Functional Characterization Of A Rice Phosphate Transporter OsPHT1;3

Phosphorus(P) is one of the major macro-nutrients indispensable for plant growth and development,and is widely involved in important physiological and biochemical processes,such as material synthesis,energy transfer and signal transduction in plants.At the same time,as one of the important life elements,P is also a key structural component of many biological macro-molecules,such as nucleic acids,phospholipids,adenosine phosphate(ATP),and P-containing enzymes.The major form of P for plants to acquire from the soil is inorganic orthophosphate(Pi).Owing to its unique chemical properties,Pi is easily fixed by metal cations such as calcium,iron,aluminum or converted to organic form by soil microorganisms.The mobility and availability of Pi in the soil is very poor.In addition,there is a large fluctuated Pi levels in the soil,ranging from only a few micromoles in the soil of the natural ecosystem to a several hundred micromoles in the soil of the farmland ecosystem.In order to adapt to the huge fluctuations of Pi in soils,plants have evolved a suite of physiological and biochemical responses including through modification of the root architecture,enhancing secretion of organic acids in roots,forming mutually beneficial symbiosis with arbuscular mycorrhizal(AM)fungi,as well as inducing phosphate transporters(PTs)to increase the availability of P and enhance the ability to acquire P in the soil.Expressing a series of phosphate transporters on the plasma membrane of roots is the major approach for plants to acquire and transport P.To date,plant phosphate transporters belonging to the PHT1 family are thought to play a crucial role in Pi uptake and translocation.There is a total of 13 members belonging to the PHT1 family in rice.Although most members of rice PHT1 have been identified and characterized,several members are still functionally uncharacterized.Moreover,PHT1 members who play key roles under an extremely low-phosphate regimes are still unknown.Since rice is one of the most important crops in China,the intensive study on the rice PHT1 genes is of great importance to decipher the mechanism in regulating Pi uptake and translocation during plants adapt to the natural environment with poor availability of P and to breed cultivars with high PUE(P use efficiency).In this study,another member of rice PHT1 family OsPHT1;3 was functionally characterized by using RT-qPCR,CRISPR-Cas9 gene editing system,yeast and oocytes heterologous expression systems.The main results obtained are summarized as follows:1.The bioinformatics analysis showed that OsPHT1;3 gene is located on chromosome 10 in the rice genome.The full-length of genomic DNA and CDS(coding sequence)are 1977 bp and 1581 bp,which encodes a total of 526 amino acids.OsPHT1;3 has a single copy in the rice genome and contains no introns.Subcellular localization analysis indicated that OsPHT1;3 is a plasma membrane-localized protein.2.The results of yeast and oocytes heterologous expression systems showed that OsPHT1;3 was able to complement a yeast mutant strain defective in Pi uptake and mediate Pi influx in Xenopus laevis oocytes only at high Pi levels,indicating that OsPHT1;3 has Pi transport activity and shows low-affinity in heterologous system.In addition,OsPHT1;3 overexpression lines were cultured hydroponically treated with different Pi levels and the growth performance and Pi accumulation were determined and analyzed.The results showed that OsPHT1;3 overexpression plants displayed a phenotype of decreased biomass and growth retardation compared to WT(wild-type)under both high-Pi(HP,200 μM)and low-Pi(LP,10 μM)conditions.Overexpression of OsPHT1;3 significantly enhanced the ability of rice roots to absorb and transport Pi.The concentration of inorganic phosphate and total P in roots and shoots of OsPHT1;3 overexpression plaIts were 2-5-fold as high as that in WT.The 32P-labelled Pi uptake assay also indicated that overexpression of OsPHT1;3 led to a significant increase in root Pi uptake rate at both high and low Pi supply levels.3.The spatio-temporal expression pattern of OsPHT1;3 was investigated by using gene chip,RT-qPCR technique and transgenic rice plants harboring the GUS reporter gene driven by OsPHT1;3 promoter.The results showed that OsPHT1;3 was strongly induced by phosphate starvation and had the highest expression abundance in rice roots while its expression was barely detectable when sufficient Pi was supplied.The transcript abundance of OsPHT1;3 gradually increased with the duration of phosphate starvation,and then decreased rapidly to a basal level comparable with that under Pi-sufficient conditions after resupply of Pi.OsPHT1;3 was also significantly induced by phosphate starvation in old leaf blades,leaf sheaths and basal nodes whereas expression induction in new leaf blades was much lower.Interestingly,in basal nodes,OsPHT1;3 expression was constrained in the phloem regions of both enlarged vascular bundle(EVB)and regular vascular bundle(RVB).In addition,the expression of OsPHT1;3 was detectable also in reproductive organs,such as geminated seeds,anthers,lemmas,and paleas.4.There was no significant difference in Pi concentration both in roots and shoots between ospht1;3 mutant and WT plants under either HP(200 μM Pi)or LP(10 μM Pi)conditions while the Pi concentration in the shoots and the short-time 32P-labelled Pi uptake in the roots of ospht1;3 mutants were significantly decreased as compared with that in WT plants only when external Pi concentration was 5 μM or 1 μM,indicating that mutation of OsPHT1;3 impaired Pi uptake and root-to-shoot Pi translocation only under extremely low Pi conditions.An isotope labelling experiment with 32P showed that ospht1;3 mutant lines were impaired in remobilization of Pi from old leaves(source organs)to newly developed leaves(sink organs).5.The expression of OsPHT1;3 was examined in OsPHR2 overexpression and mutant plants.It was found that OsPHT1;3 was down-regulated and up-regulated,respectively,by mutation and overexpression of OsPHR2.The electrophoretic mobility shift assay(EMSA)showed that OsPHR2 could bind to both P1BS cis-elements in the OsPHT1;3 promoter region.The above results indicate that OsPHT1;3 is directly regulated by OsPHR2.6.RT-qPCR analysis showed that four PHT1 genes(OsPHT1;2,OsPHT1;4,OsPHT1;9 and OsPHT1;10)were up-regulated and down-regulated,respectively,in Pi-starved roots of ospht1;3 mutants and OsPHT1;3 overexpression lines compared with that of WT plants.Phylogenetic analysis showed that OsPHT1;3 and OsPHT1;2 are the two members of rice PHT1 family that are strongly induced by phosphate starvation as well as shared with the highest amino acid sequence identity.Split-ubiquitin yeast-two-hybrid assay(Y2H),bimolecular fluorescence complementation(BiFC),and co-immunoprecipitation(Co-IP)all confirmed that PHT1;2 and PHT1;3 interacted with each other at the plasma membrane.Taken together,we functionally characterized a rice phosphate transporter belonging to the PHT1 family,OsPHT1;3,that mediates Pi uptake,translocation,and remobilization under extremely low phosphate regimes.Our findings provide the important clues to decipher the mechanism in regulating Pi uptake and translocation during plants adapt to the natural environment with poor availability of P and to breed cultivars with high PUE.