Natural and induced variations in transcriptional regulator genes result in low-nicotine phenotypes in tobacco.

In tobacco, the homologous ETHYLENE RESPONSE FACTOR (ERF) transcription factors ERF199 and ERF189 coordinate the transcription of multiple metabolic genes involved in nicotine biosynthesis. Natural alleles at the NIC1 and NIC2 loci greatly affect alkaloid accumulation and overlap with ERF199 and ERF189 in the tobacco genome, respectively. In this study, we identified several low-nicotine tobacco varieties lacking ERF199 or ERF189 from a tobacco germplasm collection. We characterized the sequence of these new nic1 and nic2 alleles, as well as the previously defined alleles nic1-1 and nic2-1. Moreover, we examined the influence of different nic alleles on alkaloid contents and expression levels of genes related to nicotine biosynthesis. We also demonstrated that the deletion of a distal genomic region attenuates ERF199 expression, resulting in a moderately negative effect on the alkaloid phenotype. Our study provides new insights into the regulation of nicotine biosynthesis and novel genetic resources to breed low-nicotine tobacco.

Suppression of pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine in leaves of tobacco (N. tabacum L.).

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.

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.

Dissection of Cancer Mutational Signatures with Individual Components of Cigarette Smoking.

Tobacco smoke delivers a complex mixture of hazardous and potentially hazardous chemicals. Some of these may induce the formation of DNA mutations, which increases the risk of various cancers that display characteristic patterns of accumulated mutations arising from the causative exposures. Tracking the contributions of individual mutagens to mutational signatures present in human cancers can help understand cancer etiology and advance disease prevention strategies. To characterize the potential contributions of individual constituents of tobacco smoke to tobacco exposure-associated mutational signatures, we first assessed the toxic potential of 13 tobacco-relevant compounds by determining their impact on the viability of a human bronchial lung epithelial cell line (BEAS-2B). Experimentally derived high-resolution mutational profiles were characterized for the seven most potent compounds by sequencing the genomes of clonally expanded mutants that arose after exposure to the individual chemicals. Analogous to the classification of mutagenic processes on the basis of signatures from human cancers, we extracted mutational signatures from the mutant clones. We confirmed the formation of previously characterized benzo[a]pyrene mutational signatures. Furthermore, we discovered three novel mutational signatures. The mutational signatures arising from benzo[a]pyrene and norharmane were similar to human lung cancer signatures attributed to tobacco smoking. However, the signatures arising from N-methyl-N'-nitro-N-nitrosoguanidine and 4-(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone were not directly related to known tobacco-linked mutational signatures from human cancers. This new data set expands the scope of the in vitro mutational signature catalog and advances understanding of how environmental agents mutate DNA.

Tobacco Alkaloid Assessment in a DSS-Induced Colitis Mouse Model with a Fully Humanized Immune System.

Inflammatory bowel disease (IBD) refers to chronic intestinal immune-mediated diseases including two main disease manifestations: ulcerative colitis (UC) and Crohn's disease (CD). Epidemiological, clinical, and preclinical evidence has highlighted the potential anti-inflammatory properties of naturally occurring alkaloids. In the present study, we investigated the potential anti-inflammatory activities of the tobacco alkaloids nicotine and anatabine in a dextran sulfate sodium (DSS)-induced UC mouse model with a fully humanized immune system. Our results show that nicotine significantly reduced all acute colitis symptoms and improved colitis-specific endpoints, including histopathologically assessed colon inflammation, tissue damage, and mononuclear cell infiltration. The tobacco alkaloid anatabine showed similar effectiveness trends, although they were generally weaker or not significant. Gene expression analysis in the context of biological network models of IBD further pinpointed a possible mechanism by which nicotine attenuated DSS-induced colitis in humanized mice. The current study enables further investigation of possible molecular mechanisms by which tobacco alkaloids attenuate UC symptoms.

Crowdsourced benchmarking of taxonomic metagenome profilers: lessons learned from the sbv IMPROVER Microbiomics challenge.

