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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]

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]

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]

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]

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]

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]

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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]

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]

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]

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]

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 FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plant-derived peptides represent a promising group of natural compounds with broad industrial and pharmaceutical applications. Low-efficiency production level is the major obstacle to the commercial production of such bioactive peptides. Today, recombinant techniques have been developed for fast and cost-effective production of high-quality peptides for various applications in the chemical and food industries. The roseltide rT1 is a plant peptide with different antimicrobial properties and therapeutic applications in the prevention and treatment of inflammatory lung diseases by inhibiting human neutrophil elastases. Here, we report the expression of functional recombinant roseltide rT1 peptide in tobacco plants. Transgenic plants were generated by the Agrobacterium-mediated transformation method followed by molecular analysis of transgenic plants to demonstrate successful integration and expression of recombinant rT1 peptide. Protein extracts of transgenic plants expressing a single-copy rT1 gene showed efficient antimicrobial properties as verified by growth inhibition of different bacterial strains. Our results illustrate that plant-derived recombinant rT1 peptide is a promising alternative for rapid and cost-effective production of this important antimicrobial peptide for application in therapeutic and food industries. • Transgenic tobacco plants expressing recombinant roseltide rT1 were generated by Agrobacterium-mediated transformation. • Single-copy transgenic lines were selected and verified by different molecular methods. • The expressed recombinant peptide performed efficient antibacterial properties. • Plant system is presented as a promising alternative for rapid and cost-effective production of recombinant rT1 peptide. [ABSTRACT FROM AUTHOR]

2-acetyl-1-pyrroline (2AP) is a highly effective volatile compound that gives fragrance to numerous plant species and food. Mutation(s) in the betaine aldehyde dehydrogenase 2 (BADH2) gene results in the accumulation of 2AP. However, the function of BADH genes in tobacco (Nicotiana tabacum L.) remains poorly understood. In this study, we successfully obtained four betaine aldehyde dehydrogenase (BADH) genes from tobacco. Phylogenetic analysis of the protein sequences showed that two of the four BADH genes were closely related to the wolfberry (Lycium barbarum) BADH gene (LbBADH1), so we named them NtBADH1a and NtBADH1b, respectively. The other two BADH genes were orthologues of the tomato (Solanum lycopersicum) aminoaldehyde dehydrogenase 2 (SlAMADH2) gene, and were named NtBADH2a and NtBADH2b, respectively. Expression analysis revealed that the biological functions of NtBADH1a and NtBADH1b were different from those of genes NtBADH2a and NtBADH2b. We introduced mutations into NtBADH1a, NtBADH1b, NtBADH2a and NtBADH2b in tobacco using the CRISPR/Cas9 system and identified transgenic Ntbadh mutant tobacco lines. Single mutants (Ntbadh1a, Ntbadh1b, Ntbadh2a and Ntbadh2b) and double mutants (Ntbadh1a-Ntbadh1b and Ntbadh2a-Ntbadh2b) harbored deletion or insertion of nucleotides, both of which led to the production of a frameshift, preventing protein accumulation. A popcorn-like scent was noticeable in tobacco leaves from the Ntbadh2a-Ntbadh2b double mutant, but not from any single mutant or the Ntbadh1a-Ntbadh1b double mutant or the wild type. Consistent with this observation, we only detected 2AP in fresh leaves from the Ntbadh2a-Ntbadh2b double mutant. These findings indicate that only the combined inactivation of NtBADH2a and NtBADH2b results in 2AP accumulation in tobacco, which was not related to NtBADH1. [ABSTRACT FROM AUTHOR]

Chlorogenic acid (CGA) and flavonoids are important secondary metabolites, which modulate plant growth and development, and contribute to plant resistance to various environmental stresses. ERF4 has been shown to be a repressor of anthocyanin accumulation in grape, but its full roles in regulating the biosynthesis of other phenylpropanoid compounds still needs to be further studied. In the present study, two NtERF4 genes were identified from N. tabacum genome. The expression level of NtERF4a was higher than that of NtERF4b in all the tobacco tissues examined. Over-expression of NtERF4a significantly promoted the accumulation of CGA and flavonoids in tobacco leaves, while silencing of NtERF4a significantly repressed the biosynthesis of CGA and flavonoids. RNA-seq analysis of NtERF4a-OE and WT plants revealed 8 phenylpropanoids-related differentially expressed genes (DEGs), including 4 NtPAL genes that encode key enzymes in the phenylpropanoid pathway. Activation of NtERF4a-GR fusion protein in tobacco significantly induced the transcription of NtPAL1 and NtPAL2 in the presence of protein synthesis inhibitor. Chromatin immunoprecipitation and Dual-Luc assays further indicated that NtERF4a could bind to the GCC box presented in the promoters of NtPAL1 and NtPAL2, thereby activating their transcription. Moreover, ectopic expression of NtERF4a induced the transcription of NtGSK1, NtMYC2, and NtJAZ3 genes, and enhanced the resistance of tobacco seedlings to salt and drought stresses, indicating multiple roles of NtERF4a in plants. Our findings revealed new roles of NtERF4a in modulating the accumulation of phenylpropanoid compounds in tobacco, and provided a putative target for improving phenylpropanoids synthesis and stress resistance in plants. [ABSTRACT FROM AUTHOR]

