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Tomato yellow leaf curl China virus (TYLCCNV) viruliferous Mediterranean (MED) whitefly infestation in tobacco plants elevated hydrogen peroxide (H2O2) levels more than non-viruliferous ones. To unravel the underlying mechanisms behind the phenomenon, comparative analyses of plant transcriptome and enzyme activities of reactive oxygen species (ROS) scavengers were conducted in this study. Compared to the control, a total of 4032 and 6853 differentially expressed genes (DEGs) were identified in the plants following the infestations of non-viruliferous and TYLCCNV-viruliferous MED, respectively. Gene ontology analysis of DEGs suggested substantial transcriptional reprogramming of diverse cellular processes, particularly in the biotic stress response pathways of tobacco plants. Respiratory burst oxidase homologs related genes and important kinases putatively involved in plant defense were differentially modulated in tobacco plants in response to the non-viruliferous and TYLCCNV-viruliferous whitefly MED infestation. The analysis reported here lays out a strong foundation for future investigations into the mechanisms of ROS-associated plant defense mediating the interactions between B. tabaci and begomoviruses. [ABSTRACT FROM AUTHOR]

The present study explored the impact of rolB gene transformation on biological activities of Nicotiana tabacum. PCR, and RT-qPCR were used to validate the integration of rolB gene and its expression. HPLC analysis was used for phytochemical profiling, and various in-vitro and in-vivo assays were used to assess pharmacological activities of wild-type and transgenic extracts using ANOVA and Tukey's multiple comparison method for data analysis. Our findings indicated increased total phenolic content (24.4 ± 0.2 mg GAE/g DW of extract) and total flavonoid content (58.8 ± 1.1 mg QE/g DW of extract) in transgenic plants. In case of antioxidant activities, an increase in reducing power (51.82 ± 0.4 AAE mg/g), and radical scavenging (IC50 188 ± 2.7 µg/mL) was observed. Similarly, transgenic lines exhibited DNA protection against damage caused by free radicals. Moreover, we detected significant antileishmanial potential (IC50 350.7 ± 8.3 µg/mL), butyrylcholinesterase (IC50 268.6 ± 4.7 µg/mL), and acetylcholinesterase inhibitions (IC50 421.6 ± 3.7 µg/mL) in transgenic plants. Similarly, compared with the non-transgenic plants, the rolB-transgenic plants showed increased analgesic (71.4%), anti-inflammatory activities (77.8%), and 35.6% decrease in depression compared to non-transgenic plants. The improved pharmacological activities in transgenic plants have been linked to the level of phytochemicals that increase the therapeutic and curative properties of N. tabacum plants. [ABSTRACT FROM AUTHOR]

Tobacco BY-2 cell culture is one of the most widely used models in plant biology. The main advantage of BY-2 suspension cultures is the synchronization of cell development and the appearance of polar elongation. In batch culture, BY-2 cells passed through the lag, proliferation, elongation, and stationary phases. During this process, the composition of the growth medium changed dramatically. Sucrose was rapidly eliminated; hexose first accumulated and then depleted. The medium's pH initially decreased and then rose with aging. As a result of the crosstalk between the internal and external stimuli, cells pass through complicated systemic rearrangements, which cause metabolomic alterations. The early stages were characterized by high levels of amino acids and sterols, which could be interpreted as the result of synthetic activity. The most intense rearrangements occurred between the proliferation and active elongation stages, including repression of amino acid accumulation and up-regulation of sugar metabolism. Later stages were distinguished by higher levels of secondary metabolites, which may be a non-specific response to deteriorating conditions. Senescence was followed by some increase in fatty acids and sterols as well as amino acids, and probably led to self-destructive processes. A correlation analysis revealed relationships between metabolites' covariation, their biochemical ratio, and the growth phase. [ABSTRACT FROM AUTHOR]

Tobacco (Nicotiana tabacum) is a globally cultivated crop, with its quality closely associated with the color and chemical composition of cured tobacco leaves. In this experiment, the effects of spraying exogenous 2, 4-epibrassinolide (EBR) and melatonin (MT) on the development of tobacco leaves at maturity stage and the quality after curing were investigated. Both EBR and MT treatments significantly enhanced the appearance quality of tobacco leaves at the stem-drying stage. Following preharvest applications, the sugar-to-alkali ratio and potassium content increased, while the contents of starch, total alkaloids, and proteins decreased. The levels of conventional chemical components were improved, enhancing the overall coordination of the tobacco. Transcriptome analysis revealed that EBR treatment down-regulated the chlorophyll biosynthetic genes hemA, MgPEC, and ChlD, while up-regulating the chlorophyll degradation genes CHL2, SGR, and PAOs. Similarly, MT treatment down-regulated the chlorophyll biosynthetic genes FC2 and MgPEC and up-regulated the degradation genes CHL2 and SGR, thus promoting chlorophyll degradation. Furthermore, in the downstream carotenoid biosynthetic pathway, both EBR and MT treatments regulated abscisic acid-related genes, with NCEDs being up-regulated and CYP707A1s down-regulated, thereby promoting the leaf ripening. Metabolomics analysis indicated that EBR treatment primarily regulated alkaloids, terpenoids, and flavonoids, while MT treatment mainly affected flavonoids. Both treatments also reduced the accumulation of the harmful substance aristolochic acid B. Comprehensive evaluations of appearance quality, physiological parameters, transcriptome, and metabolomics analyses demonstrated that exogenous spraying of EBR and MT treatments improved the maturity and quality of cured tobacco leaves, with EBR treatment exhibiting a greater effect than MT treatment. [ABSTRACT FROM AUTHOR]

Potassium sulfate (K2SO4) or 2,4-Epibrassinolide (EBL) mediated response to cadmium (Cd) stress in plants has been widely reported, but the joint effect both on plants in response to Cd stress remains obscure. Herein, our results showed the combined application of K2SO4 (10 mM) and EBL (0.2 μM) on tobacco plants under Cd stress (CdCl2, 100 μM) relieved Cd toxicity by improving the activities of AOEs and reducing the contents of decreasing superoxide (H2O2 and O2−) and cadmium ion. As a result, the synergetic application of K2SO4 and EBL protected the integrity of chloroplast, which ensured a normal process of photosynthesis without a significant decrease in photosynthetic pigment content. Additionally, the combined application of K2SO4 and EBL also inhibited the expression of the Cd transport-related gene NtNramp1 and enhanced the Cd efflux-related gene NtHMA2. Moreover, the combined treatment of EBL associated with K2SO4 also effectively reduced the Cd accumulation in tobacco leaves under Cd stress compared with the groups treated separately, which implied a potentially synergistic role of EBL and K2SO4 in alleviating Cd toxicity in tobacco plants. This study provides a theoretical reference for the further analysis of the molecular mechanism of K2SO4 and EBL in jointly mediating the response to heavy metals in plants. [ABSTRACT FROM AUTHOR]

