| As an essential element for crop growth,Nitrogen（N）is also one of the limiting factors for tobacco yield.Nitrate accumulation in tobacco leaves not only reduces nitrogen use efficiency,but also directly affects the formation of tobacco-specific nitrosamines（TSNAs）,an important harmful component in tobacco.Due to the impaired nitrogen use efficiency（NUE）,burley tobacco generally requires three to five times more N fertilization than flue-cured tobacco to achieve a comparable yield,which results in higher nitrate accumulation in burley tobacco leaves.As one of the indispensable raw materials for cigarette production,midrib plays an important role in reducing coking and formulation costs of cigarettes.However,the disadvantage of midrib is also obvious,among which is substantial higher levels of nitrate content and subsequent higher formation and accumulation of TSNAs,which greatly reduces the safety of tobacco products.Based on this,understanding the differences in nitrate accumulation and NUE of contrasting tobacco genotypes,clarifying the biological and molecular mechanism of efficient N utilization,and develop regulatory measures to increase NUE and reduce nitrate would best address these problems.However,current researches in this area are still not comprehensive.In this study,the nitrate accumulation rules and differences of different types of tobacco and the same tobacco lamina and midrib were studied.Then we compared the physiological characteristics of NUE and nitrate content between two tobacco genotypes in hydroponic experiment,aiming to screen the key factors causing the different nitrate level between tobacco genotypes.In addition,the physiological characteristics between lamina and midrib of flue-cured tobacco were analyzed.Transcriptome sequencing and single cell RNA-sequencing were used to analyze and screen candidate genes closely related to NUE and nitrate accumulation in tobacco at molecular level.In addition,leaf color mutants were obtained by EMS mutagenesis technology to verify the relationship between pigment synthesis,photosynthesis and nitrate accumulation.Finally,regulatory techniques of grafting and exogenous regulator spraying were developed to reduce nitrate and TSNAs in tobacco leaves.The main results are as follows:1.In order to explore the differences in nitrate accumulation in leaves of different types of tobacco leaves,two accessions with contrasting N efficiency（flue-cured tobacco vaiety K326 with high efficiency,and burley tobacco variety TN90 with low efficiency）were used as materials in field and hydroponic experiments.The differences in NO3-N content,NO3-N assimilation and reutilization,and physiological indices related to photosynthetic C metabolism and transcriptome involved were compared between the two accessions under normal N（CK,5 mmol/L）and low N（LN,0.5 mmol/L）nutrient solutions respectively.At the same time,the 15N isotope tracer technique was applied to study the uptake,transport,and reutilization capacity of NO3-N.Results showed that the NO3-N accumulation in the leaves of burley tobacco was higher than in flue-cured tobacco during the growth and development process.And the NO3-N and TSNAs contents in cured burley tobacco were higher compared to flue-cured tobacco.N-efficient accession K326 had significantly higher plant biomass than that of TN90 under normal and low N conditions.15N isotope tracer test also showed that the ability for NO3-N absorption,transportation,and reutilization of K326were also greater.Also,activities of nitrate reductase and glutamine synthetase of K326 were higher.Meanwhile,photosynthetic pigment content and enzyme activities related to carbon assimilation of K326 leaves were all higher than those of TN90.Transcriptomic analysis revealed that genes involved in photosynthesis,carbon fixation,and nitrogen metabolism had a higher expression level in K326 than that in TN90.Further analysis found that TPIP1,GAPB,HEMB,PGK3,PSBO,PSBP2,PSAG,and GLN2 may be the key genes affecting NUE.There results suggest that variation of NUE is closely related to the carbon metabolism efficiency,nitrogen translocation and metalism.The more advantageous changes in N uptake,N assimilation,N remobilization in combination with carbon metabolism contributed to the higher NUE.2.To study the link between a lack of Chl and abnormal nitrate accumulation,a stable albino leaf mutant（Al）and