Tobacco (Nicotiana tabacum L.) is a major industrial crop that has being cultivated for centuries for the manufacturing of cigarettes, cigars, and other smoking products. Due to its negative effects on both human health and the environment, the European Union has adopted strict policies that aspire to reduce the consumption of tobacco. Herbal cigarettes are alternative smoking products that are often advertised as healthier than conventional tobacco cigarettes and are especially popular in Asian markets. Even though the available literature suggests that they are equally detrimental to human health, the introduction of tobacco-alternative crops (TACs) to the European tobacco industry could smoothen the abandonment of tobacco, and eventually smoking products altogether, in the EU. The aim of the present systematic review was to compile a list of possible TACs that could be incorporated in the European smoking industry, and highlight their strengths and weaknesses. The most dominant crops in the literature (and in the existing market products) were calendula (Calendula officinalis L.), mullein (Verbascum thapsus L.), ginseng (Panax ginseng C.A.Mey.), tea (Camellia sinensis (L.) Kuntze), chamomile (Matricaria chamomilla L.), and mentha (Mentha spp.). Even though these crops are promising, further research is required for their incorporation in the European tobacco industry. [ABSTRACT FROM AUTHOR]

SUMMARY: We investigated the basis for better performance of transgenic Nicotiana tabacum plants with G6PDH‐isoenzyme replacement in the cytosol (Xanthi::cP2::cytRNAi, Scharte et al., 2009). After six generations of selfing, infiltration of Phytophthora nicotianae zoospores into source leaves confirmed that defence responses (ROS, callose) are accelerated, showing as fast cell death of the infected tissue. Yet, stress‐related hormone profiles resembled susceptible Xanthi and not resistant cultivar SNN, hinting at mainly metabolic adjustments in the transgenic lines. Leaves of non‐stressed plants contained twofold elevated fructose‐2,6‐bisphosphate (F2,6P2) levels, leading to partial sugar retention (soluble sugars, starch) and elevated hexose‐to‐sucrose ratios, but also more lipids. Above‐ground biomass lay in between susceptible Xanthi and resistant SNN, with photo‐assimilates preferentially allocated to inflorescences. Seeds were heavier with higher lipid‐to‐carbohydrate ratios, resulting in increased harvest yields ‐ also under water limitation. Abiotic stress tolerance (salt, drought) was improved during germination, and in floated leaf disks of non‐stressed plants. In leaves of salt‐watered plants, proline accumulated to higher levels during illumination, concomitant with efficient NADP(H) use and recycling. Non‐stressed plants showed enhanced PSII‐induction kinetics (upon dark–light transition) with little differences at the stationary phase. Leaf exudates contained 10% less sucrose, similar amino acids, but more fatty acids – especially in the light. Export of specific fatty acids via the phloem may contribute to both, earlier flowering and higher seed yields of the Xanthi‐cP2 lines. Apparently, metabolic priming by F2,6P2—combined with sustained NADP(H) turnover—bypasses the genetically fixed growth–defence trade‐off, rendering tobacco plants more stress‐resilient and productive. [ABSTRACT FROM AUTHOR]

The current study evaluated the effect of methanol and water extracts of tobacco (Nicotiana tabacum L.), wild marigold (Tagetes minuta L.), and garlic (Allium sativum L.) at 50 mg·mL−1 applied at different frequencies (weekly, biweekly, and monthly) on the control of potato bacterial wilt under field conditions (two growing seasons in 2021). Weekly and biweekly application of methanol extracts from tobacco and wild marigold showed higher efficacy of biological control of the pathogen (58% and 57%) on aerial parts of potato plants than monthly application (48%). In potato tubers, weekly and biweekly application of methanol extracts caused higher biological control efficacy (75.92% and 67.39%) than monthly application (52.49%). Weekly and biweekly application of methanol extracts of tobacco and wild marigold also reduced postharvest infection and postharvest yield losses caused by potato bacterial wilt during the storage period, among other treatments. These experiments conclude that weekly or biweekly application of methanol extract from tobacco and wild marigold at 50 mg·mL−1 is a practical approach to control potato bacterial wilt in the field and storage. [ABSTRACT FROM AUTHOR]

