Supragingival microbiome alternations as a consequence of smoking different tobacco types and its relation to dental caries.

This study aimed to assess the effect of smoking different tobacco types on the supragingival microbiome and its relation to dental caries. Forty supragingival plaque samples were collected from smokers of a single tobacco type and non-smokers seeking treatment at the University Dental Hospital Sharjah, UAE. DMFT (decayed, missing and filled teeth) was determined for all participants who were divided into two groups: no-low caries (NC-LC: DMFT = 0-4; n = 18) and moderate-high caries (MC-HC: DMFT = 5-20; n = 22). 16S rRNA gene was sequenced using third-generation sequencing with Nanopore technology. Microbiome composition and diversity were compared. Caries was most common among cigarette smokers. Supragingival microbiota were significantly altered among smokers of different tobacco types. In cigarette smokers, cariogenic bacteria from genus Streptococcus (including S. mutans) were significantly more among subjects with NC-LC, while Lactobacilli (including L. fermentum) were more among subjects with MC-HC. In medwakh smokers, several periodontopathogens were significantly elevated in subjects with NC-LC, while other pathogenic bacteria (as Klebsiella pneumoniae) were more in those with MC-HC. Cigarette and alternative tobacco smoking had a significant impact on the supragingival microbiome. Indeed, further studies are required to unravel the consequences of oral dysbiosis triggered by smoking. This could pave the way for microbiota-based interventional measures for restoring a healthy oral microbiome which could be a promising strategy to prevent dental caries.

Analysis of the possible cytogenetic mechanism for overcoming hybrid lethality in an interspecific cross between Nicotiana suaveolens and Nicotiana tabacum.

Hybrid lethality is a type of reproductive isolation in which hybrids die before maturation, due to the interaction between the two causative genes derived from each of the hybrid parents. The interspecific hybrid of Nicotiana suaveolens × Nicotiana tabacum is a model plant used in studies on hybrid lethality. While most of the progeny produced from such a cross die, some individuals grow normally and mature. Separately, a technique for producing mature hybrids by artificial culture has been developed. However, the mechanism by which hybrids overcome lethality, either spontaneously or by artificial culture, remains unclear. In the present study, we found that some hybrids that overcome lethality, either spontaneously or by artificial culture, lack the distal part of the Q chromosome, a region that includes the gene responsible for lethality. Quantitative polymerase chain reaction results suggested that the distal deletion of the Q chromosome, detected in some hybrid seedlings that overcome lethality, is caused by reciprocal translocations between homoeologous chromosomes. The results showed that chromosomal instability during meiosis in amphidiploid N. tabacum as well as during artificial culturing of hybrid seedlings is involved in overcoming hybrid lethality in interspecific crosses of the genus Nicotiana.

The ten amino acids of the oxygen-evolving enhancer of tobacco is sufficient as the peptide residues for protein transport to the chloroplast thylakoid.

KEY MESSAGE: The thylakoid transit peptide of tobacco oxygen-evolving enhancer protein contains a minimal ten amino acid sequences for thylakoid lumen transports. This ten amino acids do not contain twin-arginine, which is required for typical chloroplast lumen translocation. Chloroplasts are intracellular organelles responsible for photosynthesis to produce organic carbon for all organisms. Numerous proteins must be transported from the cytosol to chloroplasts to support photosynthesis. This transport is facilitated by chloroplast transit peptides (TPs). Four chloroplast thylakoid lumen TPs were isolated from Nicotiana tabacum and were functionally analyzed as thylakoid lumen TPs. Typical chloroplast stroma-transit peptides and thylakoid lumen transit peptides (tTPs) are found in N. tabacum transit peptides (NtTPs) and the functions of these peptides are confirmed with TP-GFP fusion proteins under fluorescence microscopy and chloroplast fractionation, followed by Western blot analysis. During the functional analysis of tTPs, we uncovered the minimum 10 amino acid sequence is sufficient for thylakoid lumen transport. These ten amino acids can efficiently translocate GFP protein, even if they do not contain the twin-arginine residues required for the twin-arginine translocation (Tat) pathway, which is a typical thylakoid lumen transport. Further, thylakoid lumen transporting processes through the Tat pathway was examined by analyzing tTP sequence functions and we demonstrate that the importance of hydrophobic core for the tTP cleavage and target protein translocation.

