Background Chilling stress severely compromises tobacco production by reducing leaf biomass accumulation and deteriorating leaf quality. Lipoxygenase (LOX) plays a key role in the oxidative metabolism of linoleic and linolenic acids, which serves as the initial step for jasmonic acid (JA) biosynthesis. However, its specific function in mediating cold-induced JA accumulation and cold tolerance in Nicotiana tabacum remains elusive. Results Our results found that cold stress significantly induces NtLOX2 expression in N. tabacum. Transgenic tobacco plants overexpressing NtLOX2 (OE#NtLOX2-1 and OE#NtLOX2-2) exhibited improved cold tolerance, which was associated with increased activities of antioxidant enzymes and improved reactive oxygen species (ROS) scavenging capacity. RNA-seq analysis revealed that NtLOX2-overexpressing lines displayed upregulation of key cold-responsive genes, including COR, ERF, LRR-RLK, GST, POD, DREB, and NCED. Furthermore, genes involved in JA biosynthesis, such as ADH and OPR1, were also upregulated, concomitant with elevated levels of both JA and MeJA as confirmed by metabolic profiling. Conclusions Our results demonstrate that NtLOX2 enhances cold tolerance by stimulating JA biosynthesis and activating a downstream transcriptional network of cold-responsive and antioxidant genes. These findings posit NtLOX2 as a promising genetic target for improving cold tolerance in crops.

Lipoxygenase (LOX) catalyzes the oxidation of linoleic acids and linolenic acids, yet their specific functions in carotenoid metabolism and regulation of methyl jasmonate (MeJA) biosynthesis in Nicotiana tabacum remain poorly understood. Expression characterization revealed LOX2 grouped in 13-LOX subfamily as the dominant isoform in N. tabacum, exhibiting strong induction upon MeJA treatment. Virus-induced gene silencing of LOX2 in N. benthamiana increased carotenoid contents, including β-carotene, lutein, violaxanthin, and neoxanthin, along with elevated levels of chlorophyll a and chlorophyll b. Conversely, LOX2-overexpression transgenic lines (OE#LOX2-1 and OE#LOX2-2) exhibited reduced carotenoid content, and enhanced emission of volatiles compounds. Furthermore, these transgenic lines showed elevated 2-Hexenal and MeJA levels compared to wild-type plants, accompanied by upregulated expression of key biosynthetic genes. Our results indicates that LOX2 not only co-oxidizes carotenoids but also critically modulates MeJA biosynthesis, establishing a functional connection between carotenoid degradation and MeJA signaling. Targeted manipulation of LOX2 could serve as a promising strategy for enhancing stress resilience and flavor quality in tobacco.

It has been known for many years that ethylene represses Jasmonic acid (JA)-induced nicotine biosynthesis. However, it is unclear whether ethylene (ET) alone can inhibit nicotine biosynthesis, and if so, how it achieves this. In this study, we found that ethylene alone can suppress nicotine accumulation and the expression of nicotine biosynthesis and transport genes in tobacco. We then performed transcriptome analysis to explore the underlying molecular mechanism. Results showed that over 6000 differentially expressed genes (DEGs) after 2, 4, 8 and 24 h treatment of ethephon, an ethylene-releasing compound, were identified. GO and KEGG enrichment analysis revealed that DEGs were enriched in pathways related to nicotine biosynthesis, including “pyridine nucleotide metabolic process” and “pyridine-containing compound metabolic process” 4 h after ethephon treatment. Further analysis revealed that expression of key regulators of JA-mediated nicotine biosynthesis was significantly altered by ethephon treatment. Specifically, CORONATINE-INSENSITIVE 1 (COI1) genes COI1/COI1L and ETHYLENE RESPONSE FACTOR (ERF) genes ERF189/199, which are two major positive regulators of nicotine biosynthesis, were downregulated, whereas four JASMONATE-ZIM-DOMAIN (JAZ) genes JAZ1/2b/7a/10 were upregulated, indicating that ET suppresses nicotine biosynthesis, at least in part, by repressing the JA signaling pathway. In addition, we identified a set of ET-responsive candidate regulatory genes co-expressed with ERF189/199, providing a resource for future functional studies of nicotine biosynthesis regulation in tobacco roots.

