| The symbiotic nitrogen fixation between soybean and rhizobia provides 23～65%of total nitrogen for plant growth and development.It is of great significance to explore and clarify the functions and mechanisms of nodulation and nitrogen fixation-related genes and finally to create new soybean germplasms and breed of new varieties with high efficiency in nitrogen fixation.In view of this,in this study,via analyzing the proteomes of the nodules at different developmental stages,we screened nodule formation and nitrogen fixation-related genes and analyzed the temporal and spatial expression of these genes by qRT-PCR,promoter GUS staining and in situ hybridization analysis.Overexpression and RNAi technologies were used to study gene functions and transgenic soybean mutants were constructed by CRISPR/Cas9 technology to further clarify the biological functions of genes in nodulation and nitrogen fixation.Interacting proteins were identified and screened by liquid chromatography-tandem mass spectrometry(LC-MS/MS),and further verified by pull-down,BiFC,Co-IP and Y2H methods.In combination with the functional analysis of the interaction proteins,the regulatory mechanisms of genes in nitrogen fixation were analyzed.The main results are as follows:1.Nodule formation and nitrogen fixation related small heat shock proteins GmHSP17.9 and GmHSP17.1 were screened from nodule proteome at different developmental stages.Analysis of the proteome database of soybean nodules of Williams82 at different developmental stages showed that the number of proteins involved in regulating nodule development increased gradually with the development of nodules.Compared with 0 day after inoculation with rhizobia,10 d nodules had 665 differentially expressed proteins,17 d nodules had 1065 differentially expressed proteins and 1279 proteins were differently expressed in the 28 d mature nodules.Among them,the small heat shock proteins GmHSP 17.9 and GmHSP 17.1 were highly expressed in 17 d and 28 d nodules with the highest expression level at 28 day post inoculation with rhizobia.2.The biological function of GmHSP17.9 in nodule formation and nitrogen fixation was clarified by composite transgenic soybean plants.GmHSP17.9 was 878 bp in length and contained one exon without intron on chromosome 4.The CDS sequence of GmHSP 17.9 was 477 bp,encoding 158 amino acids.GmHSP 17.9 was located in cytoplasm and belonged to the CII subfamily.Promoter GUS staining together with qRT-PCR showed that GmHSP17.9 was predominantly expressed in nodules,and the highest expression level was found in mature nodules at 28 d.Section of promoter GUS staining and in situ hybridization showed that GmHSP17.9 was specifically expressed in the infected region of nodules.It was confirmed that overexpression of GmHSP17.9 significantly enhanced nodule formation and nitrogen fixation ability of soybean,while RNAi inhibition of GmHSP1 7.9 greatly reduced nodule formation and nitrogen fixation ability of soybean.3.The key role of GmHSP17.9 in nodulation and nitrogen fixation was further verified in mutant soybean plants.Two mutant lines hsp17.9-1 and hsp17.9-2 were obtained by CRISPR/Cas9 technique.In hsp 17.9-1,one base was inserted in Target 1,five bases were deleted in Target 2,and there was no change in Target 1 of hsp17.9-2,who had 98 bases insertion and 6 base deletion in Target 2,resulting in frameshift and premature termination of translation.The results showed that the growth and development of nodules of hsp17.9 were significantly inhibited,and the nitrogenase activity of nodules was significantly decreased by 29.2%and 37.5%compared with the wild type,respectively.The plant growth and development were also inhibited,and the yield per plant was greatly decreased by 41.4%and 46.0%,respectively.Ultrastructure of hsp17.9 nodules was observed by transmission electron microscope and showed that nodule symbiosis was severely damaged,and the PHB content of bacteroid was significantly reduced.4.It was confirmed that GmHSP17.9 regulated nodule formation and nitrogen fixation of soybean through interacting with sucrose synthase GmNOD100 as molecular chaperone.MDH thermal aggregation and DTT-induced insulin aggregation assaysin vitro confirmed that GmHSP17.9 had molecular chaperone activity.Native-PAGE and Arabidopsis protoplast BiFC confirmed that GmHSP17.9 could form polymers.Pull-down combined with LC-MS/MS showed that sucrose synthase GmNOD100 was a potential interaction protein of GmHSP17.9,and its interaction was further confirmed by Y2H,BiFC,CoIP and pull-down assay.Further analysis showed that GmNOD100 was highly expressed in nodules,and the expression level was highest at 28 d,and the expression site was mainly detected in the infected area of nodules.Subcellular localization revealed that GmNOD100 was a cytoplasmic protein.Overexpression of GmNOD100 significantly increased nodulation and nitrogen fixation ability,with increased sucrose synthase activity,UDP-glucose content,acetyl-CoA content and SDH activity,while inhibition of GmNOD100 expression showed the opposite phenotype with overexpression.In vitro experiments confirmed that GmHSP17.9 protected GmNOD100 sucrose synthase activity as molecular chaperone,but had no significant effect on its expression level.In addition,sucrose synthase activity was significantly decreased by 14.3%and 20.9%in hsp17.9,with decreased UDP-glucose and acetyl-coA contents and SDH activity.5.The biological function of GmHSP17.1 in nodule formation and nitrogen fixation was confirmed by transgenic composite soybean plants.GmHSP17.1.located on chromosome 6.contained an exon without intron and encoded a small cytoplasmic heat shock protein composed of 150 amino acids.qRT-PCR and promoter GUS staining analysis showed that GmHSP17.1 was predominantly expressed in nodules with highest expression level in mature nodules at 28 d.GmHSP17.1 transgenic composite soybean plants showed that overexpression of GmHSP17.1 significantly enhanced nodule formation and nitrogen fixation ability,improved plant growth and increased nitrogen content;suppression of GmHSP 1 7.1 significantly inhibited the nodulation and nitrogen fixation ability of plants,and the plant nitrogen content was also significantly reduced.6.GmHSP 17.1 regulated nodule formation and nitrogen fixation of soybean by interacting with peroxidase GmRIP1 as molecular chaperone.MDH thermal aggregation and DTT-induced insulin aggregation assaysin vitro confirmed that GmHSP 17.1 had molecular chaperone activity.Pull-down combined with LC-MS/MS showed that GmRIP1 was a potential interaction protein of GmHSP 17.1,and their interaction was further confirmed by Y2H,BiFC and pull-down assays.GmRIP1 was a cytoplasmic protein and highly expressed in nodules,and its expression gradually increased with the development of root nodules with the expression level peakedat 28 d.The POD activity was consistent with the expression level of GmRIP1 in nodules.Overexpression of GmHSP 17.1 increased POD activity by 23.2%and decreased ROS content by 33.1%,while POD activity of RNAi lines decreased by 46.3%and ROS content increased by 17.1%.In conclusion,in this study through analysis of proteoms of nodules,two small heat shock proteins GmHSP 17.9 and GmHSP 17.1 highly expressed in soybean nodules were screened.The essential role of GmHSP 17.9 and GmHSP 17.1 in nodule formation and nitrogen fixation were confirmed in transgenic soybean plants.GmHSP 17.9 regulated sucrose synthase activity of GmNOD 100 and energy supply during nodule formation and development,thus controlling nodule formation and nitrogen fixation.GmHSP 17.1,as the molecular chaperone of peroxidase GmRIP1,affected POD activity and ROS content in root nodules,thus regulating nodule formation and nitrogen fixation in soybean. |
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