuoles and it is very essential for keeping phosphate homeostasis in plant cell,whereas, the transporter responsible for vacuolar phosphate sequestration is still unclear.As an analogue of Pi, arsenate [As(V)] is also easily uptaked by phosphate transporters in plants, and further threatens human’s health. Nowadays, arsenic contamination has become a serious global problem, and reducing arsenic accumulation in crops has been an important topic waiting for us to solve urgently. So far, there are many studies have shown that As(V) is uptaked by PHT1 family members in plants, but it is still unknown whether the phosphate transporters located on endomembranes also could influence the As(V) uptake when they judge the plant phosphate homeostasis.Here, we first found a transporter responsible for vacuolar phosphate sequestration in Arabidopsis thaliana using bioinformatics, plant molecular genetics, fluorescent protein localization, patch-clamp technology and other methods, and named it as VPT1 (Vacuolar Phosphate Transporter 1). Further, through gene knock-out or over-expression plants, an inducible expression system and other methods, we first found the endomembrane located phosphate transporter VPT1 also largely influenced the arsenic tolerasnce, and arsenic uptake and accumulation of plants, and we further explained it from the feedback regulation of PHT1s expression by VPT1. The main results showed as follows:1. The evolutionary conserved SPX-MFS protein VPT1 is an essential member for Arabidopsis adaptation to variable Pi status in the environment. Considering phosphate is a macroelement essential for growth and development of various organisms, the phosphate transporters may be very conserve during evolutionary process. Thus, according to the results of phylogenetic analysis, we screened the T-DNA insertional mutants of the candidate genes coding vacuolar phosphate transporters.A. The three members in Arabidopsis SPX-MFS family were predicted to be the vacuolar phosphate transporters. The three members in Arabidopsis SPX-MFS family were most close to the yeast vacuolar transporter ScVTCs and ScPHO91 in the cladogram and they have 10～11 transmembrane structures, and thus, we guessed the SPX-MFS proteins were also likely to be vacuolar phosphate transporters.B. The mutant of SPX-MFS family protein VPT1 was stunted under normal growth conditions in soil. First, we obtained the knockout mutants of Atlg63010 (VPT1) and At4g22990. Under normal conditions in soil, the vpt1 mutant was stunted, and thus, we focused on this mutant in our latter works. Further, we successfully complemented the vptl growth defects using the genomic fragment of the VPT1 gene.C. Lack of VPT1 caused growth defects under both low-and high-Pi conditions. First, we carried out a phosphorus gradient experiment in a hydroponic system, and found the vptl mutant was sensitive to high Pi conditions. Furhter, we obtained a similar phenotype on the 1/2 MS agar plates. If we transferred the seedlings grown in Pi replete hydroponic conditions into a Pi deficient condition, the vptl mutant plants would be suffered more than wild type, and correspondingly, accumulated more anthocyanin in leaves.2. VPT1 is required for Pi accumulation and distribution in Arabidopsis. In order to identify the molecular founction of VPT1, we determined the Pi content in the vptl mutant plants, and then, analysed the physiological role of VPT1 in combination with VPT1 gene expression pattern.A. The vptl mutant plants consistently retained less Pi than the wild type:In all cases, the vptl mutant plants consistently retained less Pi compared with the wild type, regardless of Pi concentrations in the culture medium. With the increased Pi concentration in the growth medium, the Pi content in wild type plants steadily increased, but the Pi content in vptl mutant plants quickly plateaued at a rather low level.B. VPT1 was expressed primarily in younger tissues under normal conditions, and it was strongly induced by high-Pi conditions in older leaves to avoid Pi toxicity to young leaves:According to the results of histochemical β-glucuronidase (GUS) staining of transgenic plants expressing a VPT1 promoter-GUS fusion, the VPT1 promoter was more active in the younger tissues in both leaves and roots during vegetative growth. Correspondingly, the Pi content was higher in the younger leaves than in the older leaves. With the increasing Pi concentration