The enzyme quinolinate phosphoribosyltransferase (QPT), encoded by a small gene family in tobacco plant, plays a critical role in the biosynthesis of nicotine, a defensive pyridine alkaloid in Nicotiana species, in addition to its vital function in the NAD(P)(H) synthesis. Previous studies have demonstrated that two NtQPT genes (NtQPT1 and NtQPT2) are present in N. tabacum genome, and it has been believed that NtQPT1 is responsible for NAD(P)(H) synthesis and thus essential for primary metabolism, while NtQPT2 is specifically involved in nicotine biosynthesis. In this study, we generated knockout tobacco lines for NtQPT1 and NtQPT2 respectively using the CRISPR/Cas9-based genome-editing technology and found that knockout of NtQPT2 caused both dramatic reduction of nicotine biosynthesis and a retardation of plant development, indicating that NtQPT2 is important not only to nicotine biosynthesis, but also to the development of tobacco plant. Like NtQPT2, NtQPT1 was also found to contribute to nicotine biosynthesis although to a much lesser extent than NtQPT2. Meanwhile, knockout of NtQPT1 did not significantly affect plant growth. Together with the observation that NtQPT2's expression is remarkably higher than that of NtQPT1 in root, leaf, stem and flower of tobacco plant, it is reasonable to infer that their functional diversification on nicotine biosynthesis and tobacco plant growth may be attributed largely to their markedly different transcript abundance.

Introduction Different types of tobacco cultivars have been used as raw material in heated tobacco to maximize the sensory richness. However, the volatile profiles of different tobacco cultivars and their correlation with the sensory quality of heated tobacco remain unknown. Methods In this study, untargeted metabolomics profiling followed by partial least squares-discriminant analysis (VIP>1 and p<0.05) was performed to identify 446 and 445 volatile metabolites that were statistically different among three tobacco cultivars in the reconstituted leaf and the aerosol, respectively. Flue-cured tobacco K326 was rated the highest in the sensory evaluation, followed by air-cured tobaccos Badahe and Leye. Results Correlation analysis revealed that 56 aerosol volatiles including aldehydes, alkenes, ketones, esters and other compounds have strong relevance with the sensory attributes. Conclusion The identified volatile compounds can be used to assist formula design of heated tobacco and establish sensory-related breeding objectives.

As the global cash crops, the formation of distinct aroma profiles in Nicotiana tabacum have garnered significant research interest. Lipids as precursors of aroma compounds in plants, play an essential role in defining the unique aroma characteristics of tobacco. In this study, lipidomics analysis was performed to identify a total of 471 lipid components in leaves of strong fragrance tobacco (SF) and mild fragrance tobacco (MF). Comparative analysis of SF and MF revealed 70 differentially changed lipid compounds, with 26 up-regulated and 44 down-regulated lipids. The up-regulated lipids were predominantly glycoglycerolipids (GL) and glycerophospholipids (GP), while the down-regulated lipids were enriched in sphingolipids (SP) and fatty acyls (FA). Gas chromatography-mass spectrometry (GC-MS) revealed that SF exhibited relatively higher levels of cis-3-hexenal and cis-3-hexenol compared to MF. Transcriptomics analysis identified a total of 905 differential expression genes (DEGs), with 370 up-regulated and 535 down-regulated DEGs. Notably, a robust correlation was observed between the expression of the key gene lipoxygenase 5 (LOX5) and content of phosphatidylcholine (PC) (15:0_18:3), a precursors of linolenic acid. Additionally, a positive correlation was detected between LOX5 expression and the degradation products of linolenic acid, including cis-3-hexenal and cis-3-hexenol. Transient over-expression of LOX5 in tobacco leaves resulted in an enhanced accumulation of these linolenic acid degradation products. Our findings elucidate the mechanism underlying the formation of diverse aroma profiles in tobacco, and provide a theoretical basis for breeding high-quality tobacco crop.

