Tobacco (Nicotiana tabacum, 2n = 48) is a key non-food economic crop, yet its stress response and gene regulatory mechanisms remain poorly understood. By analyzing 603 transcriptome datasets, this study identified 1405 tissue-specific genes, revealing tissue-specific synthesis of terpenoids and other ecologically important secondary metabolites in sepals and other tissues. Comparative stress-response analysis highlighted distinct gene expression patterns in leaves and roots under biotic and abiotic stresses. Additionally, 28,396 expression quantitative trait loci (eQTLs) were mapped in leaves, offering valuable genetic regulatory markers. These findings provide crucial insights into tobacco's gene expression characteristics and their functional implications, serving as a foundation for future research.

Antimicrobial peptides (AMPs) have emerged as a potential novel class of antimicrobial agents due to their broad microbial targeting and low resistance risks. Although AMPs have limited applications in agriculture, their potential to replace chemical pesticides could address food security and environmental concerns. Members of Bacillus sp., abundant in soil and plant microbiomes, are recognized as important sources of AMPs for their resistance and strong antimicrobial properties, making them ideal candidates for biocontrol in sustainable agriculture. To harness this potential, this study employed deep learning models to predict AMPs derived from Bacillus genomes, aiming to identify candidates with high activity against phytopathogens. Subsequently, the antimicrobial efficacy of selected candidate AMPs was experimentally validated. More than 6700 Bacillus genomes were collected to identify a broad range of short peptides (10–100 amino acids), which were analyzed using advanced deep learning models, including BERT, Mamba, CNN-LSTM, and CNN-Attention. These models demonstrated enhanced predictive accuracy and reliability over existing methods, and resulted in 4,993,389 potential AMPs from Bacillus genomes. Among these AMPs, two high-confidence AMPs (cAMP_1 and cAMP_2) were selected by cross-validation, and their structural stability and activity were evaluated and verified by molecular dynamics simulations and experimental assays, respectively. Both of them exhibited antimicrobial activity against Escherichia coli, Staphylococcus aureus, and various common agricultural fungal and bacterial pathogens. This high-throughput deep learning pipeline successfully uncovered novel AMPs from Bacillus genomes, which underscored the efficiency of deep learning models in identifying functional peptides. This approach could accelerate the discovery of potential AMPs for biocontrol applications in plant disease management, contributing to sustainable agriculture and reduced dependency on traditional antibiotics.

Nicotiana tabacum (2n = 4x = 48), an economically important non-food crop and a model plant for genetic studies, faces challenges in efficient genotyping of novel germplasm. To address this, we developed the Ta-LD-SC, a 20K SNP Affymetrix Axiom array, based on resequencing data from 150 tobacco accessions. A total of 20,213 unique SNPs were carefully selected, achieving coverage of over 90% of the tobacco genome (Nitab4.5 and NtaSR1) with a uniform probe distribution, limiting density to no more than 5 SNPs per 200 kb. The array underwent extensive validation using 866 tobacco accessions (NP panel) and 288 F2 individuals from a cross between K326 and Oxford 26 (GP panel). Performance metrics demonstrated its robustness, with high SNP call rates (93.6%–99.8%), a low technical error rate (< 1%), and a superior PolyHighResolution SNP rate (79.79%) compared to other crop SNP arrays. Population structure analysis of the NP panel revealed two major introductions of foreign germplasm that have significantly influenced the genetic diversity of Chinese tobacco resources. Using the array, a genome-wide association study (GWAS) identified 62 genes linked to eight agronomic traits, and a high-density genetic map encompassing 4553 SNPs across 6606.08 cM was constructed. The Ta-LD-SC array provides a valuable tool for rapid, high-quality genotyping offering supporting marker annotations that may benefit genetic research and breeding of tobacco.

Alternative splicing (AS) expands the transcriptome diversity by selectively splicing exons and introns from pre-mRNAs to generate different protein isoforms. This mechanism is widespread in eukaryotes and plays a crucial role in development, environmental adaptation, and stress resistance. In this study, we collected 599 tobacco RNA-seq datasets from 35 projects. 207,689 transcripts were identified in this study, of which 35,519 were annotated in the reference genome, while 172,170 transcripts were newly annotated. Additionally, tissue-specific analysis revealed 4,585 transcripts that were uniquely expressed in different tissues, highlighting the complexity and specialization of tobacco gene expression. The analysis of AS events (ASEs) across different tissues showed significant variability in the expression levels of ASE-derived transcripts, with some of these transcripts being associated with stress resistance, such as the geranyl diphosphate synthase (GGPPS). Moreover, we identified 21,763 splicing quantitative trait locus (sQTLs), which were enriched in genes involved in biological processes such as histone acetylation. Furthermore, sQTLs involved genes related to plant hormone signal transduction, terpenoid backbone biosynthesis, and other resistance pathways. These findings not only reveal the diversity of gene expression in tobacco but also provide new insights and strategies for improving tobacco quality and resistance.

