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Genome-wide Identification and Expression Analysis of the ADH Gene Family in Tobacco (Nicotiana tabacum L.)

Abstract Background Alcohol dehydrogenases (ADHs) are the crucial enzymes involved in plant development, biotic and abiotic stresses. Tobacco is one of the most important model plants, which will be of great application significance to analyze the ADH gene family and expression profile under various stresses in tobacco. Results A total of 53 ADH genes were identified in tobacco (Nicotiana tabacum L.) genome and were grouped into 6 subfamilies based on phylogenetic analysis. Gene structure (exon/intron) and protein motifs were highly conserved among the NtADH genes, especially the members within the same subfamily. A total of 5 gene pairs of tandem duplication, and 3 gene pairs of segmental duplication were identified based on the analysis of gene duplication events. Cis-regulatory elements of the NtADH promoters participated in cell development, plant hormones, environmental stress, and light responsiveness. Expression profile analysis showed that NtADH genes were widely expressed in different maturity tobacco leaves, as well as in leaves after topping, however, the expression patterns of different members appeared to be diversed. The qRT-PCR analysis of 13 NtADH genes confirmed their differential expression in response to the bacterial pathogen Ralstonia solanacearum L. infection. Metabolomics analysis revealed that NtADH genes were primarily associated with carbohydrate metabolism, and moreover, four NtADH genes (NtADH20/24/48/51) were notably involved in the pathway of alpha-linolenic acid metabolism. Conclusion The genome-wide identification, evolutionary and expression analysis of NtADH genes have provided valuable insights into the role of these genes in response to abiotic and biotic stresses. Our results have also laid a solid foundation for further functional study on ADH genes in tobacco, especially for the understanding of NtADHs under more stress conditions.

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Microbial and enzymatic changes in cigar tobacco leaves during air-curing and fermentation

Metabolic enzyme activity and microbial composition of the air-curing and fermentation processes determine the quality of cigar tobacco leaves (CTLs). In this study, we reveal the evolution of the dominant microorganisms and microbial community structure at different stages of the air-curing and fermentation processes of CTLs. The results showed that the changes in metabolic enzymes occurred mainly during the air-curing phase, with polyphenol oxidase (PPO) being the most active at the browning phase. Pseudomonas, Bacteroides, Vibrio, Monographella, Bipolaris, and Aspergillus were the key microorganisms in the air-curing and fermentation processes. Principal coordinate analysis revealed significant separation of microbial communities between the air-curing and fermentation phases. Redundancy analysis showed that bacteria such as Proteobacteria, Firmicutes, Bacteroidota, and Acidobacteriota and fungi such as Ascomycota and Basidiomycota were correlated with enzyme activity and temperature and humidity. Bacteria mainly act in sugar metabolism, lipid metabolism, and amino acid metabolism, while fungi mainly degrade lignin, cellulose, and pectin through saprophytic action. Spearman correlation network analysis showed that Firmicutes, Proteobacteria, and Actinobacteria were the key bacterial taxa, while Dothideomycetes, Sordariomycetes, and Eurotiomycetes were the key fungal taxa. This research provides the basis for improving the quality of cigars by improving the air-curing and fermentation processes.Key points• Changes in POD and PPO activity control the color change of CTLs at the air-curing stage.• Monographella, Aspergillus, Pseudomonas, and Vibrio play an important role in air-curing and fermentation.• Environmental temperature and humidity mainly affect the fermentation process, whereas bacteria such as Proteobacteria, Firmicutes, Bacteroidota, and Acidobacteriota and fungi such as Ascomycota and Basidiomycota are associated with enzyme activity and temperature and humidity.Graphical

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Physiochemical Properties and Microflora of the Rhizosphere Soil of Tobacco Plants with and without Bacterial Wilt