The majority of plant viruses rely on insect vectors for inter-plant transmission. Amid virus transmission, vector-borne viruses such as begomoviruses may significantly modulate host plants in various ways and, in turn, plant palatability to insect vectors. While many case studies on monopartite begomoviruses are available, bipartite begomoviruses are understudied. More importantly, detailed elucidation of the molecular mechanisms involved is limited. Here, we report the mechanisms by which an emerging bipartite begomovirus, the Sri Lankan cassava mosaic virus (SLCMV), modulates plant defenses against whitefly. SLCMV infection of tobacco (Nicotiana tabacum) plants significantly downregulated defenses against whitefly, as whitefly survival and fecundity increased significantly on virus-infected plants when compared to the controls. We then profiled SLCMV-induced transcriptomic changes in plants and identified a repertoire of differentially expressed genes (DEGs). GO enrichment analysis of DEGs demonstrated that the term defense response was significantly enriched. Functional analysis of DEGs associated with defense response revealed that four downregulated DEGs, including putative late blight resistance protein homolog R1B-17 (R1B-17), polygalacturonase inhibitor-like (PGI), serine/threonine protein kinase CDL1-like (CDL1), and Systemin B, directly contributed to plant defenses against whitefly. Taken together, our findings elucidate the role of novel plant factors involved in the modulation of plant defenses against whitefly by a bipartite begomovirus and shed new light on insect vector–virus–host plant tripartite interactions. [ABSTRACT FROM AUTHOR]

In this study, the fatty acid content, phenolic compounds, and antimicrobial activity of seed oils from Ege Özbaş (EO), Sarıbağlar 407 (S407) and Akhisar 98 (A98) tobacco plants grown in the Aegean region were investigated. The ozonation process altered fatty acid and phenolic compound compositions and affected antimicrobial activities against Gram-positive and Gram-negative bacteria in the oils. Tobacco seed oils (TSOs) were extracted via cold pressing (CP) and Soxhlet extraction (SE). Fatty acids and phenolic compounds were analyzed using GC-FID and LC-MS/MS, respectively. Non-ozonated oils averaged 73% linoleic acid, 13% oleic acid, 9% palmitic acid, and 3% stearic acid, while ozonated oils contained 41% linoleic acid, 18% oleic acid, 22% palmitic acid, and 11% stearic acid. The presence of 33 phenolic compounds was investigated and 22 common phenolic compounds were detected in both ozonated and non-ozonated TSOs, including gallic acid, protocatechuic acid, chlorogenic acid, 3-hydroxybenzoic acid, (-)-epicatechin, 2,5-dihydroxybenzoic acid, caffeic acid, verbascoside, luteolin-7-glucoside, hesperidin, and rosmarinic acid. The increased antimicrobial activity of fatty acids and phenolic compounds following ozonation suggests the potential for developing creams to treat skin diseases and wounds caused by antibiotic-resistant pathogens, and to reduce the effects of aging skin wrinkles. [ABSTRACT FROM AUTHOR]

Two new aromatic sesquiterpenes, 2-(3-isopropyl-4,5-dimethoxybenzyl)-4-methylfuran (1) and (5-(3-isopropyl-4,5-dimethoxybenzyl)furan-3-yl)methanol (2) were isolated from the stems of cigar tobacco (a variety of Nicotiana tabacum). Their structures were determined by spectroscopic methods, including extensive 1D and 2D NMR techniques. Interestingly, compound 2 has a weak, sweet fragrance. They can enhance the sweet aroma style of cigar smoke and have potential value for use as an essence for cigar tobacco. [ABSTRACT FROM AUTHOR]

It is still difficult to manipulate the nucleotide-binding site-leucine-rich repeat (NB-LRR) class disease resistance genes because of their large multigenic family. Here, we report the successful application of CRISPR/Cas9 to resurrect a functional allele from a susceptible allele of the N′ tobamovirus resistance gene. The susceptible alleles of N′ from some Nicotiana tabacum cultivars (Nt-n′) have a partial duplication of the N′ coding sequence upstream of a complete coding sequence of the N′ gene, which likely abolished the N′-mediated resistance. We first established a transgenic tobacco line expressing Cas9 under the control of a chemically inducible promoter. The plant line was retransformed with a construct expressing a guide RNA targeting the sequences common to the duplicated partial sequence in the upstream and the complete sequence in the downstream. The T0 transformants had different ratios of the sequences devoid of the duplicated partial sequence. Sequencing proved that some of them had sequences identical to that of the functional N′ gene, suggesting the successful resurrection of the functional N′ gene. The resurrected allele, N′-R, was inherited by a few T1 progenies and subsequent generations with the least mutation at the target site under Cas9-uninduced conditions. The plants homozygous for N′-R showed resistance to a tobamovirus, indicating that the resurrected N′-R allele is functional. [ABSTRACT FROM AUTHOR]

Hydraulic conductance of plant tissues depend on the presence of water channels aquaporins in the membranes. Previous experiments showed that over-expression of aquaporin genes in transgenic plants improved plant vigor under favorable growth conditions. However, when growing such plants under stressful conditions, contradictory results were obtained. We hypothesized that, to explain this discrepancy, attention should be paid to the deposition of lignin and suberin, which influence conductivity of the alternative pathway for water passage through the apoplast. Transgenic tobacco (Nicotiana tabacum L.) plants that carry the gene encoding barley plasma membrane aquaporin (HvPIP2;1) and control transgenic plants transformed with an empty vector were grown under normal and salt stress conditions. Under favorable conditions, hydraulic conductance was higher in plants expressing HvPIP2;1 gene accompanied by increased transpiration and growth rate. Salinity decreased hydraulic conductance in the plants of both genotypes, which helped plants to economize water under the salt caused water shortage. In the transgenic plants transformed with the empty vector, this decline in hydraulic conductance was associated with the down-regulation of NtPIP2;1 gene. Although the mRNA abundancy of the gene encoding NtPIP2;1 was also reduced by salinity in plants carrying HvPIP2;1, transgene expression remained high in plants experiencing salinity stress, while the decrease in hydraulic conductance was likely due to strengthened apoplastic barriers. Since apoplastic barriers reduce the entry of toxic sodium ions into plants, their enhancement may explain the increased salt tolerance found in transgenic plants expressing HvPIP2;1, as evidenced by increased Fv/Fm and growth rate under salinity conditions. [ABSTRACT FROM AUTHOR]

An attempt is made to create transgenic plants using rol genes from A4, 15834, and K599 strains of A. rhizogenes. Strains A4 and 15834 transformed tobacco plants are found to display better root growth than their wild types under normal conditions and at elevated concentrations of sodium chloride or cadmium acetate. At the same time, the rol genes acquired from the K599 strain negatively affect root growth under both normal and stressful conditions (e.g., salinization, hypothermia, or excess cadmium acetate). The levels of total protein, proline, total glutathione, and superoxide dismutase activity are higher in the roots of the transgenic plants with rol genes from the A4 and 15834 strains than in nontransgenic control plants under both optimum conditions and salinization. When using rol transgenes from the K599 strain, the activities of superoxide dismutase, guaiacol peroxidase, and glutathione-S-transferase are enhanced in the roots subjected to salinization. The positive effect rol genes have on the root growth of transgenic plants could therefore be explained by their influence on components of the antioxidant system. Results suggest that using rol genes from the A4 and 15834 strains of A. rhizogenes are promising for breeding plant cultivars and liness with improved parameters of root growth and stress tolerance. At the same time, rol genes from the K599 strain appear to be inadequate for this task. [ABSTRACT FROM AUTHOR]