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Tobacco black shank induced by Phytophthora nicotianae causes significant yield losses in tobacco plants. MicroRNAs (miRNAs) play a pivotal role in plant biotic stress responses and have great potential in tobacco breeding for disease resistance. However, the roles of miRNAs in tobacco plants in response to P. nicotianae infection has not been well characterized. In this study, we found that Nta-miR6155, a miRNA specific to Solanaceae crops, was significantly induced in P. nicotianae infected tobacco. Some of predicted target genes of Nta-miR6155 were also observed to be involved in disease resistance. To further investigate the function of miR6155 in tobacco during P. nicotianae infection, Nta-miR6155 overexpression plants (miR6155-OE) were generated in the Honghua Dajinyuan tobacco variety (HD, the main cultivated tobacco variety in China). We found that the Nta-miR6155 overexpression enhanced the resistance in tobacco towards P. nicotianae infections. The level of reactive oxygen species (ROS) was significantly lower and antioxidant enzyme activities were significantly higher in miR6155-OE plants than those in control HD plants during P. nicotianae infection. In addition, we found that the accumulation of salicylic acid and the expression of salicylic acid biosynthesis and signal transduction-related genes is significantly higher in miR6155-OE plants in comparison to the control HD plants. Furthermore, we found that Nta-miR6155 cleaved target genes NtCIPK18 to modulate resistance towards P. nicotianae in tobacco plants. Additionally, phenotypic analysis of miR6155-OE plants showed that Nta-miR6155 could inhibit the growth of tobacco by suppressing nitrogen uptake and photosynthesis. In conclusion, our findings indicated that miR6155 plays a crucial role in the regulation of growth and resistance against P. nicotianae infections in tobacco plants. [ABSTRACT FROM AUTHOR]

Stomatal movement can be regulated by ABA signaling through synthesis of reactive oxygen species (ROS) in guard cells. By contrast, ethylene triggers the biosynthesis of antioxidant flavonols to suppress ROS accumulation and prevent ABA-induced stomatal closure; however, the underlying mechanism remains largely unknown. In this study, we isolated and characterized the tobacco (Nicotiana tabacum) R2R3-MYB transcription factor NtMYB184, which belongs to the flavonol-specific SG7 subgroup. RNAi suppression and CRISPR/Cas9 mutation (myb184) of NtMYB184 in tobacco caused down-regulation of flavonol biosynthetic genes and decreased the concentration of flavonols in the leaves. Yeast one-hybrid assays, transactivation assays, EMSAs, and ChIP-qPCR demonstrated that NtMYB184 specifically binds to the promoters of flavonol biosynthetic genes via MYBPLANT motifs. NtMYB184 regulated flavonol biosynthesis in guard cells to modulate ROS homeostasis and stomatal aperture. ABA-induced ROS production was accompanied by the suppression of NtMYB184 and flavonol biosynthesis, which may accelerate ABA-induced stomatal closure. Furthermore, ethylene stimulated NtMYB184 expression and flavonol biosynthesis to suppress ROS accumulation and curb ABA-induced stomatal closure. In myb184 , however, neither the flavonol and ROS concentrations nor the stomatal aperture varied between the ABA and ABA+ethylene treatments, indicating that NtMYB184 was indispensable for the antagonism between ethylene and ABA via regulating flavonol and ROS concentrations in the guard cells. [ABSTRACT FROM AUTHOR]

Two new benzofulvene sesquiterpenoids, tabanoids A and B (1 and 2) as well as five known sesquiterpenoids (3–7) were isolated from the stem of Nicotiana tabacum. Their structures were determined mainly by spectroscopic methods, including extensive 1D and 2D NMR techniques. Compounds 1 and 2 were tested for their anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity. The results revealed that compounds 1 and 2 showed good inhibition with IZD of 15.2 ± 2.5, and 12.8 ± 2.2 mm, respectively. [ABSTRACT FROM AUTHOR]