Bacterial wilt is a destructive soilborne disease caused by Ralstonia solanacearum, posing a severe threat to plants in the Solanaceae family. It impacts on tobacco productivity worldwide. This study was conducted to analyze the changes in the soil’s physical and chemical properties, the number of microbes, and the bacterial diversity of the rhizosphere soil before and after the wilt disease. The rhizosphere soil of healthy and diseased tobacco plants was collected from Pucheng, Nanping, Fujian Province, Southern China. The results revealed significant differences in the trends of physical and chemical properties of the soil of healthy and diseased plants. The soil pH, available potassium (K), available phosphorous (P), and organic matter contents (SOM) were lower in the rhizosphere soil for healthy plants than for pre-diseased plants (HW). Only the available P, among all physical and chemical properties in the rhizosphere of diseased plants (HS), was significantly lower than those for pre-diseased plants (HW), changing from 149.59 mg/kg to 59.19 mg/kg. The order of numbers of the three main microbes in the rhizosphere soil for healthy plants (HC) and pre-diseased plants was the following: bacteria > actinomycetes > fungi. The number of actinomycetes in the soil of the diseased tobacco plants increased significantly. A comparison of the rhizosphere soil of diseased and healthy tobacco plants showed that the relative abundance of the bacterial community in the rhizosphere soil of the pathogenic tobacco plants changed significantly. The community diversity was increased, and the Pseudomonadaceae, to which the bacterial pathogen of bacterial wilt belonged, rose to a certain extent. Both pre-diseased and healthy plants showed changes in the physical and chemical properties, microbial quantity, and microbial diversity, thus proving that tobacco disease was closely related to the soil’s ecological environment.

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Knockdown of NtCPS2 promotes plant growth and reduces drought tolerance in Nicotiana tabacum.

Drought stress is one of the primary environmental stress factors that gravely threaten crop growth, development, and yields. After drought stress, plants can regulate the content and proportion of various hormones to adjust their growth and development, and in some cases to minimize the adverse effects of drought stress. In our previous study, the tobacco cis-abienol synthesis gene (NtCPS2) was found to affect hormone synthesis in tobacco plants. Unfortunately, the role of NtCPS2 genes in the response to abiotic stress has not yet been investigated. Here, we present data supporting the role of NtCPS2 genes in drought stress and the possible underlying molecular mechanisms. NtCPS2 gene expression was induced by polyethylene glycol, high-temperature, and virus treatments. The results of subcellular localization showed that NtCPS2 was localized in the cell membrane. The NtCPS2-knockdown plants exhibited higher levels of gibberellin (GA) content and synthesis pathway genes expression but lower abscisic acid (ABA) content and synthesis pathway genes expression in response to drought stress. In addition, the transgenic tobacco lines showed higher leaf water loss and electrolyte loss, lower soluble protein and reactive oxygen species content (ROS), and lower antioxidant enzyme activity after drought treatment compared to wild type plants (WT). In summary, NtCPS2 positively regulates drought stress tolerance possibly by modulating the ratio of GA to ABA, which was confirmed by evidence of related phenotypic and physiological indicators. This study may provide evidence for the feedback regulation of hormone to abiotic and biotic stresses.

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Effect of soil management systems on the rhizosphere bacterial community structure of tobacco: Continuous cropping vs. paddy-upland rotation

Rhizosphere bacteria play important role in soil nutrient cycling and plant growth, and their richness and diversity are influenced by soil management systems. However, the specific changes in tobacco rhizosphere bacterial community structure in continuous and tobacco-rice rotation cropping systems remain uninvestigated. In this study, soil properties and the composition of the rhizosphere bacterial community in tobacco monocropping and tobacco-rice rotation cropping systems were analyzed. Moreover, the comparison of rhizosphere bacterial community structure between tobacco continuous and tobacco-rice rotation cropping systems was performed via high-throughput sequencing. The changes in the composition of the rhizosphere bacterial community were investigated at different tobacco growth stages. The results showed that continuous tobacco cropping increased the soil soluble organic carbon (SOC), total nitrogen (TN), and the content of other nutrients (e.g., available phosphorus and available potassium) compared to tobacco-rice rotation cropping. However, monocropping decreased bacterial alpha-diversity and altered the community composition when compared to the rotation cropping system. At the phylum level, the relative abundance of Proteobacteria, Gemmatimonadetes, and Bacteroidetes increased in the continuous cropping soil, while that of Acidobacteria, Firmicutes, and Actinobacteria decreased. At the genera level, the average abundance of the dominant genus Bacillus varied from 12.96% in continuous cropping libraries to 6.33% in the rotation cropping libraries (p < 0.05). Additionally, several other taxa, such as o_Acidobacteriales and Candidatus_Solibacter decreased from 7.63 to 6.62% (p < 0.05) and 4.52 to 2.91% (p < 0.05), respectively. However, the relative abundance of f_Gemmatimonadaceae and c_Subgroup_6 showed an increase of 1.46% (p < 0.05) and 1.63% (p < 0.05) in the tobacco-rice rotation cropping system, respectively. The results of NMDS indicated that the rhizobacteria community structure differed in the two cropping systems. In tobacco, the rhizosphere bacterial community structure showed no significant changes in the prosperous long-term stage and topping stage, but the composition changed significantly in the mature stage.