BACKGROUND: Amino acids on the surface of enzymes play a vital role in their catalytic activity and functional properties, influencing enzymatic stability and inducing conformational changes. RESULTS: In this research, we demonstrate that surface mutations in geranylgeranyl diphosphate synthase1 from Nicotiana tabacum (NtGGPPS1) improve carotenoid biosynthesis and increase drought tolerance. We introduced three mutations at key surface sites: the active center V154A, the enzyme surface site V233E, and the surface complex sites V233E/I209S, creating transgenic lines OE#V154A, OE#V233E and OE#V233E/I209S, respectively. Interestingly, under standard tobacco cultivation conditions, these overexpression lines displayed improved growth and development phenotypes relative to wild-type plants, characterized by enhanced pigmentation, whereas the ntggpps1 mutant materials exhibited the opposite trends. Our studies showed that mutations in the enzyme surface sites promote plant growth, upregulate carotenoid biosynthesis-related genes, and increase drought resistance. Notably, the OE#V233E displayed significantly higher level expression of carotenoid biosynthesis genes compared to OE#NtGGPPS1 transgenic lines, effectively directing GGPP towards carotene and downstream abscisic acid production. Furthermore, it can modulate photosynthesis, promote plant growth and development, enhance antioxidant capacity, and improve drought tolerance. CONCLUSIONS: These findings provide new insights into GGPPS1 engineering and open avenues for developing drought-resistant crops. Our results pave the way for the structure-guided rational design and application of elite genes in higher plants.

Melatonin enhances photosynthesis efficiency in high plants. However, the underlying molecular mechanism remain largely unexplored. Our study provides novel insights into the multifaceted role of melatonin in Nicotiana tabacum, an important industrial crop. Foliar application of melatonin improved the photosynthetic capacity, promoted antioxidant enzymes activity, and enhanced tolerance to reactive oxygen species (ROS). Compared with control, melatonin-treated tobacco exhibited the increase in carotenoid contents including lutein, β-carotene, neoxanthin and violaxanthin, along with the higher levels of chlorophyll a and chlorophyll b. RNA-seq analysis indicated that melatonin robustly stimulated pathways related to carotenoid biosynthesis, biosynthesis of amino acids, photosynthesis, carbon fixation in photosynthetic organism, fatty acid biosynthesis, and glutathione metabolism. Co-expression network indicated the melatonin activated key transcription factors, including bHLH, WRKY, MYB, NAC, MADS6 and AP2/ERF. These transcription factors likely enhanced carotenoid biosynthesis and carbon fixation. Our results highlight the potential of melatonin as a novel eco-friendly agrochemical. Its application could address agricultural challenges by improving photosynthetic efficiency, thereby offering a sustainable solution for crop enhancement.

Tobacco cembranoids, known for their anti-insect and antifungal properties, were shown to be mainly present on the surface of leaves and flowers, being biosynthesized by their trichomes. It remains unclear whether they could be biosynthesized in other organs without trichomes. Cembratrien-ol synthases (CBTSs) catalyze the conversion of GGPP to CBT-ols and thus play an important role in cembranoid biosynthesis. This study identified the CBTS family genes in tobacco and examined their spatiotemporal expression patterns. The CBTS genes showed diverse expression patterns in tobacco organs, with the majority highly expressed in leaves and a few highly expressed in flowers. The expression of CBTS genes were also correlated with the development of tobacco plants, and most of them showed the highest expression level at the budding stage. Furthermore, their expression is mediated by the JA (jasmonate) signaling in all tobacco organs. Several CBTS genes were found to be highly expressed in tobacco roots that have no trichomes, which prompted us to determine the cembranoid production in roots and other organs. GC-MS and UPLC assays revealed that cembranoids were produced in all tobacco organs, which was supported by the bioactivity assay results that almost all these CBTS enzymes could catalyze CBT-ol biosyntheis in yeast, and that the content ratio of CBT-ols and CBT-diols in tobacco roots was different to that in leaves. This work sheds insights into the expression profiles of tobacco CBTS genes and provides a feasibility to engineer tobacco roots for industrial production of cembranoids.