Background: Nicotiana tabacum is an important economic crop, which is widely planted in the world. Lignin is very important for maintaining the physiological and stress-resistant functions of tobacco. However, higher lignin content will produce lignin gas, which is not conducive to the formation of tobacco quality. To date, how to precisely fine-tune lignin content or composition remains unclear. Results: Here, we annotated and screened 14 CCoAOMTs in Nicotiana tabacum and obtained homozygous double mutants of CCoAOMT6 and CCoAOMT6L through CRSIPR/Cas9 technology. The phenotype showed that the double mutants have better growth than the wild type whereas the S/G ratio increased and the total sugar decreased. Resistance against the pathogen test and the extract inhibition test showed that the transgenic tobacco has stronger resistance to tobacco bacterial wilt and brown spot disease, which are infected by Ralstonia solanacearum and Alternaria alternata, respectively. The combined analysis of metabolome and transcriptome in the leaves and roots suggested that the changes of phenylpropane and terpene metabolism are mainly responsible for these phenotypes. Furthermore, the molecular docking indicated that the upregulated metabolites, such as soyasaponin Bb, improve the disease resistance due to highly stable binding with tyrosyl-tRNA synthetase targets in Ralstonia solanacearum and Alternaria alternata. Conclusions: CAFFEOYL-COA 3-O-METHYLTRANSFERASE 6/6L can regulate the S/G ratio of lignin monomers and may affect tobacco bacterial wilt and brown spot disease resistance by disturbing phenylpropane and terpene metabolism in leaves and roots of Nicotiana tabacum, such as soyasaponin Bb. [ABSTRACT FROM AUTHOR]

Novel lines of tobacco (Nicotiana tabacum L.), highly expressing the AtNDB2 gene (NDB2 from Arabidopsis thaliana (L.) Heynh.), were produced with the help of agrobacterial transformation followed by a selection. The transgenic 13s line, possessing typical exterior and the AtNDB2 expression level, was compared with the initial wild type of N. tabacum regarding the parameters of growth and respiratory activity under optimal and suboptimal temperatures. It was found that the total and alternative respiration increased and the superoxide generation decreased in the 13s plants under the suboptimal temperature. The growth rate was decreased in the plants highly expressing the AtNDB2 gene in comparison with the control wild type plants, especially at the temperature below the optimum. Possible causes of the found changes are discussed. [ABSTRACT FROM AUTHOR]

Chymosin is an important milk clotting enzyme, massively used in the dairy industry. Due to the limited amount of natural chymosin, the recombinant enzyme produced in different organisms is the main source of chymosin. Plants have several advantages for large scale cost-effective production of recombinant chymosin. Here, we used a synthetic codon-optimized version of bovine prochymosin gene for ideal expression in the tobacco host. In this study, we utilized biolistic co-transformation method to introduce the synthetic bovine prochymosin gene into the tobacco genome. Several transgenic plants were regenerated on a selective medium. Molecular analysis confirmed successful integration and expression of prochymosin gene in several transgenic candidates. Total soluble protein extracts from transgenic plants were successfully applied for milk coagulation experiments, demonstrating the production of functional prochymosin in transgenic lines. In conclusion, here, we report successful expression of functional chymosin in tobacco plants from a novel synthetic version of the bovine prochymosin gene. Our experimental data support that plants could serve as a reliable source for safe and cost-effective production of recombinant chymosin enzyme for industrial usage. [ABSTRACT FROM AUTHOR]

5-Aminolevulinic acid (ALA), as a new natural plant growth regulator, has been proved to regulate protein phosphatase 2A (PP2A) activity to promote stomatal opening in apple (Malus domestica) leaves. However, the molecular mechanisms underlying remain unclear. Here, we cloned and transformed MdPTPA, MdPP2AC, and MdSnRK2.6 of apple into tobaccos (Nicotiana tabacum) and found that over-expression (OE)-MdPTPA or OE-MdPP2AC promoted stomatal aperture while OE-MdSnRK2.6 induced stomatal closure under normal or drought condition. The Ca2+ and H2O2 levels in the guard cells of OE-MdPTPA and OE-MdPP2AC was decreased but flavonols increased, and the results in OESnRK2.6 was contrary. Exogenous ALA stimulated PP2A activity but depressed SnRK2.6 activity in transgenic tobaccos, leading to less Ca2+, H2O2 and more flavonols in guard cells, and consequently stomatal opening. OE-MdPTPA improved stomatal opening and plant growth but impaired drought tolerance, while OE-MdSnRK2.6 improved drought tolerance but depressed the leaf Pn. Only OE-MdPP2AC improved stomatal opening, leaf Pn, plant growth, as well as drought tolerance. These suggest that the three genes involved in ALAregulating stomatal movement have their respective unique biological functions. Yeast two-hybrid (Y2H) assays showed that MdPP2AC interacted with MdPTPA or MdSnRK2.6, respectively, but no interaction of MdPTPA with MdSnRK2.6 was found. Yeast three-hybrid (Y3H) assay showed that MdPTPA promoted the interactions between MdPP2AC and MdSnRK2.6. Therefore, we propose a regulatory module of PTPA-PP2AC-SnRK2.6 that may be involved in mediating the ALA-inducing stomatal aperture in green plants. [ABSTRACT FROM AUTHOR]