slight-green leaf mutant（SG）obtained from the common flue-cured tobacco（Nicotiana tabacum L.）cultivar’Zhongyan 100’（ZY100）by mutagenesis with ethyl methanesulfonate（EMS）were used as materials.The differences between the Chl-deficient mutants and the wild-type（WT）were analyzed in terms of biomass,photosynthetic fluorescence parameters,and carbon-and nitrogen-related physiological parameters.RNA sequencing（RNA-seq）and WGCNA were used to explore the key pathways and candidate genes regulating differentiated chlorophyll and nitrate content.The results showed that,when compared to the WT,the Chl content and biomass of mutant plants were considerably lower while the NO3-N content was substantially elevated.The net photosynthetic rate,photosynthetic fluorescence parameters,carbohydrate,soluble protein,and carbon-and nitrogen-related enzyme activities all decreased in leaves of mutants and the development of chloroplasts was abnormal.Applying more nitrogen improved the growth and development of mutants,whereas NO3-N content distinctively increased compared with that of the WT.Through transcriptome sequencing,the downregulated genes in mutants were enriched in plant hormone signal transduction and nitrogen metabolism,which are involved in pigment biosynthesis and the carbon fixation pathway.In addition,two hub genes and seven transcription factors identified from the blue module through WGCNA were likely to be key candidate factors involved in chlorophyll synthesis and nitrate accumulation.Our results demonstrated that there is a close relationship between the leaf color and NO3-N content.Some genes affected photosynthetic carbon fixation and enzyme activities related to nitrogen metabolism by regulating chloroplast development and chloroplast metabolism pathways,and thus resulting in the accumulation of nitrate.3.With a view to sustainable tobacco agriculture and nitrogen use efficiency,developing new genotypes with high productivity under low N conditions is an important approach.To further clarify how tobacco leaves respond to low-N treatment and explore the N-responsive genes,we characterized the transcriptomes in tobacco leaves grown in normal nitrogen and nitrogen deficiency conditions by performing single‐cell RNA sequencing.We identified 10cell types with 17 transcriptionally distinct cell clusters with the assistance of reported marker genes and constructed the first single‐cell atlas of tobacco leaves.Distinct gene expression patterns of the cell clusters were observed under low-N conditions,and the mesophyll cells were the most important responsive cell type and displayed heterogeneity responses of its three subtypes.And pseudo time trajectory analysis revealed low-N stress decelerates the differentiation towards mesophyll cells.In combination with sc RNA-seq,WGCNA,and bulk RNA-seq results,we found that genes involved in porphyrin metabolism,and nitrogen metabolism play an essential role in response to low-N conditions.Gene coexpression network analysis and correlation analysis showed that COL16,GATA24,MYB73,and GLK1to be the key TFs in the regulation of N-responsive genes.And transcription regulation of genes responsible for these pathways could be the possible mechanism of high NUE in tobacco.4.In order to explore the differences in nitrate accumulation in lamina and midrib of the same tobacco variety,the differences and changes of nitrate accumulation during the development of tobacco were studied using flue-cured tobacco and burley tobacco as materials.In addition,lamina and midrib of flue-cured tobacco were used as materials in pot experiment to study the physiological indexes and transcriptome differences.The NO3-N content showed a remarkable difference between flue-cured tobacco lamina and midrib.The results of pot experiment showed that three times higher NO3-N content was observed in midrib than that in the lamina,along with lower pigment,NH4-N content,NRA,SSA,and GSA in midrib.Transcriptome analysis revealed that expression of genes involved in photosynthesis-antenna proteins,carbon fixation,nitrogen metabolism,carotenoid biosynthesis,and biosynthesis of amino acids were significantly lower in midrib than in lamina.q RT-PCR results showed that the expression level of nitrate transporter and reallocation-related genes LOC107782967,LOC107806749,LOC107775674,LOC107829632,LOC107799198,LOC107768465decreased by 2.74,1.81,49.5,3.5,2.64 