Potassium ion (K+) is one of the most essential nutrients for the growth and development of tobacco (Nicotiana tabacum L.), however, the molecular regulation of K+ concentration in tobacco remains unclear. In this study, a two-pore K (TPK) channel gene NtTPKa was cloned from tobacco, and NtTPKa protein contains the unique K+ selection motif GYGD and its transmembrane region primarily locates in the tonoplast membrane. The expression of NtTPKa gene was significantly increased under low-potassium stress conditions. The concentrations of K+ in tobacco were significantly increased in the NtTPKa RNA interference lines and CRISPR/Cas9 knockout mutants. In addition, the transport of K+ by NtTPKa was validated using patch clamp technique, and the results showed that NtTPKa channel protein exclusively transported K+ in a concentration-dependent manner. Together, our results strongly suggested that NtTPKa is a key gene in maintaining K+ homeostasis in tobacco, and it could provide a new genetic resource for increasing the concentration of K+ in tobacco.

Stomatal movement can be regulated by ABA signaling through synthesis of reactive oxygen species (ROS) in guard cells. By contrast, ethylene triggers the biosynthesis of antioxidant flavonols to suppress ROS accumulation and prevent ABA-induced stomatal closure; however, the underlying mechanism remains largely unknown. In this study, we isolated and characterized the tobacco (Nicotiana tabacum) R2R3-MYB transcription factor NtMYB184, which belongs to the flavonol-specific SG7 subgroup. RNAi suppression and CRISPR/Cas9 mutation (myb184) of NtMYB184 in tobacco caused down-regulation of flavonol biosynthetic genes and decreased the concentration of flavonols in the leaves. Yeast one-hybrid assays, transactivation assays, EMSAs, and ChIP-qPCR demonstrated that NtMYB184 specifically binds to the promoters of flavonol biosynthetic genes via MYBPLANT motifs. NtMYB184 regulated flavonol biosynthesis in guard cells to modulate ROS homeostasis and stomatal aperture. ABA-induced ROS production was accompanied by the suppression of NtMYB184 and flavonol biosynthesis, which may accelerate ABA-induced stomatal closure. Furthermore, ethylene stimulated NtMYB184 expression and flavonol biosynthesis to suppress ROS accumulation and curb ABA-induced stomatal closure. In myb184, however, neither the flavonol and ROS concentrations nor the stomatal aperture varied between the ABA and ABA+ethylene treatments, indicating that NtMYB184 was indispensable for the antagonism between ethylene and ABA via regulating flavonol and ROS concentrations in the guard cells.

Introduction Heated tobacco (Nicotiana tabacum L.) products are heating tobacco plug at a temperature of 350°C and produce different emissions in aerosol and sensory perceptions of tobacco leaf compared with combustible tobacco. Previous study assessed different tobacco varieties in heated tobacco for sensory quality and analyzed the links between sensory scores of the final products and certain chemical classes in tobacco leaf. However, contribution of individual metabolites to sensory quality of heated tobacco remains largely open for investigation. Methods In present study, five tobacco varieties were evaluated as heated tobacco for sensory quality by an expert panel and the volatile and non-volatile metabolites were analyzed by non-targeted metabolomics profiling. Results The five tobacco varieties had distinct sensory qualities and can be classified into higher and lower sensory rating classes. Principle component analysis and hierarchical cluster analysis showed that leaf volatile and non-volatile metabolome annotated were grouped and clustered by sensory ratings of heated tobacco. Orthogonal projections to latent structures discriminant analysis followed by variable importance in projection and fold-change analysis revealed 13 volatiles and 345 non-volatiles able to discriminate the tobacco varieties with higher and lower sensory ratings. Some compounds such as β-damascenone, scopoletin, chlorogenic acids, neochlorogenic acids, and flavonol glycosyl derivatives had strong contribution to the prediction of sensory quality of heated tobacco. Several lyso-phosphatidylcholine and lyso-phosphatidylethanolamine lipid species, and reducing and non-reducing sugar molecules were also positively related to sensory quality. Discussion Taken together, these discriminating volatile and non-volatile metabolites support the role of leaf metabolites in affecting the sensory quality of heated tobacco and provide new information on the types of leaf metabolites that can be used to predict applicability of tobacco varieties for heated tobacco products.

