Responsive Of Tobacco Photosynthetic Apparatus To Atmospheric NO₂ And Functional Of GSNOR Metabolic Pathway

With the rapid development of China's economy,the number of motor vehicles increases suddenly,the emission of active nitrogen increases,and continues to settle to the land and aquatic ecosystem,that is,nitrogen deposition,which has changed the pattern of natural ecosystem,causing a series of serious ecological problems such as water acidification,eutrophication and the loss of ecosystem diversity.What is more serious is that the oxidation product NO2 of nitrogen oxide causes the sharp increase of PM2.5 content in the atmosphere,causing serious haze and serious environmental pollution.Nitrogen deposition includes wet deposition and dry deposition.In recent years,people pay more attention to wet deposition,but more than 70%dry deposition of nitrogen deposition has been ignored for a long time.Atmospheric nitrogen dioxide(NO2)is the main component of dry deposition of nitrogen,accounting for about 90%.It mainly comes from nitrogen oxides(NOx)and is an important substance of acid rain and photochemical smog,as well as an important factor of ozone depletion.Plants can absorb NO2 in the atmosphere and then enter the leaves to participate in the nitrogen metabolism in plants,which affects the carbon and nitrogen metabolism and their coordination.However,we do not know how the uptake of atmospheric NO2 by plants affects the photosynthetic apparatus.In this study,the model plant Nicotiana Tabacum L.was used as experimental material,and a self-designed automatic control device was used to simulate the dry deposition of NO2 by gas fumigation,to explore the response mechanism of tobacco photosynthesis to atmospheric NO2 from plant phenotype,physiology and biochemistry,as well as proteome and transcriptome techniques.At the same time,we found a new pathway of NO2 metabolism,namely S-nitrosoglutathione reductase(GSNOR)metabolic pathway.By constructing GSNOR mutant and using NO2 fumigation method,we observed the phenotypic characteristics and metabolic function of mutant and wild-type,in order to lay a foundation for the study of NO2 stress regulation and NO2 tolerance mechanism of plants,to reveal the regulation mechanism of plant NO2 absorption and promote the comprehensive understanding of plant nitrogen sources.(1)Tobacco seedlings were fumigated with 1?L·L-1,4 ?L·L-1,8 ?L·L-1 and 16 ?L·L-1 NO2 to determine the optimal and critical concentrations of NO2 fumigated tobacco leaves.According to the surface characteristics,nitrogen metabolism and photosynthetic parameters of tobacco,1 ?L·L-1 and 4 ?L·L-1 NO2 promoted the growth,which was in the appropriate range of concentration.Each index of 8 ?L·L-1 NO2 showed a threshold state,which was the critical concentration,while NO2 ranges from 8 ?L·L-1 up to 16 ?L·L-1,tobacco injury or even death.(2)When tobacco seedlings were fumigated with the optimal NO2 concentration of 4.0?L·L-1,the relative variable fluorescence at points J(2 ms)and I(30 ms)on the chlorophyll a fluorescence dynamic curve(OJIP)of fumigated leaves decreased significantly.The results showed that the electron transfer ability of primary quinone receptor(QA)to secondary quinone receptor(QB)and the electron receiving ability of plastid quinone(PQ)were enhanced in PS? reaction center.The potential photochemical activity(Fv/Fo)and Fv/Fm of PS? in leaves of NO2 fumigation were significantly higher than those of CK,indicating that NO2 fumigation improved the activity and opening degree of PS? reaction center in tobacco leaves,and improved the photochemical efficiency.At the same time,the maximum redox capacity of PSI(?I/Io)of NO2 fumigated leaves was also increased,and the ability to accept electrons was enhanced.After NO2 fumigation,the light energy absorbed by the PS? reaction center of leaves was more used for photochemical reaction.At the 15th day of fumigation,the number of inactivated PS? reaction centers was increased,and the yield of heat dissipator for the inactivated PS? reaction center was increased.(3)Tobacco seedlings were fumigated with NO2 critical concentration 8.0 ?L·L-1,the number of leaves per plant and weight of leaves were increased,and the leaves became dark green and crimped after 10 days of fumigation.The changes of photosynthetic mechanism parameters were different.During 15 days of fumigation,the chlorophyll content,Fv/Fm,electron transfer rate(ETR)and non-photochemical quenching(NPQ)of older leaves(Lmax)increased the most,but decreased the activity of PS?(?I/Io).The Fv/Fm,ETR and NPQ of Lmin decreased.The critical concentration of NO2 increased PS? activity in tobacco leaves and inhibited PS? activity in fully developed leaves.