The Biological Functions And The Mechanism Of Alleviating Ammonium Toxicity Of PagGS1;2 And PagNADH-GOGAT2 In Populus Alba×P.glandulosa

Nitrogen is one of the macronutrients required by plants to maintain normal life activities and one of the main factors limiting plant growth and crop yield.95%of inorganic nitrogen is assimilated into organic nitrogen by glutamine synthase（GS）and glutamate synthase（GOGAT）cycle（GS/GOGAT cycle）,so GS/GOGAT cycle plays a role in nitrogen metabolism.It plays an important role in plant growth,development and stress resistance,and is also an important link in determining plant nitrogen use efficiency（NUE）.Excessive ammonium content in the soil can lead to plant ammonium toxicity and seriously affect the growth and development of plants.At present,there is a few studies on the mechanism of GS/GOGAT cycle in enhancing NUE and alleviating ammonium toxicity in woody plants.In this study,taking 84K poplar（Populus alba×P.glandulosa）as experimental material,we measured the growth and physiological indexes of carbon and nitrogen metabolism of Pag GS1;2 and Pag NADH-GOGAT2 transgenic lines.In addition,transcriptomics and metabolomics were used to determine the biological functions of Pag GS1;2 and Pag NADH-GOGAT2 in 84K poplar and the mechanisms of Pag GS1;2 for alleviating ammonium toxicity.The results are as followes:（1）Analysis of tissue-specific expression and expression pattern in response to nitrogen showed that Pag GS1;2 exhibited the highest expression level in roots and was significantly induced by NH₄NO₃.We cloned Pag GS1;2 coding sequence from 84K poplar,then constructed vectors and carried out the stable genetic transformation of 84K poplar.Transgenic studies showed that the plant height,fresh weight,dry weight and NUE of the Pag GS1;2-OE lines were significantly higher than that in wild type（WT）under high and low concentration nitrogen conditions,while the growth indexes and NUE in roots of the Pag GS1;2-RNAi lines were lower than that in WT.（2）Compared with WT,under the low nitrogen condition,the GS enzyme activity,net photosynthetic rate,stomatal conductance and other photosynthetic parameters and carbohydrate content of the Pag GS1;2-OE lines increased significantly;under the high nitrogen condition,the Pag GS1;2-OE lines nitrogen assimilation products,photosynthesis intensity and carbohydrate accumulation were significantly increased,while the nitrogen assimilation capacity and carbon fixation capacity of Pag GS1;2-RNAi lines were significantly decreased.（3）The determination of the content of lignin and cellulose in the stem of Pag GS1;2transgenic plants showed that under low nitrogen conditions,the content of cellulose in the stems of Pag GS1;2-OE lines was significantly higher than WT.Under high nitrogen conditions,the cellulose content of Pag GS1;2-OE lines was not significantly different from WT,but was significantly higher than Pag GS1;2-RNAi lines.（4）High ammonium would inhibit the growth of 84K poplar,and at the same time the leaves would appear chlorosis and other toxicity phenomenons.The results of real-time quantitative PCR showed that high ammonium could induce Pag GS1;2 gene expression in 84k poplar roots.Transgenic studies showed that the leaves of Pag GS1;2-OE lines were less chlorotic and the dry weight of shoots and roots were significantly higher than WT at 21 d of high ammonium treatment,showing higher tolerance to high ammonium;while the leaves of Pag GS1;2-RNAi lines showed yellow spots and wilting under high ammonium treatment,showing the sensitivity to high ammonium.Overexpression of Pag GS1;2 significantly increased SOD and POD activities and decreased MDA content in roots under ammonium stress.Compared with WT,under high ammonium stress,the activities of GS and GOGAT,the content of free amino acids,soluble protein,total nitrogen and chlorophyll in the Pag GS1;2-OE line were significantly increased,and the content of NH₄⁺was significantly decreased,the oxidative capacity and ammonium assimilation capacity of Pag GS1;2-RNAi lines were reduced.The total carbon content and carbon-nitrogen ratio（C/N）of the roots and shoots of Pag GS1;2-OE lines under high ammonium treatment were significantly higher than WT.（5）Transcriptomic studies showed that under normal conditions,3 914 and 973differentially expressed genes were identified in the roots and leaves of the Pag GS1;2-OE lines,respectively;After high ammonium stress,the number of differentially expressed genes increased significantly,and the number of differentially expressed genes in roots and leaves increased to 7 020 and 1 310,respectively.These DEGs were mainly enriched in the KEGG pathway of tyrosine metabolism,starch and sucrose metabolism,galactose metabolism,alpha-linolenic acid metabolism,phenylpropanoid biosynthesis and so on.Related genes involved in amino acid biosynthesis（Arog、CM、Pro DH、ALS、AGT、AACT）,sucrose starch degradation related genes（INV、PGAM、BAM、GP）,galactose metabolism related genes（GGP、Gol S、SIP、DIN10）,and antioxidation related genes（ROXY、GRX、APX）in roots of Pag GS1;2-OE lines were significantly up-regulated compared with WT under high ammonium treatment.（6）Metabolomic analysis showed that 270 and 328 differential metabolites were identified in the roots of the Pag GS1;2-OE lines under normal conditions and high ammonium stress,respectively.KEGG enrichment analysis showed that the differential metabolites in the roots of Pag GS1;2-OE lines under high ammonium stress were mainly enriched in the pathways of amino acid biosynthesis,carbon metabolism,tryptophan metabolism,fructose and mannose metabolism.Overexpression of Pag GS1;2 promoted the accumulation of 3-methylaspartic acid,glutamate,proline,serine,histidine and other amino acids,decreasing the content of carbohydrates such as fructose,starch,galactose and increased the content of sugar alcohol compounds such as sedum heptulose,maltose,D-mannitol,galactitol,D-sorbitol in roots 84K poplar under high ammonium stress.（7）Compared with WT,under high nitrogen conditions,the biomass,NUE,nitrogen assimilation enzyme activity and product content,photosynthesis intensity and carbohydrate content of Pag NADH-GOGAT2-OE lines were significantly increased,the aboveground dry weight,NUE and carbon and nitrogen metabolism relating indexes of Pag NADH-GOGAT2-RNAi lines were decreased.The cellulose content of Pag NADH-GOGAT2-OE lines was significantly higher than WT and Pag NADH-GOGAT2-RNAi lines under both nitrogen supply levels.（8）Pag NADH-GOGAT2 and Pag GS1;2 transgenic lines showed similar phenotypes under high ammonium stress.Compared with WT,under high ammonium stress conditions,the activities of SOD and POD in the roots of Pag NADH-GOGAT2-OE lines were significantly increased,and the content of MDA decreased.The overexpression of Pag NADH-GOGAT2could enhance the ammonium assimilation of GS/GOGAT cycle,increase the accumulation of nitrogen assimilation products and carbohydrates,and maintain the balance of carbon and nitrogen in 84K poplar under high ammonium environment.In conclusion,Pag GS1;2 and Pag NADH-GOGAT2 could improve nitrogen use efficiency and alleviate ammonium toxicity by affecting carbon and nitrogen metabolism in 84K poplar.This study provides an important theoretical basis for regulating the GS/GOGAT cycle by means of genetic engineering,improving the nitrogen use efficiency of forest trees and ammonium tolerant forest tree varieties.