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-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.

Nicotiana noctiflora Hook. Genome Contains Two Cellular T-DNAs with Functional Genes.

Agrobacterium (Rhizobium)-mediated transformation leads to the formation of crown galls or hairy roots on infected plants. These effects develop due to the activity of T-DNA genes, gathered on a big plasmid, acquired from agrobacteria during horizontal gene transfer. However, a lot of plant species are known to contain such sequences, called cellular T-DNAs (cT-DNAs), and maintain normal phenotypes. Some of the genes remain intact, which leads to the conclusion of their functional role in plants. In this study, we present a comprehensive analysis of the cT-DNAs in the Nicotiana noctiflora Hook. genome, including gene expression and opine identification. Deep sequencing of the Nicotiana noctiflora genome revealed the presence of two different cT-DNAs, NnT-DNA1 and NnT-DNA2, which contain the intact genes iaaM, iaaH, acs, orf13, orf13a, and orf14. According to the expression analysis results, all these genes are most active in roots in comparison with other organs, which is consistent with data on cT-DNA gene expression in other plant species. We also used genetic engineering approaches and HPTLC and HPLC-MS methods to investigate the product of the acs gene (agrocinopine synthase), which turned out to be similar to agrocinopine A. Overall, this study expands our knowledge of cT-DNAs in plants and brings us closer to understanding their possible functions. Further research of cT-DNAs in different species and their functional implications could contribute to advancements in plant genetics and potentially unveil novel traits with practical applications in agriculture and other fields.

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.

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.

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.

Comparative analysis of the Squamosa Promoter Binding-Like (SPL) gene family in Nicotiana benthamiana and Nicotiana tabacum.

SQUAMOSA PROMOTER BINDING-LIKE (SPL) proteins constitute a large family of transcription factors known to play key roles in growth and developmental processes, including juvenile-to-adult and vegetative-to-reproductive phase transitions. This makes SPLs interesting targets for precision breeding in plants of the Nicotiana genus used as e.g. recombinant biofactories. We report the identification of 49 SPL genes in Nicotiana tabacum cv. K326 and 43 SPL genes in Nicotiana benthamiana LAB strain, which were classified into eight phylogenetic groups according to the SPL classification in Arabidopsis. Exon-intron gene structure and DNA-binding domains were highly conserved between homeologues and orthologues. Thirty of the NbSPL genes and 33 of the NtSPL genes were found to be possible targets of microRNA 156. The expression of SPL genes in leaves was analysed by RNA-seq at three different stages, revealing that genes not under miR156 control were in general constitutively expressed at high levels, whereas miR156-regulated genes showed lower expression, often developmentally regulated. We selected the N. benthamiana SPL13_1a gene as target for a CRISPR/Cas9 knock-out experiment. We show here that a full knock-out in this single gene leads to a significant delay in flowering time, a trait that could be exploited to increase biomass for recombinant protein production.

Acclimation strategies of the green alga Chlorella vulgaris to different light regimes revealed by physiological and comparative proteomic analyses.

Acclimation to different light regimes is at the basis of survival for photosynthetic organisms, regardless of their evolutionary origin. Previous research efforts largely focused on acclimation events occurring at the level of the photosynthetic apparatus and often highlighted species-specific mechanisms. Here, we investigated the consequences of acclimation to different irradiances in Chlorella vulgaris, a green alga that is one of the most promising species for industrial application, focusing on both photosynthetic and mitochondrial activities. Moreover, proteomic analysis of cells acclimated to high light (HL) or low light (LL) allowed identification of the main targets of acclimation in terms of differentially expressed proteins. The results obtained demonstrate photosynthetic adaptation to HL versus LL that was only partially consistent with previous findings in Chlamydomonas reinhardtii, a model organism for green algae, but in many cases similar to vascular plant acclimation events. Increased mitochondrial respiration measured in HL-acclimated cells mainly relied on alternative oxidative pathway dissipating the excessive reducing power produced due to enhanced carbon flow. Finally, proteins involved in cell metabolism, intracellular transport, gene expression, and signaling-including a heliorhodopsin homolog-were identified as strongly differentially expressed in HL versus LL, suggesting their key roles in acclimation to different light regimes.

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.

Results and lessons learned from the sbv IMPROVER metagenomics diagnostics for inflammatory bowel disease challenge.

