Functional Characterization Of Two Phosphate Transporters OsPHT1;7 And OsPHT1;5 In Phosphate Distribution In Rice

Phosphorus(P)is one of macroelement nutrients necessary for plant growth and development.It is widely involved in important physiological and biochemical processes such as substance synthesis,energy transmission,and signal transduction in plants.At the same time,phosphorus is also a key component of many biological macromolecules such as nucleic acids,proteins,phospholipids,adenosine phosphate(ATP),and phosphorus plays an indispensable role in the growth and development of plants.The chemical form of phosphorus obtained by plant roots from the soil is mainly orthophosphate(Pi).Most of the phosphorus in the natural soil exists in the form of organic matter.The remaining inorganic phosphorus can easily combine with iron,magnesium,aluminum,calcium and other metal ions to form insoluble compounds or be converted to organic phosphorus by soil microorganisms.This results in low efficiency and low mobility of phosphorus in the soil,which cannot satify the needs of crop growth and development.In agricultural production,to maintain crop yields,phosphate fertilizers need to be continuously applied.In cereal crops,most of the phosphorus absorbed by plant roots is finally distributed to the grains,removed from the farmland system during harvest and finally excreted into natural water bodies.Reducing phosphorus loss from fields casued by crop harvesting is conducive to the full and sustainable use of phosphorus fertilizer.To achieve this goal,it is necessary to explore the phosphorus distribution rule in different tissues and organs of plants,and clarify the function of related genes involved in phosphorus distribution in crops,so as to improve the distribution ratio of phosphorus in different parts of crops.In order to cope with the lack of available phosphate,plants have formed a comprehensive and efficient adaptation mechanism during the evolution process,including changing root architecture,forming a mycorrhizal fungal symbiosis system,producing root secretas and inducing the expression of high-affinity phosphate transporters to enhance the absorption of phosphate in the soil.The movement of phosphate in plant can be roughly divided into root absorption,transportation to the shoot,(re)distribution between the shoot organs,and intracellular transportation between subcellular organelles.All of these processes require the involvement of phosphate transporters.The functions of most members of the rice phosphate transporter OsPHT1 family have been identified,but there are still some members whose functions have not yet been ascertained.The functions of OsPHT1 in rice phosphate redistribution and phosphate transport to reproductive organs have not yet been determined,which needs more and in-depth research.Our laboratory had conducted preliminary studies on rice phosphate transporters OsPHT1;7 and OsPHT1;5 in past time.However,due to the limitations of the technology at that time,only gene overexpression materials were used to study the gene functions,and mutants of OsPHT1;7 and OsPHT1;5 were not used for in-depth research.In this study,OsPHT1;5 and OsPHT1;7 were used as the research objects,and rice varieties Nipponbare and Dongjin were used as expermental materials.Real-time fluorescent quantitative PCR(RT-q PCR),CRISPR-Cas9 technology,yeast or Xenopus oocyte heterologous expression systems and other experimental methods were used to study the expression characteristics and gene functions of OsPHT1;5 and OsPHT1;7.The main results obtained are as follows:1.The OsPHT1;5 gene is located on chromosome 04 of the rice genome,the gene accession number is LOC＿Os04g10690,and the full length of the coding sequence is 1647 bp.The OsPHT1;7 gene is located on chromosome 03 of the rice genome,the gene accession number is LOC＿Os03g04360,and the full length of the coding sequence is 1581 bp.2.Subcellular localization analysis was conducted by using rice protoplast transfection,and it was found that OsPHT1;5 and OsPHT1;7 were localizaed on the plasma membrane.The yeast heterologous expression system was used to verify that OsPHT1;5 has phosphate transporter activity,and OsPHT1;5 can help to recover the growth of the phosphate-uptake yeast mutant in 50 μM Pi medium.Yeast and Xenopus oocyte heterologous expression system were used to verify that OsPHT1;7 has phosphate transporter activity.OsPHT1;7 can helip to recover the growth of the phosphate-uptake yeast mutant in 50 μM Pi medium,and only functions in