Study On The Physiological Mechanism Of Nitrogen Nutrition Regulating Soybean Nodulation And Nitrogen Fixation

Soybean(Glycine max L.)is an important grain and oil crop in China,and it is a valuable resource with rich nutritional value and wide range of uses.With the rapid development of China's economy,the consumption of soybeans has also increased rapidly.Realizing stable and high yield of soybeans has become an important part of China's food security strategy.Soybean and hizobia form nodules through complex interactions,which can fix nitrogen for their own growth.There is a combination of cooperation and contradiction between nitrogen and nodule nitrogen fixation.Excessive fertilizer nitrogen will adversely affect nodule nitrogen fixation,and the mechanism of fertilizer nitrogen affecting soybean nodulation and nitrogen fixation is still unclear.The study was performed at the Northeast Agricultural University Experimental Station from2017 to 2020.The dual-root soybean materials(single nodulation side/both nodulation sides nodules)and upper and lower layered soybean materials were prepared.Single or both sides nitrogen supply experiments,layered nitrogen supply experiments,nitrogen supply concentration change experiments and non-nodulation side nitrogen supply experiments were conducted respectively.The ¹⁵N labeling technology and local nitrogen supply method were used to study the effect of nitrogen on the nodule growth and nitrogen fixation ability of soybeans.The regulatory substances,differential proteins,differential metabolites,and gene expression related to nodule nitrogen fixation were measured to analyze the mechanism of nitrogen regulation of soybean nodulation and nitrogen fixation.The results showed that:(1)In the dual-root soybeans and the upper and lower layered soybeans,the application of fertilizer nitrogen to local roots can promote the growth and nitrogen accumulation of shoots and roots in soybean plants.The effect in the nitrogen supplied part was more obvious than the non-nitrogen supplied part,and the application of fertilizer nitrogen reduced the nitrogen accumulation in root nodules.The ¹⁵N tracer method was used to study the distribution of fertilizer nitrogen in roots and nodules after it was absorbed to the shoots,and the distribution amount was proportional to the concentration of fertilizer nitrogen.The high nitrogen application level significantly reduced the nitrogen fixation rate of nodules,while the accumulation of nitrogen fixation in nodules did not change significantly.The increase in total nitrogen accumulation in soybean plants was mainly due to the increase in fertilizer nitrogen accumulation.(2)In dual-root soybeans and upper and lower layered soybeans,fertilizer nitrogen had systemic regulation on nodule growth and nitrogenase activity,and the regulation could be restored.The nodules number,weight and nitrogenase activity of the whole soybean plants were significantly reduced by applying fertilizer nitrogen in local root parts,and the reduction of roots in the nitrogen supply parts was greater than that in the non-nitrogen parts.After the fertilizer nitrogen was withdrawn,the number,weight and nitrogenase activity of the nodules were restored.In dual-root soybeans with a single side nodulation,fertilizer nitrogen was supplied to the non-nodulation side and non-nitrogen was supplied to the nodulation side.Short-term(V₃-V₄)and long-term(V₃-R₄)nitrogen treatments were performed,there was no change in the nodules number and weight of non-nitrogen supply side on supply fertilizer nitrogen for a short time,and the reduction of SNA resulted in the suppression of ARA.The SNA on the side where fertilizer nitrogen was not supplied for a long time basically recovered the same,and the growth of nodules was inhibited which was the reason for the decline in ARA.(3)Comparing the relationship between ARA(determined by acetylene reduction method),nodules nitrogen fixation rate(¹⁵N calculation),and total nodules nitrogen fixation amount(¹⁵N calculation).It was considered that ARA was reliable to reflect the nodule nitrogen fixation rate,but it could not be used as an indicator to evaluate the nodule nitrogen fixation amount of the whole plant.ARA could only represent the nodules nitrogen fixation ability of soybean at a specific time,and could not represent the nodule nitrogen fixation accumulation of soybean at a growth stage.(4)In dual-root soybeans with both sides nodulation,fertilizer nitrogen also has a systematic regulatory effect on the regulatory substances of nodule nitrogen fixation.Partially supplied fertilizer nitrogen(100 mg/L)to one side root,the contents of daidzein,genistein and abscisic acid decreased in both sides of soybeans,while the contents of auxin,zeatin nucleoside and zeatin increased in both sides of soybeans.The contents of six regulatory substances in the nitrogen supply side were greater than those in the non-nitrogen supply side.The contents of soybean isoflavones and phytohormones were restored when the nitrogen supply was withdrawn.(5)In dual-root soybeans with a single side nodulation,the nodulation side was supplied with fertilizer nitrogen(100 mg/L),while the nodulation side was supplied non-nitrogen,compared with the nodulation treatment on both sides.There were 52 differential proteins in the nodules tissue,112 differential proteins in rhizobia,and 53 differential metabolites in nodules.The main differential proteins and differential metabolites were directly or indirectly involved in starch and sucrose metabolism,organic acid metabolism,sulfur metabolism,nitrogen metabolism,amino acid metabolism,and energy metabolism.(6)In dual-root soybeans with both sides nodulation,one side of roots was supplied with fertilizer nitrogen(100 mg/L),while the other side was supplied non-nitrogen.Compared with the nodulation on both sides of the root,the gene expression levels of nodulation factor recognition(GmNFR1A,GmN1N1a and GmN1N2a),isoflavone synthesis(GmCHS,GmIFS and GmCHR)and auxin response(GmCH3)in the roots at V₁stage were changed.In dual-root soybeans with a single side nodulation,the nodulation side roots were supplied with fertilizer nitrogen(100 mg/L)and the nodulation side roots were supplied non-nitrogen.Compared with no nitrogen treatment on both sides,the gene expression levels of nodulation formation(GmENOD93,GmN36A and GmENOD40),isoflavone synthesis(GmCHS,GmIFS and GmCHR)and nitrogenase activity(nifH,nifD and nifK)in the nodulation side nodules were changed.