BACKGROUND: Selection of optimal computational strategies for analyzing metagenomics data is a decisive step in determining the microbial composition of a sample, and this procedure is complex because of the numerous tools currently available. The aim of this research was to summarize the results of crowdsourced sbv IMPROVER Microbiomics Challenge designed to evaluate the performance of off-the-shelf metagenomics software as well as to investigate the robustness of these results by the extended post-challenge analysis. In total 21 off-the-shelf taxonomic metagenome profiling pipelines were benchmarked for their capacity to identify the microbiome composition at various taxon levels across 104 shotgun metagenomics datasets of bacterial genomes (representative of various microbiome samples) from public databases. Performance was determined by comparing predicted taxonomy profiles with the gold standard. RESULTS: Most taxonomic profilers performed homogeneously well at the phylum level but generated intermediate and heterogeneous scores at the genus and species levels, respectively. kmer-based pipelines using Kraken with and without Bracken or using CLARK-S performed best overall, but they exhibited lower precision than the two marker-gene-based methods MetaPhlAn and mOTU. Filtering out the 1% least abundance species-which were not reliably predicted-helped increase the performance of most profilers by increasing precision but at the cost of recall. However, the use of adaptive filtering thresholds determined from the sample's Shannon index increased the performance of most kmer-based profilers while mitigating the tradeoff between precision and recall. CONCLUSIONS: kmer-based metagenomic pipelines using Kraken/Bracken or CLARK-S performed most robustly across a large variety of microbiome datasets. Removing non-reliably predicted low-abundance species by using diversity-dependent adaptive filtering thresholds further enhanced the performance of these tools. This work demonstrates the applicability of computational pipelines for accurately determining taxonomic profiles in clinical and environmental contexts and exemplifies the power of crowdsourcing for unbiased evaluation.

Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence.

BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction-modification systems, is also active against BREX. In contrast to the wild-type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R-M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.

Inhibition of Arabidopsis thaliana CIN-like TCP transcription factors by Agrobacterium T-DNA-encoded 6B proteins.

Agrobacterium T-DNA-encoded 6B proteins cause remarkable growth effects in plants. Nicotiana otophora carries two cellular T-DNAs with three slightly divergent 6b genes (TE-1-6b-L, TE-1-6b-R and TE-2-6b) originating from a natural transformation event. In Arabidopsis thaliana, expression of 2×35S:TE-2-6b, but not 2×35S:TE-1-6b-L or 2×35S:TE-1-6b-R, led to plants with crinkly leaves, which strongly resembled mutants of the miR319a/TCP module. This module is composed of MIR319A and five CIN-like TCP (TEOSINTHE BRANCHED1, CYCLOIDEA and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR) genes (TCP2, TCP3, TCP4, TCP10 and TCP24) targeted by miR319a. The CIN-like TCP genes encode transcription factors and are required for cell division arrest at leaf margins during development. MIR319A overexpression causes excessive growth and crinkly leaves. TE-2-6b plants did not show increased miR319a levels, but the mRNA levels of the TCP4 target gene LOX2 were decreased, as in jaw-D plants. Co-expression of green fluorescent protein (GFP)-tagged TCPs with native or red fluorescent protein (RFP)-tagged TE-6B proteins led to an increase in TCP protein levels and formation of numerous cytoplasmic dots containing 6B and TCP proteins. Yeast double-hybrid experiments confirmed 6B/TCP binding and showed that TE-1-6B-L and TE-1-6B-R bind a smaller set of TCP proteins than TE-2-6B. A single nucleotide mutation in TE-1-6B-R enlarged its TCP-binding repertoire to that of TE-2-6B and caused a crinkly phenotype in Arabidopsis. Deletion analysis showed that TE-2-6B targets the TCP4 DNA-binding domain and directly interferes with transcriptional activation. Taken together, these results provide detailed insights into the mechanism of action of the N. otophora TE-encoded 6b genes.

A 6-month inhalation toxicology study in Apoe-/- mice demonstrates substantially lower effects of e-vapor aerosol compared with cigarette smoke in the respiratory tract.