Metabolic networks of the Nicotiana genus in the spotlight: content, progress and outlook.

Manually curated metabolic databases residing at the Sol Genomics Network comprise two taxon-specific databases for the Solanaceae family, i.e. SolanaCyc and the genus Nicotiana, i.e. NicotianaCyc as well as six species-specific databases for Nicotiana tabacum TN90, N. tabacum K326, Nicotiana benthamiana, N. sylvestris, N. tomentosiformis and N. attenuata. New pathways were created through the extraction, examination and verification of related data from the literature and the aid of external database guided by an expert-led curation process. Here we describe the curation progress that has been achieved in these databases since the first release version 1.0 in 2016, the curation flow and the curation process using the example metabolic pathway for cholesterol in plants. The current content of our databases comprises 266 pathways and 36 superpathways in SolanaCyc and 143 pathways plus 21 superpathways in NicotianaCyc, manually curated and validated specifically for the Solanaceae family and Nicotiana genus, respectively. The curated data have been propagated to the respective Nicotiana-specific databases, which resulted in the enrichment and more accurate presentation of their metabolic networks. The quality and coverage in those databases have been compared with related external databases and discussed in terms of literature support and metabolic content.

Construction of a highly error-prone DNA polymerase for developing organelle mutation systems.

A novel family of DNA polymerases replicates organelle genomes in a wide distribution of taxa encompassing plants and protozoans. Making error-prone mutator versions of gamma DNA polymerases revolutionised our understanding of animal mitochondrial genomes but similar advances have not been made for the organelle DNA polymerases present in plant mitochondria and chloroplasts. We tested the fidelities of error prone tobacco organelle DNA polymerases using a novel positive selection method involving replication of the phage lambda cI repressor gene. Unlike gamma DNA polymerases, ablation of 3'-5' exonuclease function resulted in a modest 5-8-fold error rate increase. Combining exonuclease deficiency with a polymerisation domain substitution raised the organelle DNA polymerase error rate by 140-fold relative to the wild type enzyme. This high error rate compares favourably with error-rates of mutator versions of animal gamma DNA polymerases. The error prone organelle DNA polymerase introduced mutations at multiple locations ranging from two to seven sites in half of the mutant cI genes studied. Single base substitutions predominated including frequent A:A (template: dNMP) mispairings. High error rate and semi-dominance to the wild type enzyme in vitro make the error prone organelle DNA polymerase suitable for elevating mutation rates in chloroplasts and mitochondria.

On the performance of fusion based planet-scope and Sentinel-2 data for crop classification using inception inspired deep convolutional neural network.

This research work aims to develop a deep learning-based crop classification framework for remotely sensed time series data. Tobacco is a major revenue generating crop of Khyber Pakhtunkhwa (KP) province of Pakistan, with over 90% of the country's Tobacco production. In order to analyze the performance of the developed classification framework, a pilot sub-region named Yar Hussain is selected for experimentation work. Yar Hussain is a tehsil of district Swabi, within KP province of Pakistan, having highest contribution to the gross production of the KP Tobacco crop. KP generally consists of a diverse crop land with different varieties of vegetation, having similar phenology which makes crop classification a challenging task. In this study, a temporal convolutional neural network (TempCNNs) model is implemented for crop classification, while considering remotely sensed imagery of the selected pilot region with specific focus on the Tobacco crop. In order to improve the performance of the proposed classification framework, instead of using the prevailing concept of utilizing a single satellite imagery, both Sentinel-2 and Planet-Scope imageries are stacked together to assist in providing more diverse features to the proposed classification framework. Furthermore, instead of using a single date satellite imagery, multiple satellite imageries with respect to the phenological cycle of Tobacco crop are temporally stacked together which resulted in a higher temporal resolution of the employed satellite imagery. The developed framework is trained using the ground truth data. The final output is obtained as an outcome of the SoftMax function of the developed model in the form of probabilistic values, for the classification of the selected classes. The proposed deep learning-based crop classification framework, while utilizing multi-satellite temporally stacked imagery resulted in an overall classification accuracy of 98.15%. Furthermore, as the developed classification framework evolved with specific focus on Tobacco crop, it resulted in best Tobacco crop classification accuracy of 99%.