Starch metabolism is regulated by a complex catalytic network, one of the key enzymes of which is the plastid starch phosphorylase PHO1. In this study, using the CRISPR-Cas9 system, we obtained tobacco (Nicotiana tabacum L.) plants with a partial knockout of the NtPHO1-L1 gene due to deletion variants of the catalytic domain of the NtPHO1-L1 protein, leading to the formation of nonfunctional forms of the enzyme. The edited lines differed from wild-type plants by increased starch accumulation and decreased content of sugars, chlorophylls, and carotenoids in the leaves. It was shown that, compared to the control, the edited plants were characterized by differential expression of starch (NtPHO1-L1, NtGWD, NtBAM1, NtBAM9, NtAI) and carotenoid (NtPSY2, NtPDS, NtZDS, NtCRTISO, NtVDE) metabolism genes, as well as genes encoding MADS-domain transcription factors (NtFUL1, NtSEP1, NtSEP2, NtSEP3), which are presumably involved in the regulation of transcription of the studied metabolic genes. These data suggest that partial knockout of NtPHO1-L1 alters the functional activity of tobacco starch phosphorylase. This, in turn, may influence the coordinated activity of starch catabolism enzymes, as well as chlorophyll and carotenoid synthesis enzymes, possibly through differential expression of MADS-box genes. The results highlight the critical regulatory role of plastid starch phosphorylase in transient starch metabolism and in stimulating plant photosynthesis. [ABSTRACT FROM AUTHOR]

Bread wheat (Triticum aestivum L.) is one of the most important cereals and staple food crops worldwide and is grown quite often in arid regions of the world where water shortages and salinity cause serious crop losses. The TaNAC69 gene of bread wheat encodes one of the most important transcription factors involved in the regulation of resistance to water deficit caused by drought and hypothermia, but the role of this gene under salinity is poorly understood. This study aimed to clone the TaNAC69 gene of bread wheat in a binary vector with the 35S CaMV promoter and to create hairy roots (HRs) of Nicotiana tabacum L. with constitutive expression of the TaNAC69 gene to assess the effectiveness of the target genetically engineered construct under salinity. As the result of Agrobacterium-mediated transformation of tobacco leaf discs using Agrobacterium rhizogenes, 15 lines of HR cultures were created, in six of which the presence and high level of expression of the target gene were proven. Tobacco HRs with constitutive expression of the TaNAC69 gene were characterized by faster growth rates, greater branching, and higher fresh and dry weights when exposed to 150 mM NaCl than control tobacco HRs without the target gene. Analysis of the antioxidant system revealed increased activity of the enzymes guaiacol peroxidase and glutathione-S-transferase, higher content of proline and water-soluble sugars, and higher total antioxidant capacity in HR with the TaNAC69 gene compared to the untransformed control. The TaNAC69 with the 35S CaMV promoter construct can be recommended for the genetic transformation of crops to increase their resistance to abiotic stresses. [ABSTRACT FROM AUTHOR]

Abstract Background Ribosome profiling (or Ribo-seq) is a technique that provides genome-wide information on the translational landscape (translatome). Across different plant studies, variable methodological setups have been described which raises questions about the general comparability of data that were generated from diverging methodologies. Furthermore, a common problem when performing Ribo-seq are abundant rRNA fragments that are wastefully incorporated into the libraries and dramatically reduce sequencing depth. To remove these rRNA contaminants, it is common to perform preliminary trials to identify these fragments because they are thought to vary depending on nuclease treatment, tissue source, and plant species. Results Here, we compile valuable insights gathered over years of generating Ribo-seq datasets from different species and experimental setups. We highlight which technical steps are important for maintaining cross experiment comparability and describe a highly efficient approach for rRNA removal. Furthermore, we provide evidence that many rRNA fragments are structurally preserved over diverse nuclease regimes, as well as across plant species. Using a recently published cryo-electron microscopy (cryo-EM) structure of the tobacco 80S ribosome, we show that the most abundant rRNA fragments are spatially derived from the solvent-exposed surface of the ribosome. Conclusion The guidelines presented here shall aid newcomers in establishing ribosome profiling in new plant species and provide insights that will help in customizing the methodology for individual research goals.

Abstract Background Biochar, a carbon-rich source and natural growth stimulant, is usually produced by the pyrolysis of agricultural biomass. It is widely used to enhance plant growth, enzyme activity, and crop productivity. However, there are no conclusive studies on how different levels of biochar application influence these systems. Methods and results The present study elucidated the dose-dependent effects of biochar application on the physiological performance, enzyme activity, and dry matter accumulation of tobacco plants via field experiments. In addition, transcriptome analysis was performed on 60-day-old (early growth stage) and 100-day-old (late growth stage) tobacco leaves to determine the changes in transcript levels at the molecular level under various biochar application levels (0, 600, and 1800 kg/ha). The results demonstrated that optimum biochar application enhances plant growth, regulates enzymatic activity, and promotes biomass accumulation in tobacco plants, while higher biochar doses had adverse effects. Furthermore, transcriptome analysis revealed a total of 6561 differentially expressed genes (DEGs) that were up- or down-regulated in the groupwise comparison under different treatments. KEGG pathways analysis demonstrated that carbon fixation in photosynthetic organisms (ko00710), photosynthesis (ko00195), and starch and sucrose metabolism (ko00500) pathways were significantly up-regulated under the optimal biochar dosage (600 kg/ha) and down-regulated under the higher biochar dosage (1800 kg/ha). Conclusion Collectively, these results indicate that biochar application at an optimal rate (600 kg/ha) could positively affect photosynthesis and carbon fixation, which in turn increased the synthesis and accumulation of sucrose and starch, thus promoting the growth and dry matter accumulation of tobacco plants. However, a higher biochar dosage (1800 kg/ha) disturbs the crucial source-sink balance of organic compounds and inhibits the growth of tobacco plants.

Background: Ribosome profiling (or Ribo-seq) is a technique that provides genome-wide information on the translational landscape (translatome). Across different plant studies, variable methodological setups have been described which raises questions about the general comparability of data that were generated from diverging methodologies. Furthermore, a common problem when performing Ribo-seq are abundant rRNA fragments that are wastefully incorporated into the libraries and dramatically reduce sequencing depth. To remove these rRNA contaminants, it is common to perform preliminary trials to identify these fragments because they are thought to vary depending on nuclease treatment, tissue source, and plant species. Results: Here, we compile valuable insights gathered over years of generating Ribo-seq datasets from different species and experimental setups. We highlight which technical steps are important for maintaining cross experiment comparability and describe a highly efficient approach for rRNA removal. Furthermore, we provide evidence that many rRNA fragments are structurally preserved over diverse nuclease regimes, as well as across plant species. Using a recently published cryo-electron microscopy (cryo-EM) structure of the tobacco 80S ribosome, we show that the most abundant rRNA fragments are spatially derived from the solvent-exposed surface of the ribosome. Conclusion: The guidelines presented here shall aid newcomers in establishing ribosome profiling in new plant species and provide insights that will help in customizing the methodology for individual research goals. [ABSTRACT FROM AUTHOR]