Cigarette smoking is the major cause of chronic obstructive pulmonary disease. Considerable attention has been paid to the reduced harm potential of nicotine-containing inhalable products such as electronic cigarettes (e-cigarettes). We investigated the effects of mainstream cigarette smoke (CS) and e-vapor aerosols (containing nicotine and flavor) generated by a capillary aerosol generator on emphysematous changes, lung function, and molecular alterations in the respiratory system of female Apoe-/- mice. Mice were exposed daily (3 h/day, 5 days/week) for 6 months to aerosols from three different e-vapor formulations-(1) carrier (propylene glycol and vegetable glycerol), (2) base (carrier and nicotine), or (3) test (base and flavor)-or to CS from 3R4F reference cigarettes. The CS and base/test aerosol concentrations were matched at 35 µg nicotine/L. CS exposure, but not e-vapor exposure, led to impairment of lung function (pressure-volume loop area, A and K parameters, quasi-static elastance and compliance) and caused marked lung inflammation and emphysematous changes, which were confirmed histopathologically and morphometrically. CS exposure caused lung transcriptome (activation of oxidative stress and inflammatory responses), lipidome, and proteome dysregulation and changes in DNA methylation; in contrast, these effects were substantially reduced in response to the e-vapor aerosol exposure. Compared with sham, aerosol exposure (carrier, base, and test) caused a slight impact on lung inflammation and epithelia irritation. Our results demonstrated that, in comparison with CS, e-vapor aerosols induced substantially lower biological and pathological changes in the respiratory tract associated with chronic inflammation and emphysema.

BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site.

Prokaryotes evolved numerous systems that defend against predation by bacteriophages. In addition to well-known restriction-modification and CRISPR-Cas immunity systems, many poorly characterized systems exist. One class of such systems, named BREX, consists of a putative phosphatase, a methyltransferase and four other proteins. A Bacillus cereus BREX system provides resistance to several unrelated phages and leads to modification of specific motif in host DNA. Here, we study the action of BREX system from a natural Escherichia coli isolate. We show that while it makes cells resistant to phage λ infection, induction of λ prophage from cells carrying BREX leads to production of viruses that overcome the defense. The induced phage DNA contains a methylated adenine residue in a specific motif. The same modification is found in the genome of BREX-carrying cells. The results establish, for the first time, that immunity to BREX system defense is provided by an epigenetic modification.

Organization of the TC and TE cellular T-DNA regions in Nicotiana otophora and functional analysis of three diverged TE-6b genes.

Nicotiana otophora contains Agrobacterium-derived T-DNA sequences introduced by horizontal gene transfer (Chen et al., 2014). Sixty-nine contigs were assembled into four different cellular T-DNAs (cT-DNAs) totalling 83 kb. TC and TE result from two successive transformation events, each followed by duplication, yielding two TC and two TE inserts. TC is also found in other Nicotiana species, whereas TE is unique to N. otophora. Both cT-DNA regions are partially duplicated inverted repeats. Analysis of the cT-DNA divergence patterns allowed reconstruction of the evolution of the TC and TE regions. TC and TE carry 10 intact open reading frames. Three of these are TE-6b genes, derived from a single 6b gene carried by the Agrobacterium strain which inserted TE in the N. otophora ancestor. 6b genes have so far only been found in Agrobacterium tumefaciens or Agrobacterium vitis T-DNAs and strongly modify plant growth (Chen and Otten, 2016). The TE-6b genes were expressed in Nicotiana tabacum under the constitutive 2 × 35S promoter. TE-1-6b-R and TE-2-6b led to shorter plants, dark-green leaves, a strong increase in leaf vein development and modified petiole wings. TE-1-6b-L expression led to a similar phenotype, but in addition leaves show outgrowths at the margins, flowers were modified and plants became viviparous, i.e. embryos germinated in the capsules at an early stage of their development. Embryos could be rescued by culture in vitro. The TE-6b phenotypes are very different from the earlier described 6b phenotypes and could provide new insight into the mode of action of the 6b genes.

Genomic Insights into the Evolution of the Nicotine Biosynthesis Pathway in Tobacco.

In tobacco (Nicotiana tabacum), nicotine is the predominant alkaloid. It is produced in the roots and accumulated mainly in the leaves. Jasmonates play a central signaling role in damage-induced nicotine formation. The genome sequence of tobacco provides us an almost complete inventory of structural and regulatory genes involved in nicotine pathway. Phylogenetic and expression analyses revealed a series of structural genes of the nicotine pathway, forming a regulon, under the control of jasmonate-responsive ETHYLENE RESPONSE FACTOR (ERF) transcription factors. The duplication of NAD and polyamine metabolic pathways and the subsequent recruitment of duplicated primary metabolic genes into the nicotine biosynthesis regulon were suggested to be the drivers for pyridine and pyrrolidine ring formation steps early in the pathway. Transcriptional regulation by ERF and cooperatively acting MYC2 transcription factors are corroborated by the frequent occurrence of cognate cis-regulatory elements of the factors in the promoter regions of the downstream structural genes. The allotetraploid tobacco has homologous clusters of ERF genes on different chromosomes, which are possibly derived from two ancestral diploids and include either nicotine-controlling ERF189 or ERF199 A large chromosomal deletion was found within one allele of the nicotine-controlling NICOTINE2 locus, which is part of one of the ERF gene clusters, and which has been used to breed tobacco cultivars with a low-nicotine content.

