Tobacco Mutant Research Articles

A P450 superfamily member NtCYP82C4 promotes nicotine biosynthesis in Nicotiana tabacum

In plants, cytochrome P450s are monooxygenase that play key roles in the synthesis and degradation of intracellular substances. In tobacco, the majority of studies examining the P450 superfamily have concentrated on the CYP82E subfamily, where multiple family members function as demethylases, facilitating the synthesis of nornicotine. In this study, NtCYP82C4, a tobacco P450 superfamily member, was identified from a gene-edited tobacco mutant that nicotine biosynthesis in tobacco leaves is evidently reduced. Compared to the wild-type plants, the knockout of NtCYP82C4 resulted in a significantly lower nicotine content and biomass in tobacco leaves. Transcriptome and metabolome analyses indicated that the knockout of NtCYP82C4 inhibites secondary metabolic processes in tobacco plants, leading to the accumulation of some important precursors in the nicotine synthesis process, including aspartic acid and nicotinic acid, and increases nitrogen partitioning associated with those processes such as amino acid synthesis and utilization. It is speculated that NtCYP82C4 may function as an important catalase downstream of the nicotine synthesis. Currently, most of the steps and enzymes involved in the nicotine biosynthesis process in tobacco have been elucidated. Here, our study deepens the current understanding of nicotine biosynthesis process and provides new enzyme targets for nicotine synthesis in tobacco plants.

Naringenin restricts the colonization and growth of Ralstonia solanacearum in tobacco mutant KCB-1.

Bacterial wilt severely jeopardizes plant growth and causes enormous economic loss in the production of many crops, including tobacco (Nicotiana tabacum). Here, we first demonstrated that the roots of bacterial wilt-resistant tobacco mutant KCB-1 can limit the growth and reproduction of Ralstonia solanacearum. Secondly, we demonstrated that KCB-1 specifically induced an upregulation of naringenin content in root metabolites and root secretions. Further experiments showed that naringenin can disrupt the structure of R. solanacearum, inhibit the growth and reproduction of R. solanacearum, and exert a controlling effect on bacterial wilt. Exogenous naringenin application activated the resistance response in tobacco by inducing the burst of reactive oxygen species and salicylic acid deposition, leading to transcriptional reprogramming in tobacco roots. Additionally, both external application of naringenin in CB-1 and overexpression of the Nicotiana tabacum chalcone isomerase (NtCHI) gene, which regulates naringenin biosynthesis, in CB-1 resulted in a higher complexity of their inter-root bacterial communities than in untreated CB-1. Further analysis showed that naringenin could be used as a marker for resistant tobacco. The present study provides a reference for analyzing the resistance mechanism of bacterial wilt-resistant tobacco and controlling tobacco bacterial wilt.

CRISPR/Cas9-Mediated Targeted Mutagenesis of Betaine Aldehyde Dehydrogenase 2 (BADH2) in Tobacco Affects 2-Acetyl-1-pyrroline