Tobacco bacterial wilt, caused by Ralstonia solanacearum, severely affects tobacco crops, leading to significant economic losses. We performed integrated transcriptomic and metabolomic analyses to identify key genes and metabolites that govern tobacco's resistance to bacterial wilt. Resistant (Qiongzhongwuzhishan, R1), moderately resistant (Fandisanhao-bing, R2), and susceptible (Honghuadajinyuan, S) genotypes were sampled at 3, 24, and 48 h post-inoculation. RNA-seq identified 16,295 differentially expressed genes (DEGs) (FDR < 0.05, |log(2)FC= > 1). In R1 (treated vs. control), 3046, 2332, and 2003 genes were upregulated, while 1403, 3099, and 2895 genes were downregulated at 3 h, 24 h, and 48 h, respectively. In R1 (treated) vs. R2 (treated), 1900, 3867, and 3553 genes were upregulated, while 1321, 6818, and 3490 genes were downregulated at 3 h, 24 h, and 48 h, respectively. Similarly, in R1 (treated) vs. S (treated), 1236, 1333, and 1689 genes were upregulated, and 836, 2496, and 1292 genes were downregulated at the corresponding time points. Time-course analysis using maSigPro identified 11,121 genes with significant temporal expression changes. Metabolomic profiling detected 69 differentially accumulated metabolites, from which the 20 most significant (p < 0.05) were selected. Weighted Gene Co-expression Network Analysis (WGCNA) clustered genes into 17 modules; five were significantly correlated with key metabolites and overlapped with DESeq2 and maSigPro gene sets. A total of 39 candidate genes were consistently identified through differential expression, functional enrichment, and network analyses, and six were validated by qRT-PCR. These results provide a foundational framework for future studies into the genetic architecture and molecular basis of bacterial wilt resistance in tobacco.

Based on kompetitive allele specific PCR（KASP）technology platform, the tobacco SNP（single nucleotide polymorphism）markers and corresponding primers were developed and validated,which could assist tobacco germplasm evaluation, genetic diversity analysis,and core germplasm screening. Using Python and Perl tools, 1 179 154 SNPs covering tobacco genome were designed and screened for KASP primers, and their accuracy and applicability were verified by experiment. As results, 217 621 SNPs were enough for designing corresponding KASP primers. Then 1 378 SNPs were selected for experimental validation, and 732 were approved for SNP marker. Further 48 SNP markers,with an average PIC of 0.36 and an average MAF of 0.39, were finally identified as core markers, and they were evenly distributed on 24 chromosomes of the tobacco genome. The various germplasm, especially the genotypes of current major tobacco cultivars could be distinguished by the 48 core SNPs, and the marking is of extremely high reliability.

Potassium (K) plays important roles in the energy andsubstanceconversion of tobacco metabolism and is also regarded as one of theimportant indicators of tobacco quality evaluation. However, the Kquantitative analytical method shows poor performance in terms ofbeing easy-to-use, cost-effective, and portable. Here, we developeda rapid and simple method for the determination of K content in flue-curedtobacco leaves, including water extraction with 100 degrees C heating,purification with solid-phase extraction (SPE), and analysis withportable reflectometric spectroscopy based on K test strips. The methoddevelopment consisted of optimization of the extraction and test stripreaction conditions, screening of SPE sorbent materials, and evaluationof the matrix effect. Under the optimum conditions, good linearitywas observed in 0.20-0.90 mg/mL with a correlation coefficient>0.999. The extraction recoveries were found to be in the rangeof98.0-99.5% with a repeatability and reproducibility of 1.15-1.98%and 2.04-3.26%, respectively. The sample measured range wascalculated to be 0.76-3.68% K. Excellent agreement was foundin accuracy between the developed reflectometric spectroscopy methodand the standard method. The developed method was applied to analyzethe K content in different cultivars, and the content varied greatlyamong the samples with lowest and highest contents for Y28 and Guiyan5 cultivars, respectively. This study can provide a reliable approachfor K analysis, which may become available on-site in a quick on-farmtest.

