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.

Tomato spotted wilt virus (TSWV) is one of the most destructive viral pathogens of plants. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was identified in Nicotina alata and introgressed into cultivated tobacco (N. tabacum). However, whether the TSWV carries an avirulence (Avr) factor directed against RTSW remains obscure. In the present study, we identified the non-structural protein (NSm), the movement protein of TSWV, which is an RTSW-specific Avr factor, by using two different transient expression systems. Using amino acid (aa) substitution mutants, we demonstrated the ability to induce RTSW-mediated hypersensitive response (HR) of NSm is independent of its movement function. Moreover, key substitutions (C118Y and T120N), a 21-aa viral effector epitope, and different truncated versions of NSm, which are responsible for the recognition of the Sw-5b resistance gene of tomato, were tested for their ability to trigger HR to TSWV in tobacco. Together, our results demonstrated that RTSW-mediated resistance is triggered by NSm in the same way as by Sw-5b, however, via different elicitor active sites. Finally, an Avr gene-based diagnostic approach was established and used to determine the presence and effectiveness of resistance genes in tobacco.

Introduction: Carotenoids are important precursors of various aroma components in tobacco and play an important role in the sensory quality of tobacco. Phaffia rhodozyma is a species of Xanthophyllomyces capable of synthesizing a highly valuable carotenoid-astaxanthin, but has not yet been used in improving tobacco quality.Methods: The dynamic changes of microbial community and metabolites during tobacco fermentation were analyzed in combination with microbiome and metabolome, and the quality of tobacco after fermentation was evaluated by sensory scores.Results: P. rhodozyma could grow and produce carotenoids in tobacco extract, with a maximum biomass of 6.50 g/L and a maximum carotenoid production of 36.13 mg/L at 100 g/L tobacco extract. Meanwhile, the correlation analysis combined with microbiome and metabolomics showed that P. rhodozyma was significantly positively correlated with 11 metabolites such as 6-hydroxyluteolin and quercetin. Furthermore, the contents of alcohols, ketones and esters, which were important aromatic components in fermented tobacco, reached 77.57 μg/g, 58.28 μg/g and 73.51 μg/g, increasing 37.39%, 265.39% and 266.27% compared to the control group, respectively. Therefore, the aroma and flavor, and taste scores of fermented tobacco increased by 0.5 and 1.0 points respectively.Discussion: This study confirmed that P. rhodozyma fermentation could effectively improve the sensory evaluation of tobacco, and provided a novel microbial fermentation method to improve tobacco quality.

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.