Degradome, small RNAs and transcriptome sequencing of a high-nicotine cultivated tobacco uncovers miRNA's function in nicotine biosynthesis.

Tobacco (Nicotiana tabacum) is considered as the model plant for alkaloid research, of which nicotine accounts for 90%. Many nicotine biosynthetic genes have been identified and were known to be regulated by jasmonate-responsive transcription factors. As an important regulator in plant physiological processes, whether small RNAs are involved in nicotine biosynthesis is largely unknown. Here, we combine transcriptome, small RNAs and degradome analysis of two native tobacco germplasms YJ1 and ZY100 to investigate small RNA's function. YJ1 leaves accumulate twofold higher nicotine than ZY100. Transcriptome analysis revealed 3,865 genes which were differently expressed in leaf and root of two germplasms, including some known nicotine and jasmonate pathway genes. By small RNA sequencing, 193 miRNAs were identified to be differentially expressed between YJ1 and ZY100. Using in silico and degradome sequencing approaches, six nicotine biosynthetic genes and seven jasmonate pathway genes were predicted to be targeted by 77 miRNA loci. Three pairs among them were validated by transient expression in vivo. Combined analysis of degradome and transcriptome datasets revealed 51 novel miRNA-mRNA interactions that may regulate nicotine biosynthesis. The comprehensive analysis of our study may provide new insights into the regulatory network of nicotine biosynthesis.

GC-MS Composition and Olfactory Profile of Concretes from the Flowers of Four Nicotiana Species.

The genus Nicotiana (Solanaceae) includes over 70 species, with a long history of traditional use; many of them are nowadays used in bioengineering, biosynthesis, molecular biology, and other studies, while common tobacco, N. tabacum L., is one of the most economically important industrial crops worldwide. Although Nicotiana species have been extensively investigated, relatively less research has focused on flowers, especially research related to obtaining aromatic products for cosmetic and perfumery use. On the other hand, there is evidence that Nicotiana flowers accumulate various secondary metabolites with a distinct aroma and biological activities, and the flowers represent a biomass available in sufficient quantities. Therefore, this study aimed to determinate the chemical composition (by GC-MS) and the olfactory profiles of a specific type of natural aromatic product (concrete), obtained from the flowers of four Nicotiana species, in a direct comparison between them. The yields of extracted concrete were sufficiently high, varying between the species, 1.4% (N. rustica L.), 2.5% (N. glutinosa L.), 1.6% (N. alata Link&Otto genotype with white flowers), 2.7% (N. alata genotype with pink flowers), 3.2% (N. tabacum, Oriental type), and 5.2% (N. tabacum, Virginia type). The major components of the obtained concretes belonged to different chemical classes: N. rustica and N. tabacum (OR), the hydrocarbons n-tetratriacontane (14.5%; 15.0%) and n-triacontane (12.1%; 13.3%), and 3-methyl-pentanoic acid (11.1%; 12.2%); N. glutinosa, the diterpenes sclareol (25.9%), 3-α-hydroxy-manool (16.3%), and 13-epimanool (14.9%); N. alata (WF), the phenylpropanoid terephthalic acid and di(2-ethylhexyl) ester (42.9%); N. alata (PF), the diterpene tributyl acetylcitrate (30.7%); and N. tabacum (FCV), the hydrocarbons n-hexacosane (12.9%) and n-pentacosane (12.9%). Each of the flower concretes revealed a characteristic odor profile. This is the first report about Nicotiana species as a source for obtaining flower concretes; these initial results about the concrete yield, olfactory profile, and chemical composition are a prerequisite for the possible processing of Nicotiana flowers into new aromatic products for use in perfumery and cosmetics. The study provides new data in favor of the potential of the four Nicotiana species as aromatic plants, as well as a possible alternative use of flowers, a valuable, but discarded, plant material in other applications.

Genomic diversity analysis and identification of novel SSR markers in four tobacco varieties by high-throughput resequencing.

