Identification And Characterization Of Proteins Interacting With OsPHO2, A Major Component Of Phosphate Signaling Pathway In Rice

Phosphorus is an essential macronutrient and required for plant growth and development. However, the bioavalibility of phosphate (Pi) is often limited in soil, resulting in reduced crop production. Thus, investigation of Pi starvation response genes and molecular mechanism of Pi signaling pathway is important, and would provide molecular basis to design crops with improved Pi utilizing efficiency.PHO2, encoding an E2ubiquitin-conjugase, has been shown to be a major component of the Pi-starvation response signaling pathway, negatively regulating several downstream Pi transporters indirectly. Yet very little is known about its direct interaction partners. To gain further understanding of PHO2function in rice, using OsPHO2as bait, a yeast-two-hybrid (Y2H) screen of rice cDNA library was performed, revealing several interaction proteins of OsPHO2. Among them, GIGANTEA (OsGI) is a key regulator of flowering time. Also, two proteins involved in redox status regulation, OsCP1and OsCP2(CP, Candidate Protein of OsPHO2interaction partners), were identified. Bimolecular Fluorescence Complementation (BiFC) assays were carried out to verify the interaction between OsPHO2and OsGI or OsCP1/OsCP2in planta. The reconstructed YFP signal revealed that the interactions occurred mostly overlapping with the ER marker in rice protoplasts.Characterization of gi and pho2mutants showed that they shared similar phenotypes, such as reduced growth, deleyed leaf emergence, late flowering and over-accumulation of Pi in the leaves compared to wild-type (WT). The expression of flowering gene OsHd3a was dramatically repressed and consistently lower than that of WT over the developmental stage. Furthermore, analysis on Pi concentration in individual leaves demonstrated that gi mutant, similar as pho2mutant, was also impaired in Pi remobilization from old to young leaves, though to a lesser extent. Altogether, the interaction observed between PHO2and GI provides evidence regarding the molecular cross-talk between Pi homeostasis and the regulation of flowering time in rice.The interaction was occured between rice PHO2and CP1/CP2particularly, whilst the AtPHO2did not interact with any homologs of OsCP proteins, as revealed by Y2H assays. Furthermore, the interaction required the N-terminus (aal-633) of OsPHO2. The UBC (E2ubiquitin-conjugating) domain at the C-terminus was not involved.qRT-PCR analysis indicated that the expressions of OsCP1and OsCP2were relatively stable in both leaves and roots of WT rice seedlings, independently of Pi status in nutrient solution.In addition, Pi concentrations in leaves and roots of OsCP1over-expression and RNAi lines were not significant different from that of WT. These results implied that the interaction between OsPHO2and OsCP1was not associated with the Pi signaling pathway.In summary, this study identified three OsPHO2interacting proteins OsGI and OsCP1/OsCP2, suggesting the existence of crosstalk between Pi signaling pathway and other process regulation, such as flowering time regulation and redox-status regulation.