| Nitrogen(N)is very important for plant growth and crop yield.Soil nitrogen content is generally low and unevenly distributed in China,and blind application of nitrogen fertilizer in production will cause environmental pollution.Therefore,in agricultural production,it is necessary to cultivate crop varieties with low nitrogen tolerance and study their molecular mechanisms to reduce dependence on nitrogen fertilizer.Achnatherum inebrians is a gramineous perennial herb widely distributed in the northwest of China,and it usually grows in poor soil.It can form a mutuallybeneficial symbiotic relationship with Epichlo(?) endophytic fungi,which has strong stress resistance and competitiveness,and has evolved a unique barren tolerance mechanism,especially low nitrogen tolerance mechanism.The study on the physiological and molecular mechanism of A.inebrians under low nitrogen conditions can provide important functional gene resources for the selection of resistant varieties by endophytic fungi,and also provide theoretical basis for the development of new varieties with low nitrogen tolerance.In this study,A.inebrians carrying Epichlo(?) gansuensis endophytic fungi(E+)and A.inebrians without E.gansuensis endophytic fungi(E-)were used as materials.The seedlings of A.inebrians were treated by setting different nitrogen concentrations(7.5 m M N and0.01 m M N).The biomass,metal content,amino acid content and nitrogen use efficiency of E+ and E-were analyzed by physiological methods under different nitrogen concentrations.The molecular mechanism of endophytic fungi regulating the response of E+ and E-to low nitrogen was analyzed by metabolomics and transcriptomics.The main research results are as follows:(1)Under low N conditions(0.01 m M N),the endophytic fungi significantly increased the dry weight of leaves and roots of E+ A.inebrians by 30.5% and 25.7%,respectively,compared to E-A.inebrians.Under low N conditions,endophytic fungi regulated the uptake and utilization of N by E+ A.inebrians.Compared to E-A.inebrians,infestation of endophytic fungi significantly increased the Nitrogen Uptake Efficiency(NUp E),and improved the Total N Accumulation(TNA)and enhanced the Nitrogen Utilization Efficiency(NUt E)of E+ A.inebrians,thus improving the growth of A.inebrians under low N conditions.Under low nitrogen conditions,endophytic fungi increased the potassium(K),calcium(Ca)and iron(Fe,P < 0.01)and root potassium,calcium and sodium(Na,P < 0.05),magnesium(Mg)and iron contents of A.inebrians leaves and decreased the leaf and magnesium contents,altering the distribution of metal elements in A.inebrians.The low nitrogen treatment significantly affected the amino acid content of leaves and roots of A.inebrians(Adonis: P = 0.001),while both conditions of endophytic fungal infestation and low nitrogen treatment × endophytic fungal infestation interactions had no significant effect on the amino acid content of leaves and roots of A.inebrians.Under low nitrogen conditions,the endophytic fungus up-regulated the content of 17 amino acids in the leaves of A.inebrians,including proline,tryptophan and tyrosine,while the endophytic fungus only down-regulated the threonine content in the roots of A.inebrians.(2)The results of the metabolome analysis showed that under low nitrogen conditions,the endophytic fungus caused changes in 7(3 down-regulated,4 upregulated)and 13(5 down-regulated,8 up-regulated)differential metabolites in leaves and roots respectively;the results of the KEGG enrichment analysis showed that under low nitrogen conditions,the endophytic fungus had the strongest effect on two metabolic pathways in leaves,namely the TCA cycle and pyruvate metabolic pathway;The four metabolic pathways of roots: pyruvate metabolism,cutin,suberine and wax biosynthesis,butanoate metabolism,alanine,aspartate and glutamate metabolism may be the key pathways regulated by the endophytic fungus under low nitrogen conditions.It is suggested that amino acid and organic acid metabolism pathways are the main pathways through which endophytic fungi affect A.inebrians under low nitrogen conditions.(3)Transcriptome analysis showed that the endophytic fungus affected 1213 differentially expressed genes(DEGs)in leaves(645 up-regulated and 568 downregulated)and 483 DEGs in roots(114 up-regulated and 369 down-regulated)under low nitrogen conditions.Enrichment analysis of the biological processes of GO in leaves and roots affected by endophytic fungi under low nitrogen conditions revealed that these DEGs were mainly enriched in amino acid metabolism,organic acid metabolism,oxidative stress response,phytohormone signalling,ion transport,secondary metabolite synthesis and transcriptional and translational regulation.KEGG pathway enrichment analysis showed that DEGs up-regulated by endophytic fungi were mainly enriched in glycolysis,pentose phosphate,carbon metabolism and phenylpropyl biosynthesis in leaves under low nitrogen conditions.Down-regulated DEGs are mainly concentrated in arginine and proline metabolism,flavonoid biosynthesis,MAPK signal pathway and plant hormone signal transduction.DEGs up-regulated by endophytic fungi are mainly enriched in amino acid metabolism and fatty acid metabolism,while down-regulated differential genes are mainly enriched in plant pathogen interaction.The results of the combined analysis of transcription combination and metabolome showed that endophytic fungi reprogrammed the transcription and metabolic levels of energy metabolism and nitrogen absorption and utilization pathway,which helped the growth of A.inebrians under low nitrogen conditions.In summary,this study has shown that the endophytic fungus reorganized the transcriptional and metabolic pathways of A.inebrians under low nitrogen conditions by regulating nitrogen uptake and utilization,redistribution of metal elements and amino acid content,and improved the growth of A.inebrians under low nitrogen conditions,providing molecular-level evidence for the mechanism of low nitrogen tolerance in A.inebrians,and providing important functional genetic resources for the use of endophytic fungi to select resistant varieties. |
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