Functional Study On Phosphate Starvaion-induced Genes GmSPX And GmWRKY75 In Soybean(Glycine Max(L.)Merr.)

Among all of the important plant elements,phosphorus is an essential mineral.It involves in plant biosynthesis,signal transduction,energy transferring and other important metabolic processes.And phosphorus is a key component on regulation of many enzymatic reactions in signal transduction processes.However,very little of Phosphate(Pi)is present in ionic forms that are available to soybean,because most of it that remained in the soil is converted to organic compounds by microorganisms or becomes insoluble by interacting with cations.Therefore,the utilization of phosphorus has become an important factor limiting the high yield of soybean.In this study,we analyzed the molecular functions of GmSPX and Gm WRKY in phosphate signaling network,which selected from two aspects of signal transduction and transcriptional regulation in soybean.The proteins only containing the SPX domain are key players controlling a set of processes involved in the maintenance of an internal steady state of phosphate ions at the level of the cell,defined as Pi homeostasis.In Arabidopsis thaliana,there are four members in the SPX family,in which AtSPX1-AtSPX3 plays a negative role in the response of P starvation.Six proteins containing SPX have been reported in rice,which is similar to the regulation of SPX protein in Arabidopsis thaliana.In the legume,three SPX proteins have been reported in Phaseolus vulgaris.In addition,nine SPX proteins have been reported in soybean,in which PvSPX1 and GmSPX3 play positive regulatory role in PI signaling pathway.The WRKY transcriptional factors(TFs)are a large family of regulatory proteins in plants.There are 74 genes in Arabidopsis,109 genes in rice,and more than 64 genes in soybean.And the more and more evidences showed that WRKY was involved in the feedback regulation of biotic and abiotic stress at transcription level in plants.The main results of our study were shown in follows:1.Based on the sequence homology to Arabidopsis SPX,10 GmSPX genes were identified from the soybean genome(NCBI,http://www.ncbi.nlm.nih.gov and Phytozome 9.1,http://www.phytozome.net),and we named them as GmSPX1?GmSPX10.Bioinformatics analysis demonstrated that the GmSPX family could be classified into three groups.Genetic structure analysis revealed that each gene of GmSPX1?GmSPX10 contained three highly conserved domains.Fluorescence quantitative PCR analysis found all GmSPX1?GmSPX10 expression level in root and leaf of soybean would be induced by phosphorus starvation stress,however,the expression also declined rapidly by phosphorus supplying after a day recover,indicating that the expression level of was very sensitive to the changes in phosphorus concentration.2.GmSPXl was cloned from soybean.The fusion expression vector pJIT166-GFP-GmSPXl was constructed,and transformated into the onion epidermal cells and Arabidopsis protoplasts cell,respectively.And the results showed that GmSPX1 was located in all areas.GmSPXl had the highest homology with AtSPX3,therefore,we chose a T-DNA insertion Arabidopsis mutant of AtSPX3 for complementation.35S::GmSPXl was introduced into wild-type Arabidopsis plants and spx3 to get T3 generation transgenic seedlings,named OXSPX1 and spx3/OXSPX1.the expression levels of these phosphate starvation genes was inhibited in OXSPX1 and spx3/OXSPX1 compared with WT,only under Pi-sufficient condition.These implied that GmSPX1 might negatively control the transcription of Pi starvation responsive genes indirectly.However,there were no differences between expression levels of these PSI genes in spx3 and those in WT under-Pi conditions.These facts implied that the negative regulation of GmSPXl on PSI genes was depending on Pi concentration.3.The total P concentration was significantly lower in overexpression seedlings(OXSPX1 and spx3/OXSPX1)compared with the WT under both Pi-sufficient and Pi-deficient conditions.Using H2DCF-DA fluorescence staining,ROS levels in the root tips of the transgenic plants were detected.The results showed that ROS levels in the root tips of the overexpression GmSPX1 plants(OXSPX1 and spx3/OXSPX)were found notably higher than which in WT.It's implying that GmSPX1 play an important role in mediating ROS scavenging to enhance plant tolerance.The RSA of the WT,spx3,OXSPX1 and spx3/OXSPX1 was then exa mined.The results showed a significant reduction was found in root hair elongation and the number of root hairs in both OXSPX1 and spx3/OXSPX1.The expression levels of AtUBP14,AtPIP5K-1 and AtPIP5K-3 in roots were exa mined under Pi-deficient conditions.In OXSPX1 and spx3/OXSPX1,the transcript levels of all three genes were obviously suppressed compared with WT and spx3 plants.These results suggested that GmSPXl plays an important role in root development by negatively regulating root hair growth through a process that might suppress AtUBP14,AtPIP5K-1 and AtPIP5K-3 transcription.4.We screened a soybean root cDNA library using the full-length GmSPX1 protein by yeast two-hybrid(Y2H)assay,and found that GmSPX1 could interact with GmMYB48,which is a R2R3-MYB transcription factor,in both Y2H and BiFC assays.The qRT-PCR data showed that GmMYB48 was induced by Pi-starvation;therefore,it might be involved in the regulation of Pi homeostasis.To investigate the effect of GmSPX1 and GmMYB48 together,we chose AtMYB4,an ortholog of GmMYB48,for an experiment in Arabidopsis.In OXSPX1,the expression level of AtMYB4 decreased significantly under Pi-sufficient and Pi-deficient conditions.In contrast,the relative expression level of AtMYB4 increased significantly in spx3 under Pi-deficient conditions.This suggested that GmSPXI might function as a negative regulator of AtMYB4.5.In the study of GmWRKY75 gene function,the total P concentration was significantly higher in overexpression GmWRKY75 seedlings(OXWRKY75)compared with the WT under both Pi-sufficient and Pi-deficient conditions,while the total P concentration was significantly lower in wrky75 seedlings compared with the WT.Through the observation about root structure system,we found the development of lateral roots and root hairs are significantly suppressed in OXWRKY75,compared with the wild-type Arabidopsis.We screened a soybean root cDNA library using the full-length GmWRKY75 protein by yeast two-hybrid(Y2H)assay,and found that GmWRKY75 could interact with GmPAP22-1,which is an acid phosphatase,in both Y2H and BiFC assays.Consistent with the characteristics of these secreted proteins,subcellular detection results showed GmPAP22-1 were located on the cell membrane.Ther are two homologous genes of GmPAP22-1 in soybean,which are named as GmPAP22-2 and GmPAP22-3.We have divided these three genes to GmPAP22 subfamily.Through Quantitative PCR we found that GmPAP22s were induced by Pi-starvation.In order to investigate if GmWRKY75 could affect the expression of GmPAP22-1,we chose AtPAP22,an ortholog of of GmPAP22-1,in Arabidopsis to analyze the relative expression levels,by perfor ming BLAST analysis in Arabidopsis database Tair.Interestingly,the expression of AtPAP22 significantly reduced in roots of OXWRKY75 plant,while the expression of AtPAP22 significantly increased in roots of OXWRKY75 plant.