2-acetyl-1-pyrroline (2AP) is a highly effective volatile compound that gives fragrance to numerous plant species and food. Mutation(s) in the betaine aldehyde dehydrogenase 2 (BADH2) gene results in the accumulation of 2AP. However, the function of BADH genes in tobacco (Nicotiana tabacum L.) remains poorly understood. In this study, we successfully obtained four betaine aldehyde dehydrogenase (BADH) genes from tobacco. Phylogenetic analysis of the protein sequences showed that two of the four BADH genes were closely related to the wolfberry (Lycium barbarum) BADH gene (LbBADH1), so we named them NtBADH1a and NtBADH1b, respectively. The other two BADH genes were orthologues of the tomato (Solanum lycopersicum) aminoaldehyde dehydrogenase 2 (SlAMADH2) gene, and were named NtBADH2a and NtBADH2b, respectively. Expression analysis revealed that the biological functions of NtBADH1a and NtBADH1b were different from those of genes NtBADH2a and NtBADH2b. We introduced mutations into NtBADH1a, NtBADH1b, NtBADH2a and NtBADH2b in tobacco using the CRISPR/Cas9 system and identified transgenic Ntbadh mutant tobacco lines. Single mutants (Ntbadh1a, Ntbadh1b, Ntbadh2a and Ntbadh2b) and double mutants (Ntbadh1a-Ntbadh1b and Ntbadh2a-Ntbadh2b) harbored deletion or insertion of nucleotides, both of which led to the production of a frameshift, preventing protein accumulation. A popcorn-like scent was noticeable in tobacco leaves from the Ntbadh2a-Ntbadh2b double mutant, but not from any single mutant or the Ntbadh1a-Ntbadh1b double mutant or the wild type. Consistent with this observation, we only detected 2AP in fresh leaves from the Ntbadh2a-Ntbadh2b double mutant. These findings indicate that only the combined inactivation of NtBADH2a and NtBADH2b results in 2AP accumulation in tobacco, which was not related to NtBADH1.

Comprehensive transcriptome and WGCNA analysis reveals the potential function of anthocyanins in low-temperature resistance of a red flower mutant tobacco

The anthocyanin is a protective substance in various plants, and plays important roles in resisting to low-temperature. Here, we explored transcriptome analysis of pink flower (as CK) and the natural mutant red flower (as research objects) under low-temperature conditions, and aimed to reveal the potential functions of anthocyanins and anthocyanin-related regulatory factors in resistance to low-temperature. Our results showed that most of the differentially expressed genes (DEGs) encoding key enzymes in the late stage of anthocyanin metabolism in the mutant were significantly up-regulated. Meanwhile, several genes significantly differentially expressed in CK or mutant were obtained by classification and analysis of transcription factors (TFs), phytohormones and osmoregulators. Additionally, WGCNA was carried out to mine hub genes resistanted to low-temperature stress in flavonoid pathway. Finally, one UFGT family gene, three MYB and one bHLH were obtained as the future hub genes of this study. Combined with the above information, we concluded that the ability of the red flower mutant to grow and develop normally at low-temperatures was the result of a combination of flavonoids and cold resistance genes.

Reducing chlorophyll levels in seed-filling stages results in higher seed nitrogen without impacting canopy carbon assimilation.

Chlorophyll is the major light-absorbing pigment for plant photosynthesis. While evolution has been selected for high chlorophyll content in leaves, previous work suggests that domesticated crops grown in modern high-density agricultural environments overinvest in chlorophyll production, thereby lowering light use and nitrogen use efficiency. To investigate the potential benefits of reducing chlorophyll levels, we created ethanol-inducible RNAi tobacco mutants that suppress Mg-chelatase subunit I (CHLI) with small RNA within 3 h of induction and reduce chlorophyll within 5 days in field conditions. We initiated chlorophyll reduction later in plant development to avoid the highly sensitive seedling stage and to allow young plants to have full green leaves to maximise light interception before canopy formation. This study demonstrated that leaf chlorophyll reduction >60% during seed-filling stages increased tobacco seed nitrogen concentration by as much as 17% while canopy photosynthesis, biomass and seed yields were maintained. These results indicate that time-specific reduction of chlorophyll could be a novel strategy that decouples the inverse relationship between yield and seed nitrogen by utilising saved nitrogen from the reduction of chlorophyll while maintaining full carbon assimilation capacity.