SUMMARY: UV‐B radiation can induce the accumulation of many secondary metabolites, including flavonoids, in plants to protect them from oxidative damage. BRI1‐EMS‐SUPPRESSOR1 (BES1) has been shown to mediate the biosynthesis of flavonoids in response to UV‐B. However, the detailed mechanism by which it acts still needs to be further elucidated. Here, we revealed that UV‐B significantly inhibited the transcription of multiple transcription factor genes in tobacco, including NtMYB27, which was subsequently shown to be a repressor of flavonoids synthesis in tobacco. We further demonstrated that NtBES1 directly binds to the E‐box motifs present in the promoter of NtMYB27 to mediate its transcriptional repression upon UV‐B exposure. The UV‐B‐repressed NtMYB27 could bind to the ACCT‐containing element (ACE) in the promoters of Nt4CL and NtCHS and served as a modulator that promoted the biosynthesis of lignin and chlorogenic acid (CGA) but inhibited the accumulation of flavonoids in tobacco. The expression of NtMYB27 was also significantly repressed by heat stress, suggesting its putative roles in regulating heat‐induced flavonoids accumulation. Taken together, our results revealed the role of NtBES1 and NtMYB27 in regulating the synthesis of flavonoids during the plant response to UV‐B radiation in tobacco. Significance Statement: UV‐B radiation can induce the accumulation of flavonoids in plants to protect them from oxidative damage. BES1 (BRI1‐EMS‐SUPPRESSOR1) has been shown to mediate the biosynthesis of flavonoids in response to UV‐B. However, the detailed mechanism by which BES1 acts still needs to be further elucidated. We provided evidence to reveal the role of NtBES1 and NtMYB27 in modulating plant response to UV‐B radiation by regulating the synthesis of flavonoids in tobacco. [ABSTRACT FROM AUTHOR]

SUMMARY: The outcome of certain plant–virus interaction is symptom recovery, which is accompanied with the emergence of asymptomatic tissues in which the virus accumulation decreased dramatically. This phenomenon shows the potential to reveal critical molecular factors for controlling viral disease. MicroRNAs act as master regulators in plant growth, development, and immunity. However, the mechanism by which miRNA participates in regulating symptom recovery remains largely unknown. Here, we reported that miR172 was scavenged in the recovered tissue of tobacco mosaic virus (TMV)‐infected Nicotiana tabacum plants. Overexpression of miR172 promoted TMV infection, whereas silencing of miR172 inhibited TMV infection. Then, TARGET OF EAT3 (TOE3), an APETALA2 transcription factor, was identified as a downstream target of miR172. Overexpression of NtTOE3 significantly improved plant resistance to TMV infection, while knockout of NtTOE3 facilitated virus infection. Furthermore, transcriptome analysis indicated that TOE3 promoted the expression of defense‐related genes, such as KL1 and MLP43. Overexpression of these genes conferred resistance of plant against TMV infection. Importantly, results of dual‐luciferase assay, chromatin immunoprecipitation‐quantitative PCR, and electrophoretic mobility shift assay proved that TOE3 activated the transcription of KL1 and MLP43 by binding their promoters. Moreover, overexpression of rTOE3 (the miR172‐resistant form of TOE3) significantly reduced TMV accumulation compared to the overexpression of TOE3 (the normal form of TOE3) in miR172 overexpressing Nicotiana benthamiana plants. Taken together, our study reveals the pivotal role of miR172/TOE3 module in regulating plant immunity and in the establishment of recovery in virus‐infected tobacco plants, elucidating a regulatory mechanism integrating plant growth, development, and immune response. Significance Statement: miR172/TOE3 module is well‐known for its role in regulating reproductive process. In this study, we demonstrated that miR172/TOE3 module plays an important role in regulating the resistance of tobacco to TMV infection and the formation of symptom recovery. [ABSTRACT FROM AUTHOR]

The natural source of chymosin, a key enzyme in the dairy industry, is insufficient for rapidly growing cheese industries. Large-scale production of recombinant proteins in heterologous hosts provides an efficient alternative solution. Here, the codon-optimized synthetic prochymosin gene, which has a CAI index of 0.926, was subcloned from a cloning vector (pUC57-bCYM) into the pBI121 vector, resulting in the construct named pBI121-bCYM. CAI ranges from 0 to 1 and higher CAI improves gene expression in heterologous hosts. The overexpression of the prochymosin gene was under the control of constitutive CaMV 35S promoter and NOS terminator and was transferred into the tobacco via A. tumefaciens strain LBA4404. Explant type, regeneration method, inoculation temperature, cell density (OD600) of Agrobacterium for inoculation, and acetosyringone concentration were leaf explants, direct somatic embryogenesis, 19 °C, 0.1, and 100 µM, respectively. The successful integration and expression of the prochymosin gene, along with the bioactivity of recombinant chymosin, were confirmed by PCR, RT-PCR, and milk coagulation assay, respectively. Overall, this study reports the first successful overexpression of the codon-optimized prochymosin form of the bovine chymosin enzyme in the tobacco via indirect transformation. Production of recombinant bovine chymosin in plants can be an easy-to-scale-up, safe, and inexpensive platform. [ABSTRACT FROM AUTHOR]

SUMMARY: The RNA regulatory network is a complex and dynamic regulation in plant cells involved in mRNA modification, translation, and degradation. Ras‐GAP SH3 domain‐binding protein (G3BP) is a scaffold protein for the assembly of stress granules (SGs) and is considered an antiviral component in mammals. However, the function of G3BP during virus infection in plants is still largely unknown. In this study, four members of the G3BP‐like proteins (NtG3BPLs) were identified in Nicotiana tabacum and the expression levels of NtG3BPL1 were upregulated during chilli veinal mottle virus (ChiVMV) infection. NtG3BPL1 was localized in the nucleus and cytoplasm, forming cytoplasmic granules under transient high‐temperature treatment, whereas the abundance of cytoplasmic granules was decreased under ChiVMV infection. Overexpression of NtG3BPL1 inhibited ChiVMV infection and delayed the onset of symptoms, whereas knockout of NtG3BPL1 promoted ChiVMV infection. In addition, NtG3BPL1 directly interacted with ChiVMV 6K2 protein, whereas 6K2 protein had no effect on NtG3BPL1‐derived cytoplasmic granules. Further studies revealed that the expression of NtG3BPL1 reduced the chloroplast localization of 6K2‐GFP and the NtG3BPL1‐6K2 interaction complex was localized in the cytoplasm. Furthermore, NtG3BPL1 promoted the degradation of 6K2 through autophagy pathway, and the accumulation of 6K2 and ChiVMV was affected by autophagy activation or inhibition in plants. Taken together, our results demonstrate that NtG3BPL1 plays a positive role in tobacco resistance against ChiVMV infection, revealing a novel mechanism of plant G3BP in antiviral strategy. Significance Statement: G3BP is a key component of cytoplasmic stress granules and exerts antiviral effects in mammals, but its function during plant virus infection remains unclear. Here, we revealed that NtG3BPL1 is induced upon ChiVMV infection and interacts with ChiVMV 6K2 to disturb the chloroplast localization of 6K2, and promote the degradation of 6K2, thereby limiting ChiVMV infection. This study enhances our understanding of the function of G3BP in plant virus infection. [ABSTRACT FROM AUTHOR]

