| The symbiotic nitrogen fixation of soybean nodules can provide 50-60%of its nitrogen requirement.Soybean nodulation process requires more P compared with other growth and development stages.Low P stress affects growth and development of nodules,reduces nitrogenase activity,nitrogen fixation and further influnce plant growth,yield and quality.In view of this,low P-tolerant variety Zhonghuang15 and low P-sensitive variety Niumaohuang inoculated with rhizobia were grown in high and low phosphorus respectively,in this study.28 d post inoculation,nitrogen fixation related traits were determined and nodules from the two varieties under different P condition were used for transcriptome sequencing.By analysis of the transcriptome data,GmSPX8 and GmCCD8 were screened as P responsive genes.In this study,the function and mechanism of GmSPX8 and GmCCD8 involving in nodules under low P condition were explored,which will provide functional genes for effective nitrogen fixation in soybean breeding and theoretical basis for efficient use of high nitrogen fixation-related genes in soybean nodule under low P stress.The main results are as follows:1.There were significant differences in plant growth,nodulation and nitrogen fixation-related traits between these two soybean varieties inoculated with rhizobia at 28 d under different phosphorus treatments.Compared with normal P,under low P stress,the plant height,fresh and dry weight of ZH 15 were decreased by 13%,16%and 32%,respectively,root fresh weight and dry weight were decreased by 16.9%and 26.5%,respectively,nodule fresh and dry weight were decreased by 46.6%and 51.6%and nodule number was decreased by 30.1%,total N content of plant and nodule were decreased by 45.2%and 13.8%and P content were decreased by 70.3%and 39.8%,respectively;for NMH,the plant height,fresh and dry weight were decreased by 25.0%,42.2%and 40.9%,respectively,root fresh weight and dry weight were decreased by 28.8%and 28.5%,respectively,nodule fresh and dry weight were decreased by 60.8%and 57.9%,nodule number was decreased by 37.2%,total N content of plant and nodule were decreased by 44.2%and 13.4%and P content were decreased by 72.9%and 50.2%,respectively.2.There were significant differences in gene expression between the two soybean varieties under low P stress.Transcriptome data showed that under low P condition 7723 differentially expressed genes(DEGs)were found in nodules of ZH15 and 1440 DEGs were found in nodules of NMH.737 DEGs were up-regulated in both of the two varieties among which 19 up-regulated DEGs were selected for further qRT-PCR analysis.GmSPX8 from both of the two varieties and GmCCD8 from only ZH15 were screened as candidate genes for further functional verification.3.GmSPX8 could respond to low phosphorus stress and affect nodulation and nitrogen fixation processes.GmSPX8 was mainly expressed in nodules,and its expression increased gradually during nodule growth and development,and it was also induced by low phosphorus stress.Under low P stress,the GUS activity of transgenic nodules driven by GmSPX8 promoter was significantly higher than that of nodules in normal P condition,and the nodule number,fresh weight,nitrogenase activity and P content of GmSPX8 overexpressed transgenic nodules were significantly higher than those of wild type plants.Such as ZH15,the number of nodules,nodule fresh weight,P content and nitrogenase activity were increased by 10.4%,16.5%,35.0%and 37.8%under low P stress,respectively.In GmSPX8 RNAi transgenic nodules,nitrogen fixation-ralated traits were significantly lower than that of the wild type under low P stress.4 GmSPX8 interacted with transcription factor GmPTF1 to enhance its transcriptional activity.The interaction between GmSPX8 and GmPTF1 was verified by Y2H,BiFC and pull-down techniques.RT-PCR analysis showed that the expression level of GmPTF1 did not change during nodule growth and development,however,it was greatly induced by low P stress.Expression of GmPTF1 was significantly up-regulated in GmSPX8 overexpressed nodules suggesting that GmSPX8 is a positive regulator of GmPTF1.The transcriptional activation activity of GmPTF1 was increased in the presence of GmSPX8.5.GmCCD8 was involved in low P stress and nodule nitrogen fixation in soybean nodules.Expression of GmCCD8 under low P stress was higher than that in normal P condition,and the GUS activity driven by the promoter of GmCCD8 gradually increased during nodule development.Compared with the wild type,the expression level of GmCCD8 in nodules of overexpressed composite plants was increased by 2.3 times,the nitrogenase activity and nodule fresh weight were increased by 30.0%and 40.0%,respectively.Transmission electron microscopy showed that the vesicles in the nodules of GmCCD8 RNAi line were damaged,or the edges were irregular and oblong,and the number of bacteroids and accumulation of PHB decreased,indicating that loss of function of GmCCD8 seriously affected the formation and structure of the vesicles in the nodules,and destroyed the symbiotic interaction between rhizobia and soybean.Based on these results,we got conclusions as follows:P deficiency significantly affected the growth and nitrogen fixation of soybean nodules,and plant height,N and P content of shoot and nodules,fresh and dry weight of soybean nodules and plants,number of nodules were all decreased.GmSPX8 was mainly expressed in nodule and was induced by low P stress.Under low P stress condition,GUS activity driven by the promoter of GmSPX8 was higher than that of normal P conditon,and the nodule number,fresh weight,nitrogenase activity and P content were significantly increased in GmSPX8 overexpressed composite plants,while the nodule-related traits in GmSPX8 RNAi lines were significantly decreased.GmSPX8 interacts with transcription factor GmPTF1.Overexpression of GmSPX8 significantly increases the expression and transcription activity of GmPTF1.GmCCD8 was mainly expressed in nodules and induced by low P stress.Nitrogenase activity and nodule fresh weight were significantly increased in GmCCD8 overexpressed transgenic nodules,while nodule-related traits were significantly decreased in GmCCD8 RNAi lines. |