and 2.96 folds while nitrate store-related gene LOC107789301 increased by 8.23 folds in midrib but not in lamina.Low capacity of chlorophyll synthesis and carbon fixation,reduced ability of nitrate reallocation but the high capacity of accumulation might lead to nitrate accumulation in midrib.5.Based on previous work,influences of spraying modulators ang grafting on nitrate metabolism and TSNAs accumulation were explored.In this study,reciprocal pot and field grafting experiments were conducted using burley tobacco Eyan No.1 and flue-cured tobacco K326 to investigate whether grafting burley tobacco scions on flue-cured tobacco rootstocks could reduce the NO3-N and TSNAs level in burley tobacco leaves.Photosynthesis,carbon and nitogen-related physioligical characteristics,NO3-N and TSNAs content were assessed for flue-cured tobacco/flue-cured tobacco（K/K）,burley tobacco/burley tobacco（E/E）,burley tobacco/flue-cured tobacco（E/K）”.The results showed that grafting burley tobacco scions on flue-cured tobacco rootstocks significantly increased the photosynthetic rate,nitrate reductase and glutamine synthetase activities,compared with burley tobacco self-rooting,while decreasing the NO3-N and TSNAs level.Transcriptomic analysis revealed that grafting resulted in upregulated expression of genes involved in starch,sucrose,porphyrin,chlorophyll,and N metabolism,as well as carbon fixation and carotenoid biosynthesis.The findings suggest that grafting on high N use efficiency rootstock is an exceptionally promising means of decreasing NO3-N accumulation by improving photosynthesis and N metabolism in the scion,thereby reducing the levels of harmful TSNAs.In order to explore the effects of reduce the accumulation of nitrate and TSNAs in tobacco leaves,eciprocal pot and field grafting experiments were conducted.the burley tobacco variety TN90 were used in the pot experiments.Three treatments of CK（water）,T1（1ml/L glycerol）,T2（1ml/L glycerol and 10mg/L IAA）were arranged with three replicates.The growth indicators,pigment content,photosynthetic rate,nitrate reductase activity,nitrogen utilization efficiency and carbon and nitrogen compounds content were studied.The results showed that the leaves had higher pigment content,photosynthetic rate,nitrate reductase activity,nitrogen utilization efficiency,biomass,carbon and nitrogen compounds content when the leaves were treated with T1 and T2.Compared with CK,the physiological parameters were significantly improved with T2 treatment,the dry matter of root,stem and leaves increased by 43.75%,27.85%and 41.13%,the nitrogen utilization efficiency increased by 12.18%,and nitrate content reduced by 14.52%,respectively.Glycerol and IAA spraying was able to effectively improve the metabolism of carbon and nitrogen,and promoted the nitrogen use efficiency,thereby decreasing the content of NO3-N in tobacco seedlings.The flue-cured tobacco（Zhongyan100 and Zhongchuan208）and the burley tobacco（TN90）were used in the field experiment.The photosynthetic indexes,physical properties,conventional chemical components,NO3-N and TSNAs contents of tobacco leaves were analyzed under the condition of reducing nitrogen by 20%and spraying 2 m L/L glycerol and10 mg/L IAA.The results showed reducing nitrogen fertilizer treatment not only promote tobacco carbon metabolism of flue-cured tobacco and burley tobaccobut also improve single leaf weight,leaf quality weight,leaf length and leaf width.Meanwhile,the contents of NO3-N and TSNAs in tobacco leaves were reduced under reducing nitrogen application.The average contents of nitrate nitrogen in tobacco upper leaves Zhongyan 100,Zhongchuan 208and Eyan 1 decreased by 20.65%,20.81%and 17.03%,respectively,and contents of nitrate nitrogen in tobacco middle leaves decreased by 19.20%,22.25%and 19.43%,respectively.The average contents of total TSNA in tobacco upper leaves Zhongyan 100,Zhongchuan 208and Eyan 1 decreased by 35.21%,46.21%and 43.13%,respectively,and contents of total TSNA in tobacco middle leaves decreased by 34.13%,33.19%and 34.68%,respectively.We speculated that reducing nitrogen fertilizer and spraying glycerol and auxin could improve the photosynthetic efficiency and nitrogen utilization efficiency of tobacco leaves,which is an effective measure to reduce the NO3-N and TSNAs accumulation in tobacco leaves. |
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