Functional Study Of GmSPX6、 GmSPX8 In Soybean

Soybean is an important grain and oil crop,which is the main source of protein and oil for human and other animals.Meanwhile,soybean can symbiosis with rhizobia to fix nitrogen from air through biological nitrogen fixation.Phosphorus is one of the most essential mineralnutrient elements for soybean,which is also one of the main factors affecting soybean yield.There are large areas of acidic soil in the South China and in which low phosphate availabilable is the major problem.This is the major limiting factor for increasing soybean yield in China.Phosphate deficiency affects both biological nitrogen fixation and the yield of soybean.However,the mechanism how phosphate deficiency inhibit nodulation of soybean plant is currently unknown.In this study,we obtained Gmspx6,Gmspx6Gmspx8 mutants by CRISPR-cas9 and studied the function of GmSPX6,GmSPX8 in regulation of soybean phosphate starvation signaling and phsoapate homestasisand their function in the nodulation inhibition when grown in the phosphate deficiency conditions.The results are as follows:(1)Subcellular localization showed that GmSPX6 was located in both nucleus and cytoplasm,and GmSPX8 was mainly located in nucleus.Two GmSPX6 and GmSPX8 mutants were obtained by CRISPR-cas9 and transgenic technology.The mutation of GmSPX6 and GmSPX8 resulted in significantly lower plant height,which is caused by shorter node spacing,but not the node number.Besides,the plant height of Gmspx6 and Gmspx6Gmspx8 was also significantly lower than that of the wild type in the field trial.In addition,q RT-PCR showed that mutants of GmSPX6 and GmSPX8 induced the expression of phosphate starvation signaling genes,such as GmSPX3,GmPT7,GmIPS1 and GmSPX7.Besides,the mutations of GmSPX6 and GmSPX8 also resulted in the accumulation of phosphate in the shoots of soybean.These results suggeated that GmSPX6 and GmSPX8 were suppressors of phosphate starvation signaling in soybean plants.Four PHRs genes were identified with high expression level in nodule,which are GmPHR1,GmPHR8,GmPHR16 and GmPHR31.Through subcellular colocation and protein interaction screening,it was found that GmSPX6 could interacte and change the subcellular location of GmPHR1,GmPHR8,and GmPHR16.GmSPX8 could interacted with GmPHR1,GmPHR8,GmPHR16 and GmPHR3 in the nucleus without changing their subcellular localization.(2)The results also showed that the lateral roots of Gmspx6 and Gmspx6Gmspx8 were also significantly lower that WT plants.This indicated that GmSPX6 and GmSPX8 were also involved in the development of lateral roots.Furthermore,it was found that the stability of GmSPX6 protein was decreased and the protein was easy to degrade under low phosphate condition.Through the grafting experiments with WT,Gmspx6 and Gmspx6Gmspx8,the expression of the phosphate starvation signaling genes GmSPX3,GmPT7 and GmIPS1 were enhanced when the aboveground and underground parts were mutants compared with WT.and the soluble phosphate concentration was significantly increased when the underground parts were mutants compared with WT,indicating that the phosphate starvation signaling of the aboveground and underground parts were mutually regulated.(3)Phosphate deficiency inhibited the nodulation of soybean,and the symbiotic signaling genes GmEROD40 and GmERN1 of soybean and rhizobia were significantly inhibited.Besides,mutation of GmSPX6 inhibited the nodulation of soybean.Further the transcription level of symbiotic signaling genes GmENOD40 and GmERN1 were inhibited in the Gmspx6 mutant.Transcription levels of GmSPX3,GmSPX7 and GmSPX8 genes were induced in the Gmspx6 mutant.Furthermore,GmSPX7,GmSPX8 interacted with GmERN1,GmNFY-A and GmNIN1 in the nucleus.Taken together,we speculated that degradation of GmSPX6 under phosphate starvation condition might involved in the nodulation through inducing the expression of GmSPX7 and GmSPX8.In this study,we investigated the functions of GmSPX6 and GmSPX8 in soybean phosphate starvation signaling,phosphate homestasis and symbiotic signaling between soybean and rhizobia and reveal the potential mechansims.This will pave the way for improving the phosphate efficiency,enhancing symbiotic nitrogen fixation and increasing the yield of soybean.