Post-trnslation Regulation Of Phosphate Transporters PHT1Family In Orvza Sativa

Rice is one of the most important food crops in China and Asian countries. Inefficient utilization and executive application of phosphate (Pi) fertilizer in rice production increases farmers’cost and environmental pollution. Developing cultivars with improved Pi efficiency is essential for the sustainability of agriculture. Pi efficiency is co-regulated by Pi uptake, translocation and remobilization in plants through the functions of Pi transporters (PTs) and other factors involved in Pi translocation and physiological utilization. The function of PTs localized in plasma membrane (PM) is regulated at both transcriptional and posttranscriptional levels. The PHF1(PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1) is found to be a crucial regulator to modulate PHT1;1, a high-affinity phosphate (Pi) transporter in Arabidopsis, endoplasmic reticulum (ER)-exit to PM which is dependent on Ser-514phosphorylation status at hydrophilic C-terminal domain(CTD) ofPHT1;1.In this study, we isolated OsPHF1(LOC_Os07g0900) in rice through screening an EMS (ethyl methylsulfonate) generated rice mutant library (Oryza sativa L.ssp. japonica var. Nipponbare) under arsenate (Na3AsO4·H2O) stress (30μM) and characterized its function using the mutants and the transgenic plants with overexpressed OsPHF1. Three OsPHF1allelic mutants carrying one point mutation at the fifth WD40-repeat motif and two point mutations at the transmembrane helix, respectively, were found by map-based cloning. These mutants showed arsenate resistance and significant reduction of Pi uptake ability. The results indicate that both WD40domain at hydrophilic N-termini and transmembrane helix are important to the function of OsPHF1.Mutation of OsPHF1results in the ER retention of the low-affinity Pi transporter OsPT2(LOC_Os03g05640) and high-affinity Pi transporter OsPT8(LOC_Os10g30790). Because of the conserved Ser-514motif in PHT1family members in Arabidopsis and Rice, the effect of OsPHF1on the ER-exit of OsPT2/8may be extended to other OsPTs.Mutation of OsPHF1remarkably impairs Pi uptake ability and reduced Pi accumulation in plants under OsPHR2-overexpressed background. Overexpression of OsPHF1results in Pi accumulation in Pi-supplied solution cultures. These results indicate the role of OsPHF1for multiple OsPTs and determine Pi uptake and translocation.The similar function of PHF1 required facilitating PHT1transit through ER to PM between Arabidopsis and rice provides an example of expectations from what one would deduce from sequence comparisons to extend knowledge from Arabidopsis to crops.Further study through screening a rice root cDNA library, we find an OsPT8interactor, protein kinase OsCK2component OsCK2β3(LOC_Os07g02350). OsCK2is a conserved tetrameric Ser/Thr phosphotransferase in Eukaryon. Four OsCK2components, OsCK2a2(LOC_Os07g02350),OsCK2a3(LOC_Os03g10940), OsCK2(31(LOC_Os10g41250), OsCK2β3(LOC_Os07g31280) in rice have been identified. In vitro and in vivo experiments indicate that OsCK2β3directly interacts with OsPT2/8, and is necessary for interaction between OsCK2α3and OsPT2/8, which is consistent with the previous report that β subunit plays as anchor for binding of the holoenzyme to the substrate. ER-retention of OsPT2/8resulted from overexpression of both OsCK2α3and OsCK2β3was observed in rice protoplast cells and transgenic plants. These results provide evidence that catalytic subunit OsCK2α3and regulatory subunit OsCK2β3come into play as a holoenzyme.The genetic perturbation of both OsCK2a3/β3by using CAMV35S promoter drived expression (Ox) and RNA interfering (Ri) of the two genes affects Pi uptake ability in respective transgenic plants, provides additive evidence to the holoenzyme character.Subcellular localization of OsCK2α3/β3showed that both subunits targeted in nucleus and cytoplasm. The co-expression of OsCK2α3/β3with ER marker (OsPHF1) and cis-Golgi marker (GmManl) indicated that the two subunits localized in early trafficking pathway. The multiple subcellular localization of OsCK2α3/β3implied the pleiotropic function of OsCK2.In vitro phosphorylation assay showed no kinase ability of the regulatory subunit β3, while the catalytic subunit OsCK2α3was able to phosphorylate OsPT8CTD all by itself. Site-directed mutagenesis of the three potential CK2phosphorylation sites to Ala confirmed that Ser517is the only phosphorylation site for CK2. In vivo phosphorylation experiments using extracts from plant cells further proved the conclusion.In vitro and in vivo data showed that OsPHF1interacts with wild type or mimic unphosphorylated OsPTs, but not the mimic phosphoryted ones. Phosphorylation modification of OsPT8inhibits its interation with OsPHF1was also confirmed by in vitro phosphorylation analysis. By contrast, genetic perturbation of OsCK2α3/β3can’t change the PM-location of mimic unphosphorylated OsPTs and the impairment of Pi uptake ability in phfl mutant. Thus, we proposed that ER-exit of PTs is under antagonistic control of PHF1and CK2. That is, PHF1interacts with unphosphorylated PT and facilitates its trafficking from ER to PM; CK2phosphorylates PT and inhibits interaction between PHF1and PT. Conclusively, the facilitation of PHF1is necessary for PT exit from ER regarding phosphorylation status of PT. In addition, we proposed the possible mechanism under phosphorylation of PT responsive to cellular Pi status:Pi-starvation enhanced ER-exit of PT could be attributed to accumulation of OsPHF1protein, reduced OsCK2β3in Pi-deplete condition, which blocked CK2holoenzyme interaction with PTs.The present study provides a potential strategy for breeding crops with enhanced Pi uptake ability through site-directed mutagenesis (change the Ser to Ala). The field experiments are required to test the Pi-efficiency in different rice varieties under different Pi fertilizer application levels. Screening point mutation plants for Ala mutation in important PTs from EMS-mutagenized seeds may also be feasible.