Chemical Composition of Tobacco Seed Oils and Their Antioxidant, Anti-Inflammatory, and Whitening Activities.

Tobacco seeds are a valuable food oil resource, and tobacco seed oil is rich in nutrients, especially polyunsaturated fatty acids. The aim of this work was to perform a comprehensive study on the chemical constituents, and the antioxidant, anti-inflammatory, and whitening activities of tobacco seed oils (NC89 and BS4). A GC/MS analysis revealed that NC89 and BS4 had 11 and 6 volatile compounds, respectively. The PUFA contents in NC89 and BS4 were 74.98% and 72.84%, respectively. These two tobacco seed oils also presented good radical scavenging capacities with the neutralization of ABTS, OH-, and superoxide (O2-) radicals in a concentration-dependent manner. Meanwhile, NC89 and BS4 inhibited reactive oxygen species (ROS) accumulation and cell apoptosis, enhanced SOD and CAT activities, and increased the GSH content in H2O2-induced HepG2 cells. In addition, NC89 and BS4 exhibited significant anti-inflammatory activities by inhibiting the expressions of NO, TNF-α, IL-1ß, and IL-6 in LPS-induced RAW.264.7 cells through the regulation of the MAPK signaling pathway. Moreover, NC89 and BS4 expressed whitening activities by inhibiting tyrosinase activity and intracellular melanin production. Therefore, tobacco seed oils could be used as an important oil resource for the development of high value-added products.

Design of high-oleic tobacco (Nicotiana tabacum L.) seed oil by CRISPR-Cas9-mediated knockout of NtFAD2-2.

BACKGROUND: Tobacco seed oil could be used as an appropriate feedstock for biodiesel production. However, the high linoleic acid content of tobacco seed oil makes it susceptible to oxidation. Altering the fatty acid profile by increasing the content of oleic acid could improve the properties of biodiesel produced from tobacco seed oil. RESULTS: Four FAD2 genes, NtFAD2-1a, NtFAD2-1b, NtFAD2-2a, and NtFAD2-2b, were identified in allotetraploid tobacco genome. Phylogenetic analysis of protein sequences showed that NtFAD2-1a and NtFAD2-2a originated from N. tomentosiformis, while NtFAD2-1b and NtFAD2-2b from N. sylvestris. Expression analysis revealed that NtFAD2-2a and NtFAD2-2b transcripts were more abundant in developing seeds than in other tissues, while NtFAD2-1a and NtFAD2-1b showed low transcript levels in developing seed. Phylogenic analysis showed that NtFAD2-2a and NtFAD2-2b were seed-type FAD2 genes. Heterologous expression in yeast cells demonstrated that both NtFAD2-2a and NtFAD2-2b protein could introduce a double bond at the Δ12 position of fatty acid chain. The fatty acid profile analysis of tobacco fad2-2 mutant seeds derived from CRISPR-Cas9 edited plants showed dramatic increase of oleic acid content from 11% to over 79%, whereas linoleic acid decreased from 72 to 7%. In addition, the fatty acid composition of leaf was not affected in fad2-2 mutant plants. CONCLUSION: Our data showed that knockout of seed-type FAD2 genes in tobacco could significantly increase the oleic acid content in seed oil. This research suggests that CRISPR-Cas9 system offers a rapid and highly efficient method in the tobacco seed lipid engineering programs.

High accumulation in tobacco seeds of hemagglutinin antigen from avian (H5N1) influenza.

Tobacco seeds can be used as a cost effective system for production of recombinant vaccines. Avian influenza is an important respiratory pathogen that causes a high degree of mortality and becomes a serious threat for the poultry industry. A safe vaccine against avian flu produced at low cost could help to prevent future outbreaks. We have genetically engineered tobacco plants to express extracellular domain of hemagglutinin protein from H5N1 avian influenza virus as an inexpensive alternative for production purposes. Two regulatory sequences of seed storage protein genes from Phaseolus vulgaris L. were used to direct the expression, yielding 3.0 mg of the viral antigen per g of seeds. The production and stability of seed-produced recombinant HA protein was characterized by different molecular techniques. The aqueous extract of tobacco seed proteins was used for subcutaneous immunization of chickens, which developed antibodies that inhibited the agglutination of erythrocytes after the second application of the antigen. The feasibility of using tobacco seeds as a vaccine carrier is discussed.