Physiological and phosphoproteomic analyses revealed that the NtPOD63 L knockout mutant enhances drought tolerance in tobacco

Drought is an important environmental factor affecting plant growth and development. Tobacco is a widely cultivated and economically important crop worldwide. Drought can decrease the yield and quality of tobacco. Peroxidase III can maintain the balance of peroxy free radicals and H2O2 in plants and participate in a variety of physiological processes, including lignin biosynthesis and auxin catabolism. In this study, we first found that a CRISPR/Cas9-mediated peroxidase 63-like (NtPOD63 L) knockout mutant had high drought tolerance, such as reduced stomatal apertures, increased lignin content and interfascicular fiber number, and induced ABA-related gene expression. Then, a quantitative phosphoproteomic analysis of tobacco mutant leaves was conducted after PEG treatment. Phosphoproteomic analysis revealed 609 phosphorylated proteins, among which 266 were upregulated and 343 were downregulated. KEGG pathway analysis indicated that the cluster with “Pyruvate metabolism” contained the most upregulated pathways. The cluster with “RNA degradation”, “Cysteine and methionine metabolism,” and “Tryptophan metabolism contained the three primary downregulated pathways. Sixty-seven kinases were found to change phosphorylation levels. Our study provides new information for understanding NtPOD63 L function in drought stress and describes the molecular regulation mechanisms of the tobacco response to drought at the posttranslational level.

Morphological and physiological mechanism of cytoplasmic inheritance stigma exsertion trait expression in tobacco (Nicotiana tabacum)

Stigma exsertion is an essential outcrossing trait that can improve hybrid seed production efficiencies. In this study, the morphological and physiological mechanisms of cytoplasmic inheritance stigma exsertion trait expression in a tobacco line (MSK326SE) which generated from a stigma exsertion tobacco mutant through continuous backcross were investigated. Compared with its homonuclear-heteroplasmic lines (MSK326 and K326 with inserted stigmas), the exserted stigma phenotype of MSK326SE was mainly caused by corolla shortening, while was stable under different environmental temperature. The different responses of mainly endogenous hormones and expression of cell division- and expansion-related genes caused the differences in cell division and expansion in different flower organs, which further determined the lengths of the corolla. Furthermore, the significant decrease of MSK326SE corolla epidermal cell size caused corolla shortening and finally resulting in stigma exsertion. Exogenous JA could shorten the corolla and more effective increased stigma exsertion degree of MSK326SE, suggesting a potential relationship between stigma exsertion and high JA levels during early bud development. The hybrid seed production efficiency could be improved in tobacco. Our results provide a basis for elucidating the cytoplasmic inheritance stigma exsertion trait expression in tobacco while helping to improve hybrid seed production efficiency.

Analysis of rhizosphere bacterial communities of tobacco resistant and non-resistant to bacterial wilt in different regions

Tobacco bacterial wilt has seriously affected tobacco production. Ethyl methanesulfonate (EMS) induced tobacco bacterial wilt resistant mutants are important for the control of tobacco bacterial wilt. High-throughput sequencing technology was used to study the rhizosphere bacterial community assemblages of bacterial wilt resistant mutant tobacco rhizosphere soil (namely KS), bacterial wilt susceptible tobacco rhizosphere soil (namely GS) and bulk soil (namely BS) in Xuancheng, Huanxi, Yibin and Luzhou. Alpha analysis showed that the bacterial community diversity and richness of KS and GS in the four regions were not significantly different. However, analysis of intergroup variation in the top 15 bacterial communities in terms of abundance showed that the bacterial communities of KS and GS were significantly different from BS, respectively. In addition, pH, alkali-hydrolysable nitrogen (AN) and soil organic carbon (SOC) were positively correlated with the bacterial community of KS and negatively correlated with GS in the other three regions except Huanxi. Network analysis showed that the three soils in the four regions did not show a consistent pattern of network complexity. PICRUSt functional prediction analysis showed that the COG functions were similar in all samples. All colonies were involved in RNA processing and modification, chromatin structure and dynamics, etc. In conclusion, our experiments showed that rhizosphere bacterial communities of tobacco in different regions have different compositional patterns, which are strongly related to soil factors.

Species variation in the hydrogen isotope composition of leaf cellulose is mostly driven by isotopic variation in leaf sucrose.