Background and aims: Nitrogen (N) distribution in plants is intricately linked to key physiological functions, including respiration, photosynthesis, structural development, and nitrogen storage. However, the specific effects of different N morphologies on N accumulation and plant growth are poorly understood. Our research specifically focused on determining how different N morphologies affect N absorption and biomass accumulation. Methods: This study elucidated the impact of different application rates (CK: 0 g N/plant; T1: 4 g N/plant; T2: 8 g N/plant) of N fertilizer on N and biomass accumulation in tobacco cultivars Hongda and K326 at different growth stages. Results: Ourfindings emphasize the critical role of N distribution invarious plantparts, including leaves, stems, and roots, in determining the complexmechanisms of N and biomass accumulation in tobacco. We found that in relation tototalN, a greater ratio of water-soluble N (Nw) in leaves facilitated N accumulation in leaves. In contrast, an increased ratio of SDS (detergent)-insoluble N (Nin-SDS) in leaves and non-protein N (Nnp) in roots hindered this increase. Additionally, our results indicate that a greater proportion of Nnp in leaves has a negative impact on biomass accumulation in leaves. Furthermore, elevated levels of Nin-SDS, Nw, and Nnp in roots, and Nnp in leaves adversely affected biomass accumulation in tobacco leaves. The Hongda cultivar exhibited greater biomass and N accumulation abilities as compared to K326. Conclusions: Our findings highlight the significant role of distribution of N morphologies on plant growth, as well as N and biomass accumulation in tobacco plants. Understanding N distribution allows farmers to optimize N application, minimizing environmental losses and maximizing yield for specific cultivars. These insights advance sustainable agriculture by promoting efficient resource use and reducing environmental impact. [ABSTRACT FROM AUTHOR]

Water supply limitations will likely impose increasing restrictions on future crop production, underlining a need for crops that use less water per mass of yield. Water use efficiency (WUE) therefore becomes a key consideration in developing resilient and productive crops. In this study, we hypothesized that it is possible to improve WUE under drought conditions via modulation of chloroplast signals for stomatal opening by up-regulation of non-photochemical quenching (NPQ). Nicotiana tabacum plants with strong overexpression of the PsbS gene encoding PHOTOSYSTEM II SUBUNIT S, a key protein in NPQ, were grown under differing levels of drought. The PsbS -overexpressing lines lost 11% less water per unit CO2 fixed under drought and this did not have a significant effect on plant size. Depending on growth conditions, the PsbS -overexpressing lines consumed from 4–30% less water at the whole-plant level than the corresponding wild type. Leaf water and chlorophyll contents showed a positive relation with the level of NPQ. This study therefore provides proof of concept that up-regulation of NPQ can increase WUE, and as such is an important step towards future engineering of crops with improved performance under drought. [ABSTRACT FROM AUTHOR]

In current study, anti-parasitic properties of Nicotiana tabacum leaves (N. tabacum L.) and their traditional use in ethno-veterinary therapy were investigated using both in-vitro and in-vivo approaches. Crude aqueous extracts (CAE) of N. tabacum L. were used to evaluate in-vitro antiparasitic effect of Haemonchus (H.) placei that had been directly retrieved from the abomasum of cows. The nematodes' death or paralysis within three hours subsequent to being exposed to different quantities 25, 50 and 100 mg/ml of CAE proved to be statistically significant (P<0.05) indicator. Cattle infected with gastrointestinal nematodes, specifically H. placei, were orally administered with crude aqueous extracts (CAE) and crude methanolic extracts (CME) at gradually higher dosage of 1.0 and 3.0 gm/kg, respectively to evaluate in-vivo anthelmintic efficiency. When administered with 3.0 gm/kg of the body weight, CME showed an ideal decrease of 75.8 and 79.03% parasitic infection but CAE showed 48.88 and 51.11% reduction after 5 and 10 days of treatment, respectively (P<0.05). The overall reduction in eggs per gram was 98.68% in positive controls treated with levamisole HCl. Being an initial investigation of its kind in Pakistan, the outcomes suggested that N. tabacum L. have potential to be used as anti-parasitic agent in ethno-veterinary medication. [ABSTRACT FROM AUTHOR]

Ticks are ectoparasites belonging to the phylum Arachnida infesting humans and animals. Besides their parasitic role, ticks also spread multiple bacterial, viral, and parasitic diseases because of their vector role. Hyalomma is a major genus of ticks containing species of zoonotic and pathogenic significance. Control of the ticks is the primary importance, but chemical acaricides are being avoided because of resistance and one health-related issue. In this study, acaricidal and repellent activities of the ethanolic and ethyl acetate extracts of Tobacco (Nicotiana tabacum) have been evaluated against the ticks of Hyalomma genus. Each extract was serially diluted to have 1.25, 2.5, 5, and 10% concentrations. The parameters taken were adult mortality, larval mortality, egg hatchability and product effectiveness. The results suggested that the ethyl acetate and ethanolic extracts had statistically comparable (P>0.05) effects on adult mortality (tick immersion), larval mortality (larval immersion test), egg hatchability and tick repellency parameters. This research also revealed that extracts of N. tabacum were also comparable to (P>0.05) standard medicated control at the highest concentrations. Ethanolic and ethyl acetate extracts had significantly (P<0.05) different acaricidal effects from the blank control at 10% concentration. This research suggests that the N. tabacum can be used as an acaricidal and tick-repellent agent. [ABSTRACT FROM AUTHOR]

Copyright of Revista Chapingo Serie Horticultura is the property of Universidad Autonoma Chapingo and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Salvia guaranitica is considered one of the most significant medicinal and aromatic herbs in terms of nutritional and medical benefits due to its wealth of important active components. Among these compounds, terpenoids are the most prominent and abundant, particularly monoterpenes (C10), sesquiterpenes (C15), and diterpenes (C20). They are biologically advantageous to plants and perform a multitude of functions. The current study aimed to clone the S. guaranitica gene that encodes for geranyllinalool synthases (SgGES, EC: 4.2.3.144), with consideration for these features. The open reading frame of the 867-amino-acid protein encoded by SgGES consists of 2.721 base pairs. In addition, the SgGES protein has five domains that belong to the terpene synthase family, which are related to the terpene and terpenoid synthase domains. We manipulated and overexpressed the SgGES gene in Nicotiana tabacum to explore its function. When compared to the GUS control, the transgenic N. tabacum plants displayed an increase in leaf production and diameter when compared with the wild-type plants. Finally, analysis of transgenic plants using gas chromatography/mass spectrometry (GC-MS) showed that SgGES is responsible for producing various terpene species, especially diterpenes. [ABSTRACT FROM AUTHOR]

Kunitz trypsin inhibitor genes play important roles in stress resistance. In this study, we investigated RpKTI2 cloned from Robinia pseudoacacia and its effect on tobacco. RpKTI2 was introduced into the tobacco cultivar NC89 using Agrobacterium-mediated transformation. Six RpKTI2-overexpressing lines were obtained. Transgenic and wild-type tobacco plants were then compared for photosynthetic characteristics and endogenous hormone levels. Transgenic tobacco showed minor changes in chlorophyll content, fluorescence, and photosynthetic functions. However, the maximum photochemical efficiency (Fv/Fm) increased significantly while intercellular CO2 concentration (Ci) decreased significantly. Stomatal size and hormone content (indole-3-acetic acid, zeatin riboside, gibberellin, and indole-3-propionic acid) were reduced, while brassinosteroid content increased. Random forest regression revealed that RpKTI2 overexpression had the biggest impact on carotenoid content, initial fluorescence, Ci, stomatal area, and indole-3-acetic acid. Overall, RpKTI2 overexpression minimally affected chlorophyll synthesis and photosynthetic system characteristics but influenced stomatal development and likely enhanced the antioxidant capacity of tobacco. These findings provide a basis for future in-depth research on RpKTI2. [ABSTRACT FROM AUTHOR]

The INDETERMINATE DOMAIN (IDD) gene family, encoding a class of C2H2 transcription factor, played diverse roles in land plants. The IDD family in tobacco (Nicotiana tabacum) has not been characterized. In this study, 26 NtIDDs were identified in the tobacco genome. Phylogenetic analysis showed that NtIDDs were divided into five groups. Motif analysis revealed that the ID domain was conserved in NtIDDs. Gene duplication analysis demonstrated that segmental/whole-genome duplication and dispersed duplication would have occurred in NtIDDs. Cis-element analysis predicted that hormone-, stress-, and development-related elements are located in NtIDD promoters. Expression analysis revealed tissue preference patterns and differential hormone responses in NtIDDs. Further investigations on the function of NtIDD9 exhibited increased leaf angle degrees in RNA silencing plants. Cellular localization suggested that NtIDD9 expressed in the endodermis of the leaf petiole base. Subcellular localization analysis revealed that the NtIDD9 protein was located in the nucleus. Hormone quantification found that the levels of auxin, ABA, JA, and GA were significantly changed in NtIDD9-silenced plants. Thus, the study suggested that NtIDD9 played a crucial role in modulation of leaf angle development. Overall, these findings lay foundations for future function and mechanism research on IDDs in tobacco.

