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Tobacco (Nicotiana tabacum) is cultivated and consumed worldwide. It requires great amounts of nitrogen (N) to achieve the best yield and quality. With a view to sustainable and environmentally friendly agriculture, developing new genotypes with high productivity under low N conditions is an important approach. It is unclear how genes in tobacco are expressed at the cellular level and the precise mechanisms by which cells respond to environmental stress, especially in the case of low N. Here, we characterized the transcriptomes in tobacco leaves grown in normal and low‐N conditions by performing scRNA‐seq. We identified 10 cell types with 17 transcriptionally distinct cell clusters with the assistance of marker genes and constructed the first single‐cell atlas of tobacco leaves. Distinct gene expression patterns of cell clusters were observed under low‐N conditions, and the mesophyll cells were the most important responsive cell type and displayed heterogene responses among its three subtypes. Pseudo‐time trajectory analysis revealed low‐N stress decelerates the differentiation towards mesophyll cells. In combination with scRNA‐seq, WGCNA, and bulk RNA‐seq results, we found that genes involved in porphyrin metabolism, nitrogen metabolism, carbon fixation, photosynthesis, and photosynthesis‐antenna pathway play an essential role in response to low N. Moreover, we identified COL16, GATA24, MYB73, and GLK1 as key TFs in the regulation of N‐responsive genes. Collectively, our findings are the first observation of the cellular and molecular responses of tobacco leaves under low N stress and lay the cornerstone for future tobacco scRNA‐seq investigations. [ABSTRACT FROM AUTHOR]

Nitrosation of pyridine alkaloids in tobacco generates tobacco‐specific nitrosamines (TSNAs), which are notable toxicants in tobacco products and smoke. Burley tobacco, a chloroplast‐ and nitrogen (N)‐deficient phenotype that accumulates high levels of nitrate‐nitrogen (NO3‐N) in its leaves, is particularly susceptible to TSNAs formation. In this study, reciprocal pot and field grafting experiments were conducted using burley tobacco Eyan No.1 and flue‐cured tobacco K326 to investigate whether grafting burley tobacco scions on flue‐cured tobacco rootstocks could enhance pigment biosynthesis and photosynthesis, while reducing the NO3‐N level in burley tobacco leaves. Grafting burley tobacco scions on flue‐cured tobacco rootstocks significantly increased the total pigment content, photosynthetic rate, biomass, nitrate reductase and glutamine synthetase activities, as well as ammonium‐nitrogen (NH4‐N), total soluble and reducing sugar, and soluble protein levels in burley tobacco leaves compared with burley tobacco self‐rooting, while decreasing the NO3‐N level and nitrate‐N to total N ratio. Transcriptomic analysis revealed that grafting resulted in upregulated expression of genes involved in starch, sucrose, porphyrin, chlorophyll, and N metabolism, as well as carbon fixation and carotenoid biosynthesis. The findings suggest that grafting on high N use efficiency rootstock is an exceptionally promising means of decreasing NO3‐N accumulation by improving photosynthesis and N metabolism in the scion, thereby reducing the levels of harmful TSNAs. [ABSTRACT FROM AUTHOR]

Background: Photosynthetic pigments in higher plants, including chlorophyll (Chl) and carotenoids, are crucial for photosynthesis and photoprotection. Chl-deficient tobacco seedlings generally have a lower photosynthesis rate and higher nitrate-nitrogen (NO3-N) content, which causes a profound influence on tobacco yield and quality. In this study, a stable albino leaf mutant (Al) and slight-green leaf mutant (SG) obtained from the common flue-cured tobacco (Nicotiana tabacum L.) cultivar 'Zhongyan 100' (ZY100) by mutagenesis with ethyl methanesulfonate (EMS) were used as materials. The differences between the Chl-deficient mutants and the wild-type (WT) were analyzed in terms of biomass, photosynthetic fluorescence parameters, and carbon- and nitrogen-related physiological parameters. RNA sequencing (RNA-seq) and weighted gene co-expression network analysis (WGCNA) were used to explore the key pathways and candidate genes regulating differentiated chlorophyll and nitrate content. Results: The results showed that, when compared to the WT, the Chl content and biomass of mutant plants were considerably lower while the NO3-N content was substantially elevated. The net photosynthetic rate, photosynthetic fluorescence parameters, carbohydrate, soluble protein, and carbon- and nitrogen-related enzyme activities all decreased in leaves of mutants and the development of chloroplasts was abnormal. Applying more nitrogen improved the growth and development of mutants, whereas NO3-N content distinctively increased compared with that of the WT. Through transcriptome sequencing, the downregulated genes in mutants were enriched in plant hormone signal transduction and nitrogen metabolism, which are involved in pigment biosynthesis and the carbon fixation pathway. In addition, two hub genes and seven transcription factors identified from the blue module through WGCNA were likely to be key candidate factors involved in chlorophyll synthesis and nitrate accumulation. Conclusion: Our results demonstrated that differences in chlorophyll and nitrate content were caused by the combined effects of chloroplast development, photosynthesis, as well as related biological activity. In addition, transcriptome results provide a bioinformatics resource for further functional identification of key pathways and genes responsible for differences in chlorophyll and nitrate content in tobacco plants. [ABSTRACT FROM AUTHOR]