in the growth medium, the expression of VPT1 was strongly induced in both roots and leaves. Interestingly, a detailed analysis of VPT1 expression in leaves of different ages indicated that Pi induction occurred more strongly in old leaves than in young leaves, supporting the role of old leaves as "detoxifying organs" for the protection of the young leaves.Correspondingly, the increasing Pi accumulation occurred mainly in the older leaves under high-Pi conditions.3. VPT1 is a tonoplast phosphate transporter for vacuolar Pi uptake. Genetic analysis of the vptl mutant suggested that VPT1 functions in vacuolar sequestration. To further support this hypothesis, we conducted experiments to show that VPT1 protein is indeed located in the vacuolar membrane. We also measured the activity of VPT1 in plant vacuoles using a patch-clamp procedure.A. VPT1 was localized on the tonoplast. First, we observed the green fluorescence signal in the transgenic plants that expressed VPT-GFP fusion protein using confocal laser scanning microscope, and found it was completely unmerged with the red fluorescence signal emissed by plasma membrane fluorochrome FM4-64. Second, we observed the transgenic plants that expressed VPT1-GFP and tonoplast maker γ-TIP-mCherry fusion protein, and found their fluorescence signals were well merged. Third, we isolated the intact vacuoles from mesophyll cells that expressed VPT-GFP, and it more clearly showed that VPT1 was really localized on the tonoplast.B. VPT1 was transporter essential for vacuolar Pi sequestration. Patch-clamp analysis of isolated vacuoles showed that the Pi influx current was severely reduced in vptl compared with wild type plants. When ectopically expressed in Nicotiana benthamiana mesophyll cells, VPT1 mediates vacuolar influx of anions, including Pi, SO42-, NO3-, Cl-, and malate with Pi as that preferred anion. The VPT1-mediated Pi current amplitude was dependent on cytosolic phosphate concentration. Single-channel analysis showed that the open probability of VPT1 was increased with the increase in transtonoplast potential. We conclude that VPT1 is a transporter responsible for vacuolar Pi storage and is essential for Pi adaptation in Arabidopsis.4. The feedback regulation of arsenate uptake and accumutation by genetic manipulation of VPT1 in Arabidopsis. Defect of VPT1 would lead cytoplastic Pi retention, especially under high phosphate conditions.Whereas, high cytoplastic Pi level couldn’t only antagonize the competitive toxicity of As(V), but also could inhibit the gene expression of the plasma membrane localized PHT1 proteins in a feedback manner, and then reduce plant As(V) uptake.A. Lack of VPT1 caused a feedback inhibit of PHTls expression. Under Pi sufficient conditions, two genes coding Pi transporters located on plasma membrane essential for Pi /As(V) uptake, PHTL;1 and PHT1;4, were both expressed at a much lower level in vptl mutant than in wild type. Correspondingly, the transcript inhibitor WRKY6 and the transcript activator WRKY45 of PHT1;1 were expressed at a higher and lower level in the vptl mutant than in the wild type, respectively.B. VPT1 influenced the As(V) tolerance of Arabidopsis in a phosphate dependent manner:As compared with wild type, the vptl mutant plants were more tolerance to As(V) stress under Pi sufficient conditions, but the VPT1 over-expression plants were more sensitive. Whereas, these changes of As(V) sensitivity caused by the genetic modification of VPT1 expression disappeared under Pi deficient conditions.C. VPT1 influenced the As(V) uptake and arsenic accumulation of Arabidopsis:Under Pi sufficient conditions, as compared with the wild type, the vptl mutant accumulated less arsenic in whole plants after a short-term exposure of As(V), but the VPT1 over-expression plants accumulated much more arsenic. After a long-term exposure in low dose of As(V), the vptl mutant accumulated less arsenic in the siliques, but it was completely contrast with the case in the VPT1 over-expression plant siliques.Thus, the vacuolar phosphate transporter VPT1 isn’t only essential for Pi homeostasis under different phosphate conditions, but also is important for controlling As(V) uptake and accumulation of plants by a feedback regulation manner. This study could provide a new pathway for engineering crops that can better adapt to variable Pi availability in the soil and accumulate less arsenic in the edible parts.