Background: Anthocyanins determinate the flower color of many plants. Tobacco is a model plant for studying the molecular regulation of flower coloration. We investigated the mechanism underlying flower coloration in tobacco by profiling flavonoid metabolites,expression of anthocyanin biosynthetic structural genes and their regulator genes in the pink-flowered tobacco cultivar Yunyan 87 and white-flowered Yunyan 87 mutant.Result: Significant down-accumulation of anthocyanins, including cyanidin 3-O-glucoside, cyanin, cyanidin 3-O-rutinoside, pelargonidin 3-O-beta-D-glucoside, cyanidin O-syringic acid, pelargonin, and pelargonidin 3-O-malonylhexoside (log₂fold change < − 10), endowed the flower color mutation in Yunyan 87 mutant. Transcriptome analysis showed that the coordinately down-regulated anthocyanin biosynthetic genes including chalcone isomerase, naringenin 3-dioxygenase, dihydroflavonol 4-reductase and UDP-glucose:flavonoid 3-O-glucosyltransferase played critical roles in suppressing the formation of the aforesaid anthocyanins. Several genes encoding MYB and bHLH transcription factors were also found down-regulated, and probably the reason for the suppression of structural genes.Conclusion: This is the first study of tobacco flower coloration combining metabolome and transcriptome analyses, and the results shed a light on the systematic regulation mechanisms of flower coloration in tobacco. The obtained information will aid in developing strategies to modify flower color through genetic transformation.

To characterize volatile organic compounds (VOCs) and aromatic sensory properties in Yun cigar, 27 samples from four origins were analyzed using SPME–HS–GC/MS and sensory analysis. The investigation results were analyzed using principal component analysis (PCA), Fisher linear discriminant analysis (LDA), and Pearson correlation analysis. In Yunnan cigars, the content of nicotine and neophytadiene accounted for over 90% of the total VOC content. Nicotine was significantly positively correlated with neophytadiene and phytol. The cigars from four origins were clearly classified by the PCA of VOCs. Four region discrimination functions were established through the LDA of 14 compounds, and the validation accuracy was 100%. The sensory descriptors with the highest geometric mean were woody, roasted, fresh‐sweet, bean, and scorched. Acetophenone, megastigmatrienone A, and thunbergene were positively correlated with multiple aroma descriptors, while nicotine was negatively correlated with multiple aroma descriptors.

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.