Whole genome profiling physical map and ancestral annotation of tobacco Hicks Broadleaf.

Genomics-based breeding of economically important crops such as banana, coffee, cotton, potato, tobacco and wheat is often hampered by genome size, polyploidy and high repeat content. We adapted sequence-based whole-genome profiling (WGP™) technology to obtain insight into the polyploidy of the model plant Nicotiana tabacum (tobacco). N. tabacum is assumed to originate from a hybridization event between ancestors of Nicotiana sylvestris and Nicotiana tomentosiformis approximately 200,000 years ago. This resulted in tobacco having a haploid genome size of 4500 million base pairs, approximately four times larger than the related tomato (Solanum lycopersicum) and potato (Solanum tuberosum) genomes. In this study, a physical map containing 9750 contigs of bacterial artificial chromosomes (BACs) was constructed. The mean contig size was 462 kbp, and the calculated genome coverage equaled the estimated tobacco genome size. We used a method for determination of the ancestral origin of the genome by annotation of WGP sequence tags. This assignment agreed with the ancestral annotation available from the tobacco genetic map, and may be used to investigate the evolution of homoeologous genome segments after polyploidization. The map generated is an essential scaffold for the tobacco genome. We propose the combination of WGP physical mapping technology and tag profiling of ancestral lines as a generally applicable method to elucidate the ancestral origin of genome segments of polyploid species. The physical mapping of genes and their origins will enable application of biotechnology to polyploid plants aimed at accelerating and increasing the precision of breeding for abiotic and biotic stress resistance.

Chromosome-level genome assemblies of Nicotiana tabacum, Nicotiana sylvestris, and Nicotiana tomentosiformis.

The Solanaceae species Nicotiana tabacum, an economically important crop plant cultivated worldwide, is an allotetraploid species that appeared about 200,000 years ago as the result of the hybridization of diploid ancestors of Nicotiana sylvestris and Nicotiana tomentosiformis. The previously published genome assemblies for these three species relied primarily on short-reads, and the obtained pseudochromosomes only partially covered the genomes. In this study, we generated annotated de novo chromosome-level genomes of N. tabacum, N. sylvestris, and N. tomentosiformis, which contain 3.99 Gb, 2.32 Gb, and 1.74 Gb, respectively of sequence data, with 97.6%, 99.5%, and 95.9% aligned in chromosomes, and represent 99.2%, 98.3%, and 98.5% of the near-universal single-copy orthologs Solanaceae genes. The completion levels of these chromosome-level genomes for N. tabacum, N. sylvestris, and N. tomentosiformis are comparable to other reference Solanaceae genomes, enabling more efficient synteny-based cross-species research.

Correction to "Quantification and Mapping of Alkylation in the Human Genome Reveal Single Nucleotide Resolution Precursors of Mutational Signatures".

[This corrects the article DOI: 10.1021/acscentsci.2c01100.].

Blood and urine multi-omics analysis of the impact of e-vaping, smoking, and cessation: from exposome to molecular responses.

Cigarette smoking is a major preventable cause of morbidity and mortality. While quitting smoking is the best option, switching from cigarettes to non-combustible alternatives (NCAs) such as e-vapor products is a viable harm reduction approach for smokers who would otherwise continue to smoke. A key challenge for the clinical assessment of NCAs is that self-reported product use can be unreliable, compromising the proper evaluation of their risk reduction potential. In this cross-sectional study of 205 healthy volunteers, we combined comprehensive exposure characterization with in-depth multi-omics profiling to compare effects across four study groups: cigarette smokers (CS), e-vapor users (EV), former smokers (FS), and never smokers (NS). Multi-omics analyses included metabolomics, transcriptomics, DNA methylomics, proteomics, and lipidomics. Comparison of the molecular effects between CS and NS recapitulated several previous observations, such as increased inflammatory markers in CS. Generally, FS and EV demonstrated intermediate molecular effects between the NS and CS groups. Stratification of the FS and EV by combustion exposure markers suggested that this position on the spectrum between CS and NS was partially driven by non-compliance/dual use. Overall, this study highlights the importance of in-depth exposure characterization before biological effect characterization for any NCA assessment study.