(4)Tobacco seedlings were fumigated at high concentration of 16.0 ?L·L-1NO2,phenotype and photosynthetic metabolism were damaged.The main reason was that high concentration of NO2 increased the superoxide anion(O2-)production rate and the content of peroxynitrite anion(ONOO-)in leaves,and the membrane peroxidation and electrolyte leakage.Chlorophyll content and net photosynthetic rate decreased,chloroplast was damaged,and the photosynthetic mechanism of leaves was destroyed.After NO2 fumigation,the activity of PS?reaction center decreased,the oxygen release complex was damaged,and the electron transfer from the PS? donor side to the acceptor side was inhibited,and the electron transfer from QA to QB on the acceptor side was also blocked,leading to the dissociation of thylakoid membrane.The activity of PSI reaction center also decreased,and the ability to accept electrons was weakened,and the electron transfer from PS? to PSI was inhibited,which increased the damage of PS? to tobacco seedling leaves at high concentration of NO2.(5)Exogenous melatonin(Mel)can alleviate the damage of high concentration NO2 to leaves by alleviating chlorophyll degradation and increasing the expression of key proteins in the process of chlorophyll synthesis.In addition to that,PS? donor-side proteins(PsbO,PsbP1,PsbP3 and PsbQ)and receptor-side proteins(PsbA,PsbC,PsbD and PsbL),PSB27-H1 and Psb28)and PSI reaction center proteins(PsaB,PsaC,PsaD,PsaEa,PsaEb,PsaF,PsaN,PsaG and PsaO)were up-regulated after exogenous Mel treatment.Mel pretreatment also enhanced the expression of enzymes and proteins related to ascorbate-glutathione(AsA-GSH)and thioredoxin peroxiredoxin(Trx-Prx)pathways in NO2 fumigated leaves,and regulated the redox balance of leaves.Mel pretreatment mediated polyamine(PAs)synthesis pathway,and increased the expression of SAMS 1,SAMS2 and SAMS3 key enzymes in PAs synthesis pathway in NO2 fumigated leaves.Mel regulates abscisic acid(ABA)signal transduction and increases the expression of calmodulin-binding transcription factor CAMTA12 and NtCaM calmodulin NtCaM2 in Ca2+signal transduction.(6)In order to further clarify the characteristics of NO2 metabolism in leaves,Illumina HiSeq sequencing technology and bioinformatics analysis were used to study the transcriptome characteristics of tobacco leaves in response to the atmospheric NO2 optimal concentration of 4.0 ?L·L-1.The differentially expressed genes(DEGs)in CK and NO2 fumigated leaves were screened and 2463 DEGs were obtained.Compared with CK,1248 DEGs were significantly up-regulated and 1215 DEGs were significantly down-regulated in NO2 fumigation leaves.GO enrichment was performed on DEGs to clarify the biological processes,cell components and molecular functions of these specifically expressed genes.The KEGG pathway enrichment analysis of DEGs showed that carbon pathway was the KEGG pathway with the most significant difference under NO2 fumigation,and nitrogen metabolism and glycolysis/gluconeogenesis were also significantly enriched.GDH-A,GDH-B,NR,GS and GSNOR expression were up-regulated in NO2 fumigation leaves,and GSNOR expression was the most significantly up-regulated.(7)In order to verify the GSNOR pathway of NO2,GSNOR transgenic tobacco lines were constructed and fumigated with the optimum concentration of 4.0 ?L·L-1NO2 to overexpress GSNOR(OE)and inhibit GSNOR(IE)seedlings.After fumigation,the content of glutathione(GSH)and the activity of nitroso glutathione reductase(GSNOR)in OE leaves were increased,which made OE leaves have multiple mechanisms to remove excess nitric oxide(NO)in vivo,while the content of NO in IE leaves increased,causing serious S-nitrosylation damage.NO2 increased the glutamine synthase/glutamate synthase cycle(GS/GOGAT)in OE leaves and improved the ammonia assimilation capacity of organic nitrogen sources,while the GS/GOGAT cycle level in IE leaves decreased.NO2 can damage IE leaves,but it can improve the adaptability to NO2 by regulating the osmotic pressure of cytoplasm and stabilizing the biological macromolecular structure.Inhibition of GSNOR resulted in S-nitrosylation and ROS outbreak.NO2 fumigation increased the chlorophyll content of OE leaves,but decreased the chlorophyll content of IE leaves and its ability to capture light energy.NO2 fumigation inhibited the electron transfer and oxygen evolution complex(OEC)activity from QA to QB on the PS? receptor side of IE leaves,and destroyed the stability of thylakoid membrane.In conclusion,this study creatively determined the optimal concentration and critical concentration of NO2 in tobacco leaves,the photosynthetic response characteristics and mechanism of optimal,critical and high concentration NO2,and the mechanism of exogenous Mel alleviating the injury of high concentration NO2,proposed a new way of plant metabolism of NO2-GSNOR pathway,and analyzed it from the perspective of molecular biology.It provides a reference for us to understand the effects of atmospheric NO2 on plant growth and photosynthetic characteristics,and also broadens our vision for a new understanding of nitrogen metabolism from different sources in plants.