Vacuolar Phosphate Transporter Family Members Are Essential For Phosphate Homeostasis In Arabidopsis

Phosphorus(P),one of the essential macronutrients for plant growth and development,plays important roles in most of bio-metabolism in the cells.Phosphate(Pi)is available form that can be utilized by plant,but the concentration of Pi in soil is limited(about 10 ?M).So Pi is always added in fertilizers for sustainable production in agriculture.However,phosphate rock that is used to produce Pi fertilizers are unsustainable around the world.We should improve P nutrient use efficiency before we find the sustainable Pi resources.Therefore,detecting the genetic network that induces Pi transport in plants is pivotal and worthwhile.Previous studies have identified that VPT1(also termed PHT5;1),a SPX-MFS protein,mainly contributes to vacuolar Pi sequestration in Arabidopsis.The SPX-MFS family consists of three members(VPT1,VPT2 and VPT3)which are all localized to tonoplast.The functions of VPT2 and VPT3 are still need to be elucidated.To further explore the function of VPT genes,we generated various-double mutants(vptl/vpt2,vpt1/vpt3,vpt2/vpt3)and triple mutant(vpt1/pt2/vpt3).The phenotype and Pi contents assays revealed that vpt1/vpt2 and vptl were archived in one genotype,and vpt2/vpt3 were consistent with wild type plants.However,the vpt1/vpt3 double mutant displayed lower Pi content as compared with vptl single mutant,and this kind of disordered Pi accumulation could be complemented by expressing genome sequence of VPT1 or VPT3.Patch clamp data indicated Pi currents from mesophyll cell vacuole of vptl/vpt3 mutant were severely decreased as compared to vptl,demonstrating that defected vacuolar Pi sequestration was responsible for lower Pi content of vptl/vpt3.Nevertheless,Pi current density of vpt2/vpt3 vacuole had no difference with the control seedling.Thus,VPT1 is the major Pi transporter that induces Pi accumulated in vacuole,while VPT3 is another one in context of lacking VPT1 in Arabidopsis.Moreover,vptl/vpt3 double mutants showed more disordered Pi homeostasis than vptl which indicated by adaption ability to the fluctuation of Pi concentration in the environment.vptl/vpt2/vpt3 exhibited similar phenotype with vpt1/vpt3.Additionally,the most obvious phenotype of vptl/vpt3 was related to reproductive development.vptl/vpt3 generated shorter siliques and the seed formation was impaired while other vpt double mutants(vptl/vpt2 and vpt2/vpt3)were normal.We used scanning electron microscope to detect if the pollen and stigma were not well developed that leads to the defected silique in vptl/vpt3 double mutant.The scanning results showed there was no difference between Col and vptl/vpt3.In addition,Hochest staining of pollen grains revealed that vptl/vpt3 had three nuclei,and in-vitro pollen tube growth assay indicated the pollen germination rate and pollen tube length of vptl/vpt3 were consistent with the wild type seedlings.All these data demonstrated that the pollen and pistil of vptl/vpt3 were well developed.Pi content analysis brought up the unexpected result.Although Pi content of leaves and roots were lower in vptl/vpt3 than in wild type plants,the vptl/vpt3 double mutant flower organ Pi content was much higher than the control seedling.Moreover,the higher Pi concentration of culture solution was,the shorter vptl/vpt3 silique was,the fewer mature seeds could be generated,and expressing VPT1 or VPT3 could compensate this phenotype.So Pi toxicity should be the reason why the seed set of vptl/vpt3 is impaired.Pi content measurements revealed that all components(including pistil,pollen,petal and sepal)of vptl/vpt3 flower organ contained more Pi than wild type seedlings.To explore which part(pollen or pistil)was responsible for the defected seed set,reciprocal crosses were performed between the wild type and vptl/vpt3.The results suggested that the lack of flower set and defected seed formation in vptl/vpt3 might not be due to lack of pollen viability,the pistil of vpt1/vpt3 should be considered.In addition,Col seedlings were used as the pollen donor to pollinate Col and vpt1/vpt3 under various