The assembly of the rhizosphere community, even the diazotroph community, is mainly shaped by soil environmental factors (including soil climate and physiochemical characteristics) and plant selection. To better understand the driving forces on the active overall and nitrogen-fixing bacterial community compositions, we characterized the communities of tobacco rhizosphere soil collected from three sampling sites with a large geographic scale (> 600 km). The results indicate that the diversity and community composition of the overall bacterial and diazotroph communities are obviously differed according to the sampling sites. Still, no significant difference is found between the communities in rootzone and rhizosphere samples. Climate variables including mean annual precipitation (MAP) and mean annual temperature (MAT), soil physiochemical characteristics including available nitrogen (AN), available potassium (AK) and pH are main factors that affect the bacterial and diazotroph community structures in the three sampling sites. Furthermore, MAP and MAT, AN and available phosphorus (AP), total nitrogen (TN) and organic carbon (OC), AK and electrical conductivity (EC) showed similar effects, but pH showed independent effect on the composition of the overall bacteria and diazotroph communities. However, the alpha diversity indices of active overall and nitrogen-fixing bacteria in the rhizosphere are obviously higher than in the rootzone samples, and no significant differences are observed among different sampling sites. Proteobacteria is the predominant active phylum of all samples for overall and nitrogen-fixing bacteria. Escherichia-Shigella, Achromobacter, Streptomyces and Sphingomonas are the dominant active bacterial genera, and Bradyrhizobium, Skermanella and Extensimonas are dominant active nitrogen-fixing bacteria genera in rhizosphere. Furthermore, the high active abundance of Escherichia-Shigella but low abundance of Ralstonia in all three sampling sites indicate high root-knot nematode infection and low wilt disease endemic risk. These results indicate that soil environmental factors contribute more to the tobacco rhizosphere bacterial community assemblage, but the rhizosphere contributes more to the diversity of active overall bacteria and nitrogen-fixing bacteria in the community. Our study provides novel knowledge for the assemble of rhizosphere bacterial and active bacteria communities across a large geographical scale.

Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.

Salinity is one of the most severe forms of abiotic stress and affects crop yields worldwide. Plants respond to salinity stress via a sophisticated mechanism at the physiological, transcriptional and metabolic levels. However, the molecular regulatory networks involved in salt and alkali tolerance have not yet been elucidated. We developed an RNA-seq technique to perform mRNA and small RNA (sRNA) sequencing of plants under salt (NaCl) and alkali (NaHCO₃) stress in tobacco. Overall, 8064 differentially expressed genes (DEGs) and 33 differentially expressed microRNAs (DE miRNAs) were identified in response to salt and alkali stress. A total of 1578 overlapping DEGs, which exhibit the same expression patterns and are involved in ion channel, aquaporin (AQP) and antioxidant activities, were identified. Furthermore, genes involved in several biological processes, such as “photosynthesis” and “starch and sucrose metabolism,” were specifically enriched under NaHCO₃ treatment. We also identified 15 and 22 miRNAs that were differentially expressed in response to NaCl and NaHCO₃, respectively. Analysis of inverse correlations between miRNAs and target mRNAs revealed 26 mRNA-miRNA interactions under NaCl treatment and 139 mRNA-miRNA interactions under NaHCO₃ treatment. This study provides new insights into the molecular mechanisms underlying the response of tobacco to salinity stress.

Tobacco plants (Nicotiana tabacum L.) exhibit considerable potential for phytoremediation of soil cadmium (Cd) pollutants, owing to their substantial biomass and efficient metal accumulation capabilities. The reduction of Cd accumulation in tobacco holds promise for minimizing Cd intake in individuals exposed to cigar smoking. NRAMP transporters are pivotal in the processes of Cd accumulation and resistance in plants; however, limited research has explored the functions of NRAMPs in tobacco plants. In this investigation, we focused on NtNRAMP6c, one of the three homologs of NRAMP6 in tobacco. We observed a robust induction of NtNRAMP6c expression in response to both Cd toxicity and iron (Fe) deficiency, with the highest expression levels detected in the roots. Subsequent subcellular localization and heterologous expression analyses disclosed that NtNRAMP6c functions as a plasma membrane-localized Cd transporter. Moreover, its overexpression significantly heightened the sensitivity of yeast cells to Cd toxicity. Through CRISPR-Cas9-mediated knockout of NtNRAMP6c, we achieved a reduction in Cd accumulation and an enhancement in Cd resistance in tobacco plants. Comparative transcriptomic analysis unveiled substantial alterations in the transcriptional profiles of genes associated with metal ion transport, photosynthesis, and macromolecule catabolism upon NtNRAMP6c knockout. Furthermore, our study employed plant metabolomics and rhizosphere metagenomics to demonstrate that NtNRAMP6c knockout led to changes in phytohormone homeostasis, as well as shifts in the composition and abundance of microbial communities. These findings bear significant biological implications for the utilization of tobacco in phytoremediation strategies targeting Cd pollutants in contaminated soils, and concurrently, in mitigating Cd accumulation in tobacco production destined for cigar consumption.

The changes of microbial community on tobacco leaves are affected by several factors during fermentation. However, the relative contribution of different factors in determining microbial community is not clear. This study investigated the effects of fermentation time (fermentation for 0, 3, 6, 9 and 12 months), leaf position (middle and top tobacco leaves) and fermentation site (Longyan and Xiamen warehouses) on bacterial community of tobacco leaves using 16 S rDNA sequencing. The results demonstrated that fermentation time had a much stronger impact on bacterial diversity, composition, co-occurrence network and functional profiles than leaf position and fermentation site. With the fermentation progressed, the difference of bacterial community between middle and top tobacco leaves was gradually reduced or even disappeared. The bacterial community diversity and network complexity at three, six and nine months of fermentation were significantly lower than those at fermentation initiation. Specific bacterial genera with desired functions were recruited at different fermentation stages, such as Terribacillus, Pantoea and Franconibacter at three or six months of fermentation and Pseudomonas at nine months of fermentation. The recruited microorganisms would form biofilms on tobacco leaves and compete for polysaccharide or protein substances to accelerate the degradation of tobacco macromolecular substances. In conclusion, fermentation time was an important factor in determining the composition and function of microbial community on tobacco leaves during the fermentation process.

Background: Carbon nano sol (CNS) can markedly affect the plant growth and development. However, few systematic analyses have been conducted on the underlying regulatory mechanisms in plants, including tobacco (Nicotiana tabacum L.). Results: Integrated analyses of phenome, ionome, transcriptome, and metabolome were performed in this study to elucidate the physiological and molecular mechanisms underlying the CNS-promoting growth of tobacco plants. We found that 0.3% CNS, facilitating the shoot and root growth of tobacco plants, significantly increased shoot potassium concentrations. Antioxidant, metabolite, and phytohormone profiles showed that 0.3% CNS obviously reduced reactive oxygen species production and increased antioxidant enzyme activity and auxin accumulation. Comparative transcriptomics revealed that the GO and KEGG terms involving responses to oxidative stress, DNA binding, and photosynthesis were highly enriched in response to exogenous CNS application. Differential expression profiling showed that NtNPF7.3/NtNRT1.5, potentially involved in potassium/auxin transport, was significantly upregulated under the 0.3% CNS treatment. High-resolution metabolic fingerprints showed that 141 and 163 metabolites, some of which were proposed as growth regulators, were differentially accumulated in the roots and shoots under the 0.3% CNS treatment, respectively. Conclusions: Taken together, this study revealed the physiological and molecular mechanism underlying CNS-mediated growth promotion in tobacco plants, and these findings provide potential support for improving plant growth through the use of CNS.