The Mechanism Of OsSPX4Regulating Phosphate Central Regulator OsPHR2in Rice

Systemic phosphate (Pi) starvation signaling and homeostasis is regulated by the conserved central regulators, transcription factor AtPHRl and its partial functional redundant members in Arabidopsis. AtPHR1is a transcprition fator with MYB-CC domain. OsPHR2, as a homologue of AtPHRl in rice has been reported to play the role in central regulation.In rice there are six SPX-domain genes (defined as the proteins exclusively contain SPX-domain) desiganated as OsSPX1-6have been identified. All of the genes, with exception of OsSPX4, are response to Pi-starvation signaling at transcription level, which let us reason that OsSPX4may function with other different way as the other SPX genes. The previous work demonstrated excessive shoot Pi accumulation and upregulation of Pi-starvation-induced (PSI) genes in spx4mutant. On the contrast, overexpression of OsSPX4(desiganated OxSPX4) reduces expressions of PSI genes responsive to Pi-starvation.To genetically confirm the negative effect of OsSPX4on OsPHR2function, we developed double overexpression of OsSPX4and OsPHR2plants (designated as OxSPX4/OxPHR2). We found that the upregulated PSI gene expressions and shoot excessive Pi accumulation caused by overexpression of OsPHR2can be rescued by overexpressed of OsSPX4. The results indicated that OsSPX4is a repressor of OsPHR2function.Becauese OsSPX4is not responses to Pi-starvation at transcription level. We suspected OsSPX4protein may responses to Pi-statues in planr cells. To determine it, we used the transgenic plants with OsSPX4p-OsSPX4-GUS and OsSPX4p-OsSPX4-GFP fusions to investigate the OsSPX4protein responses to Pi statues in plants. The results showed that the fusion protein of OsSPX4is reduced with deficiency of Pi. Ferther, We performed cell-free degradation analysis to examine the stability of GST-SPX4proteins. The result showed that the half-life of GST-SPX4 incubated with totoal peotein exteacted fron the seedlings grown in Pi-deficient condition (-P) is shorter than that incubated with extracts from the seedlings grown in Pi-supplied (+P) condition.In addition, the exogenous Pi (NaH2PO4,Pi) and phosphite (NaHPO3, Phi) could stabilize GST-SPX4, while the exogenous addtions of nitrate (NaNO3) and sulfate (Na2SO4) could don’t. The results indicate the specific effect of Pi on stabilization of OsSPX4.To determine whether OsSPX4interacts with OsPHR2, we performed yeast two-hybrid assays and co-immunoprecipitation (Co-IP) assays. The results indicate that OsSPX4physically interacts with OsPHR2. Further analysis showed that N-terminus of OsSPX4containing SPX-domain can interact with C-terminus of OsPHR2with MYB-CC domains. Chromatin immunoprecipitation analysis (Chip) and Electrophoretic mobility shift assay (EMSA) indicate that the interaction of OsSPX4with OsPHR2inhibits the binding ability of OsPHR2to its cis-element P1BS (PHR1Binding Sequence) in the promoters of PSI genes’in vivo or in vitro.Using bimolecular fluorescence complementation (BiFC) analysis, strong signals for the interaction of OsSPX4and OsPHR2was observed in the in the cytoplasm, while also weak signals can be detected in the nucleus. We transiently coexpressed35S-GFP or35S-OsSPX4-GFP together with35S-OsPHR2-mCherry in rice protoplasts. The red fluorescence signal of OsPHR2-mCherry was predominantly found in the nucleus when transiently alone or simultaneously expressed with GFP in the protoplasts, while with coexpression of OsSPX4-GFP, the OsPHR2-mCherry signal was retained in the cytoplasm, indicating interaction of OsSPX4with OsPHR2inhibits the OsPHR2into nuclear.To further confirm the results from BiFC and rice protoplasts,The genetic plants with OsPHR2p-OsPHR2-FLAG/spx4and OsPHR2p-OsPHR2-FLAGIOxSPX4were developed by introducing the OsPHR2p-OsPHR2-FLAG fusion into spx4mutant and OxSPX4plants by crossing.In the spx4mutant background, the OsPHR2protein level increased significantly both in nuclear and cytoplasmic fractions, but much more strikingly in the nuclear fraction. By contrast, the OsPHR2protein level was reduced remarkably in the nuclear fraction, but no significant change in the cytoplasmic fraction in OxSPX4background.In summary, we found that OsSPX4negatively regulates OsPHR2function through its interaction with OsPHR2under Pi-supplied condition. OsSPX4protein is responses to Pi-starvation. With cell Pi concentration decreasion, OsSPX4protein is degradated in a dosage manner, therefore releasing OsPHR2into nuclear to binding its cis-element and triggers its downstream PSI genes. Because this central reglator function is highly conserved in planta, our finding has a general importance to the manipulation of crops adaptive to Pi conditions.