Background: Anatabine, although being one of four major tobacco alkaloids, is never accumulated in high quantity in any of the naturally occurring species from the Nicotiana genus. Previous studies therefore focused on transgenic approaches to synthetize anatabine, most notably by generating transgenic lines with suppressed putrescine methyltransferase (PMT) activity. This led to promising results, but the global gene expression of plants with such distinct metabolism has not been analyzed. In the current study, we describe how these plants respond to topping and the downstream effects on alkaloid biosynthesis. Results: The surge in anatabine accumulation in PMT transgenic lines after topping treatment and its effects on gene expression changes were analyzed. The results revealed increases in expression of isoflavone reductase-like (A622) and berberine bridge-like enzymes (BBLs) oxidoreductase genes, previously shown to be crucial for the final steps of nicotine biosynthesis. We also observed significantly higher methylputrescine oxidase (MPO) expression in all plants subjected to topping treatment. In order to investigate if MPO suppression would have the same effects as that of PMT, we generated transgenic plants. These plants with suppressed MPO expression showed an almost complete drop in leaf nicotine content, whereas leaf anatabine was observed to increase by a factor of ~ 1.6X. Conclusion: Our results are the first concrete evidence that suppression of MPO leads to decreased nicotine in favor of anatabine in tobacco roots and that this anatabine is successfully transported to tobacco leaves. Alkaloid transport in plants remains to be investigated to higher detail due to high variation of its efficiency among Nicotiana species and varieties of tobacco. Our research adds important step to better understand pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine. [ABSTRACT FROM AUTHOR]

Pollen germination in vivo on wet stigmas is assisted by the receptive fluid—stigma exudate. Its exact composition is still unknown because only some components have been studied. For the first time, hormonal screening was carried out, and the fatty acid (FA) composition of lipid-rich (Nicotiana tabacum) and sugar-rich (Lilium longiflorum) exudates was studied. Screening of exudate for the presence of plant hormones using HPLC-MS revealed abscisic acid (ABA) in tobacco stigma exudate at the two stages of development, at pre-maturity and in mature stigmas awaiting pollination, increasing at the fertile stage. To assess physiological significance of ABA on stigma, we tested the effect of this hormone in vitro. ABA concentration found in the exudate strongly stimulated the germination of tobacco pollen, a lower concentration had a weaker effect, increasing the concentration did not increase the effect. GC-MS analysis showed that both types of exudate are characterized by a predominance of saturated FAs. The lipids of tobacco stigma exudate contain significantly more myristic, oleic, and linoleic acids, resulting in a higher unsaturation index relative to lily stigma exudate lipids. The latter, in turn, contain more 14-hexadecenoic and arachidic acids. Both exudates were found to contain significant amounts of squalene. The possible involvement of saturated FAs, ABA, and squalene in various exudate functions, as well as their potential relationship on the stigma, is discussed. [ABSTRACT FROM AUTHOR]

Copyright of Bulletin of Botanical Research is the property of Bulletin of Botanical Research Editorial Department 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.)

High accumulation of a single high-mannose glycan structure is important to ensure the quality of therapeutic proteins. We developed a glyco-engineering strategy for ensuring high accumulation of the Man 5 GlcNAc 2 structure by combining N -acetylglucosaminyltransferase I (GnT I) gene suppression and mannosidase I (Man I) gene overexpression. Nicotiana tabacum SR1 was used as the glyco-engineered host owing to the lower risk of pathogenic contamination than that in mammalian cells. We generated three glyco-engineered plant strains (gnt, gnt-MANA1, and gnt-MANA2) with suppression of GnT I or the combined suppression of GnT I and overexpression of Man I A1 or A2. The quantitative reverse transcriptase-PCR analysis showed a higher level of upregulation of Man I expression in gnt-MANA1/A2 plants than in the wild-type plants. Man I activity assay showed that the gnt-MANA1 plants had a higher Man I activity than did the wild-type and gnt-MANA2 plants. N -glycan analysis independently performed on two plants of each plant strain showed that gnt-MANA1 plants had a low abundance of the Man 6-9 GlcNAc 2 structure (2.8%, 7.1%) and high abundance of the Man 5 GlcNAc 2 structure (80.0%, 82.8%) compared with those in the wild-type and gnt plants. These results indicated that GnT I knockdown suppressed further modification of the Man 5 GlcNAc 2 structure, and Man I overexpression enhanced the conversion of Man 6-9 GlcNAc 2 structures to the Man 5 GlcNAc 2 structure. The developed glyco-engineered plants have potential for serving as novel expression hosts for therapeutic proteins. [ABSTRACT FROM AUTHOR]