为提高抗PVY烟草品种的分子育种效率,本研究针对抗病种质资源半坤村晒烟中隐性抗病基因eIF4E1的SNP位点G149C建立一种简单快速的双向等位基因特异性PCR(Bi-directional PCR amplification of specific alleles,Bi-PASA)检测方法,并对该检测方法的特异性、准确度及实际应用效果进行验证.结果表明,建立的Bi-PASA检测方法能够在一个PCR反应中有效区分eIF4E1基因G149C位点3种基因型:野生型GG、杂合突变型GC、纯合突变型CC.利用Bi-PASA检测方法可以对K326×半坤村晒烟F2分离群体的基因型进行有效鉴别,且鉴定结果与普通的等位基因特异性PCR(allele-specific PCR,AS-PCR)以及直接测序法的鉴定结果一致.综上所述,本研究建立的Bi-PASA检测方法特异性强、准确度高、操作简便,可更好地应用于eIF4E1基因的分子标记辅助育种.

为提高烟草白粉病隐性抗病基因(感病基因)NtMLO1(M1)和NtMLO2(M2)在分子标记辅助选择育种中基因型的选择效率,根据已报道的M1和M2突变基因序列,设计4对单基因特异性引物,组成2个双重PCR反应体系,分别用于同时扩增M1和M2突变型或M1和M2野生型等位基因.结果表明,建立的2个双重PCR体系扩增的特异性片段与单重PCR体系扩增的片段完全吻合,突变基因纯合体只在突变基因多重PCR体系中产生191和306 bp的特异性片段,野生基因纯合体只在野生基因多重PCR体系中产生437和652 bp的特异性片段,杂合体在2个反应体系中均有特异性片段.利用该体系可经过1次或2次PCR扩增准确检测出回交转育过程中回交或自交后代M1和M2的基因型,加快抗病品种育种进程.

The tobacco eIF4E1.S gene is a well-recognized susceptibility factor for potato virus Y (PVY) and a dominant target locus for current PVY resistance breeding in tobacco. In this study, over 900 tobacco landraces from China were screened for resistance to PVY and the DNA sequence of eIF4E1.S was analysed for all resistant accessions. Eight PVY-resistant lines were identified from this screen, and a naturally occurring mutant allele of eIF4E1.S carrying a single nucleotide insertion was discovered in seven, including a landrace called Kaiyangxiaoheiyan (Kaixiao), and designated eIF4E1.Kai. Knowledge of the mutant gene sequence enabled the development of a codominant PCR-based marker that can distinguish eIF4E1.S/eIF4E1.Kai heterozygotes from both homozygous classes. Using this marker, we genotyped F-2 plants segregating for the eIF4E1.Kai mutant and confirmed the relationship between genotype and phenotype. The results described here provide valuable genetic insights into Chinese tobacco landraces as a source of naturally occurring PVY resistance, as well as a powerful tool for the molecular marker-assisted breeding of tobacco varieties containing the resistance source eIF4E1.Kai.

[背景和目的]广东连江县晒烟品种"塘蓬烟"是我国特有的烟草隐性遗传白粉病抗性种质资源,但到目前为止对其抗病机制未见深入报道.[方法]利用塘蓬烟花粉与抗白粉病烟草品种Kutsaga E1及感病品种K326杂交进行抗病基因的等位性检测;利用等位基因分子标记检测塘蓬烟中感白粉病基因NtMLO1/2的突变情况;利用PCR克隆技术获得塘蓬烟NtMLO1/2基因的eDNA和gDNA序列信息,明确基因突变类型.[结果](1)根据等位性测验结果推断塘蓬烟白粉病抗性由NtMLO1/2隐性突变基因控制.(2)等位基因分子标记检测和PCR克隆测序结果发现塘蓬烟NtMLO2基因的转录本发生外显子重复突变,形成一个新的可变剪接变异体.[结论]本研究从分子水平阐述了地方烟草品种塘蓬烟的抗白粉病机制,其结果不仅丰富了烟草抗白粉病基因资源信息,而且为日后抗性基因资源的深入研究和合理利用提供了参考依据.