Black shank disease, caused by Phytophthora nicotianae, is one of the major causes of yield loss in tobacco production. The present study aimed to explore the dynamic transcriptome in tobacco genotypes resistant or susceptible to black shank disease and to understand the defense response of tobacco to P. nicotianae infection. Roots and stems were sampled from two resistant and two susceptible materials at 0, 12, 24, 48, and 72 h post infection and used in RNA-sequencing. Conventional approaches that identify differentially expressed genes are not the best way for handling the complex datasets, so a new method that calculates the standard deviation among samples was applied to identify transcripts with variable expression levels in roots and stems of the four materials at different time points. Of the total of 229,501 transcripts, 7,261 were found to be variable transcripts, with many of them annotated to be related to defense responses against pathogen infection. These variable transcripts showed expression patterns that varied significantly between roots and stems as well as between the resistant and the susceptible materials. Several transcripts were identified to be potential candidates for further functional characterization. Our findings provide invaluable insights into the dynamic transcriptome in defense responses of tobacco against P. nicotianae infection.

Tomato yellow leaf curl China virus (TYLCCNV) viruliferous Mediterranean (MED) whitefly infestation in tobacco plants elevated hydrogen peroxide (H2O2) levels more than non-viruliferous ones. To unravel the underlying mechanisms behind the phenomenon, comparative analyses of plant transcriptome and enzyme activities of reactive oxygen species (ROS) scavengers were conducted in this study. Compared to the control, a total of 4032 and 6853 differentially expressed genes (DEGs) were identified in the plants following the infestations of non-viruliferous and TYLCCNV-viruliferous MED, respectively. Gene ontology analysis of DEGs suggested substantial transcriptional reprogramming of diverse cellular processes, particularly in the biotic stress response pathways of tobacco plants. Respiratory burst oxidase homologs related genes and important kinases putatively involved in plant defense were differentially modulated in tobacco plants in response to the non-viruliferous and TYLCCNV-viruliferous whitefly MED infestation. The analysis reported here lays out a strong foundation for future investigations into the mechanisms of ROS-associated plant defense mediating the interactions between B. tabaci and begomoviruses.

Identifying molecular markers linked to tobacco bacterial wilt resistance is crucial for developing resistant tobacco varieties, thereby enhancing tobacco production and quality. In this two-year study, we evaluated the tobacco bacterial wilt disease index (TBWDI) in a mapping population of 78 tobacco accessions using SSR/InDel markers across 1377 marker loci. Two association models, GLM_Q and MLM_Q + Kinship, were used for association analysis. By considering multiple environments, selection thresholds, and phenotype values, we identified 19 reliable marker loci (P = 7.07-E05–4.98-E02) that explained 5.37–19.10 % of the phenotypic variation. Among these, we selected 11 accessions with high resistance to tobacco bacterial wilt, each containing at least one excellent locus. The models predicted five hybrid combinations whose offspring aggregated additional excellent loci. Additionally, one locus was identified during quantitative trait loci mapping, with accessions carrying this locus exhibiting significantly different TBWDI values from those without it, confirming its reliability and stability. A candidate gene (LOC107795335) was also identified downstream at 17.58 Kbp of the locus. Our study pinpointed reliable loci associated with bacterial wilt resistance, identified important materials for breeding resistant tobacco varieities, and predicted the best hybrid combinations for low disease indexes. The results of our study offer invaluable theoretical insights for breeding varieties with high and stable resistance.

Nicotiana tabacum is a kind of plant cultivated for its leaves used for manufacturing medicine and cigarettes. With the common name, the Tobacco plant is grown in many countries including China, Indonesia, Malawi and Tanzania just to mention a few. Literatures suggest a technical gap in the proper identification of grade labels for various parts of the plant. In addition, manual grading has resulted in various gaps and biases. To mitigate this, a data-driven grading solution is necessary. However, relevant datasets to train grade classifiers from various countries become of the essence. This article presents images concentrated on tobacco leaf plant position namely Leaf position which normally carries 23 grade labels. Due to high rainfall which swiped away the applied fertilizer on the tobacco plants in the farms, we failed to get images of one grade. Therefore, this research could capture and label 22 grade labels. Images of tobacco leaves based on the tobacco plant position were collected in Tanzania through participatory community research. Canon 5D mark III cameras with 100 mm micro lens were used to take pictures of tobacco leaves based on the tobacco plant position. Domain experts were used for image labelling and cleaning according to tobacco grade labels identified in Tanzania. The dataset carries 49,779 images, which can be used to develop machine learning models for tobacco leaf grade label identification. The collected dataset can be used to train models and enhance the performance of pre-trained models in any country of interest.

The coastal areas of western Taiwan feature acidic and saline soils with low fertility. Sodium bicarbonate (NaHCO3), produced readily by carbon capture and storage technologies, could be suitable for the neutralization of acidic soils, but its effects on plant growth and the ability of Bacillus subtilis var. natto to confer salinity tolerance remain unclear. In this study, we examined the potential of sodium bicarbonate and B. subtilis var. natto (NTU18) to improve the growth of tobacco (Nicotiana tabacum L.) under salt stress conditions. We found that salt stress was the main factor affecting tobacco growth, resulting in shorter roots and shoots, a reduced leaf area and leaf number, and clustered dark green leaves. The addition of sodium bicarbonate exacerbated the symptoms of salinity stress. Inoculating the soil with B. subtilis did not enhance salinity tolerance, but intriguingly it increased shoot and root growth under normal conditions and in the presence of sodium bicarbonate. The mechanism of growth promotion mediated by the bacteria is unknown and should be investigated in more detail.