Genome resequencing was carried out on two varieties of flue-cured tobacco (LY1306 and Qinyan 96), one variety of sun-cured tobacco (Wanmao 3), and one variety of air-cured Maryland tobacco (Wufeng 1), for a comparative analysis of genomic variation across the four varieties. Single nucleotide polymorphisms (SNPs), insertions and deletions (InDels), structural variations (SVs), and copy-number variations (CNVs) were then identified in each tobacco variety. Furthermore, a functional analysis of mutated genes was carried out. Through in-depth comparative analysis of genomes of different tobacco varieties, we identified genome variations in a number of SNPs, InDels, SVs, and CNVs, respectively. Computational analysis to predict the function of mutated genes containing these differential SNPs, InDels, SVs, and CNVs showed that they were mainly involved in different functions, such as carbohydrate metabolism and secondary metabolites biosynthesis. We mainly focused on genes that were involved in the biosynthesis of secondary metabolites and nicotine metabolism. In addition, we identified five simple sequence repeat (SSR)-based markers and verified them by PCR amplification in 10 tobacco varieties. Taken together, our study increases the understanding of genetic differences between tobacco types or varieties and identifies five SSR markers to classify tobacco varieties or types.

Bioluminescence Detection of Superoxide Anion Using Aequorin.

Although the superoxide anion (O2-·) is generated during normal cellular respiration and has fundamental roles in a wide range of cellular processes, such as cell proliferation, migration, apoptosis, and homeostasis, its dysregulation is associated with a variety of diseases. Regarding these prominent roles in biological systems, the development of accurate methods for quantification of superoxide anion has attracted tremendous research attention. Here, we evaluated aequorin, a calcium-dependent photoprotein, as a potential bioluminescent reporter protein of superoxide anion. The mechanism is based on the measurement of aequorin bioluminescence, where the lower the concentration of coelenterazine under the oxidation of superoxide anion, the lower the amount aequorin regeneration, leading to a decrease in bioluminescence. The bioluminescence intensity of aequorin was proportional to the concentration of superoxide anion in the range from 4 to 40 000 pM with a detection limit (S/N = 3) of 1.2 pM, which was 5000-fold lower than those of the chemiluminescence methods. The proposed method exhibited high sensitivity and has been successfully applied to the determination of superoxide anion in the plant cell samples. The results could suggest a photoprotein-based bioluminescence system as a highly sensitive, specific, and simple bioluminescent probe for in vitro detection of superoxide anion.

Genome-Wide Identification and Expression Analysis of HD-ZIP I Gene Subfamily in Nicotiana tabacum.

The homeodomain-leucine zipper (HD-Zip) gene family, whose members play vital roles in plant growth and development, and participate in responding to various stresses, is an important class of transcription factors currently only found in plants. Although the HD-Zip gene family, especially the HD-Zip I subfamily, has been extensively studied in many plant species, the systematic report on HD-Zip I subfamily in cultivated tobacco (Nicotiana tabacum) is lacking. In this study, 39 HD-Zip I genes were systematically identified in N. tabacum (Nt). Interestingly, that 64.5% of the 31 genes with definite chromosome location information were found to originate from N. tomentosoformis, one of the two ancestral species of allotetraploid N. tabacum. Phylogenetic analysis divided the NtHD-Zip I subfamily into eight clades. Analysis of gene structures showed that NtHD-Zip I proteins contained conserved homeodomain and leucine-zipper domains. Three-dimensional structure analysis revealed that most NtHD-Zip I proteins in each clade, except for those in clade η, share a similar structure to their counterparts in Arabidopsis. Prediction of cis-regulatory elements showed that a number of elements responding to abscisic acid and different abiotic stresses, including low temperature, drought, and salinity, existed in the promoter region of NtHD-Zip I genes. The prediction of Arabidopsis ortholog-based protein-protein interaction network implied that NtHD-Zip I proteins have complex connections. The expression profile of these genes showed that different NtHD-Zip I genes were highly expressed in different tissues and could respond to abscisic acid and low-temperature treatments. Our study provides insights into the evolution and expression patterns of NtHD-Zip I genes in N. tabacum and will be useful for further functional characterization of NtHD-Zip I genes in the future.

Antiparasitic properties of leaf extracts derived from selected Nicotiana species and Nicotiana tabacum varieties.

