Genetic And Molecular Mechanisms Of GmINS1 In Regulating Nodule Development In Soybean

Nitrogen（N）is an essential macronutrient for plant growth and development.Legumes can form a new nitrogen（N₂）-fixing organ-nodules,by establishing symbiosis with rhizobia,which can fix N₂ and then provide available N source to plants.Symbiotic N₂fixation is a quantitative trait controlled by multiple genes,and the nodulation including rhizobia infection,nodule primordia initiation and nodule development,are strictly regulated by both rhizobia and hosts.However,the specific genetic and molecular mechanisms are still not very clear.The expansion of nodule primordia is critical for nodule formation and N₂ fixation,but the research progresses in regulating nodule development are very slow and the main genetic and molecular regulatory mechanisms have not yet been elucidated.In this study,the controlling nodule size related QTL loci was identified through forward genetics technique in soybean（Glycine max）.The candidate gene,INCREASING NODULE SIZE1（GmINS1）for the QTL loci was cloned and functionally analyzed.The main results were shown as follows:（1）In this study,we identified a QTL,qBNF-11,for nodule size by using 175 F_9:11RILs（Recombinant inbred lines）which derived from a P1×P2 cross and have been established genetic map in soybean.Through the prediction and screening of the candidate genes,we cloned the major gene controlling the nodule size in qBNF-11,which was identified as INCREASING NODULE SIZE1（GmINS1）.（2）The results from bioinformatics analysis showed that GmINS1 belongs to the cell wallβ-expansins family and encodes cell wall expansin.This family has 9 members in total.Previously,GmEXPB2 was found involvement in regulation of soybean nodulation,and GmINS1 and GmEXPB2 exhibited 78%amino acid sequence similarity and were classified into the same subgroup through phylogenetic analysis.（3）Qualitative PCR detection revealed that four single-nucleotide polymorphisms（SNPs）distinguishing the two parents were found in the GmINS1 promoter region.Among them,harboring SNP A/C had significant effects on GmINS1 expression in the parental genotype P2,based onβ-glucuronidase activity and promoter deletion analysis.The expression of GmINS1 contain SNP C from P2 genotype were strongly associated with nodule development,not only in the parents and in the 40 progeny lines but also in the 40genotypes selected from a soybean core collection,suggesting that the expression of GmINS1 is closely associated with nodule development.（4）Histochemical localization results showed that GmINS1 expression started upon the initiation of nodule primordia,which was followed by the expression in nodule vascular bundle（NVB）tissues during early nodule development and then extended to the nodule cortex and parenchymatous cell（PA）in elder nodules.The results suggested that GmINS1may be mainly involved in soybean nodule development.（5）Results from GmINS1 involved in nodule development showed that overexpression of GmINS1 resulted in increases in the number,biomass,infection cell abundance,and nitrogenase activity of large nodules and subsequently changed the N content and biomass in soybean plants.Suppression of GmINS1 via RNA interference resulted in a significant reduction in the number and weight of large nodules,the number and size of infected cells,and a significant decrease in nodule nitrogenase activity.（6）Expansins play an important role in the nodulation of legumes and are essential for the determinate nodule development which depend largely on cell expansion and cell wall extension.The results from functional analysis showed that double suppression of GmEXPB2 and GmINS1 dramatically inhibited soybean nodulation,the nodulation parameters including the large nodules number and weight,single nodule size,infection cells development and nodule nitrogenase activity were significantly inhibited.In summary,we identified and cloned a candidate gene for controlling nodule size,GmINS1,by map-based cloning.Our study demonstrated that GmISN1 is a critical gene encoding a cell wallβ-expansin,and plays important roles in regulating nodule development.GmINS1 and GmEXPB2 synergistically control soybean nodulation.Our findings provide a theoretical basis and a candidate gene for optimizing BNF capacity through molecular breeding in soybean.