Study On The Pathways Of Soybean Assimilation Of Nitrate Nitrogen And Regulating Of Nodule Nitrogen Fixation

Soybean is an important grain and oil crop in China,which is rich in nutritional value and widely used.In recent years,Chinese soybean imports continue to increase,the import volume was more than 100 million tons in 2020,the self-sufficiency rate is seriously inadequate,improving the yield of soybean is one of the core content to ensure food security in China.The symbiotic interaction between soybean and rhizobium can fix nitrogen in the air to provide nitrogen for soybean growth,but soybean still need to apply nitrogen to achieve high yield.Most of the nitrogen fertilizer added to the soil are taken up by soybean as nitrate nitrogen,but higher concentrations of nitrate nitrogen reduce the nitrogen fixation capacity of nodules.The mechanisms of nitrate nitrogen transport and assimilated pathways within the soybean plant and inhibition of nodulation and nitrogen fixation remain unclear.The experiments were conducted in Northeast Agricultural University from 2017 to 2021.The experiments were prepared on dual-root soybean with both sides nodulated and single side nodulated.Four experiments were conducted separately,including supplying nitrate nitrogen to single side or both sides,changing nitrate nitrogen concentrations on the nitrogen supply side,supply of nitrate nitrogen on the non-nodulated side after girdling of the root base phloem⁺(including epidermis,cortex and phloem)on the nodulated side,and 95%shading of the whole soybean.Using ¹⁵N and¹³C labeling techniques,we studied local nitrate nitrogen supplied and then the transport pathways of nitrate nitrogen and its assimilations in soybean,nitrate nitrogen on the distribution of carbon in soybean,and the nitrogen fixation ability of nodules.We also observed the nodule microstructures,submicroscopic structures,determination of relative expression of genes related to nitrate nitrogen transport and carbohydrate synthesis.The results showed that:(1)In dual-root soybean when one side of the roots supplied ¹⁵N-labeled nitrate nitrogen,¹⁵N abundance was detected in shoots,nodules,roots,and xylem⁺(including xylem and pith)and phloem⁺of both root bases;changing nitrate nitrogen concentration on nitrogen supplied side,the concentrations of nitrate nitrogen in shoots,roots,nodules,xylem⁺and phloem⁺of both root bases were changed.The results indicated that nitrate nitrogen absorbed by roots can be transported directly in the soybean plant and down through shoots to the other side of the roots and nodules,with both xylem⁺and phloem⁺being the main channels.(2)Nitrate nitrogen was supplied to one side of dual-root soybean with both sides nodulated,and nitrate reductase and glutamine synthetase activities were measured in the root tip,the glutamine synthetase activity in the root tip of both sides and the asparagine content in roots,nodules,root basal xylem⁺and phloem⁺all varied with the concentration of supplied nitrate nitrogen.The results indicated that nitrate nitrogen absorbed by roots can be assimilated through the GS-GOGAT(glutamine synthetase/glutamate synthetase)pathway and eventually transported within the soybean plant in the form of asparagine.(3)Nitrate nitrogen was supplied to one side of dual-root soybean with both sides nodulated,nodule nitrogenase activity,nodule number,and dry matter weight on the nitrogen supplied side was decreased,and the same pattern of changes on the nitrogen-free supplied side was observed.The results indicated that the systematic regulation of nodulation and nitrogen fixation by nitrate nitrogen in soybean.In dual-root soybean with single side nodulated,gridling the nodular side of root base phloem⁺and whole soybean shading resulted in reduced nodule nitrogenase activity,nodule number and dry matter weight of nodules on the nodulated side,indicating that blocked photosynthetic product synthesis and transport affected nodulation and nitrogen fixation in soybean.(4)In dual-root soybean with single side nodulated,nitrate nitrogen supply to the non-nodulated side reduced the distribution of plant assimilated ¹³C to roots and nodules on nitrogen-free side,but the distribution of assimilated ¹³C to roots and nodules of nitrogen-free side was not significantly affected by the nitrogen concentration supplied to the non-nodulated side.The results indicated that distribution of photosynthetic carbon assimilates was N-tending.(5)In dual-root soybean with single side nodulated,nitrogen suppled on the non-nodulated side reduced poly-?-hydroxybutyrate(PHB),which provided carbon source for rhizobium of nodules on nodulated side.The nucleus of the infected cells in the rhizobium disappeared after the root base phloem⁺girdling on the nodulated side,and the shading treatment performed similarly to this result.The results indicated that the supply of nitrate nitrogen reduced the nodule carbon source,leading to changes in the internal structure and affecting the nodule's nitrogen fixation ability.(6)In dual-root soybean with single side nodulated,nitrate nitrogen supplied on the non-nodulated side or root basal phloem⁺gridling on the nodulated side reduced starch concentration in nodules;in the shade treatment,both sucrose and starch concentrations in nodules on nodulated side were reduced.The results indicated that the inhibition of nodules nitrogen fixation under conditions of supplying nitrate nitrogen or obstruction of photosynthetic product synthesis and transport was associated with the reduction of sucrose and starch in nodules.(7)In dual-root soybean with single side nodulated,nitrate nitrogen supplied on the non-nodulated side and nitrate nitrogen supplied on the non-nodulated side after root basal phloem⁺girdling on the nodulated side both resulted in altered relative expression of genes assimilated with nitrate-transport(GmNRT1-2,GmNRT2,and GmNIR)in leaf and nodules on nodulated side,and carbohydrate synthesis transport-related genes(GmSS,GmSS,GmGBSS1,GmSTP1,GmSUT1)in both sides roots and nodules on nodulated side.Shade treatments of whole soybean plant resulted in altered relative expression of carbohydrate synthesis related genes(GmSS and GmGBSS1)in both sides roots and nodules on nodulated side.The above research results aim to clarify that nitrate nitrogen can be transported in both directions in soybean plants,and part of nitrate nitrogen was assimilated into asparagine in the roots through the GS-GOGAT pathway and was transported in soybeans.The supply of nitrate nitrogen hinders the transportation of carbon sources to soybean nodules,resulting in changes in the nodule structure and reduction of PHB,which was the energy substance of nodule nitrogen fixation,thereby reducing the nodule's nitrogen-fixing ability.