Within the traditional pharmacopeia, tobacco (Nicotiana spp.) is often cited as an efficient pesticide. This activity is generally attributed to nicotine, but tobacco plants contain other alkaloids that could potentially contribute to this effect. In this study, we tested methanolic extracts of N. glutinosa, N. glauca, N. debneyi, and N. tabacum (putrescine N-methyltransferase line, burley TN90 and Stella, Virginia ITB 683 and K326), selected according to alkaloid content. Their antiparasitic activity was evaluated in bioassays against adult fleas (Ctenocephalides felis), blowfly (Lucilia cuprina) larvae, nematodes (Caenorhabditis elegans), and ticks (Rhipicephalus sanguineus larvae and adults, Ixodes ricinus nymphs). None of the extracts killed fleas and blowfly larvae effectively at the concentrations tested. Only N. tabacum K326 and N. glutinosa exhibited moderate anthelmintic activity. All extracts significantly repelled R. sanguineus ticks, but not I. ricinus, and the nicotine-rich extracts rapidly knocked down all tick species and stages at high concentrations. The link between nicotine and tick knockdown was confirmed by successfully testing the pure alkaloid at concentrations found in the tobacco extracts. In contrast, repellent activity could not be correlated to the individually tested alkaloids (nicotine, nornicotine, anabasine, anatabine), although anatabine and nornicotine were active in the tick bioassay at high concentrations.

Comparison of TSNAs concentration in saliva according to type of tobacco smoked.

OBJECTIVE: To compare tobacco-specific nitrosamines (TSNAs) measured in saliva according to different types of tobacco smoked in a sample of smokers of the city of Barcelona (Spain). METHODS: We used data from a cross-sectional study of a sample of the adult smoking population of Barcelona, Spain in 2013-2014 (n = 165). We classified smokers in five groups according to the type of tobacco smoked: a) manufactured cigarettes only, b) roll-your-own (RYO) cigarettes only, c) dual smokers (both manufactured and RYO cigarettes), d) manufactured plus other types of tobacco products different from RYO and e) other types of tobacco products different from manufactured and RYO cigarettes. We calculated the geometric mean (GM) and geometric standard deviation (GSD) of TSNAs concentration in saliva (pg/mL), including N'-nitroaonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) according to the five tobacco groups. We also described all TSNAs concentration in each tobacco group stratified by the number of cigarettes smoked per day. RESULTS: Smokers from the RYO cigarette group had higher TSNAs concentration than smokers from the manufactured cigarette group: 13 pg/mL vs 4.9 pg/mL of NNN, 1.9 pg/mL vs 1.7 pg/mL in NNK and 1.1 pg/mL vs 0.9 pg/mL of NNAL. There were significant differences in NNN concentrations between smokers of RYO vs manufactured cigarettes. The higher the number of cigarettes smoked, the higher the TSNAs concentrations. After adjusted by number of cigarettes smoked, there were not statistically significant differences in TSNAs between RYO and manufactured cigarettes. CONCLUSIONS: Our data shows that RYO cigarette is at least as hazardous as manufactured cigarettes. Regulating RYO tobacco prices could be an effective strategy to control tobacco use.

In silico and in vivo analyses of the mutated human tissue plasminogen activator (mtPA) and the antithetical effects of P19 silencing suppressor on its expression in two Nicotiana species.

Human tissue-type plasminogen activator is one of the most important therapeutic proteins involved in the breakdown of blood clots following the stroke. A mutation was found at position 1541 bp (G514E) and the mutated form was cloned into the binary vector pTRAc-ERH. In silico analysis showed that this mutation might have no significant effect on the active site of the tissue plasminogen activator enzyme. Accordingly, zymography assay confirmed the serine protease activity of the mutated form and its derivatives. The expression of the mutated form was verified with/without co-agroinjection of the P19 gene silencing suppressor in both Nicotiana tabacum and N. benthamiana. The ELISA results showed that the concentration of the mutated form in the absence of P19 was 0.65% and 0.74% of total soluble protein versus 0.141% and 1.36% in the presence of P19 in N. benthamiana and N. tabacum, respectively. In N. tabacum, co-agroinjection of P19 had the synergistic effect and increased the mutated tissue plasminogen activator production two-fold higher. However, in N. benthamiana, the presence of P19 had the adverse effect of five-fold reduction in the concentration. Moreover, results showed that the activity of the mutated form and its derivatives was more than that of the purified commercial tissue plasminogen activator.

Influence of Smoking Puff Parameters and Tobacco Varieties on Free Radicals Yields in Cigarette Mainstream Smoke.

