Regulatory Mechanism Of OsPHT1;1 By OsWRKY-P2 And OsWRKY-P3 And Function In Phosphate Homeostasis In Rice

Phosphorus is one of the major mineral nutrient elements which is indispensable for plant growth and development process,and is widely involved in physiological and biochemical processes in plants such as biosynthesis,energy transfer,signal transduction.Plants mainly absorb the inorganic orthophosphate（Pi）from soil.However,the content of inorganic orthophosphate is lower than 10 μM,due to its unique chemical properties,easily fixed by metal cations such as Ca and Mg,and converted to organic forms by soil microorganisms.To cope with phosphorus stress,plant has already evolved a series of complex mechanism to maintain its normal growth and development.Transcription factors play important roles in transcriptional regulation for phosphate starvation response genes.So far,it has formed a set of phosphorus signaling pathway centered around PHR regulator in plants.Nevertheless,the molecular regulatory mechanisms of plants under phosphorus sufficient condition are still unknown.Plant phosphate transporters are responsible for Pi uptake,translocation and redistrbution.Among them,PHT1（Phosphate Transporter 1）family transporters play the vital role.PHT1 genes are mostly expressed in root,and they are probably the only transporters responsible for Pi uptake from soil.In addition,most PHT1 genes would be up-regulated differently under phosphate deficient condition.There are 13 genes（OsPHT1;1～13）in rice PHT1 family.Among them,besides two members induced by mycorrihizal（OsPHT1;11 and OsPHT1;13）,7 genes of the rest which have been reported（OsPHT1;2、OsPHT1;3、OsPHT1;4、OsPHT1;6,OsPHT1;8、OsPHT1;9 and OsPHT1;10）are all induced expressed by phosphate deficiency.But unlike them,OsPHT1;1 is strongly expressed under both Pi sufficient and deficient conditions,and it is insensitive to phosphate starvation.The physiological function of OsPHT1;1 has not been studied via a null allele mutant.Moreover,its unique expression pattern indicates that its transcription expression is regulated by a signaling pathway independent of PHR2（rice PHR transcription factor）.Therefore,we carried on research on OsPHT1;1,and on the basis of its further physiological function analysis,we screened out a transcription factor OsWRKY-P2,which could directly bind to its promoter and promote its expression,via Yeast One-hybrid screening system.Another WRKY family transcription factor OsWRKY-P3 was found interacted with OsWRKY-P2 via Yeast Two-hybrid system.Finally,the molecular mechanisms and physiological function of two transcription factors regulating OsPHT1;1 were studied via genetic,physiology and molecular biology analysis.The main results obtained were summarized as follows:1.OsPHT1;1 knockout mutant lines were obtained via CRISPR/Cas9 gene editing technology and the hydroponic experiments supplied with different Pi concentrations were performed to study its Pi accumulation status.The result showed that the Pi content in root of ospht1;1 mutant was lower than that of wild type under Pi supplied conditions（normal phosphate condition,Ctrl=90 μM or high phosphate condition,HP=300 μM Pi）;and under 300 μM Pi supplied condition,the Pi content in shoot of mutants was as well lower than that of wild type.These results indicated that knocking out OsPHT1;1 would effect rice phosphate uptake process under Pi replete condition.2.In order to find suitable promoter fragment applied to Yeast One-hybrid system,several truncated fragments of OsPHT1;1 promoter were generated and fused to GUS reporter gene,the results showed that four truncated fragment（2768 bp,2147 bp,1613 bp,1164 bp）fused GUS reporter gene displayed almost the same GUS activity,which indicated that 1164-bp promoter framgment contained the cis-elements that maintained OsPHT1;1 constitutive expression.Subsequently,we performed a yeast one-hybrid screening with part of this fragment（TATA-box upstream,-1164 bp to-112 bp）.Twenty eight positive clones were isolated.Among them,three clones correspond to the same gene encoding a WRKY transcription factor OsWRKY-P2.The one to one yeast one-hybrid,EMSA and ChIP-qPCR assays all confirmed that OsWRKY-P2 could interact with OsPHT1;1 promoter and could specifically bind to-606 bp～-601 bp W-box cis-element of OsPHT1;1 promoter.3.OsWRKY-P2 is located on chromosome 1 in the rice genome.It is consisted of two exons and one intron,and belongs to a Group Ⅲ type WRKY transcription factor.The transcriptional activation activity of OsWRKY-P2 was confirmed in Y2H system,which indicated that the exist of 45 amino acids in C terminal of OsWRKY-P2 protein were necessary for its transcriptional activation activity.The subcellular localization analysis showed that OsWRKY-P2 was a nuclear localized protein.The spatio-temporal expression pattern of OsWRKY-P2 was investigated by RT-qPCR and OsWRKY-P2 tissue localization transgenic plants.The RT-qPCR analysis showed that OsWRKY-P2 was expressed in different tissue organs during the whole growing