Drought causes a decrease in tobacco yield and quality as well as a reduction in net photosynthetic rate. Salicylic acid (SA) is involved in the regulation of many physiological processes and has been widely used to improve plant drought tolerance. The hydroponic experiment was carried out on fluecured tobacco variety K326. SA solutions (0.3 mM) were sprayed on the leaves every day for three days, after which 15% PEG-6000 was used to simulate moderate drought. The results showed that drought stress reduced the photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr), while it enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), malondialdehyde (MDA), proline and protein content. Compared with PEG drought stress, SA improved the Pn, Gs and Tr by 45.74%, 26.82%, and 52.22% respectively. Besides, the activities of SOD, POD, CAT, protein and proline content increased by 44.27%, 50.18%, 25%, 34.69% and 24.88%, respectively, while the MDA content decreased by 23.89%. Furthermore, SA spraying increased the drought tolerance of tobacco by modulating the expression level of the genes associated with photosynthesis, carbon metabolism, and photosynthesis-antenna proteins. Hence, SA spraying reduced the inhibition of drought on photosynthesis and stimulated the antioxidant system, thus improving the drought resistance in tobacco. [ABSTRACT FROM AUTHOR]

Nitrate is an important precursor of tobacco-specific nitrosamines (TSNAs) and a remarkable difference in nitrate accumulation between lamina and midrib of flue-cured tobacco has long been observed. However, the physiological and molecular mechanisms underpinning this difference remain poorly understood. In this study, physiological and genetic factors impacting nitrate accumulation were identified in pot experiments using flue-cured tobacco K326 with contrasting nitrate content between lamina and midrib. The results showed that three times higher NO3-N content was observed in midrib than that in the lamina, along with lower pigment, NH4-N content, nitrate reductase activity (NRA), sucrose synthetase activity (SSA), and glutamine synthetase activity (GSA) in midrib. Transcriptome analysis revealed that expression of genes involved in porphyrin and chlorophyll metabolism, carotenoid biosynthesis, photosynthesis-antenna proteins, photosynthesis, carbon fixation in photosynthetic organisms, starch and sucrose metabolism, nitrogen metabolism, and biosynthesis of amino acids were significantly lower in midrib than in lamina. qRT-PCR results showed that the expression level of nitrate transporter genes LOC107782967, LOC107806749, LOC107775674, LOC107829632, LOC107799198, LOC107768465 decreased by 2.74, 1.81, 49.5, 3.5, 2.64 and 2.96-folds while LOC107789301 increased by 8.23-folds in midrib but not in lamina. Reduced chlorophyll content might result in low carbohydrate formation which is the source of energy and carbon skeleton supply, then the low capacity of nitrogen reduction, assimilation and transportation, and the poor ability of nitrate reallocation but the high capacity of accumulation might lead to nitrate accumulation in midrib. The results laid the foundation for reducing nitrate content and TSNA formation in tobacco midribs and their products. [ABSTRACT FROM AUTHOR]

The purpose of this study was to explore the effects of biochar application on soils in the main tobacco-producing areas in China. The study was conducted in four study regions in China, where the same three experimental treatments were set up in each area, including a control (CK), a treatment involving the application of chemical fertilizer (F), and a treatment involving the application of biochar (B). We analyzed the basic physical and chemical properties, microbial diversity, and root system of tobacco plants. The results show that: Biochar increased the soil pH, which was most obvious in the study site in Shaowu City, Fujian Province (FUS), where the soil pH increased by 22.64% and 27.49% compared with soil under the CK and F treatments, respectively. Biochar increased the microbial biomass carbon (MBC) content, and increased the soil content of available nitrogen, phosphorus, and potassium; this effect was most obvious in FUS. The root activity in plots treated with biochar increased by 6.95% and 13.72% compared to the CK and F plots, respectively. Similarly, the number of root tips increased by 89.76% and 21.48% compared to the CK and F plots, respectively. In short, biochar improved the physical soil structure, increased the soil pH, and promoted the effectiveness of soil nutrients. Furthermore, biochar improved the bacterial soil diversity, enriched the population structure of soil bacteria, and promoted the healthy development of flue-cured tobacco roots. However, the demand for and types of biochar suitable for use in different tobacco-planting soils need further study. [ABSTRACT FROM AUTHOR]

This study aimed to explore a new way to address the burning of agricultural waste in China while achieving the sustainable use of it. Three agricultural wastes (Wheat straw, peanut shell, and rice husk) were slowly pyrolyzed into biochar, which was subsequently added to the soil to reduce CO2 emissions from the soil, and to improve soil fertility as well as microbial community structure. The biochar and raw materials were added to the soil and cultured under controlled conditions, and then the CO2 emissions produced from the mixing. At the same time, this study used pot experiments to determine the effects of biochar on tobacco soil physical and chemical properties and, therefore, the microbial communities of the soil. This study suggests that (1) biochar can effectively reduce soil CO2 emission rate. Compared with the control, peanut shell biochar could reduce the total CO2 emissions of soil by 33.41%, and the total CO2 emissions of wheat straw biochar treatment was 90.25% lower than that of wheat straw treatment. (2) The soil's physical and chemical properties were improved. The soil bulk density of wheat straw biochar treatment kept 34.57% lower than that of the control as well as 21.15% lower than that of wheat straw treatment. The soil's organic carbon of peanut shell biochar treatment was 87.62% more than that of peanut shell treatment. (3) Biochar changed soil microbial community structure. (4) Biochar is suitable for tobacco growth. Peanut husk biochar significantly increased the total biomass of tobacco, and wheat straw biochar significantly increased tobacco root vigor. This study concluded that processing Chinese agricultural waste into biochar and adding it to the soil instead of burning it directly would be an effective means to reduce greenhouse gas emissions, to improve soil, and to promote crop growth. [ABSTRACT FROM AUTHOR]