Experimental approaches to isolate drivers of variation in the carbon-bound hydrogen isotope composition (δ2 H) of plant cellulose are rare and current models are limited in their application. This is in part due to a lack in understanding of how 2 H-fractionations in carbohydrates differ between species. We analysed, for the first time, the δ2 H of leaf sucrose along with the δ2 H and δ18 O of leaf cellulose and leaf and xylem water across seven herbaceous species and a starchless mutant of tobacco. The δ2 H of sucrose explained 66% of the δ2 H variation in cellulose (R2 = 0.66), which was associated with species differences in the 2 H enrichment of sucrose above leaf water ( : -126% to -192‰) rather than by variation in leaf water δ2 H itself. was positively related to dark respiration (R2 = 0.27), and isotopic exchange of hydrogen in sugars was positively related to the turnover time of carbohydrates (R2 = 0.38), but only when was fixed to the literature accepted value of ‰. No relation was found between isotopic exchange of hydrogen and oxygen, suggesting large differences in the processes shaping post-photosynthetic fractionation between elements. Our results strongly advocate that for robust applications of the leaf cellulose hydrogen isotope model, parameterization utilizing δ2 H of sugars is needed.

Using CRISPR-Cas9 Technology to Eliminate Xyloglucan in Tobacco Cell Walls and Change the Uptake and Translocation of Inorganic Arsenic.

Xyloglucan is a quantitatively major polysaccharide in the primary cell walls of flowering plants and has been reported to affect plants’ ability to tolerate toxic elements. However, it is not known if altering the amounts of xyloglucan in the wall influences the uptake and translocation of inorganic arsenic (As). Here, we identified two Nicotiana tabacum genes that encode xyloglucan-specific xylosyltransferases (XXT), which we named NtXXT1 and NtXXT2. We used CRISPR-Cas9 technology to generate ntxxt1, ntxxt2, and ntxxt1/2 mutant tobacco plants to determine if preventing xyloglucan synthesis affects plant growth and their ability to accumulate As. We show that NtXXT1 and NtXXT2 are required for xyloglucan biosynthesis because no discernible amounts of xyloglucan were present in the cell walls of the ntxxt1/2 double mutant. The tobacco double mutant (ntxxt1/2) and the corresponding Arabidopsis mutant (atxxt1/2) do not have severe growth defects but do have a short root hair phenotype and a slow growth rate. This phenotype is rescued by overexpressing NtXXT1 or NtXXT2 in atxxt1/2. Growing ntxxt mutants in the presence of AsIII or AsV showed that the absence of cell wall xyloglucan affects the accumulation and translocation of As. Most notably, root retention of As increased substantially and the amounts of As translocated to the shoots decreased in ntxxt1/2. Our results suggest that xyloglucan-deficient plants provide a strategy for the phytoremediation of As contaminated soils.

Lack of plastid-encoded Ycf10, a homolog of the nuclear-encoded DLDG1 and the cyanobacterial PxcA, enhances the induction of non-photochemical quenching in tobacco.

pH homeostasis in the chloroplast is crucial for the control of photosynthesis and other metabolic processes in plants. Recently, nuclear‐encoded Day‐Length‐dependent Delayed Greening1 (DLDG1) and Fluctuating‐Light Acclimation Protein1 (FLAP1) that are required for the light‐inducible optimization of plastidial pH in Arabidopsis thaliana were identified. DLDG1 and FLAP1 homologs are specifically conserved in oxygenic phototrophs, and a DLDG1 homolog, Ycf10, is encoded in the chloroplast genome in plant cells. However, the function of Ycf10 and its physiological significance are unknown. To address this, we constructed ycf10 tobacco Nicotiana tabacum mutants and characterized their phenotypes. The ycf10 tobacco mutants grown under continuous‐light conditions showed a pale‐green phenotype only in developing leaves, and it was suppressed in short‐day conditions. The ycf10 mutants also induced excessive non‐photochemical quenching (NPQ) compared with those in the wild‐type at the induction stage of photosynthesis. These phenotypes resemble those of Arabidopsis dldg1 mutants, suggesting that they have similar functions. However, there are distinct differences between the two mutant phenotypes: The highly induced NPQ in tobacco ycf10 and the Arabidopsis dldg1 mutants are diminished and enhanced, respectively, with increasing duration of the fluctuating actinic‐light illumination. Ycf10 and DLDG1 were previously shown to localize in chloroplast envelope‐membranes, suggesting that Ycf10 and DLDG1 differentially control H+ exchange across these membranes in a light‐dependent manner to control photosynthesis.