Nicotiana Tabacum; the annual herb; known as Tobacco from the Solanaceae family was known for its alkaloids and especially nicotine (NCT) content; smoking products, insecticides, anthelminthic activity and clinically proven therapeutics are examples of its uses. Herein two different methods were applied in an attempt to quantify the total alkaloids and NCT content in Nicotiana tabacum cultivated in Lebanon. Total alkaloids were investigated through the formation of a complex with bromocresol green under a simple spectrophotometric method. Whilst HPLC-DAD was the choice for the quantitation of NCT levels. The column was Lichrospher select B (5 µm, 250x4 mm), the temperature was set at 29 ⁰C and the wavelength at 260 nm. The mobile phase consisted of 2 M O-phosphoric acid and methanol (60:40, v/v) using isocratic elution at 1 mL/min. A linear relationship was proved under both instruments. The extraction yield of alkaloid totum ranges between 2.1 ± 0.25 and 6.8% ± 0.58 and alkaloids contents range from 12.14 ± 2.01 to 53.12 ± 4.54 mg of AE/g of extract for Ghandouriyeh and Danniye samples respectively. On the other side among the different areas which cultivated Tobacco in Lebanon, Danniye was found to have the highest NCT concentration of dry weight (2.64%) while Al-Hissa possesses the lowest content (0.75%). Even if the results were generally similar to other countries, the study showed a difference in values from one region to another. [ABSTRACT FROM AUTHOR]

Vaccines can reduce the use of antibiotics by preventing specific infective diseases in pigs. Plant-based edible vaccines are particularly attractive because, upon oral ingestion via feed, they can elicit the local immune system against a foreign disease-causing organism. The aim of this study was to engineer two different independent lines of tobacco plants for the seed-specific expression of immunogenic proteins of VTEC as a model of an edible vaccine. For each antigen, fifty Nicotiana tabacum L. cv Xanthi leaf disks were transformed by agroinfection for the seed-specific expression of the structural parts of the fimbrial subunit FedF of F18 and the B-subunit of Vt2e genes. The synthetic genes, optimized by the codon adaptation index for their expression in tobacco, were inserted into expression cassettes under the control of β-conglycinin promoter. Regenerated tobacco plants (T0) were characterized by molecular and immunoenzymatic techniques. Our results showed that both FedF and Vt2eB genes were integrated into tobacco genome efficiently (> 80%) and they are also maintained in the second generation (T1). Western blotting analyses carried out on the positive producing lines, showed the tissue-specific expression in seeds and the temporal protein accumulation in the mid-late maturation phases. The enzyme-linked immunosorbent assay showed seed expression levels of 0.09 to 0.29% (from 138 to 444 µg/g of seeds) and 0.21 to 0.43% (from 321 to 658 µg/g of seeds) of total soluble protein for the FedF and Vt2eB antigens, respectively. This study confirmed the seed-specific expression of the selected antigens in plant seeds. The expression level is suitable for seed-based edible vaccination systems, which could represent a cost-effective way to prevent VTEC infection. Our findings encourage further in vivo studies focused on the activation of the local immune response. [ABSTRACT FROM AUTHOR]

Promoters play an important role in controlling the expression of genes in transgenic plants. Inducible promoters regulated by stressful conditions are valuable tools for the temporal expression of stress-tolerant genes. Exploring new promoters aims to fine-tune gene expression. Overexpression of a gene in transgenic research using strong and constitutive promoters is always an energy barrier to the cell and can lead to gene silencing, making mild expression preferable, especially when controlling a cascade of genes using transcriptional factors. Although generalized promoters are available, their tissue specificity and controlled level of induction remains questionable. Inducible promoters express genes only when coded proteins are needed under specific conditions. Therefore, exploration of stress inducible promoters and determining their level of expression is deeply desired in transgenic research for a desired level of gene expression. In this study, three monocot stress-inducible promoters controlling the dehydrin gene in Hordeum vulgare, HSP101 in Oryza sativa, and ZmPIS in Zea mays were isolated and compared for transient β-glucuronidase (GUS) production under different stress conditions. The transient expression of each promoter was analyzed by GUS histochemical staining and fluorometric assays after mimicking drought and salinity stresses using polyethylene glycol (PEG-6000), abscisic acid (ABA), and 200mM NaCl salt treatments, respectively. The results indicated that drought stress induced by PEG-6000 caused highest expression of GUS by all the three promoters followed by ABA and salt stress was the least to induce these promoters. Furthermore, PlantCARE and PLACE analyses of the promoter DNA sequences highlighted various cis-regulatory elements, such as DREB, MYB, ABRE, and WRKY, as key motifs common to these stress-inducible promoters. These studies indicate that dehydrin, HSP101, and ZmPIS promoters respond to drought from the highest to moderate levels in sequential order. It was concluded that all three promoters reported here responded well to drought stress. [ABSTRACT FROM AUTHOR]

Transcription factors belonging to the family of ethylene response factors (ERFs) in plants function in disease resistance to pathogens. One of the tobacco ERF genes, NtERF5, inhibits the accumulation of tobacco mosaic virus (genus Tobamovirus) when overexpressed in tobacco carrying the resistance gene N. Here, we report that NtERF5 can function as a transcriptional activator through binding of the GCC-box in planta. We then determined whether overexpression of NtERF5 in tobacco lacking the N gene (nn tobacco) inhibited infection by potato virus X (PVX: genus Potexvirus). Transient overexpression of NtERF5 inhibited the accumulation of PVX without affecting virus spread and increased the transcript levels of NtPRB-1b and NtPR6, which are jasmonic acid/ethylene-signaling marker genes encoding pathogenesis-related (PR) proteins. Stable overexpression of NtERF5 in nn tobacco also resulted in decreased PVX accumulation and the induction of the expression of NtPRB-1b and NtPR6. Our results revealed that overexpression of NtERF5 limited successful infection and accumulation of PVX, suggesting the role of NtERF5 as a positive regulator of basal defense against virus infection. [ABSTRACT FROM AUTHOR]

A comparative analysis was performed under in vitro conditions using Nicotiana tabacum lines overexpressing the genes of arginine decarboxylase (FvADC) and spermidine synthase (FvSPDS) enzymes. The transgenic and native lines were tested under controlled conditions and exposed to long-term treatment of arginine (150 mg/L), putrescine (10 mg/L), and spermidine (10 mg/L). Chlorophyll and lignin contents of the samples were measured spectrophotometrically, while proline, putrescine, spermidine, and spermine contents were determined using high-performance liquid chromatography methods. The experimental results showed that the arginine decarboxylase enzyme affects polyamine metabolism. As it is involved in several other biosynthetic pathways, this effect is significant but not outstanding. Spermidine synthase is more abundant: it directly enhances the accumulation of higher polyamine forms (spermidine and spermine) without a direct negative feedback. A complex regulatory mechanism plays an important role in the precise adjustment of the amount and proportion of polyamines, an equilibrium that cannot be disrupted by minor influences. [ABSTRACT FROM AUTHOR]

Two new aromatic sesquiterpenes, 7-isopropyl-2,2,5-trimethyl-2H-chromene (1) and 7-isopropyl-5- (methoxymethyl)-2,2-dimethyl-2H-chromene (2), were isolated from the leaves of Nicotiana tabacum (a mutant tobacco for vitamin E synthesis gene knockout by CRISPR-Cas9 gene editing). Their structures were determined by spectroscopic methods, including extensive 1D and 2D NMR techniques. Interestingly, compounds 1 and 2 have a weak bean fragrance, and they have the potential value for use as an essence for Heat Not Burning (HNB) cigarettes. [ABSTRACT FROM AUTHOR]