stage,and the expression of OsWRKY-P2 in root would be induced by high Pi condition.Histochemical staining for GUS activity result showed that Os WRKY-P2 was expressed everywhere except the root cap,meristematic zone and partial elongation region.Additionally,we obtained the Os WRKY-P2 overexpression and knocked out transgenic lines.The hydroponical experiment results demonstrated that OsWRKY-P2 overexpression plants displayed a phenotype of dwarf and growth retardation,but an excess Pi accumulation,compared with wild type plants.However,there was no significant difference between oswrky-p2 mutant and wild type plants.4.WRKY transcription factors often interact with other memebers of its family or even with other regulators to joint their function.Hereby,the rice cDNA library was constructed and yeast two-hybrid screening system was performed to seek out potential proteins interacted with OsWRKY-P2.Finally,100 positive clones were isolated and four clones corresponded to the same gene encoding another WRKY transcription factor OsWRKY-P3.The one to one Y2H,BiFC and pull-down assays were performed to confirm that OsWRKYP2 could physically interact with OsWRKY-P3.5.OsWRKY-P3 is also located on chromosome 1 in the rice genome,and it is consisted of three exons and two introns,and as well belongs to a Group Ⅲ type WRKY transcription factor.The research results had proved that OsWRKY-P3 and OsWRKY-P2 have extremely similar gene function in transcriptional activation activity,subcellular localization,spatiotemporal expression pattern and physiological and biochemical phenotypes of overexpression and knocked out transgenic lines,which meaned that OsWRKY-P3 overexpression plants displayed an excess Pi accumulation phenotype but mutants displayed none significant difference compared with wild type.Meanwhile,the results of Y1H,EMSA and ChIP-qPCR analysis proved that OsWRKY-P3 could specifically bind to the-606～-601 bp and-587～-5 82 bp W-box cis-elements of OsPHT1;1promoter.6.RT-qPCR analysis showed that four genes of PHT1 famiy（PHT1;1,PHT1;2,PHT1;4 and PHT1;8）were up-regulated differently in Os WRKY-P2 and OsWRKY-P3 overexpression transgenic plants,specially the OsPHT1;1 were significantly up-regulated in both shoot and root of overexpression plants under Pi sufficient/deficient condition.It is worth noting that PHT1;1,PHT1;2,PHT1;4 and PHT1;8 are expressed with highest enrichment among PHT1 members under Pi sufficient condition.The inorganic Pi content analysis in plant leaf bade of isolated populations of different genotypes in F2 generation obtained by OsWRKYP2/OsWRKY-P3 overexpression plants and ospht1;1 mutants hybridization,showed that OsPHT1;1 mutation could lead to decreased Pi accumulation in OsWRKY-P2 or Os WRKYP3 overexpression plants.It on the one hand provided the genetic evidence that OsWRKY-P2 and Os WRKY-P3 could regulate OsPHT1;1 in rice,and on the other hand,it further indicated that the excessive Pi accumulations in OsWRKY-P2 and OsWRKY-P3 overexpression plants were caused by OsPHT1;1 up-regulation.7.The oswrky-p2 oswrky-p3 double mutants were generated and the hydroponic experiments supplied with different Pi concentrations were performed.The results showed that only under 1 mM Pi treatment condition,the inorganic Pi content in root of wrky-p2 wrky-p3 double mutants were significantly lower than that of wild type plants.RT-qPCR analysis proved as well that the expression of OsPHT1;1,OsPHT1;2 and OsPHT1;4 were remarkably decreased in root of double mutants under this situation.Besides,in order to overcome the function redundency of WRKY transcription factors in regulating OsPHT1;1,OsWRKY-P2 chimeric repressor（Pro35S:WRKY-P2-SRDX）transgenic plant was generated.It displayed the similar phenotype with ospht1;1 mutant plants.Compared with wild type,the inorganic Pi content in root of OsWRKY-P2 chimeric repressor transgenic plants was decreased dramatically.These results suggested that there existed other WRKY family transcription factors had function redundency with OsWRKY-P2 and OsWRKY-P3 in rice.8.In mature stage,the total P content in ospht1;1 rice seeds was remarkably lower than that of wild type,but the total P content in the vegetable organs below Node I of ospht1;1 mutant was significantly increased.Meanwhile,the preliminary experiment results showed that total P content in seeds of oswrky-p2 oswrky-p3 double mutant would be as well lower than that of wild type plants,but the regulatory mechanism needs to be further studied.Based on the results above,two upstream transcription factors of OsPHT1;1,OsWRKYP2 and OsWRKY-P3,were obtained by independent screening,and the regulation mechanism was identified in this study.The WRKY-PHT1;1 regulation pathway is independent of OsPHR2 regulation pathway,and it plays an important role in maintaining Pi homeostasis in rice under Pi replete condition.This study further improves the crop phosphorus signal transduction network and provides theoretical basis and candidate genes for the cultivation of phosphorus efficient crop.