Main conclusionThe role of six alkaloid biosynthesis genes in the process of nicotine accumulation in tobacco was investigated. Downregulation of ornithine decarboxylase, arginine decarboxylase, and aspartate oxidase resulted in viable plants with a significantly lower nicotine content. Attenuation of nicotine accumulation in Nicotiana tabacum was addressed upon the application of RNAi technologies. The approach entailed a downregulation in the expression of six different alkaloid biosynthesis genes encoding upstream enzymes that are thought to function in the pathway of alkaloid and nicotine biosynthesis. Nine different RNAi constructs were designed to lower the expression level of the genes that encode the enzymes arginine decarboxylase, agmatine deiminase, aspartate oxidase, arginase, ornithine decarboxylase, and SAM synthase. Agrobacterium-based transformation of tobacco leaves was applied, and upon kanamycin selection, T0 and subsequently T1 generation seeds were produced. Mature T1 plants in the greenhouse were topped to prevent flowering and leaf nos. 3 and 4 below the topping point were tested for transcript levels and product accumulation. Down-regulation in arginine decarboxylase, aspartate oxidase, and ornithine decarboxylase consistently resulted in lower levels of nicotine in the leaves of the corresponding plants. Transformants with the aspartate oxidase RNAi construct showed the lowest nicotine level in the leaves, which varied from below the limit of quantification (20 μg per g dry leaf weight) to 1.3 mg per g dry leaf weight. The amount of putrescine, the main polyamine related to nicotine biosynthesis, showed a qualitative correlation with the nicotine content in the arginine decarboxylase and ornithine decarboxylase RNAi-expressing transformants. A putative early senescence phenotype and lower viability of the older leaves was observed in some of the transformant lines. The results are discussed in terms of the role of the above-mentioned genes in the alkaloid biosynthetic pathway and may serve to guide efforts to attenuate nicotine content in tobacco leaves.

To improve the potential of cadmium phytoremediation, distant hybridization between tobacco (Nicotiana tabacum L. var. 78–04), a high-biomass crop, and Perilla frutescens var. frutescens, a wild Cd-hyperaccumulator, was carried out, developing a new variety N. tabacum L. var. ZSY. Seedlings at the six-leaf stage were grown in hydroponics and treated with 0 (control), 10 µM, 180 µM, and 360 µM CdCl2 for 7 days; then, the differences in Cd tolerance and accumulation and physiological and metabolic responses were evaluated among "ZSY" and its parents. At high Cd dose, the growth of "ZSY," such as fresh weight, plant height, and root length, was evidently better than "78–04." In contrast to P. frutescens and "78–04," "ZSY" could accumulate more Cd in shoots than roots. Under the same treatment, "ZSY" accumulated greater amounts of Cd in both shoots (195–1523 mg kg−1) and roots (140–1281 mg kg−1) than "78–04" (shoots: 35–89 mg kg−1, roots: 39–252 mg kg−1), followed by P. frutescens (shoots: 156–454 mg kg−1, roots: 103–761 mg kg−1). BCF and TF values of "ZSY" reached 38–195 and 1.2–1.4, which were far higher than those of "78–04" (BCF: 2.2–35.3, TF: 0.35–0.9). Perilla frutescens was found with BCF and TF of 11–156 and 0.5–1.5. Cd stress obviously promoted the production of ROS and MDA in seedlings but reduced chlorophyll contents, especially in "78–04." As a response to Cd stress, "ZSY" had higher SOD and CAT activities when compared to P. frutescens and "78–04," while "78–04" produced more POD and proline than those of P. frutescens and "ZSY." Cd stress could affect the production and accumulation of alkaloids and phenolic compounds in root (endodermis and cortex) and mesophyll. At high Cd doses, P. frutescens and "ZSY" had more alkaloids in tissues than "78–04." Phenolic compounds in "78–04" were more obviously inhibited compared with P. frutescens and "ZSY." These secondary metabolites may play an important role in eliminating oxidative damage and enhancing Cd tolerance and accumulation in "ZSY" and P. frutescens. Results indicated that distant hybridization could be one of effective methods for introducing excellent genes from metal-hyperaccumulators into high biomass species, creating plants with superior phytoremediation potential. [ABSTRACT FROM AUTHOR]

