Leaf stacking fermentation involves enzymatic actions of many microorganisms and is an efficient and environmentally benign process for degrading macromolecular organic compounds. We investigated the dynamics of metabolite profiles, bacterial and fungal communities and their interactions during fermentation using cigar leaves from three geographic regions. The results showed that the contents of total sugar, reducing sugar, starch, cellulose, lignin, pectin, polyphenol and protein in cigar tobacco leaves was significantly decreased during fermentation. Notably, the furfural, neophytadiene, pyridine, benzyl alcohol, geranylacetone, 3-hydroxy-2-butanone, N-hexanal, 3-Methyl-1-butanol and 2,3-pentanedione were important features volatile aroma compounds during fermentation. The α-diversity of fungi and bacteria initially increased and then decreased during fermentation. An analysis of variance showed that microbial diversity was influenced by fermentation stages and growing locations, in which the all stages had greater impacts on α- and β-diversity than all regions. Microbiome profiling had identified several core bacteria including Sphingomonas, Bacillus, Staphylococcus, Pseudomonas, Ralstonia, Massilia and Fibrobacter. Fungal biomarkers included Aspergillus, Penicillium, Fusarium, Cladosporium and Trichomonascus. Interestingly, the molecular ecological networks showed that the core taxa had significant correlations with metabolic enzymes and physicochemical properties; bacteria and fungi jointly participated in the carbohydrate and nitrogen compound degrading and volatile aroma compound chemosynthesis processes during fermentation. These studies provide insights into the coupling of material conversion and microbial community succession during leaf fermentation. Graphical abstract

The purpose of this study was to elucidate how the microbiota affects the metabolic state and characteristic flavor development of cigar tobacco leaves (CTL) in the fermentation process through microbial metabolism and co-metabolism with the host. The results showed that core bacterial communities in the fermentation process were Cyanobacteria , Pseudomonas , and Staphylococcus , and Aspergillus , Penicillium , and Inocybe were the most metabolically active fungi. Pseudomonas fulva , Staphylococcus nepalensis , Bacillus subtilis , Stenotrophomonas rhizophila , Alternaria alternata , and Aspergillus cristatus could degrade carbon and nitrogen compounds, such as protein, starch, lignin, and nicotine. There were 12 common non-volatile metabolites and three common volatile metabolites in DH, LC, PE, and YX before and after fermentation, of which menatetrenone, solanidine, γ-glutamylphenylalanine, carotol, and phenol, 4-(1,1,3,3-tetramethylbutyl) were significantly different before and after fermentation. The synthesis and degradation metabolism of various amino acids, alkaloids, and flavonoids are the key metabolic pathways of characteristic flavor development during CTL fermentation. Co-occurrence and interaction patterns showed that seven bacteria and 12 fungi were strictly linearly positively or negatively correlated with 72 and 55 volatile compounds, respectively. In conclusion, this study preliminarily confirmed that CTL fermentation is a microbially mediated carbon–nitrogen coupling metabolism. The carbohydrates in tobacco leaves were largely decomposed and consumed, providing energy sources for microorganisms and the carbon skeleton required for cell construction. The nitrogen-containing macromolecular compounds were degraded to form volatile compounds or flavor precursors with typical flavor. IMPORTANCE: The development of the metabolic state and characteristic flavor of cigar tobacco during fermentation is the key to process control. Innovative discoveries in the development of the metabolic state and characteristic flavor of cigar tobacco during fermentation are the key to process control. Innovative discoveries of core functional microorganisms and key metabolites were made during fermentation, suggesting potential pathways for carbon and nitrogen metabolisms. We demonstrate for the first time that cigar tobacco leaf fermentation is a microbially mediated carbon–nitrogen coupling metabolism. Many carbohydrates in tobacco leaves are decomposed and consumed to provide energy source and carbon skeleton for the construction of microbial cells, and nitrogen-containing macromolecular compounds are degraded to form volatile compounds or flavor precursors with typical flavors.

Phytochemical investigations of the leaves of Garcinia paucinervis resulted in the isolation of three new xanthones 1–3 and five known analogues 4–8. Structural elucidations of 1–3 were performed by spectral methods such as 1D and 2D (HMQC, HMBC, and ROESY) NMR spectroscopy, in addition to high resolution mass spectrometry. Compounds 1–3 showed anti-TMV activities, with inhibition rates above 20%, especially for 1, which had a lower IC₅₀ value of 21.4 µM.

Background: The quality of cigar tobacco leaves was closely related to fermentation. To investigate the substance changes in cigar tobacco leaves during their fermentation, metabolomics was determined at different fermentation stages. In this study, the metabolic profile among different regions and fermentation periods of cigar tobacco leaf were evaluated. Results: A total of 1103 metabolites were identified in cigar tobacco leaf samples. A total of 293, 105 and 199 metabolites showed differential accumulation in the cigar tobacco leaf among different regions (PEF0 vs. LCF0, PEF0 vs. DHF0, PEF0 vs. YXF0) and 216, 242, 220, 227 and 198 metabolites showed differential accumulation in the different fermentation (LCF0 vs. LCF1, LCF0 vs. LCF2, LCF0 vs. LCF3, LCF0 vs. LCF4, LCF0 vs. LCF5). The main upregulated compounds were flavonoids, phenolic acids and lignans and coumarins, and the main downregulated compounds were organic acids, phenolic acids and amino acids and derivatives in the fermentation comparison group. Conclusions: These results provide valuable information for accurately grasping the end time of fermentation and improve efficiency of cigar tobacco leaf fermentation. Graphical

Tobacco (Nicotiana tabacum L.) is one of the world’s most economically important crops, with nicotine content being a crucial factor in determining its quality and economic value. By integrating plant physiology with transcriptomics and metabolomics analyses, we gained insight into the regulation of nicotine synthesis and metabolism in tobacco seedlings under different forms of nitrogen supply. Our findings revealed that ammonium nitrogen promotes nitrogen transformation in the root system of tobacco seedlings but inhibits root growth, whereas nitrate nitrogen inhibits biochemical processes but enhances root growth. Additionally, we observed that nitrate nitrogen facilitates the NAD + pathway in nicotine biosynthesis but inhibits the polyamine pathway, whereas ammonium nitrogen has the opposite effect. Overall, our comprehensive analysis indicates that the co-application of ammonium nitrate is conducive to nicotine synthesis. These results not only contribute to identifying key genes and metabolites involved in nicotine synthesis, but also provide a theoretical basis for establishing efficient regulation theories and pathways for tobacco's nitrogen utilization. Furthermore, they offer scientific support for future efforts aimed at regulating tobacco nicotine synthesis.

In this study, the agricultural traits, alkaloids content and Fourier transform infrared spectroscopy (FT-IR) and two-dimensional correlation infrared spectroscopy (2DCOS-IR) analysis of the tobacco after Berberine Bridge Enzyme-Like Proteins (BBLs) knockout were investigated. The knockout of BBLs has limited effect on tobacco agricultural traits. After the BBLs knockout, nicotine and most alkaloids are significantly reduced, but the content of myosmine and its derivatives increases dramatically. In order to identify the gene editing of tobacco, principal component analysis (PCA) was performed on the FT-IR and 2DCOS-IR spectroscopy data. The results showed that FT-IR can distinguish between tobacco roots and leaves but cannot classify the gene mutation tobacco from the wild one. 2DCOS-IR can enhance the characteristics of the samples due to the increased apparent resolution of the spectra. Using the autopeaks in the synchronous map for PCA analysis, we successfully identified the mutants with an accuracy of over 90%.