A critical role of the soybean evening complex in the control of photoperiod sensitivity and adaptation

Photoperiod sensitivity is a key factor in plant adaptation and crop production. In the short-day plant soybean, adaptation to low latitude environments is provided by mutations at the J locus, which confer extended flowering phase and thereby improve yield. The identity of J as an ortholog of Arabidopsis ELF3, a component of the circadian evening complex (EC), implies that orthologs of other EC components may have similar roles. Here we show that the two soybean homeologs of LUX ARRYTHMO interact with J to form a soybean EC. Characterization of mutants reveals that these genes are highly redundant in function but together are critical for flowering under short day, where the lux1 lux2 double mutant shows extremely late flowering and a massively extended flowering phase. This phenotype exceeds that of any soybean flowering mutant reported to date, and is strongly reminiscent of the "Maryland Mammoth" tobacco mutant that featured in the seminal 1920 study of plant photoperiodism by Garner and Allard [W. W. Garner, H. A. Allard, J. Agric. Res. 18, 553-606 (1920)]. We further demonstrate that the J-LUX complex suppresses transcription of the key flowering repressor E1 and its two homologs via LUX binding sites in their promoters. These results indicate that the EC-E1 interaction has a central role in soybean photoperiod sensitivity, a phenomenon also first described by Garner and Allard. EC and E1 family genes may therefore constitute key targets for customized breeding of soybean varieties with precise flowering time adaptation, either by introgression of natural variation or generation of new mutants by gene editing.

Development of a Rapid Selection System for Salt-Resistant Mutants of Nicotiana benthamiana through Protoplast Culture after Gamma Irradiation.

We aimed to develop a novel technology capable of rapidly selecting mutant plant cell lines. Salt resistance was chosen as a rapid selection trait that is easily applicable to protoplast-derived cell colonies. Mesophyll protoplasts were cultured in a medium supplemented with 0, 50, 100, 150, 200, 250, and 300 mM NaCl. At NaCl concentrations ≥ 100 mM, cell colony formation was strongly inhibited after 4 weeks of culture. Tobacco protoplasts irradiated with 0, 50, 100, 200, and 400 Gy were then cultured to investigate the effects of radiation intensity on cell division. The optimal radiation intensity was 50 Gy. To develop salt-resistant tobacco mutant plants, protoplasts irradiated with 50 Gy were cultured in a medium containing 100 mM NaCl. The efficiency of cell colony formation from these protoplasts was approximately 0.002%. A salt-resistant mutant callus was selected and proliferated in the same medium and then transferred to a shoot inducing medium for adventitious shoot formation. The obtained shoots were then cultured in a medium supplemented with 200 mM NaCl and developed into normal plantlets. This rapid selection technology for generating salt-resistant tobacco mutants will be useful for the development of crop varieties resistant to environmental stresses.

Performing photosynthesis without β-carotene.

Research on mutant tobacco plants shows that a pigment called β-carotene is not necessary for photosynthesis.

NAC transcription factor involves in regulating bacterial wilt resistance in potato.