Pi conditions.Both Col and vpt1/vpt3 displayed regular pollen tube growth under low Pi condition.However,when Pi concentration of the culture solution was upregulated,the pollen tube elongation was inhibited.These data suggested that toxic level of Pi in pistil would lead to the inhibition of pollen tube growth,which was also proved by in-vitro assays.So what is the reason that toxic level of Pi could be accumulated in vpt1/vpt3 flower organ?All Pi that distributed in various parts of plant are derived from root-uptake Pi.So it was realized that more Pi was transported upwardly during flowering in double mutant.To validate this point,we gathered xylem sap of vpt1/vpt3 double mutant and wild type separately for Pi measurements.The experiment results showed Pi level of the vpt1/vpt3 double mutant xylem sap was much higher than that of wild type plants.Then we generatedvpt1/vpt3/phol triple mutant for further validation of this disordered Pi distribution.PHO1 is the major transport protein that facilitates Pi loading into xylem for long distance transport.The phenotyping assay demonstrated that knockout of PHO1 in the context of vpt1/vpt3 could complement this reproductive development defect which means reducing the content of Pi that transported to flower organ contributes to normal silique formation in vpt1/vpt3 double mutant.So loss of VPT1 and VPT3 induces more Pi transported upwardly to flower organ that reaches a toxic level for reproduction.VPTs serve as a stabilizator to maintain Pi homeostasis at the whole plant level,they systemically guide Pi distribution in a proper manner for growth and development.The toxicity of arsenate[As(V)]is derived from its close chemical similarity to phosphate(Pi),which allows the metalloid to be easily transported into plant through the Pi transport system.Lacking genes that are associated with Pi homeostasis always alters the sensitivity to As(V)in plant.Current findings referred to VPT family members are the major Pi transporter that responsible for vacuolar Pi accumulation which means Pi homeostasis of the vpt mutants is severely disordered.To detect if VPTs are involved in As(V)tolerance in Arabidopsis,we stressed various vpt mutants with toxic level of As(V).The vptl mutants(including vptl,vptl/vpt2,vptl/vpt3,vptl/vpt2/vpt3)displayed resistance to As(V),while the vpt2/vpt3 did not show any phenotype as compared with wild type.On the contrary,overexpressing VPT1 could lead to more sensitive to As(V)toxicity with higher level of arsenic content in seedlings.Then it was suggested that VPTs are involved in the process that is responding to As(V)toxicity in plant.In this episode,we focused on the VPT1 which is the primary vacuolar Pi transporter.Firstly,a yeast system was used to validate that VPT1 had no As(V)transport activity,which indicates VPT1 cannot deal with As(V)toxicity in an direct manner.Previous studies suggested that more Pi would be accumulated in cytosol in vptl mutant,which could induce the down regulation of PHT1 family members.PHT1s are the major transporters contribute to Pi uptake and also show As(V)transport activity.Then it was realized that As(V)resistance of vptl could rely on Pi balance in cytosol.The phenotyping assay indicated vptl is not resistant to As(V)toxicity when the Pi concentration in medium is down regulated.So the vptl displays As(V)resistance with a Pi dependent manner.To specify how VPT1 participates in the As(V)tolerance process,we detected the expression pattern of VPT1 response to As(V).qPCR data showed the expression of VPT1 was not significantly regulated as it was stressed by As(V).Then,we scanned the expression pattern of VPT1 at the post-transcriptional level by using a eGFP marker.The results indicated As(V)would reduce the amount of VPT1 on vacuolar membrane without changing its subcellular localization pattern.It could be concluded that As(V)inhibits VPT1 that lead to more Pi is accumulated in the cytosol,then the expression level of plasma membrane localized Pi transporter genes are down regulated for.avoiding As(V)uptake.Thus VPT1 related Pi balance contributes to As(V)resistance in the plant cell.