Studying the genome evolution of Nicotiana has been hindered by the small chromosome and complex chromosomal number. Consequently, studying the chromosomal structure is a very important work in clarifying the genome evolution of genus Nicotiana. In the present study, the 18S and 5S rDNA sites of eight Nicotiana species were physically mapped by double-probe FISH. Results showed the following: 1) each species (including the S-genome and T-genome of N. tabacum) had one or two pairs of 5S rDNA sites and chromosome VIII of each species had one pair of 5S rDNA sites in the interstitial region of the long arm; 2) each species (including the S-genome and T-genome of N. tabacum) had one, two or three pairs of 18S rDNA sites; 3) the chromosomal distribution of 5S and 18S rDNA was same between N. setchellii, N. kawakamii and N. otophora, and between N. glutinosa and N. tomentosa; and the chromosomal distribution of 5S and 18S rDNA of N. sylvstris and N. tomentosiformis was same with the S-genome and T-genome of N. tabacum, respectively. Finally, the genome evolutions of the genus Nicotiana were inferred. The present results could provide obvious references for studies on the system and phylogenesis of genus Nicotiana and were of significance to genetic breeding and germplasm innovation for tobacco.

Potato virus Y (PVY) is one of the most problematic pathogens affecting tobacco production. Genetic resistance to PVY in cultivated tobacco is mediated by recessive mutations, including the locus designated VAM, and its derived mutant va, both of which were previously shown to contain large chromosomal deletions. Among the genes deleted in lines containing VAM or va is eIF4E1.S, a gene whose protein product has been shown to facilitate infection by certain potyviruses, including PVY. Because the extent of the deletions and the exact nature of their specific breakpoints have not been precisely established for VAM or va, it has not been possible to develop co-dominant markers to facilitate their transfer to elite varieties using molecular breeding technologies. Here, we report the discovery of a novel naturally-occurring allele of eIF4E1.S from the Chinese tobacco landrace Fuquanliuye, designated eIF4E1.Fu, which lacks a genomic region of approximately 27 kb, including the 3'-end of eIF4E1.S. Characterization of the deletion junction enabled the development of a co-dominant PCR-based marker specific for eIF4E1.Fu, which can distinguish eIF4E1.S/eIF4E1.Fu heterozygotes from both homozygous classes. Using this co-dominant marker, we genotyped F2 plants segregating for the mutant eIF4E1.Fu allele and confirmed the correlation between genotype and phenotype. This study describes a novel source of resistance and an ideal co-dominant marker that can be applied in breeding for resistance to PVY and other potyviruses.

[背景和目的]从作物种质资源中进行等位基因鉴定,特别是优异等位基因的发掘和应用,是使种质资源加速转变为基因资源的关键所在.[方法]采用苗期汁液摩擦接种的方法,对烟草种质资源的马铃薯Y病毒(Potato virus Y,PVY)抗病性进行鉴定;通过烟草隐性抗PVY基因eIF4E1(又称为感PVY基因eIF4E1)等位性测验、等位基因分子标记检测和RT-PCR扩增测序,对PVY抗病资源进行基因型分析.[结果](1)从收集到的900多份来自国内外的烟草种质资源中筛选出19份高抗PVY资源.(2)根据等位性测验结果推断19份抗源所具有的PVY抗性均由eIF4E1基因所控制.(3)等位基因分子标记检测和RT-PCR扩增测序结果将19份抗源的eIF4E1基因区分为4种突变类型.[结论]本研究结果丰富了我国对抗PVY烟草种质资源的筛选和利用的内容,为后续烟草PVY抗性优异等位基因的发掘和育种利用提供了基础材料和信息支撑.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Glandular trichomes, a specialized multicellular structure, are considered as biofactories due to their capability to synthesize and secrete a large amount of secondary metabolites. Tobacco leaves have a high density of glandular trichomes that produce huge amounts of secondary metabolites, which can be used as important industrial raw materials. However, molecular mechanism controling glandular trichome development in tobacco still remains largely unknown. In the present study, we found that NbGL3, an HD-Zip IV family gene from Nicotiana benthamiana, was highly expressed in mature leaves, and ethylene or auxin application could increase the expression of NbGL3. Virus induced gene silencing NbGL3 resulted in a decreased glandular trichome density on leaves. Putative downstream genes, such as micro trichome, cyclin B2, and wax inducer 1 like were down regulated in NbGL3 silenced plants. Our results demonstrate that NbGL3 could positively regulate initiation of glandular trichomes in tobacco.