Bacterial wilt (BW) is a serious disease that affects potato (Solanum tuberosum L.) production. Although resistance to this disease has been reported, the underlying mechanism is unknown. In this study, we identified a NAC family transcription factor (StNACb4) from potato and characterised its structure, function, expression, its localisation at the tissue and its role in BW resistance. To this end, the transgenic Nicotiana benthamiana Domin lines were generated in which the expression of NACb4 was constitutively upregulated or suppressed using RNAi. Different tobacco mutants were stained after inoculating with Ralstonia solanacearum to observe the cell death and callose deposition. The results indicated that StNACb4 could be upregulated under the induction of R. solanacearum, and salicylic acid, abscisic acid and methyl jasmonate could also induce the expression of StNACb4. Tissue localisation analysis indicated that its expression was tissue specific, and it was mainly in the phloem of the vascular system of stems and leaves. NbNACb4 gene silencing can enhance the sensitivity of tobacco to R. solanacearum; on the contrary, StNACb4 gene overexpression can enhance the tolerance of tobacco to R. solanacearum. Meanwhile, StNACb4 gene overexpression can induce cell death and callose deposition in tobacco. The upregulated expression of StNACb4 can also activate the StPR10 gene expression. Our results provide important new insights into the regulatory mechanisms of bacterial wilt resistance in potato.

Development of a PVY Resistant Flue-Cured Tobacco Line via EMS Mutagenesis of eIF4E

Recessive resistance against potyviruses, such as Potato virus Y (PVY), relies on mutations in the eukaryotic translation initiation factor 4E (eIF4E) or one of its isoforms. The eIF4E1-S mutants of burley tobacco (Nicotiana tabacum L.) exhibit recessive resistance against PVY strains. Here, we developed a TILLING population of flue-cured tobacco (N. tabacum cv. Yunyan87) using ethyl methanesulfonate (EMS) to identify eIF4E1-S mutants. M3 plants homozygous for a nonsense mutation in exon 1 of the eIF4E1-S gene demonstrated resistance against PVYMN. These M3 plants were backcrossed to ‘Yunyan87’, and BC4F3 plants were screened using derived cleaved amplified polymorphic sequence (dCAPS) markers. BC4F3 plants showing agronomic traits comparable to the recurrent parent ‘Yunyan87’ and resistance against PVYO, PVYN, and PVYNTN strains were identified. These genotypes would provide useful germplasm for future tobacco improvement and would aid in basic research on PVY resistance in flue-cured tobacco.

Mitochondrial complex I dysfunction increases CO2 efflux and reconfigures metabolic fluxes of day respiration in tobacco leaves.

Mutants affected in complex I are useful to understand the role played by mitochondrial electron transport and redox metabolism in cellular homeostasis and signaling. However, their respiratory phenotype is incompletely described and a specific examination of day respiration (Rd ) is lacking. Here, we used isotopic methods and metabolomics to investigate the impact of complex I dysfunction on Rd in two respiratory mutants of forest tobacco (Nicotiana sylvestris): cytoplasmic male sterile II (CMSII) and nuclear male sterile 1 (NMS1), previously characterized for complex I disruption. Rd was higher in mutants and the inhibition of leaf respiration by light was lower. Higher Rd values were caused by increased (phosphoenol)pyruvate (PEP) metabolism at the expense of anaplerotic (PEP carboxylase (PEPc) -catalyzed) activity. Denovo synthesis of Krebs cycle intermediates in the light was larger in mutants than in the wild-type, although numerically small in all genotypes. Carbon metabolism in mutants involved alternative pathways, such as alanine synthesis, and an increase in amino acid production with the notable exception of aspartate. Our results show that the alteration of NADH re-oxidation activity by complex I does not cause a general inhibition of catabolism, but rather a re-orchestration of fluxes in day respiratory metabolism, leading to an increased CO2 efflux.