Despite the nutritional and economic importance of carotenoids and their products, regulation of carotenoid content in leaves remains to be fully elucidated. Recent findings indicate that carotenoid content are determined, at least in part, by the activity of carotenoid cleavage dioxygenase 1 (CCD1). This study examined whether NtCCD1 affects leaf carotenoids in the model plant Nicotiana tabacum . Three NtCCD1s, NtCCD1a, NtCCD1b, and NtCCD1c, were investigated including their phylogenetic relationships, conserved motifs, and exon-intron architecture. Of the three transcripts, NtCCD1c exhibited the highest expression level in various tissue as determined by real-time PCR. Confocal microscopy indicated that all NtCCD1s are located in the cytoplasm. The enzymatic activity of the NtCCD1c protein was also studied by co-expressing NtCCD1c in Escherichia coli engineered to accumulate β-carotene or lycopene. SPME-GC-MS indicated that NtCCD1c cleaves β-carotene and lycopene specifically at the 9–10/9′-10′ site to produce β-ionone and pseudoionone, respectively. Virus-induced gene silencing (VIGS) of NbCCD1 increased carotenoid contents including β-carotene, α-carotene, zeaxanthin, phytoene, lutein, violaxanthin, neoxanthin, and β-cryptoxanthin, along with a decline in reactive oxygen species (ROS). RNA-seq of silenced plants suppressed NbCCD1 but enhanced expression of essential genes encoding antioxidant enzymes including NtGST, NtCuZnSOD, NtAAO, and NtPOD, along with a decrease in expression of genes related to carbon fixation such as NtRCA1, NtRCA2, and NtRBCS. These observations suggest that NtCCD1 is a negative regulator of carotenoid content and plays an essential role in the regulation of ROS levels in tobacco leaves.

Proanthocyanidins (PAs) are important phenolic compounds and PA biosynthesis is regulated by a ternary MBW complex consisting of a R2R3-MYB regulator, a bHLH factor and a WDR protein. In this study, a tobacco R2R3-MYB factor NtMYB330 was characterized as the PA-specific regulator in which the PA biosynthesis was promoted in the flowers of NtMYB330-overexpressing lines while decreased in the flowers of ntmyb330 mutants. NtMYB330 can interact with flavonoid-related bHLH partner NtAn1b and WDR protein NtAn11-1, and the NtMYB330-NtAn1b complex is required to achieve strong transcriptional activation of the PA-related structural genes NtDFR1, NtANS1, NtLAR1 and NtANR1. Our data reveal that NtMYB330 regulates PA biosynthesis in seeds and affects seed germination, in which NtMYB330-overexpressing lines showed higher PA accumulations in seed coats and inhibited germination, while ntmyb330 mutants had reduced seed coat PAs and improved germination. NtMYB330 affects seed germination possibly through two mechanisms: modulating seed coat PAs to affect coat-imposed dormancy. In addition, NtMYB330 regulates the expressions of abscisic acid (ABA) and gibberellin acid (GA) signaling-related genes, affecting ABA-GA crosstalk and seed germination. This study reveals that NtMYB330 specifically regulates PA biosynthesis via formation of the MBW complex in tobacco flowers and affects germination through adjustment of PA concentrations and ABA/GA signaling in tobacco seeds.

The biosynthesis and transport of nicotine has been shown to be coordinately upregulated by jasmonate (JA). MYC2, a member of basic helix-loop-helix (bHLH) transcription factor family, is well-documented as the core player in the JA signalling pathway to regulate diverse plant development processes. Four MYC2 genes were found in the tobacco genome, NtMYC2a/2b and 1a/1b. In this study, we tested whether one of them, NtMYC2a, acts as a 'master switch' in the regulation of nicotine biosynthesis and transport in tobacco. We generated NtMYC2a knockout tobacco plants using the CRISPR-Cas9 technique and analysed the effect of NtMYC2a knockout on expression of the nicotine biosynthesis genes (NtAO, NtQS, NtPMT1a, NtQPT2, NtODC2, NtMPO1, NtA622 and NtBBLa) and transport genes (NtMATE2 and NtJAT1), as well as leaf accumulation of nicotine in the NtMYC2a knockout plants. We found that all the nicotine biosynthesis and transport genes tested in this study were significantly downregulated (>50% reduction compared with wild-type control) in the NtMYC2a knockout plants. Moreover, the leaf nicotine content in knockout plants was dramatically reduced by ca 80% compared with the wild-type control. These results clearly show that NtMYC2a acts as a 'master switch' to coordinate JA-induced nicotine accumulation in tobacco and suggests that NtMYC2a might play an important role in tobacco nicotine-mediated defence against herbivory.