A growing body of evidence links gut microbiota changes with inflammatory bowel disease (IBD), raising the potential benefit of exploiting metagenomics data for non-invasive IBD diagnostics. The sbv IMPROVER metagenomics diagnosis for inflammatory bowel disease challenge investigated computational metagenomics methods for discriminating IBD and nonIBD subjects. Participants in this challenge were given independent training and test metagenomics data from IBD and nonIBD subjects, which could be wither either raw read data (sub-challenge 1, SC1) or processed Taxonomy- and Function-based profiles (sub-challenge 2, SC2). A total of 81 anonymized submissions were received between September 2019 and March 2020. Most participants' predictions performed better than random predictions in classifying IBD versus nonIBD, Ulcerative Colitis (UC) versus nonIBD, and Crohn's Disease (CD) versus nonIBD. However, discrimination between UC and CD remains challenging, with the classification quality similar to the set of random predictions. We analyzed the class prediction accuracy, the metagenomics features by the teams, and computational methods used. These results will be openly shared with the scientific community to help advance IBD research and illustrate the application of a range of computational methodologies for effective metagenomic classification.

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

Chemical modifications to DNA bases, including DNA adducts arising from reactions with electrophilic chemicals, are well-known to impact cell growth, miscode during replication, and influence disease etiology. However, knowledge of how genomic sequences and structures influence the accumulation of alkylated DNA bases is not broadly characterized with high resolution, nor have these patterns been linked with overall quantities of modified bases in the genome. For benzo(a) pyrene (BaP), a ubiquitous environmental carcinogen, we developed a single-nucleotide resolution damage sequencing method to map in a human lung cell line the main mutagenic adduct arising from BaP. Furthermore, we combined this analysis with quantitative mass spectrometry to evaluate the dose-response profile of adduct formation. By comparing damage abundance with DNase hypersensitive sites, transcription levels, and other genome annotation data, we found that although overall adduct levels rose with increasing chemical exposure concentration, genomic distribution patterns consistently correlated with chromatin state and transcriptional status. Moreover, due to the single nucleotide resolution characteristics of this DNA damage map, we could determine preferred DNA triad sequence contexts for alkylation accumulation, revealing a characteristic DNA damage signature. This new BaP damage signature had a profile highly similar to mutational signatures identified previously in lung cancer genomes from smokers. Thus, these data provide insight on how genomic features shape the accumulation of alkylation products in the genome and predictive strategies for linking single-nucleotide resolution in vitro damage maps with human cancer mutations.

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.

Toxicoproteomics reveals an effect of clozapine on autophagy in human liver spheroids.

Background: Clozapine is an atypical antipsychotic drug used to treat treatment-resistant schizophrenia. Its side effects, including liver enzyme abnormalities, experienced by many patients preclude its more common use as a first-line therapy for schizophrenia. Toxicoproteomic approaches have been demonstrated to effectively guide the identification of toxicological mechanisms.Methods: To further our understanding of the molecular effects of clozapine, we performed a data-independent acquisition (DIA)-based quantitative proteomics investigation of clozapine-treated human liver spheroid cultures.Results: In total, we quantified 4479 proteins across the five treatment groups (vehicle; 15 µM, 30 µM, and 60 µM clozapine; and 10 ng/mL TNFα + IL-1ß). Clozapine (60 µM) treatment yielded 36 differentially expressed proteins (FDR < 0.05). Gene-set enrichment analysis indicated perturbation of several gene sets, including interferon gamma signaling (e.g. interferon gamma receptor 1) and prominent autophagy-related processes (e.g. upregulation of sequestosome-1 (SQSTM1), MAP1LC3B/LC3B2, GABARAPL2, and nuclear receptor coactivator 4). The effects of clozapine on autophagy were confirmed by targeted mass spectrometry and western blotting using conventional SQSTM1 and LC3B markers.Conclusions: Combined with prior literature, our work suggests a broad contribution of autophagy to both the therapeutic and side effects of clozapine. Overall, this study demonstrates how proteomics can contribute to the elucidation of physiological and toxicological mechanisms of drugs.

Shotgun Lipidomics of Rodent Tissues.

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Systems biology reveals anatabine to be an NRF2 activator.

Anatabine, an alkaloid present in plants of the So lanaceae family (including tobacco and eggplant), has been shown to ameliorate chronic inflammatory conditions in mouse models, such as Alzheimer's disease, Hashimoto's thyroiditis, multiple sclerosis, and intestinal inflammation. However, the mechanisms of action of anatabine remain unclear. To understand the impact of anatabine on cellular systems and identify the molecular pathways that are perturbed, we designed a study to examine the concentration-dependent effects of anatabine on various cell types by using a systems pharmacology approach. The resulting dataset, consisting of measurements of various omics data types at different time points, was analyzed by using multiple computational techniques. To identify concentration-dependent activated pathways, we performed linear modeling followed by gene set enrichment. To predict the functional partners of anatabine and the involved pathways, we harnessed the LINCS L1000 dataset's wealth of information and implemented integer linear programming on directed graphs, respectively. Finally, we experimentally verified our key computational predictions. Using an appropriate luciferase reporter cell system, we were able to demonstrate that anatabine treatment results in NRF2 (nuclear factor-erythroid factor 2-related factor 2) translocation, and our systematic phosphoproteomic assays showed that anatabine treatment results in activation of MAPK signaling. While there are certain areas to be explored in deciphering the exact anti-inflammatory mechanisms of action of anatabine and other NRF2 activators, we believe that anatabine constitutes an interesting molecule for its therapeutic potential in NRF2-related diseases.

