Function Of OsSPX3and OsSPX5on Pi-signaling And Pi-homeostasis In Rice

SPX domain-containing proteins have been reported to play important roles in phosphate-(Pi) signaling and Pi-homeostasis in yeast and plants. In rice, six SPX domain proteins (the proteins exclusively harboring the SPX domain) were identified. Phylogenetic relationships and structural characteristics of the Arabidopsis and rice SPX domain-containing proteins indicate that the SPX domain proteins can be classified into three groups. The first group contains two orthologs genes SPX1and SPX2, the second group contains SPX4, while the third group contains SPX3in Arabidopsis (AtSPX3) and three paralogs genes in rice, named as SPX3, SPX5and SPX6. Phylogenetic analysis using different creal crops and dicot plants indicates that the three paralogs genes are evolved in cereal crops. Further, we investigate whether the two paralog SPX proteins, SPX3and SPX5function on Pi-signaling and Pi-homeostasis.Subcellular localization showed that SPX3and SPX5were localized in nucleus and cytoplasm. qRT-PCR analysis indicates that both of SPX3and SPX5is specifically up-regulated at transcription level by Pi-starvation. To investigate whether the proteins of them are positively response to Pi-starvation, we developed the transgenic plants harboring fused gene of SPX3p-SPX3-GFP (Green fluorescent protein) or SPX5p-SPX5-GFP. The Western blotting analysis using GFP antibody showed that the levels of both proteins are increased under Pi-starvation.To examine the function of the SPX proteins, we isolated spx3mutant from rice T-DNA insertional library (CIRAD) and developed transgenic plants with knockdown SPX5using RNA interfering (RNAi). No significant effect on Pi-signaling and Pi-homeostasis in the single mutant spx3and the transgenic plants with repressed SPX5(SPX5-Ri) were found compared to the wild type plants, while significant effects were observed in the plant with spx3mutation under repressed SPX5background (designated as spx3/SPX5-Ri). The results indicate that SPX3and SPX5functions redundantly. To further determine the function of SPX3and SPX5, transgenic plants with overexpressed of the two genes, respectively, were developed under control of35S promoter of cauliflower mosaic virus (CaMV). It was found that overexpression of SPX3and SPX5similarly reduces root-to-shoot Pi translocation, reduces expressions of Pi-starvation induced (PSI) genes, and retards plant growth under both Pi-supplied and Pi-deficient conditions. Taken together, these results confirm the redundant function of the two SPX proteins in Pi-signaling and Pi-homeostasis.Similar tissue expression pattern of SPX3and SPX5genes and their protein levels in leaves and roots were indicated by in situ hybridization and the GUS reporter in the transgenic paints with SPX3-GUS or SPX5-GUS fused gene under control of their promoters, respectively. In vitro and in vivo assays using yeast two-hybrid (YTH), co-immunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC) revealed that SPX3and SPX5can form homodimer or heterodimer. The similar tissue expression pattern and the physical interaction of them support their redundant function.Genetic interaction between SPX3or SPX5and PHR2, the central regulator for Pi-signaling and Pi-homeostasis in rice, were analyzed. The plants with simultaneously overexpressions of SPX3or SPX5and PHR2showed similar shoot Pi concentration and PSI gene expressions with the wild type plants, indicating the excessive shoot Pi accumulation and up-regulated PSI genes driven by overexpressed PHR2can be counteracted by overexpression of SPX3or SPX5. By contrast, only partial rescue of shoot Pi accumulation in pho2mutant was observed by overexpression of SPX3or SPX5, indicating that SPX3and SPX5mainly functions as a repressor of PHR2. The physical interaction of SPX3and SPX5with PHR2was indicated by in vitro and in vivo evidence from yeast two-hybrid and co-IP assays.The present data provide the evidence that the central Pi-signaling pathway and the regulated Pi-homeostasis in plants is under a feedback under control of PHR2(the rice homolog of AtPHRl in Arabidopsis) and SPX proteins. Our finding in this study provides further research directions to understand the comprehensive Pi-signaling network for plants’ adaptation to environmental Pi stress, and increases our knowledge to generate plants with efficient Pi utilization.