Cigarette smoke is a major exogenous source of free radicals, and the resulting oxidative stress is one of the major causes of smoking-caused diseases. Yet, many of the factors that impact free radical delivery from cigarettes remain unclear. In this study, we machine-smoked cigarettes and measured the levels of gas- and particulate-phase radicals by electron paramagnetic resonance (EPR) spectroscopy using standardized smoking regimens (International Organization of Standardization (ISO) and Canadian Intense (CI)), puffing parameters, and tobacco blends. Radical delivery per cigarette was significantly greater in both gas (4-fold) and particulate (6-fold) phases when cigarettes were smoked under the CI protocol compared to the ISO protocol. Total puff volume per cigarette was the major factor with radical production being proportional to total volume, regardless of whether volume differences were achieved by changes in individual puff volume or puff frequency. Changing puff shape (bell vs sharp vs square) or puff duration (1-5 s), without changing volume, had no effect on radical yields. Tobacco variety did have a significant impact on free radical production, with gas-phase radicals highest in reconstituted > burley > oriental > bright tobacco and particulate-phase radicals highest in burley > bright > oriental > reconstituted tobacco. Our findings show that modifiable cigarette design features and measurable user smoking behaviors are key factors determining free radical exposure in smokers.

Genome-Wide Identification, Evolutionary and Expression Analyses of the GALACTINOL SYNTHASE Gene Family in Rapeseed and Tobacco.

Galactinol synthase (GolS) is a key enzyme in raffinose family oligosaccharide (RFO) biosynthesis. The finding that GolS accumulates in plants exposed to abiotic stresses indicates RFOs function in environmental adaptation. However, the evolutionary relationships and biological functions of GolS family in rapeseed (Brassica napus) and tobacco (Nicotiana tabacum) remain unclear. In this study, we identified 20 BnGolS and 9 NtGolS genes. Subcellular localization predictions showed that most of the proteins are localized to the cytoplasm. Phylogenetic analysis identified a lost event of an ancient GolS copy in the Solanaceae and an ancient duplication event leading to evolution of GolS4/7 in the Brassicaceae. The three-dimensional structures of two GolS proteins were conserved, with an important DxD motif for binding to UDP-galactose (uridine diphosphate-galactose) and inositol. Expression profile analysis indicated that BnGolS and NtGolS genes were expressed in most tissues and highly expressed in one or two specific tissues. Hormone treatments strongly induced the expression of most BnGolS genes and homologous genes in the same subfamilies exhibited divergent-induced expression. Our study provides a comprehensive evolutionary analysis of GolS genes among the Brassicaceae and Solanaceae as well as an insight into the biological function of GolS genes in hormone response in plants.

Molecular thresholds of ITS2 and their implications for molecular evolution and species identification in seed plants.

Although molecular data have revealed huge amounts of plant diversity, interpreting genetic diversity into entities corresponding to species is still challenging. Taxonomic ranking based on genetic distance has been used extensively, but the results have been open to dispute, while the application of the strategy to plants has been restricted to a small number of cases. Here, levels of internal transcribed spacer 2 (ITS2) sequence variation were examined from 17,203 sequences, representing 5,439 species in 113 genera of seed plants, to ascertain the association between species status and their molecular divergence. Our results showed that, although the average genetic distances of sister species (AGDS) varied among angiosperms, the mean value was 3.98% and seemed not to be influenced by higher-level hierarchical classification or life history. AGDS was also stable within the major lineages of the gymnosperms but at approximately half the value of angiosperms, except for the Gnetidae, where the AGDS almost equaled that of angiosperms. We found that these AGDS discrepancies, associated with the rates of molecular evolution, cannot simply be attributed to generation-time differences, and highlight the complex life histories of plants. Our results provide general ITS2 thresholds in seed plants, and suggest their use in species identification.

Nicotiana attenuata Data Hub (NaDH): an integrative platform for exploring genomic, transcriptomic and metabolomic data in wild tobacco.