Cytokinins are known to keep stomata open, which supports gas exchange and correlates with increased photosynthesis. However, keeping the stomata open can be detrimental if the increased transpiration is not compensated for by water supply to the shoots. In this study, we traced the effect of ipt (isopentenyl transferase) gene induction, which increases the concentration of cytokinins in transgenic tobacco plants, on transpiration and hydraulic conductivity. Since water flow depends on the conductivity of the apoplast, the deposition of lignin and suberin in the apoplast was studied by staining with berberine. The effect of an increased concentration of cytokinins on the flow of water through aquaporins (AQPs) was revealed by inhibition of AQPs with HgCl2. It was shown that an elevated concentration of cytokinins in ipt-transgenic plants increases hydraulic conductivity by enhancing the activity of aquaporins and reducing the formation of apoplastic barriers. The simultaneous effect of cytokinins on both stomatal and hydraulic conductivity makes it possible to coordinate the evaporation of water from leaves and its flow from roots to leaves, thereby maintaining the water balance and leaf hydration. [ABSTRACT FROM AUTHOR]

Lighting time plays an important role in the yield and quality of flue-cured tobacco (Nicotiana tabacum L.). This study aimed to explore the effects of different lighting time on flue-cured tobacco quality at a metabolomics level. Pot experiments were used to breed long-day and short-day flue-cured tobacco. The long-day and short-day tobacco plants were treated with 16 h sunlight/8 h dark and 8 h sunlight/16 h dark, respectively. Thereafter, the leaves of the long-day and short-day tobacco were collected for liquid chromatography-mass spectrometry and metabolomics analysis. At phenotype level, the growth process of the long-day tobacco plant was faster (more yellow leaves); whereas the short-day tobacco plant manifested delayed nitrogen metabolism and slow maturation. After metabolomics analysis, a total of 92 differential metabolites (4 up-regulated and 88 down-regulated) were identified between the long-day and short-day tobacco, including amino acids, organic acids, saccharides, and pyrimidines. Functional analyses showed that these differential metabolites were significantly enriched in 14 KEGG pathways, including "metabolic pathways", "pyrimidine metabolism", "phenylalanine metabolism", and "phenylpropanoid biosynthesis". Our work reveals the key metabolites in tobacco plants with different lighting time, and lighting time can regulate metabolic pathways, phenylpropanoid biosynthesis, pyrimidine metabolism, amino acid-related pathways and nicotinic acid-derived alkaloids biosynthesis, thereby affecting tobacco plant growth and development, and the quality of flue-cured tobacco. [ABSTRACT FROM AUTHOR]

Biolistics is one of the widely used methods to deliver nucleic acids into plant cells. In this work, we optimized the protocol for biolistic transformation of Nicotiana tabacum with the gene gun PDS-1000/He Biolistic Particle Delivery System. The Green Fluorescent Protein (GFP) gene was used as a marker. The optimal parameters for the transformation of N. tabacum leaf cells were determined as: pressure, 1350 psi; tungsten particle size, 1.3 μm; plasmid DNA purification method, ethanol re-precipitation. The results should be useful for the development of biolistic transformation protocols for plant cells, including application for plant genome editing of agricultural species. [ABSTRACT FROM AUTHOR]

Cytokinin hormones are indispensable for plant growth and development. Cytokinin oxidase/dehydrogenase (CKX) helps regulate the dynamic balance of endogenous cytokinin levels. However, little is known about the CKX genes of Nicotiana tabacum (NtCKXs). In silico analyses were used to isolate, characterize, and elucidate the phylogenetic relationships of 15 NtCKX genes. Multi-species phylogenetic tree construction placed NtCKX1–15 on five of the eight branches of the CKX phylogenetic tree. Protein structure and network analyses revealed that NtCKX genes located on the same phylogenetic branch generally contained several conserved motifs and have highly similar structures, with structural domains related to flavin adenine dinucleotide (FAD) and cytokinin-binding found on all of the predicted NtCKX-encoded proteins. The upstream promotor region of NtCKX genes contained many abiotic stress-responsive cis-acting elements, including DRE, ERE, MBS, MYB, and MYC. Gene expression analysis revealed that each NtCKX gene responded differently to salt stress and exogenous abscisic acid (ABA) treatment. Four NtCKX genes exhibited ABA-induced expression trends with varying peak times. Under salt stress, NtCKX expression was significantly suppressed in two genes and upregulated in five others. In summary, we provided basic information regarding the CKX gene family of N. tabacum and elucidated their gene expression patterns under abiotic stresses, including ABA treatment and salt stress. The findings of this work can serve as a foundation for future study of the functions of NtCKX genes. [ABSTRACT FROM AUTHOR]