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Ammonia-oxidizing archaea and ammonia-oxidizing bacteria communities respond differently in oxy-gen-limited habitats

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) are the most important ammonia oxidation functional community, while the coastal environment just provides a different oxygen environment for the ammonia oxidation process. However, few surveys concentrated on the influence of oxygen concentration on the niche specialization of AOA and AOB in the ocean intertidal zones. Here, high-throughput sequencing by Illumina MiSeq and qPCR were applied to detect the change of abundance, diversity as well as community structure of both AOA and AOB with 0–60 cm sediments depth in the intertidal zone in Qingdao, China. Results showed that the AOA/AOB amoA gene copy numbers and AOA/AOB OTU numbers rate increased as sediment depth went more profound, which indicated that AOA was more adaptive to oxygen-limited niches compared to AOB. Oxygen indeed led to the niche specialization of AOA and AOB in intertidal sediments. The dominant AOA and AOB were the clusters of Nitrosopumilus and Nitrosospira, respectively, which indicated ecological success in the intertidal zone. A significant and positive correlation (p &amp;lt; 0.01) between AOB abundance/AOB OTU numbers and Oxidation-reduction potential (ORP) was observed. In addition, both total nitrogen (TN) (p &amp;lt; 0.01) and pH (p &amp;lt; 0.05) were significantly negatively correlated to AOB abundance. TN was also significantly negatively correlated to AOB OTU numbers (p &amp;lt; 0.05). Hence, oxygen led to niche specialization of AOA and AOB, especially under anoxic conditions, AOA played a dominant role in the process of ammonia oxidation. The Nitrosopumilus and Nitrosospira clusters were the dominant AOA and AOB, respectively, representing an ecological success in the intertidal zone.

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fliA, flrB, and fliR regulate adhesion by controlling the expression of critical virulence genes in Vibrio harveyi

Bacteria adhesion to fish mucus is a crucial virulence mechanism. As the initial step of bacterial infection, adhesion is impacted by bacterial motility and environmental conditions. However, its molecular mechanism is yet unclear. In this study, a significant decrease in gene expression of adhesion-deficient Vibrio harveyi was observed when the bacteria were subjected by Cu2+(50 mg/L), Pb2+(100 mg/L), Hg2+(25 mg/L), and Zn2+(50 mg/L). The genes fliA, fliR, and flrB were responsible for flagellation; being crucial for adhesion, these genes were identified and silenced via RNAi. After silencing of these genes by RNAi technology, the ability of adhesion, biofilm formation, motility, and flagella synthesis of V. harveyi were considerably reduced. Compared with the control group, it was observed that the expression levels of fliS, fliD, flgH, and flrC were significant down-regulated in fliR-RNAi, flrB-RNAi, and fliA-RNAi. This data indicates that the expression levels of most virulence genes are affected by fliA, fliR, and flrB. Also, the expression of fliA, fliR, and flrB can be influenced by the salinity, temperature, and pH. The results show that: (1) fliA, fliR, and flrB have important roles in the adhesion of V. harveyi; (2) fliA, fliR, and flrB can regulate bacterial adhesion by affecting its motility, and biofilm formation; (3) fliA, fliR, and flrB can regulate adhesion ability of V. harveyi in different environments.

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