BACKGROUND: Nicotiana attenuata (coyote tobacco) is an ecological model for studying plant-environment interactions and plant gene function under real-world conditions. During the last decade, large amounts of genomic, transcriptomic and metabolomic data have been generated with this plant which has provided new insights into how native plants interact with herbivores, pollinators and microbes. However, an integrative and open access platform that allows for the efficient mining of these -omics data remained unavailable until now. DESCRIPTION: We present the Nicotiana attenuata Data Hub (NaDH) as a centralized platform for integrating and visualizing genomic, phylogenomic, transcriptomic and metabolomic data in N. attenuata. The NaDH currently hosts collections of predicted protein coding sequences of 11 plant species, including two recently sequenced Nicotiana species, and their functional annotations, 222 microarray datasets from 10 different experiments, a transcriptomic atlas based on 20 RNA-seq expression profiles and a metabolomic atlas based on 895 metabolite spectra analyzed by mass spectrometry. We implemented several visualization tools, including a modified version of the Electronic Fluorescent Pictograph (eFP) browser, co-expression networks and the Interactive Tree Of Life (iTOL) for studying gene expression divergence among duplicated homologous. In addition, the NaDH allows researchers to query phylogenetic trees of 16,305 gene families and provides tools for analyzing their evolutionary history. Furthermore, we also implemented tools to identify co-expressed genes and metabolites, which can be used for predicting the functions of genes. Using the transcription factor NaMYB8 as an example, we illustrate that the tools and data in NaDH can facilitate identification of candidate genes involved in the biosynthesis of specialized metabolites. CONCLUSION: The NaDH provides interactive visualization and data analysis tools that integrate the expression and evolutionary history of genes in Nicotiana, which can facilitate rapid gene discovery and comparative genomic analysis. Because N. attenuata shares many genome-wide features with other Nicotiana species including cultivated tobacco, and hence NaDH can be a resource for exploring the function and evolution of genes in Nicotiana species in general. The NaDH can be accessed at: http://nadh.ice.mpg.de/ .

Related allopolyploids display distinct floral pigment profiles and transgressive pigments.

PREMISE OF THE STUDY: Both polyploidy and shifts in floral color have marked angiosperm evolution. Here, we investigate the biochemical basis of the novel and diverse floral phenotypes seen in allopolyploids in Nicotiana (Solanaceae) and examine the extent to which the merging of distinct genomes alters flavonoid pigment production. METHODS: We analyzed flavonol and anthocyanin pigments from Nicotiana allopolyploids of different ages (N. tabacum, 0.2 million years old; several species from Nicotiana section Repandae, 4.5 million years old; and five lines of first-generation synthetic N. tabacum) as well as their diploid progenitors. KEY RESULTS: Allopolyploid floral pigment profiles tend not to overlap with their progenitors or related allopolyploids, and allopolyploids produce transgressive pigments that are not present in either progenitor. Differences in floral color among N. tabacum accessions seems mainly to be due to variation in cyanidin concentration, but changes in flavonol concentrations among accessions are also present. CONCLUSIONS: Competition for substrates within the flavonoid biosynthetic pathway to make either flavonols or anthocyanins may drive the differences seen among related allopolyploids. Some of the pigment differences observed in allopolyploids may be associated with making flowers more visible to nocturnal pollinators.

Organ- and Growing Stage-Specific Expression of Solanesol Biosynthesis Genes in Nicotiana tabacum Reveals Their Association with Solanesol Content.

Solanesol is a noncyclic terpene alcohol that is composed of nine isoprene units and mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum L.). In the present study, RNA-seq analyses of tobacco leaves, stems, and roots were used to identify putative solanesol biosynthesis genes. Six 1-deoxy-d-xylulose 5-phosphate synthase (DXS), two 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), two 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD), four 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE), two 2-C-methyl-d-erythritol 2,4-cyclo-diphosphate synthase (IspF), four 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), two 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IspH), six isopentenyl diphosphate isomerase (IPI), and two solanesyl diphosphate synthase (SPS) candidate genes were identified in the solanesol biosynthetic pathway. Furthermore, the two N. tabacum SPS proteins (NtSPS1 and NtSPS2), which possessed two conserved aspartate-rich DDxxD domains, were highly homologous with SPS enzymes from other solanaceous plant species. In addition, the solanesol contents of three organs and of leaves from four growing stages of tobacco plants corresponded with the distribution of chlorophyll. Our findings provide a comprehensive evaluation of the correlation between the expression of different biosynthesis genes and the accumulation of solanesol, thus providing valuable insight into the regulation of solanesol biosynthesis in tobacco.