Background: Carotenoids, which are secondary metabolites derived from isoprenoids, play a crucial role in photo-protection and photosynthesis, and act as precursors for abscisic acid, a hormone that plays a significant role in plant abiotic stress responses. The biosynthesis of carotenoids in higher plants initiates with the production of phytoene from two geranylgeranyl pyrophosphate molecules. Phytoene synthase (PSY), an essential catalytic enzyme in the process, regulates this crucial step in the pathway. In Daucus carota L. (carrot), two PSY genes (DcPSY1 and DcPSY2) have been identified but only DcPSY2 expression is induced by ABA. Here we show that the ectopic expression of DcPSY2 in Nicotiana tabacum L. (tobacco) produces in L3 and L6 a significant increase in total carotenoids and chlorophyll a, and a significant increment in phytoene in the T1L6 line. Tobacco transgenic T1L3 and T1L6 lines subjected to chronic NaCl stress showed an increase of between 2 and 3- and 6-fold in survival rate relative to control lines, which correlates directly with an increase in the expression of endogenous carotenogenic and abiotic-related genes, and with ABA levels. Conclusions: These results provide evidence of the functionality of DcPSY2 in conferring salt stress tolerance in transgenic tobacco T1L3 and T1L6 lines. [ABSTRACT FROM AUTHOR]

Viroids are small, non-coding, pathogenic RNAs with the ability to disturb plant developmental processes. This dysregulation redirects the morphogenesis of plant organs, significantly impairing their functionality. Citrus bark cracking viroid (CBCVd) causes detrimental developmental distortions in infected hops (Humulus lupulus) and causes significant economic losses. CBCVd can infect cells and tissues of the model plant tobacco (Nicotiana tabacum), provided it is delivered via transgenesis. The levels of CBCVd in tobacco were enhanced in plant hybrids expressing CBCVd cDNAs and either the tobacco or hop variant of TFIIIA-7ZF, a viroid-mediated splicing derivative of transcription factor IIIA, which is important for viroid replication by DNA-dependent RNA polymerase II. The TFIIIA-7ZF variants can change the tobacco morphogenesis if expressed in leaves and shoots. In addition to the splitting of shoots, the "pathomorphogenic" network in hybrid plants expressing CBCVd and HlTFIIIA-7ZF induced leaf fusions and malformations. Moreover, CBCVd can dramatically change another morphogenesis into teratomic and petal-like tissues if propagated above some limit in young transgenic tobacco microspores and anthers. By comparative RNA profiling of transgenic tobacco shoots bearing TFIIIA-7ZFs and CBCVd-transformed/infected anthers, we found a differential expression of many genes at p < 0.05. As the main common factor showing the differential up-regulation in shoot and anther tissues, a LITTLE ZIPPER 2-like transcription factor was found. We propose that this factor, which can interact as a competitive inhibitor of the also dysregulated homeobox-leucin zipper family protein (HD-ZIPIII) in apical meristem, is essential for a network responsible for some morphological changes and modifications of plant degradome within shoot meristem regulation and secondary xylem differentiation. [ABSTRACT FROM AUTHOR]

Two new sesquiterpenes, nicosesquites A and B (1 and 2) as well as four known sesquiterpenes (3–6) were isolated from the leaves of Nicotiana tabacum. Their structures were determined mainly by spectroscopic methods, including extensive 1D and 2D NMR techniques. Compounds 1 and 2 were tested for their antimethicillin-resistant Staphylococcus aureus (anti-MRSA) activity. The results revealed that compounds 1 and 2 showed good inhibition with IZD of 13.4 ± 2.2, and 14.6 ± 2.4 mm, respectively. In addition, compounds 1 and 2 also have weak floral fragrances, and they have the potential value for use as an essence for cigarettes. [ABSTRACT FROM AUTHOR]