Comparative Study On The Adaptability Of Two Ecotypes Of Wild Soybean(Glycine Soja Sieb.et Zucc.) Seedlings Under Low Nitrogen Stress

Nitrogen plays a vital role in plant life activities.Insufficient nitrogen will change the morphological structure and physiological metabolism of plants and inhibit the growth and yield of crops.The lack of nitrogen in arable land is a common problem in countries all over the world.In order to ensure that crops obtain sufficient nitrogen,a large amount of nitrogen fertilizer is often used in agricultural production.Excessive nitrogen fertilizer will cause serious pollution to the environment,which is not conducive to the sustainable development of agriculture.Improving the nitrogen use efficiency of crops and rational use of nitrogen fertilizer are considered one of the key research topics in the 21st century internationally.The wild relatives of crops are high-quality resources that improve crop resistance and genetic diversity.Glycine soja is a closely related wild species of cultivated soybean,with many excellent characters and strong stress resistance.Therefore,understanding the low nitrogen tolerance mechanism of different ecotypes of wild soybeans and improving the ability of wild soybeans to resist low-nitrogen stress is important for industry of cultivating,improving and optimizing cultivated soybeans.In this study,two ecotypes of Glycine soja,common wild soybean and poor-soil-tolerant wild soybean were used as experimental materials,and a quarter concentration of nitrogen was used to simulate a low-nitrogen stress environment.Through the determination of biomass,ionomics,transcriptomics and metabolomics,the analysis and comparison of the common wild soybean and poor-soil-tolerant wild soybean seedling leaves and roots at the physiological,ionomics,metabolomics and transcriptomics levels.The difference in response to low nitrogen stress reveals the tolerance mechanism of poor-soil-tolerant wild soybean from the perspectives of plant growth status,nutritional balance,metabolite composition and metabolic processes,and transcriptional regulation.The main conclusions of this study are as follows:（1）Compared with the control group,under low nitrogen stress,the growth of common wild soybean and poor-soil-tolerant wild soybean were inhibited,while the decline degree of common wild soybean was greater than that of poor-soil-tolerant wild soybean,showing poor-soil-tolerant wild soybean has stronger low-nitrogen tolerance.In addition,the root system of the two ecological types of wild soybeans increased,between which the poor-soil-tolerant wild soybean increased more significantly.It was conducive to absorbing more nitrogen,and the total carbon and C/N in the leaves and roots of the poor-soil-tolerant wild soybean seedlings showed a significant upward trend,which could resist low nitrogen stress.（2）Ionomics studies showed that K⁺、Mg²⁺、Ca²⁺and SO₄^2-played important roles in the process of wild soybean seedlings resisting low nitrogen stress.Under low nitrogen stress,the NO3^-content in common wild soybean seedlings leaves and roots decreased much more than that of poor-soil-tolerant wild soybean seedlings;the contents of K⁺and SO4^2-accumulated in the roots of the two ecotypes of wild soybean seedlings,and the accumulation range was greater in the poor-soil-tolerant wild soybean;Mg²⁺and Ca²⁺accumulated in the leaves and roots of poor-soil-tolerant wild soybean seedlings,but there is no such phenomenon in common wild soybean.It showed that the poor-soil-tolerant wild soybean seedlings leaves and roots maintained ion balance while accumulating beneficial ions to resist low-nitrogen stress.（3）Through the transcriptome study of two ecotypes of wild soybean seedlings,it was found that 6344 differentially expressed genes in the leaves of common wild soybean seedlings,of which 3426 were up-regulated and 2918 were down-regulated;there were550 differentially expressed genes in the roots,of which 430 were up-regulated and 120were down-regulated,there were 3614 differentially expressed genes in the leaves of poor-soil-tolerant wild soybean seedlings,of which 1688 were up-regulated and 1926 were down-regulated;there were 381 differentially expressed genes in the root system,of which140 were up-regulated and 241 were down-regulated.The related differential gene research in the entry"Cell response to nitrogen starvation（GO:0006995）"showed that the number of differential genes in poor-soil-tolerant wild soybean leaves was greater than the roots,and genes Glyma.13G361900.Wm82.a2.v1 and Glyma.19G130400.Wm82.a2.v1 were present in the leaves and roots of poor-soil-tolerant wild soybean seedlings,showing that they played an important role in resisting low-nitrogen stress.Weighted gene co-expression network analysis showed that 788 and 35 differentially expressed genes in the leaves and roots of poor-soil-tolerant wild soybean seedlings were closely related to low-nitrogen tolerance.Further cluster analysis of co-expression patterns showed that these genes showed an up-regulation trend.In addition,poor-soil-tolerant wild soybean seedlings resist low-nitrogen stress by regulating transcription factors such as b HLH,AP2/ERF,C3H and MYB.KEGG enrichment showed that the unique differential genes in the leaves and roots of poor-soil-tolerant wild soybean seedlings were jointly enriched in galactose metabolism,showing its importance in low-nitrogen tolerance.（4）Through the study of metabolomes of common wild soybean and poor-soil-tolerant wild soybean,it was found that in the two ecological types of wild soybeans,79 metabolites were significantly changed,including 52 species in leaves and roots respectively,and 14 kinds of amino acids metabolites,36 organic acid metabolites,20 sugar alcohol metabolites and 9 secondary metabolites.Under low nitrogen stress,the glycolysis,sugar alcohol metabolism and fatty acid metabolism in the two ecotypes of wild soybean seedling leaves increased,and the increase was greater in the poor-soil-tolerant wild soybean.In addition,the tricarboxylic acid cycle,shikimic acid pathway,glutamine synthetase/glutamate synthetase（GS/GOGAT）cycle and organic acid metabolism in the leaves of poor-soil-tolerant wild soybean seedlings were enhanced,but weakened in common wild soybean.Poor-soil-tolerant wild soybean seedling roots reduce energy consumption by weakening the overall amino acid metabolism,while promoting the synthesis of proline and secondary metabolites to resist low-nitrogen stress.And improving the metabolism of shikimic acid was also an important low-nitrogen tolerance mechanism of the root system of poor-soil-tolerant wild soybean seedlings.（5）Transcriptomics and low-nitrogenomics correlation analysis found that the differential genes in poor-soil-tolerant soybean leaves were significantly enriched in 7metabolic pathways,and five metabolic pathways including phenylpropane biosynthesis（ko00940）,glyoxylic acid and dicarboxylate metabolism（ko00630）,carbon fixation in photosynthetic organisms（ko00710）,nitrogen metabolism（ko00910）and carbon metabolism（ko01200）are enriched with differential metabolites;Different genes in poor-soil-tolerant wild soybean root were significantly enriched in phenylpropane biosynthesis（ko00940）and amino sugar and nucleotide sugar metabolism（ko00520）,and all of them were enriched in metabolites,showing that these metabolisms play important roles in the resistance of poor-soil-tolerant wild soybeans to resist low-nitrogen stress.This research aims to provide scientific evidence and theoretical basis for the mining and utilization of high-quality wild soybean resources,provide a theoretical basis for cultivating high-quality soybean varieties,and provide important ideas and methods for the promotion of sustainable agricultural development and friendly environment development.