Regulation Of Phosphorus Levels On Nitrogen Fixation And Related Metabolic Pathways In Soybean Nodule

Soybean is an important grain and oil crop in China.It not only has rich nutritional value,but also has a wide range of uses.The demand for soybeans continues to increase with the rapid development of China’s economy.Increasing soybean yield has become the main way to solve the contradiction between soybean supply and demand.Phosphorus is an important element that affects soybean growth and nitrogen fixation.Rational application of phosphate fertilizer can regulate the growth and development of soybean nodules,enhance the nitrogen fixation ability of root nodules,to increase soybean yield.However,the effect mechanism of phosphorus level on nitrogen fixation and phosphorus uptake and transport in soybean root nodules is still unclear.This experiment was carried out in the experimental base of Northeast Agricultural University from 2018 to 2021.Experiments were performed using normal growing single-root soybean material and grafted double-root soybean material（single nodulation side/both nodulation side）.The effects of phosphorus level on fertilizer nitrogen uptake and nodule nitrogen fixation of single root soybeans by ¹⁵N labeling technology were investigated.Local phosphorus supply experiment on dual-rootsoybeans with bothsides nodulation and singleside nodulation.Experiment on changes of phosphorus supply levels in dual-root soybeans with both sides.Analysis of the effect of phosphorus on soybean nodule growth and nitrogen fixation.Determination of the changes of regulatory substances and gene expression related to nodule nitrogen fixation and screen the differential genes,proteins and metabolites of soybean roots in response to low phosphorus stress.To systematically study the regulatory effects of phosphorus levels on soybean nodule nitrogen fixation and related metabolism,and provide reference for analyzing the mechanism of phosphorus regulation of soybean nodulation nitrogen fixation.The results showed that:（1）In dual-root soybeans with both sides nodulation,under the same phosphorus supply level,both sides phosphorus supply has a greater promoting effect on dry matter accumulation and phosphorus absorption than one side phosphorus supply.Bilateral supply of 15.5 mg/L phosphorus had the most obvious promoting effect on dry matter accumulation,and bilateral supply of 31 mg/L phosphorus significantly promoted phosphorus absorption.Phosphorus can be transported inside the plant and preferentially supplynodules to maintain its function.Supply lowconcentration phosphorus to one side of dual-root soybeans with both sides nodulation to inhibit the accumulation of dry matter and phosphorus in roots,root nodules and shoots on both sides,and the phosphorus content of the nodules in phosphorus-free side was higher than that of the roots.The change from lowconcentration phosphorus supply to highconcentration phosphorus supply promotes both root dry weight and phosphorus accumulation,and then changes to low concentration phosphorus supply and inhibits it.Then it becomes a low-concentration phosphorus supply and has an inhibitory effect.（2）Phosphorus system regulates the relative expression of isoflavones and hormones and their related genes in soybean roots.Supply low concentration phosphorus to one side of dual-root soybeans with bothsides nodulatedmainly affects the accumulation of bilateral root auxin（IAA）,cytokinin（CTK）,gibberellin（GA₃）and abscisic acid（ABA）.And it also down regulated the relative expressions of genes involved in nodulation（Gm ENOD93 and Gm N-36A）,isoflavone synthesis（Gm CHI and Gm CHS）,auxin regulation（Gm CH3）and root hair curling（Gm RJI）in roots of both sides.（3）Phosphorus system regulates soybean nodule growth,nodule nitrogenase activity and relative expression of related genes.Supply lowconcentration phosphorus to one side of dual-root soybeans with bothsides nodulated,inhibit the accumulation of nodule dry weight,nodule number,ARA,SNA and ureide accumulationin soybean on the phosphorus supply side,reduce the number of bacteroids,infected cells,downregulated relative expression of nodule cell wall synthesis（Gm EXPB2）,nodule nitrogenase activity genes（nif H,nif D,nif K）,and ureide synthesis genes（Gm ALN1,Gm UR5,Gm HIUH5,Gm ACP1,Gm PUR5）,The phosphorus-free side is also suppressed,but the degree of inhibition is less than that of phosphorus-application side.Increased phosphorus supply restored nodule dry weight,nodule number,and nitrogenase activity.Supply low concentration phosphorus to non-nodulated side of dual-root soybeans with single nodulation side,Inhibits the nodule dry weight,nodule number,ARA,SNA,and ureide accumulation,reduces the number of nodules,the number of infected cells,and down-regulates nodules Gm EXPB2,nif H,nif D,nif K,Gm ALN1,Gm UR5,Gm HIUH5,Gm ACP1,Gm PUR5relative gene expressionin nodular lateral roots of soybean.（4）The appropriate phosphorus level can promote soybean nitrogen absorption and root nitrogen fixation,the optimum level of phosphorus supply level of 31 mg/L-41 mg/L to promote nitrogen absorption of soybean fertilizer,Different growth stages of root nodule nitrogen fixation have different requirements for phosphorus supply level,the optimum concentration of phosphorus for nitrogen fixation in soybean root nodules was 31 mg/L-41 mg/L at the initial flowering stage and the seed drum stage.（5）Low phosphorus stress inhibits nonstructural carbohydrate accumulation in soybean plants.Low-concentration phosphorus supply reduces sucrose,soluble sugar,starch content,sucrose synthase and starch synthase activities in various parts of soybean plants,down-regulates sucrose synthesis and transporter-related genes（Gm STP1,Gm MST1,Gm SUT1）in root nodules,and particle binding Relative expression levels of starch synthase gene（Gm GBSS）and sucrose synthase gene（Gm GBSS）.（6）In dual-root soybeans with a single side nodulation,the nodulated lateral roots do not supply phosphorus,and the non-nodulated lateral roots supply phosphorus.Compared with the normal concentration of phosphorus supply,the nodulated lateral roots supply phosphorus through genes,proteins,The metabolites responded to low phosphorus stress together,including 4410differential genes,211 differential proteins and 47 differential metabolites."Fructose and mannose metabolism pathway","phosphoinositide metabolism"and other metabolic pathways.