Chromosome-scale assemblies of S. malaccense, S. aqueum, S. jambos, and S. syzygioides provide insights into the evolution of Syzygium genomes.

Syzygium is a large and diverse tree genus in the Myrtaceae family. Genome assemblies for clove (Syzygium aromaticum, 370 Mb) and sea apple (Syzygium grande, 405 Mb) provided the first insights into the genomic features and evolution of the Syzygium genus. Here, we present additional de novo chromosome-scale genome assemblies for Syzygium malaccense, Syzygium aqueum, Syzygium jambos, and Syzygium syzygioides. Genome profiling analyses show that S. malaccense, like S. aromaticum and S. grande, is diploid (2n = 2x = 22), while the S. aqueum, S. jambos, and S. syzygioides specimens are autotetraploid (2n = 4x = 44). The genome assemblies of S. malaccense (430 Mb), S. aqueum (392 Mb), S. jambos (426 Mb), and S. syzygioides (431 Mb) are highly complete (BUSCO scores of 98%). Comparative genomics analyses showed conserved organization of the 11 chromosomes with S. aromaticum and S. grande, and revealed species-specific evolutionary dynamics of the long terminal repeat retrotransposon elements belonging to the Gypsy and Copia lineages. This set of Syzygium genomes is a valuable resource for future structural and functional comparative genomic studies on Myrtaceae species.

Polyploid Nicotiana section Suaveolentes originated by hybridization of two ancestral Nicotiana clades.

Introduction: Nicotiana section Suaveolentes is an almost all-Australian clade of allopolyploid tobacco species that emerged through hybridization between diploid relatives of the genus. In this study, we aimed to assess the phylogenetic relationship of the Suaveolentes section with several Nicotiana diploid species based on both plastidial and nuclear genes. Methods: The Nicotiana plastome-based phylogenetic analysis representing 47 newly re-built plastid genomes suggested that an ancestor of N. section Noctiflorae is the most likely maternal donor of the Suaveolentes clade. Nevertheless, we found clear evidence of plastid recombination with an ancestor from the Sylvestres clade. We analyzed 411 maximum likelihood-based phylogenetic trees from a set of conserved nuclear diploid single copy gene families following an approach that assessed the genomic origin of each homeolog. Results: We found that Nicotiana section Suaveolentes is monophyletic with contributions from the sections Alatae, Sylvestres, Petunioides and Noctiflorae. The dating of the divergence between these sections indicates that the Suaveolentes hybridization predates the split between Alatae/Sylvestres, and Noctiflorae/Petunioides. Discussion: We propose that Nicotiana section Suaveolentes arose from the hybridization of two ancestral species from which the Noctiflorae/Petunioides and Alatae/Sylvestres sections are derived, with Noctiflorae the maternal parent. This study is a good example in which the use of genome wide data provided additional evidence about the origin of a complex polyploid clade.

Phage T3 overcomes the BREX defense through SAM cleavage and inhibition of SAM synthesis by SAM lyase.

Bacteriophage T3 encodes a SAMase that, through cleavage of S-adenosyl methionine (SAM), circumvents the SAM-dependent type I restriction-modification (R-M) defense. We show that SAMase also allows T3 to evade the BREX defense. Although SAM depletion weakly affects BREX methylation, it completely inhibits the defensive function of BREX, suggesting that SAM could be a co-factor for BREX-mediated exclusion of phage DNA, similar to its anti-defense role in type I R-M. The anti-BREX activity of T3 SAMase is mediated not just by enzymatic degradation of SAM but also by direct inhibition of MetK, the host SAM synthase. We present a 2.8 Å cryoelectron microscopy (cryo-EM) structure of the eight-subunit T3 SAMase-MetK complex. Structure-guided mutagenesis reveals that this interaction stabilizes T3 SAMase in vivo, further stimulating its anti-BREX activity. This work provides insights in the versatility of bacteriophage counterdefense mechanisms and highlights the role of SAM as a co-factor of diverse bacterial immunity systems.