文章利用湖北省2015-2016年VLF/LF三维闪电监测定位系统的探测资料,分析了湖北省闪电的总闪、云闪和地闪的 日变化、月变化和密度变化特征.根据分析结果总结了湖北省云闪和地闪主要发生的时间、次数、比例、日变化特征的一致性、空间密度分布的高密度值区 和电流强度的主要分布.分析结果为业务人员预报预警提供了参考.

MAIN CONCLUSION:After tobacco topping, changes in the auxin content could affect K+ uptake by inhibiting the activity of K+ uptake-related genes through the NtARF genes, thus causing changes in K+ content. Tobacco (Nicotiana tabacum) is a valuable industrial and commercial crop, and the leaf is its primary product. Topping (removing apical buds) is a common agronomic practice that significantly improves the yield of tobacco leaves. Potassium (K+) plays an important physiological role in tobacco growth and leaf traits, including combustibility, aroma, and safety in cigarette products, and its levels are significantly decreased after topping. Here, to present global spatial-temporal gene expression profiles and gene regulatory networks of the core elements of K+ uptake, leaves and roots from topped and untopped plants at short- and long-term time points after topping were sampled for transcriptome analysis. We found that the wounding response was initiated in leaves in the early stages after topping. Then, in the long term, processes related to metabolism and transcription regulation, as well as ion binding and transport, were altered. The expression profiles showed that core elements of K+ uptake and xylem loading were drastically suppressed in roots after topping. Finally, transient expression experiments confirmed that changes in the auxin content could affect K+ uptake by inhibiting the activity of K+ uptake-related genes through the tobacco auxin response factor (NtARF) genes, thus causing changes in the K+ content. These results suggest that some ARFs could be selected as targets to enhance the expressions of K+ uptake transporters, leading to increment of K+ contents and improvement of leaf quality in tobacco breeding.

本研究以烤烟品种'云烟97'和'变异云烟97'为亲本,分别构建P1、P2、F1和F24个世代遗传群体,利用植物数量性状主基因+多基因混合遗传模型方法对烟草的株高、节距、叶数和茎围进行遗传及相关分析.结果 表明,株高分别与节距、叶数间呈极显著正相关,与茎围间呈显著正相关;而节距与叶数间则呈现极显著负相关.株高、节距和叶数3个性状具有相同的最优遗传模型,即2对加性-显性-上位性主基因+加性-显性多基因混合遗传模型(MX2-ADI-AD),其主基因遗传率分别为95.2258％、94.2854％和99.1771％,多基因遗传率分别为4.7742％、5.5684％和0.8229％;茎围性状符合2对加性-显性主基因+加性-显性多基因混合最优遗传模型(MX2-AD-AD),其主基因和多基因遗传率分别为90.3146％和9.6854％.上述性状均受主基因+多基因混合遗传模型控制,主基因遗传率均高达90％以上且远大于多基因遗传率,受环境影响可以忽略.因此,在烟草高产育种过程中针对上述农艺性状的定向选择宜在早期世代进行.

云烟119是以云烟87为母本、77089为父本,采用杂交育种方法选育而成的烤烟新品种.该品种田间生长整齐一致,生长势较强,株式塔型,叶形长椭圆,平均打顶株高127.4 cm,有效叶数22片左右,大田生育期平均为124.2 d,中抗黑胫病、根结线虫病;主要经济性状略优于对照K 326,各指标与对照的差异均不显著;初烤烟叶外观质量略优于对照K 326,物理特性与对照相当;各化学成分含量在适宜范围之内,协调性较好;感官质量接近于对照K 326.该品种是一个较能兼顾产量、质量和抗病性的烤烟新品种,适宜我国南方主产烟区种植.