Plant senescence, as a highly integrated developmental stage, involves functional degeneration and nutrient redistribution. NAM/ATAF1/CUC (NAC) transcription factors orchestrate various senescence-related signals and mediate the fine-tuning underlying plant senescence. Previous data revealed that knockout of either NtNAC028 or NtNAC080 leads to delayed leaf senescence in tobacco (Nicotiana tabacum), which implies that NtNAC028 and NtNAC080 play respective roles in the regulation of leaf senescence, although they share 91.87% identity with each other. However, the mechanism underlying NtNAC028- and NtNAC080-regulated leaf senescence remains obscure. Here, we determined that NtNAC028 and NtNAC080 activate a putative jasmonic acid (JA) biosynthetic gene, NtLOX3 , and enhance the JA level in vivo. We found that NtNAC028 and NtNAC080 interact with each other and themselves through their NA-terminal region. Remarkably, only the dimerization between NtNAC028 and NtNAC080 stimulated the transcriptional activation activity, but not the DNA binding activity of this heterodimer on NtLOX3. Metabolome analysis indicated that overexpression of either NtNAC028 or NtNAC080 augments both biosynthesis and degradation of nicotine in the senescent stages. Thus, we conclude that NtNAC028 cooperates with NtNAC080 and forms a heterodimer to enhance NtLOX3 expression and JA biosynthesis to trigger the onset of leaf senescence and impact secondary metabolism in tobacco. [ABSTRACT FROM AUTHOR]

The pyridine alkaloid nicotine acts as one of best-studied plant resistant traits in tobacco. Previous research has shown that NtERF199 and NtERF189, actingasmaster regulators within the NIC1 and NIC2 locus, quantitatively contribute to nicotine accumulation levels in N. tabacum. Genome editing-created Nic1(Nterf199) and Nic2 (Nterf189) double mutant provides an ideal platform for precisely dissecting the defensive role of nicotine and the connection between the nicotine biosynthetic pathway with other putative metabolic networks. Taking this advantage, we performed a comparative transcriptomic analysis to reevaluate the potential physiological and metabolic changes in response to nicotine synthesis defect by comparing the nic1nic2 and NIC1NIC2 plants. Our findings revealed that nicotine reduction could systematically diminishes the expression intensities of genes associated with stimulus perception, signal transduction and regulation, as well as secondary metabolic flux. Consequently, this global expression reduction might compromise tobacco adaptions to environmental fitness, herbivore resistances, and plant growth and development. The up-regulation of a novel set of stressresponsive andmetabolic pathway genes might signify a newly establishedmetabolic reprogramming to tradeoff the detrimental effect of nicotine loss. These results offer additional compelling evidence regarding nicotine's critical defensive role in nature and highlights the tight link between nicotine biosynthesis and gene expression levels of quantitative resistance-related genes for better environmental adaptation. [ABSTRACT FROM AUTHOR]

Summary: Inducible expression systems can overcome the trade‐off between high‐level transgene expression and its pleiotropic effects on plant growth. In addition, they can facilitate the expression of biochemical pathways that produce toxic metabolites. Although a few inducible expression systems for the control of transgene expression in plastids have been developed, they all depend on chemical inducers and/or nuclear transgenes. Here we report a temperature‐inducible expression system for plastids that is based on the bacteriophage λ leftward and rightward promoters (pL/pR) and the temperature‐sensitive repressor cI857. We show that the expression of green fluorescent protein (GFP) in plastids can be efficiently repressed by cI857 under normal growth conditions, and becomes induced over time upon exposure to elevated temperatures in a light‐dependent process. We further demonstrate that by introducing into plastids an expression system based on the bacteriophage T7 RNA polymerase, the temperature‐dependent accumulation of GFP increased further and was ~24 times higher than expression driven by the pL/pR promoter alone, reaching ~0.48% of the total soluble protein. In conclusion, our heat‐inducible expression system provides a new tool for the external control of plastid (trans) gene expression that is cost‐effective and does not depend on chemical inducers. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agronomica Sinica is the property of Crop Science Society of China and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Background Stigma exsertion is an essential agricultural trait that can promote cross-pollination to improve hybrid seed production efficiency. However, the molecular mechanism controlling stigma exsertion remains unknown. Results In this study, the Nicotiana tabacum cv. K326 and its two homonuclear-heteroplasmic lines, MSK326 (male-sterile) and MSK326SE (male-sterile and stigma exserted), were used to investigate the mechanism of tobacco stigma exsertion. A comparison of the flowers between the three lines showed that the stigma exsertion of MSK326SE was mainly due to corolla shortening. Therefore, the corollas of the three lines were sampled and presented for RNA-seq analysis, which found 338 candidate genes that may cause corolla shortening. These genes were equally expressed in K326 and MSK326, but differentially expressed in MSK326SE. Among these 338 genes, 15 were involved in hormone synthesis or signal transduction pathways. Consistently, the content of auxin, dihydrozeatin, gibberellin, and jasmonic acid was significantly decreased in the MSK326SE corolla, whereas abscisic acid levels were significantly increased. Additionally, seven genes involved in cell division, cell cycle, or cell expansion were identified. Protein-protein interaction network analysis identified 45 nodes and 79 protein interactions, and the largest module contained 20 nodes and 52 protein interactions, mainly involved in the hormone signal transduction and pathogen defensive pathways. Furthermore, a putative hub gene coding a serine/threonine-protein kinase was identified for the network. Conclusions Our results suggest that hormones may play a key role in regulating tobacco stigma exsertion induced by corolla shortening.

Abstract Polyphenol oxidases (PPOs) are type-3 copper enzymes and are involved in many biological processes. However, the potential functions of PPOs in pollination are not fully understood. In this work, we have screened 13 PPO members in Nicotiana. tabacum (named NtPPO1-13, NtPPOs) to explore their characteristics and functions in pollination. The results show that NtPPOs are closely related to PPOs in Solanaceae and share conserved domains except NtPPO4. Generally, NtPPOs are diversely expressed in different tissues and are distributed in pistil and male gametes. Specifically, NtPPO9 and NtPPO10 are highly expressed in the pistil and mature anther. In addition, the expression levels and enzyme activities of NtPPOs are increased after N. tabacum self-pollination. Knockdown of NtPPOs would affect pollen growth after pollination, and the purines and flavonoid compounds are accumulated in self-pollinated pistil. Altogether, our findings demonstrate that NtPPOs potentially play a role in the pollen tube growth after pollination through purines and flavonoid compounds, and will provide new insights into the role of PPOs in plant reproduction.

[Objective] Phosphorus absorption and utilization by plants mainly depend on phosphorus transporters, among which the low-affinity phosphorus transporters encoded by the PHT2 family are responsible for the absorption, transport and reuse of phosphate by plants under normal phosphorus conditions. We aim to explore the role and expression pattern of NtPHT2;1 gene in tobacco phosphate transport, and to cultivate new tobacco varieties with efficient phosphorus utilization through NtPHT2;1. [Methods] We cloned NtPHT2;1 using the cDNA of a common tobacco K326 as a template, and conducted bioinformatics analysis and subcellular localization analysis of the protein. The expression pattern of this gene under abiotic stress including low phosphorus were analyzed by fluorescence quantitative PCR. [Results] (1) The total length of NtPHT2;1 gene was 1 764 bp, encoding 587 amino acids. (2) NtPHT2;1 protein was localized to chloroplast. (3) The homology between NtPHT2;1 and CaPHT2;1 protein was 91%. (4) Nt- PHT2;1 promoter contains cis-acting elements involved in the regulation of plant aging and stress. (5) The expression of NtPHT2;1 was the highest in leaves, and expression in new leaves was higher than that in old leaves. There was no significant difference in the expression of this gene under low phosphorus compared with normal conditions. (6) The expression of this gene was decreased significantly under salt stress and drought stress. [Conclusion] NtPHT2;1 gene is involved in phosphate transport and utilization in growth and development of tobacco plants.