Transcript profiling of salt tolerant tobacco mutants generated via mutation breeding

The main aim of the study is to identify the genes differentially, predominantly or specifically expressed in salt tolerant tobacco mutants, improved from Akhisar 97 and İzmir Özbaş varieties via mutation breeding, with respect to unstressed control plants. Seven tobacco mutants which have different salt tolerance capacities were evaluated by Gene Fishing analysis. Under stress conditions differentially expressed 100 reproducible bands were identified (74 of up-regulated and 20 of down-regulated while 6 were unknown). 75 of differentially expressed genes (DEGs) were successfully extracted from the gel and sequence analyses were performed. Functional annotation of the DEGs was performed against Blastn by interrogating their sequences. The 65 salt-regulated differentially expressed genes showed similarity with known genes, while 6 of DEGs didn't show any genetic similarities with known genes. DEGs were classified in eleven functional categories involving the abiotic stress response, biotic stress response, energy metabolism, cellular transport, catalitic activity, protein modification, amino acid metabolism and transcription factors. All the mutants were evaluated for their regulatory mechanisms against salt stress. The current data reveal that these six DEGs should be identified by next generation sequencing techniques and functional analysis should be design to understand the role of these six differentially expressed genes of tobacco mutants in further studies to improve new genetic resources.

Shine-Dalgarno Sequences Play an Essential Role in the Translation of Plastid mRNAs in Tobacco.

In prokaryotic systems, the translation initiation of many, though not all, mRNAs depends on interaction between a sequence element upstream of the start codon (the Shine-Dalgarno sequence [SD]) and a complementary sequence in the 3' end of the 16S rRNA (anti-Shine-Dalgarno sequence [aSD]). Although many chloroplast mRNAs harbor putative SDs in their 5' untranslated regions and the aSD displays strong conservation, the functional relevance of SD-aSD interactions in plastid translation is unclear. Here, by generating transplastomic tobacco (Nicotiana tabacum) mutants with point mutations in the aSD coupled with genome-wide analysis of translation by ribosome profiling, we provide a global picture of SD-dependent translation in plastids. We observed a pronounced correlation between weakened predicted SD-aSD interactions and reduced translation efficiency. However, multiple lines of evidence suggest that the strength of the SD-aSD interaction is not the only determinant of the translational output of many plastid mRNAs. Finally, the translation efficiency of mRNAs with strong secondary structures around the start codon is more dependent on the SD-aSD interaction than weakly structured mRNAs. Thus, our data reveal the importance of the aSD in plastid translation initiation, uncover chloroplast genes whose translation is influenced by SD-aSD interactions, and provide insights into determinants of translation efficiency in plastids.

RNA-sequencing predicts three novel genes in response to drought in electron beam irradiation treated tobacco

Electron beam irradiation (EBI) has protective effects on food storage. This study aimed to investigate drought tolerance of tobacco after EBI treatment. Nicotiana tabacum L. seeds were irradiated with different doses of EBI. Germination percentage (GP) was counted. Phenotype of EBI-treated M1 generation was observed in a drought field. The candidate drought-resistant tobacco line was selected and then drought treated without watering for 8 and 16 days, respectively. Samples at 8 days were collected for RNA-sequencing on the Illumina HiSeqTM 2500 platform. Then, differentially expressed genes (DEGs) were selected, followed by functional enrichment analysis. EBI with 500 Gy was selected as the optimal dose. Five lines: M1-7, M1-9, M1-10, M1-12, and M1-40 were candidate drought-resistant lines based on phenotype observation. Among them, M2-12 and M2-40 had high GPs. By comparing their M1 phenotypes, M2-12 was identified as the final candidate drought-resistant line. After drought treatment, M2-12 showed a wavier leaf margin than control and kept a well growth condition during the whole period. A total of 3547 DEGs were identified, and the up-regulated DEGs were significantly enriched in functions like “response to water deprivation (LOC107782385)”, “response to gibberellin (LOC104231633, LOC104219851)”, and “response to jasmonic acid (JA) (LOC104219851)”. Results of qRT-PCR confirmed that LOC104231633, LOC107782385, and LOC104219851 were up-regulated under drought stress. In conclusion, EBI may be a promising way to generate drought tolerant mutant tobacco. The above three genes may be drought-inducible and may involve in JA- and gibberellin-mediated signaling pathways.