The Mechanism Of Protein Kinase CK2 Involved In Phosphate Uptake And Homeostasis In Rice

Previous studies showed that phosphate concentrations of knockdown transgenic lines of OsCK2a3 and OsCK2β3 were increased, particularly the leaves of OsCK2α3 knockdown transgenic lines exhibited severe chlorosis and necrosis, which indicated that OsCK2 may involved in phosphate uptake and phosphate homeostasis in rice, through phosphorylating regulatory proteins or phosphate transporters in phosphate signaling pathways.To further investigate the role of OsCK2, the effect of external phosphate concentration on the phosphorylation level of phosphate transporter OsPHT1;8(referred as OsPT8) by OsCK2, the funcational analysis of simulation of non-phosphorylaion of PT8, and the interaction between ubiquitin E2 conjugating enzyme OsPHO2 and OsCK2 were perfomed. The results were summarized as follows:i) In vivo phosphorylation of PT8 by OsCK2 was regulated by the environmental phosphate concentration. PT8 was phosphorylated by OsCK2a3 under high phosphate condition, which required the assistance of OsCK2β3, simultaneously OsCK2β3 stability was increased by phosphorylation. While in phosphate deficiency condition, the phosphorylation level of OsCK2(33 was declined, which result in the degradation of OsCK2β3 through ubiquitination pathway, as a consequence the phosphorylation level of PT8 by OsCK2a3 was reduced.ii) The transgenic lines of PT8S517A/pt8, simulation of non-phosphorylaion of PT8, and PT8S517/pt8 as a control were generated. Under culture solutions with different phosphate concentrations, the expression level of PT8 in total membrane protein of PT8S517A/pt8 was significantly higher than PT8S517/pt8, in accordance with the dry weight and phosphate concentration of roots and leaves. These results indicated that in case the pathway of PT8 phosphorylated by OsCK2 was blocked, the efficiency of PT8S517A exported from the endoplasmic reticulum and anchored to the plasma membrane increased, which enhanced Pi uptake ability.iii) Split ubiquitin membrane yeast two-hybrid showed that OsCK2a3 interacted with ubiquitin E2 ligase OsPHO2, which was validated by in vivo Co-immunoprecipitation and in vitro Pull-down assays. OsPHO2 and OsCK2a3 were co-expressed in the endomembrane system (mainly in endoplasmic reticulum and Golgi apparatus), through the observation of subcellular location of OsPHO2-GFP and bimolecular fluorescence complementation (BiFC) assay. Meanwhile, in vitro phosphorylation experiments showed that OsCK2a3 phosphorylated OsPHO2, which may affect the stability or the function of OsPHO2.