Advancements in Tobacco (Nicotiana tabacum L.) Seed Oils for Biodiesel Production.

With the increasing demand for fossil fuels, decreasing fossil fuel reserves and deteriorating global environment, humanity urgently need to explore new clean and renewable energy to replace fossil fuel resources. Biodiesel, as an environmentally friendly fuel that has attracted considerable attention because of its renewable, biodegradable, and non-toxic superiority, seems to be a solution for future fuel production. Tobacco (Nicotiana tabacum L.), an industrial crop, is traditionally used for manufacturing cigarettes. More importantly, tobacco seed is also widely being deemed as a typical inedible oilseed crop for the production of second-generation biodiesel. Advancements in raw material and enhanced production methods are currently needed for the large-scale and sustainable production of biodiesel. To this end, this study reviews various aspects of extraction and transesterification methods, genetic and agricultural modification, and properties and application of tobacco biodiesel, while discussing the key problems in tobacco biodiesel production and application. Besides, the proposals of new ways or methods for producing biodiesel from tobacco crops are presented. Based on this review, we anticipate that this can further promote the development and application of biodiesel from tobacco seed oil by increasing the availability and reducing the costs of extraction, transesterification, and purification methods, cultivating new varieties or transgenic lines with high oilseed contents, formulating scientific agricultural norms and policies, and improving the environmental properties of biodiesel.

Reactive Oxygen Species and Gibberellin Acid Mutual Induction to Regulate Tobacco Seed Germination.

Seed germination is a complex process controlled by various mechanisms. To examine the potential contribution of reactive oxygen species (ROS) and gibberellin acid (GA) in regulating seed germination, diphenylene iodonium chloride (DPI) and uniconazole (Uni), as hydrogen peroxide (H2O2) and GA synthesis inhibitor, respectively, were exogenously applied on tobacco seeds using the seed priming method. Seed priming with DPI or Uni decreased germination percentage as compared with priming with H2O, especially the DPI + Uni combination. H2O2 and GA completely reversed the inhibition caused by DPI or Uni. The germination percentages with H2O2 + Uni and GA + DPI combinations kept the same level as with H2O. Meanwhile, GA or H2O2 increased GA content and deceased ABA content through corresponding gene expressions involving homeostasis and signal transduction. In addition, the activation of storage reserve mobilization and the enhancement of soluble sugar content and isocitrate lyase (ICL) activity were also induced by GA or H2O2. These results strongly suggested that H2O2 and GA were essential for tobacco seed germination and by downregulating the ABA/GA ratio and inducing reserve composition mobilization mutually promoted seed germination. Meanwhile, ICL activity was jointly enhanced by a lower ABA/GA ratio and a higher ROS concentration.

Ultrahigh performance supercritical fluid chromatography of lipophilic compounds with application to synthetic and commercial biodiesel.

Ultrahigh performance supercritical fluid chromatography (UHPSFC) in combination with sub-2µm particles and either diode array ultraviolet (UV), evaporative light scattering, (ELSD), or mass spectrometric (MS) detection has been shown to be a valuable technique for the determination of acylglycerols in soybean, corn, sesame, and tobacco seed oils. Excellent resolution on an un-endcapped single C18 column (3.0mm×150mm) with a mobile phase gradient of acetonitrile and carbon dioxide in as little as 10min served greatly as an improvement on first generation packed column SFC instrumentation. Unlike high resolution gas chromatography and high performance liquid chromatography with mass spectrometric detection, UHPSFC/MS was determined to be a superior analytical tool for both separation and detection of mono-, di-, and tri-acylglycerols as well as free glycerol itself in biodiesel without derivatization. Baseline separation of residual tri-, di-, and mono-acylglycerols alongside glycerol at 0.05% (w/w) was easily obtained employing packed column SFC. The new analytical methodology was applied to both commercial B100 biodiesel (i.e. fatty acid methyl esters) derived from vegetable oil and to an "in-house" synthetic biodiesel (i.e. fatty acid ethyl esters) derived from tobacco seed oil and ethanol both before and after purification via column chromatography on bare silica.