Xenopus oocytes in the acidic(p H 5.5)condition.3.Temporal and spatial expression characteristics of OsPHT1;7 were analyzed using RT-q PCR technology and GUS reporter driven by OsPHT1;7 promoter at the rice seedling stage.The expression of OsPHT1;7 in the shoots and roots of rice at the seedling stage are induced by phosphate deficiency.The expression abundances of OsPHT1;7 in old leaves and basal nodes were higher than young leaves and root,and strongly induced by phosphate starvation at the seedling stage.The expression abundances of OsPHT1;7 in young leaves were very low even if induced by phosphate deficiency.OsPHT1;7 was mainly located in the phloem of the vascular bundle in the leaves,and specifically located in the phloems of the regular vascular bundle(RVB)and enlarged vascular bundle(EVB)in the basal nodes.Knockout of OsPHT1;7 in rice results increased in the phosphate and phosphorus concentration in old leaves of seedling plants,which was casued by the inhibition of the redistribution process that phosphate molecules transfered from old leaves to new leaves at the seedling stage through phloems in leaves and shoot basal nodes.4.Temporal and spatial expression characteristics of OsPHT1;7 in the reproductive growth period of rice were analyzed using RT-q PCR technology and GUS reporter driven by OsPHT1;7 promoter.OsPHT1;7 was mainly expressed in leaves,anthers and nodes during the reproductive growth period(flowering period).OsPHT1;7 was specifically located in the phloems of diffuse vascular bundles(DVB)and enlarged vascular bundles(EVB)in rice nodes.Knockout of OsPHT1;7 affected the phosphorus distribution of rice at the flowering stage,which increased the percentage of phosphorus distributed to leaves and decreased the percentage of phosphorus distributed to the panicle.Knockout of OsPHT1;7 also affected rice anther development,decreased anther length,anther phosphorus concentration,pollen grain activity and pollen grain germination rate,and altered the distribution ratio of phosphorus to anthers in floral organs.In full ripening stage,copared with wild type plants,the heights,seed setting rates and yields per plant of the ospht1;7 mutants decreased,and the number of panicles and phosphorus concentrations in different parts increased.4.The expression characteristics of OsPHT1;5 in rice were examined by gene chip,RTq PCR technology and GUS reporter driven by OsPHT1;5 promoter.OsPHT1;5 was dully responsed to phosphate starvation or phosphate resupply in seedling rice,and expression level in shoot was weekly increased after low Pi treatment for seven days.After transplanting to field,OsPHT1;5 had a higher expression abundance in leaves from 3 to 6 weeks and from15 to 18 weeks than other stages.Moreover,the expression of OsPHT1;5 was induced by exogenous application of ABA and drought stress growing in soil.Knockout of OsPHT1;5did not affect the phosphate or phosphorus concentration in the shoots and roots of the Nipponbare seedlings under high and low phosphate supply conditions or PEG simulated drought treatments.However,the Pi concentrations in old leaf blades under normal Pi supply condition were decreased in ospht1;5 mutants compared with Nipponbare wild type.After transplanting in a paddy field for 21 days,total P concentrations in shoot of ospht1;5 mutants had no significant difference compared with Nipponbare wild type plants.However,in the Dongjin rice variety,a single pht1;5-dj mutant line had lower total phosphorus and inorganic phosphate concentrations in the shoots than the wild type when it was supplied with normal phosphate.In summary,we identified two plasma membrane phosphate transporters OsPHT1;5 and OsPHT1;7 in rice.It was found that OsPHT1;7 was involved in the phosphorus redistribution in rice at the seedling and reproductive growth stages,and OsPHT1;7 was also responsible for transporting phosphate to rice anthers for reproductive development.OsPHT1;5 affects Pi accumulation in old leaf blades of Nipponbare and in the shoot of Dongjin,so that knockout of OsPHT1;5 leads to decreases of Pi concentrations in old leaf blades of Nippobare and in shoot of Dongjin under normal Pi supply condition.According to the expression pattern of OsPHT1;5,it is speculated that OsPHT1;5 may involve in the phosphate transport in rice at the tillering or maturing stage and under upland condition.The results of the study laid the foundation for exploring mechanisms of phosphate distribution in rice plants of different growth periods and phosphate affecting plant reproductive development.