Transcriptional Regulation And Physiological Response Mechanism Of Strong Ammonium Tolerance In Aquatic Plant Myriophyllum Aquaticum

Rural areas in our country are facing serious agricultural non-point source pollution,one of its major causes is ammonia nitrogen emission from livestock and poultry breeding.As the preferred plant for constructing ecological wetlands to purify swine wastewater with high ammonium,Myriophyllum aquaticum has a strong tolerance to high ammonium concentration in the environment.However,due to the lack of gene function information,current research on M.aquaticum is mainly focused on the physiological level.To better understand the mechanism of high ammonium tolerance of M.aquaticum,this work uses Iso-seq to obtain full-length transcripts of stem,root and leaf,and annotates gene functions.Furthermore,the differentially expressed genes under different ammonium concentrations were identified by RNA-seq,and the physiological responses to different ammonium concentrations were combined to reveal the physiological and molecular mechanisms of high ammonium tolerance of M.aquaticum.The role of GS in high ammonium tolerance was elucidate by analysis of physiological and molecular responses after GS inhibition.The main results are as follow:（1）Based on single-molecule real-time sequencing,15.83,18.21 and 19.14 G of polymerase read were obtained from stem,root and leaf,respectively.A total of64046,77012 and 78262 unigenes were obtained from the three tissues after filtration,correction and redundancy removal,of which 93.03%,95.74%and 95.6%unigenes successfully annotated gene information,respectively.It is found that Vitis Vinifera was the species with the most matched genes with NR database.There are 38468,49479 and 48933 genes in stem,root and leaf,respectively,annotated into 54functional groups of biological process（BP）,cellular component（CC）and molecular function（MF）in GO database.Most genes were enriched in metabolic process,cell and binding activity of BP,CC and MF,respectively.A total of 62285,76918 and77959 coding sequences were predicted in stem,root and leaf,respectively,whose length is mainly distributed between 200-2500 nt.SNF2 and C3H families were the most predicted transcription factors in stem,root and leaf.A total of 47019,40468 and46652 SSR were detected in these tissues,and the single nucleotide repeat loci with9-12 repeats accounted for the largest proportion.（2）With 0.2m M NH₄⁺（N1）treatment as control group,31678 and 35072differentially expressed genes were identified under 10 m M（N2,ammonium tolerant concentration）and 70 m M NH₄⁺（N3,ammonium toxic concentration）treatment,respectively.Photosynthesis related pathways were significantly enriched in the leaf of M.aquaticum under high ammonium conditions.All the light-harvesting chlorophyll a/b binding（LHC）genes identified in N2 treated leaf were up-regulated,which contributed to the improvement of photosynthetic capacity of leaf,LHC genes were down-regulated under N3 treatment.The peroxisome（ko04146）is enriched significantly under high ammonium conditions,catalase（CAT）genes were up-regulated under N2 and N3 treatment,which contributed to scavenge reactive oxygen species（ROS）induced by excessive NH₄⁺in vivo.Under high NH₄⁺condition,ammonium transporter（AMT）genes in roots were down-regulated,which prevented excessive NH₄⁺absorption.Under N2 conditions,the expression levels of genes related to ammonium assimilation including glutamine synthase（GS）and glutamate synthase（GOGAT）genes in leaves and roots,glutamate dehydrogenase（GDH）and asparagine synthase（AS）genes in roots were high,which could assimilate NH₄⁺into amino acids in time and avoid the toxicity to cells caused by excessive NH₄⁺accumulation.Under N3 condition,GS and GOGAT genes were down-regulated in leaves and roots,while GDH genes in roots and AS genes in all tissues were up-regulated,suggesting that GDH and AS genes play a major role in NH₄⁺assimilation under ultra-high NH₄⁺condition.（3）After two weeks of treatments,the plants treated with N1 and N2 grew vigorously,while the N3 treated plants showed obvious NH₄⁺toxicity symptoms,showing stunted growth,withered leaf margin.The fresh weight of N2 plant was 1.16times that of N1,while the fresh weight of N3 plant was only 59.21%of N1.Chlorophyll concentrations in N2 plant was significantly higher than that in N1 plant,and that of N3 plant was the lowest.The concentrations of TN,NH₄⁺and NO₃^-in all tissues increased significantly with the increase of exogenous NH₄⁺concentrations.CAT activity increased significantly under high ammonium condition.The fluorescence intensity of ROS in roots of plants treated with N3 was significantly enhanced,the concentrations of malondialdehyde and proline in all tissues were significantly higher than those in N1 and N2 treatments,indicating that 70 m M NH₄⁺caused severe stress to M.aquaticum.GDH activity in stem and root of N3 plant was1.16 and 1.46 times than that of N1 plant,respectively.AS activity in tissues increased significantly with the increase of ammonium concentrations,suggesting that GDH and AS play an important role in ammonium assimilation under high ammonium conditions.The concentrations of asparagine（Asn）,glutamine（Gln）and glutamate in N3 treated stems were significantly increased,indicating that these three amino acids were the main forms of nitrogen transport under this condition.（4）At a concentration of 15 m M NH₄⁺,0.1 m M GS inhibitor（methionine sulfoximine,MSX）treatment seriously hindered the growth of M.aquaticum and caused the symptoms of ammonium toxicity including leaf chlorosis and withered.Short-term（5h）MSX treatment resulted in NH₄⁺accumulation only in roots,while long-term（5d）MSX treatment resulted in significant NH₄⁺accumulation in all tissues,which was more serious in leaves.Long-term GS inhibition significantly reduced the concentrations of photosynthetic pigments in M.aquaticum.After 5 days of treatment,Gln concentration in leaves,stems and roots decreased by 86.88%,63.46%and97.43%,Asn concentration decreased by 57.73%,12.37%and 76.41%,respectively.The significantly decreased reduced glutathione/oxidized glutathione ratio and glutathione reductase activity under MSX treatment indicated that the inhibition of GS interfered with the redox state in leaves and roots.The expression levels of GS1715,GOGAT-Fe and AMT genes in MSX treated leaves and GS1542,GOGAT-NADH and GDH genes in roots decreased by 21.34%to 60.13%,while the expression levels of AMT genes in roots increased by 100.92%.Regulation of the expression of these genes can exacerbate the symptoms of ammonium toxicity in plants.