Background: Nicotine biosynthesis is mainly regulated by jasmonate (JA) signaling cascade in Nicotiana tabacum . As an allotetraploid species, the regulation of nicotine biosynthesis has been genetically verified via two unlinked NIC loci (named as NIC1 and NIC2 ) which are possibly originated from its two ancestral diploids. Previously, a N. tomentosiformis originated ethylene response factor ( ERF ) gene cluster was identified as the NIC2 -locus which has been demonstrated positively regulates nicotine accumulation in N. tabacum . Results: Here, we describe the genetic mapping of NIC1 -locus, the major nicotine regulatory locus, by using a NIC1 -locus segregating population through bulked segregant analysis. We identified two linkage marker TM23004 and TM22038 were delimited the NIC1 -locus within a ~34.3-Mb genomic region at pseudochromosome 07 of tobacco genome. Genomic scan within this region revealed a NIC2 - like locus ERF gene cluster exist in. To verify this ERF gene cluster is the genetically called “ NIC1 -locus”, different functional experiments based on most of the ERFs in regulating nicotine biosynthesis and their influences on alkaloid accumulations have been carried out. Collinearity analysis showed that NIC1 -locus ERF genes are originated from N. sylvestris and exclusively expressed in root tissues. In addition, transcriptomic results indicate that NIC1 -locus ERF genes are coexpressed with the NIC2 -locus ERF genes and other nicotine biosynthetic genes and regulators after JA induction. Furthermore, the suppressed expression of four ERFs of the NIC1 -locus genes corresponding with decreased NtPMT and NtQPT expression in NtMYC2 -RNAi lines indicates the selected NIC1 -locus ERFs function in downstream of NtMYC2 in the JA signaling cascades. In the meanwhile, the alkaloid levels are also determined by the amplitude of the four ERF gene expressions in both wild type and LA mutant. Additionally, in vitro binding assays, transient activation assays, and ectopic expression in transgenic plants demonstrate that these ERF genes are able to bind the GCC-box elements residing in the step-limiting gene promoters (such as NtPMT2 , NtQPT2 ) and functional redundant but quantitatively transactivate nicotine biosynthetic gene expression. For nic1 -locus mutation, two different sizes of deletions ( nic1-S and nic1-B ) were identified which occurred at the surrounding regions of the NIC1 -locus gene cluster, which might disrupt, to some extent, chromosomal microenvironment and change gene expression around the deletion regions (including NIC1 -locus ERFs ), resulting in the decreased expression levels of NIC1 -locus ERFs (such as NtERF199 ) and reduced alkaloid accumulation in the nic1 -locus mutant. Conclusions: Our findings not only provide insight in to the mechanism of the NIC1 -locus ERFs in the regulatory network of nicotine biosynthesis, but also unraveled the theoretical basis of the nic1 -locus mutation in low nicotine mutant. These functional verified NIC1 -locus ERF genes can be further used as potential target(s) for ethyl methanesulfonate-based mutagenesis to manipulate nicotine level in tobacco variety in tobacco breeding program.

Stacking, a pivotal step in the preparation of composted organic manures, plays a crucial role in organic matter decomposition, nutrient availability, and soil microbial diversity, with effects dependent on the stacking duration. However, the mechanisms through which organic manures with different stacking times influence crop growth and development via alterations in the soil environment remain poorly understood. This study employed a pot experiment with tobacco plants, applying conventional fertilizers alongside organic cottonseed meal manures subjected to various stacking times (0, 10, 20, and 30 days). The primary aim was to examine the impact of cottonseed meal organic manures on the microbial community structure, function, and chemical properties of tobacco-growing soil, as well as to assess dry matter accumulation in tobacco plants. Results revealed that, compared to conventional fertilizers alone, the addition of cottonseed meal organic manures significantly enhanced soil organic matter content, with the 20d-stacked manure showing the highest increase (68.94%). Additionally, the composition of the soil microbial community was modified, which has increased the relative abundance of carbon and nitrogen metabolic functions. Dry matter accumulation in tobacco plants increased by 13.66%, 25.83%, 31.65%, and 9.69% across the different stacking times. In conclusion, composted organic manures regulate the soil–microbe system, promoting tobacco growth by enhancing the microbial structure of rhizosphere soil and improving nutrient availability. Cottonseed meal organic manures subjected to 20d of composting exhibited the most comprehensive benefits. The enrichment of beneficial microbes like Chryseobacterium and Humicola (potential antagonists of soil pathogens) might contribute to the observed enhancement in soil organic matter and available nutrients. This improvement in soil quality led to a balanced nutrient environment, ultimately promoting dry matter accumulation in tobacco plants. These findings offer robust theoretical support for the application of composted organic manures. IMPORTANCE: This study is important because it elucidates the crucial mechanisms by which composted organic manures, specifically through the regulation of the soil–microbe system, promote crop growth. It provides key evidence that the stacking duration is a critical factor, identifying a 20-day (d) composting period for cottonseed meal manure as optimal for delivering comprehensive benefits: (i) Enhanced soil health: significantly increasing soil organic matter and improving nutrient availability. (ii) Improved microbial community: shifting the soil microbial structure to favor beneficial bacteria and suppress pathogens, thereby enhancing metabolic functions related to carbon and nitrogen cycling. (iii) Direct crop promotion: leading to a substantial increase in dry matter accumulation in tobacco plants