Tobacco is one of the most widely cultivated nonfood cash crops, a source of income, model organism for plant molecular research, a natural pesticide and of pharmaceutical importance. First domesticated in South Americas, the modern-day tobacco (Nicotiana tabacum) is now cultivated in more than 125 countries to generate revenues worth billions of dollars each year. However, the production of this crop is highly threatened by the global presence of devastating infectious agents, which cause huge fiscal loss. These threats have been battled through breeding for acquiring disease resilience in tobacco plants, first, via conventional and now with the use of modern molecular breeding approaches. For efficacy and precision, the characterization of the genetic components underlying disease resistance is the key tool in tobacco for resistance breeding programs. The past few decades have witnessed significant progress in resilience breeding through advanced molecular techniques. The current review discusses history of tobacco breeding since its time of origin till date, highlighting the most widely used techniques and recent advances in molecular research and strategies for resistance breeding. In addition, we narrate the budding possibilities for the future. This review will provide a comprehensive and valuable information for the tobacco growers and researchers to deal with the destructive infectious diseases.

AP2/ERF类转录因子,是植物所特有的最大的一类转录因子家族,在植物的生长发育过程中,扮演着重要的角色.探究花烟草ERF转录因子的生理功能,为花烟草抵御逆境的分子机制研究提供借鉴.采用同源克隆的方法进行基因克隆.通过对花烟草进行非生物胁迫,运用qPCR的方法进行基因表达模式分析.从花烟草(Nicotiana alata)中克隆了一个属于ERF家族的基因NaERF1.该基因的开放阅读框全长为819 bp,编码了272个氨基酸.生物信息学分析结果表明,该基因编码的蛋白分子量为30.7 kD,等电点为6.07;具有AP2/ERF类转录因子家族典型的保守结构域;该基因主要定位于细胞质内,并含有多个磷酸化位点.同源性分析的结果显示,NaERF1基因与茄科植物的ERF同源性较高,并且与普通烟草的ERF亲缘关系最近.NaERF1基因的表达具有组织表达特异性,花中表达量最高,茎中次之,根和叶中表达量较低.同时,在高盐、干旱、低温、ABA、低钾及H2O2等非生物胁迫下,NaERF1的表达呈现5种模式.其中,对低钾及ABA胁迫的响应强烈.NaERF1基因属于AP2/ERF类转录因子,可能广泛参与了花烟草包括非生物胁迫响应在内的众多生理过程.

为了给烟草碳酸酐酶的功能研究提供理论依据,本研究采用同源克隆的方法,从普通烟草(Nicotianatabaccum)中克隆得到一个属于α碳酸酐酶家族的烟草碳酸酐酶基因NtCA 7.该基因CDS全长810 bp,编码了一个由270个氨基酸组成的蛋白.该蛋白分子量为31.03 kD,等电点为8.95,定位在细胞质基质中,属于亲水性蛋白,存在信号肽与磷酸位点.同源性分析结果显示,NtCA7基因与其它茄科植物的氨基酸序列相似度较高.NtCA7基因在烟草根、茎、叶、花中均有表达,其中,在叶中表达量最高.同时,NtCA7基因响应高盐、干旱、低钾、ABA、低温及H2O2的诱导表达.本研究结果表明,NtCA7基因可能在烟草非生物胁迫生理响应过程中发挥作用,对关于增强烟叶抗逆性,提高烟叶产质量的进一步研究有着重要意义.

[Introduction] Seven smoke constituents, including hydrogen cyanide (HCN), ammonia (NH3), phenol, benzo[alpha] pyrene (B[a]P), carbon monoxide (CO), crotonaldehyde, and 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanone (NNK), are proposed be the most relevant constituents for smoking-related diseases. [Methods] Different combinations of leaf stalk positions, varieties and locations were used to create variable chemistry of cigarette filler and smoke. Experimental cigarettes were measured for emission level of seven smoke toxicants and content of seventy-three filler components. [Results] The ranges of coefficient of variation (CV) for seven smoke toxicants were 15.435%-43.15%. The emission pattern of NNK and crotonaldehyde were different from that of other five smoke toxicants. Most of the seven smoke toxicants were influenced in following order: stalk position > location > variety. The leaf constitutes closely correlated with seven smoke toxicants were analyzed. [Conclusions] The results showed that seven toxicants were significantly influenced by leaf position and location, and closely correlated with leaf components, such as potassium, malate and alkaloid contents. The results provide useful and comprehensive information on the affecting factors and correlating leaf constituents for the variations of seven smoke toxicants.