Fractionation and Extraction Optimization of Potentially Valuable Compounds and Their Profiling in Six Varieties of Two Nicotiana Species.

There is an increasingly urgent call to shift industrial processes from fossil fuel feedstock to sustainable bio-based resources. This change becomes of high importance considering new budget requirements for a carbon-neutral economy. Such a transformation can be driven by traditionally used plants that are able to produce large amounts of valuable biologically relevant secondary metabolites. Tobacco plants can play a leading role in providing value-added products in remote areas of the world. In this study, we propose a non-exhaustive list of compounds with potential economic interest that can be sourced from the tobacco plant. In order to optimize extraction methodologies, we first analyzed their physico-chemical properties using rapid solubility tests and high-resolution microfractionation techniques. Next, to identify an optimal extraction for a selected list of compounds, we compared 13 different extraction method-solvent combinations. We proceeded with profiling some of these compounds in a total of six varieties from Nicotiana tabacum and Nicotiana rustica species, identifying the optimal variety for each. The estimated expected yields for each of these compounds demonstrate that tobacco plants can be a superior source of valuable compounds with diverse applications beyond nicotine. Among the most interesting results, we found high variability of anatabine content between species and varieties, ranging from 287 to 1699 µg/g. In addition, we found that CGA (1305 µg/g) and rutin (7910 µg/g) content are orders of magnitude lower in the Burley variety as compared to all others.

Application of Secondary Electrospray Ionization Coupled with High-Resolution Mass Spectrometry in Chemical Characterization of Thermally Generated Aerosols.

Inhalation as a route for administering drugs and dietary supplements has garnered significant attention over the past decade. We performed real-time analyses of aerosols using secondary electrospray ionization (SESI) technology interfaced with high-resolution mass spectrometry (HRMS), primarily developed for exhaled breath analysis with the goal to detect the main aerosol constituents. Several commercially available inhalation devices containing caffeine, melatonin, cannabidiol, and vitamin B12 were tested. Chemical characterization of the aerosols produced by these devices enabled detection of the main constituents and screening for potential contaminants, byproducts, and impurities in the aerosol. In addition, a programmable syringe pump was connected to the SESI-HRMS system to monitor aerosolized active pharmaceutical ingredients (APIs) such as chloroquine, hydroxychloroquine, and azithromycin. This setup allowed us to detect caffeine, melatonin, hydroxychloroquine, chloroquine, and cannabidiol in the produced aerosols. Azithromycin and vitamin B12 in the aerosols could not be detected; however, our instrument setup enabled the detection of vitamin B12 breakdown products that were generated during the aerosolization process. Positive control was realized by liquid chromatography-HRMS analyses. The compounds detected in the aerosol were confirmed by exact mass measurements of the protonated and/or deprotonated species, as well as their respective collision-induced dissociation tandem mass spectra. These results reveal the potential wide application of this technology for the real-time monitoring of aerosolized active pharmaceutical ingredients that can be administered through the inhalation route.

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.

The clove (Syzygium aromaticum) genome provides insights into the eugenol biosynthesis pathway.

The clove (Syzygium aromaticum) is an important tropical spice crop in global trade. Evolving environmental pressures necessitate modern characterization and selection techniques that are currently inaccessible to clove growers owing to the scarcity of genomic and genetic information. Here, we present a 370-Mb high-quality chromosome-scale genome assembly for clove. Comparative genomic analysis between S. aromaticum and Eucalyptus grandis-both species of the Myrtaceae family-reveals good genome structure conservation and intrachromosomal rearrangements on seven of the eleven chromosomes. We report genes that belong to families involved in the biosynthesis of eugenol, the major bioactive component of clove products. On the basis of our transcriptomic and metabolomic findings, we propose a hypothetical scenario in which eugenol acetate plays a key role in high eugenol accumulation in clove leaves and buds. The clove genome is a new contribution to omics resources for the Myrtaceae family and an important tool for clove research.