Microbial communities function as dynamic societies where intercellular communication governs collective behaviors. However, mapping these interaction networks has remained a fundamental challenge in microbiology. This study aims to decode the social networks of complex bacterial communities at single-cell resolution by developing BACON, a computational framework that infers quorum sensing-mediated communication from single-microbe transcriptomic data. The approach combines a curated database of signaling systems with a statistical model that quantifies communication strength through coordinated expression of signal synthesis and receptor genes. Validation in model systems demonstrated BACON's precision in reconstructing density-dependent communication trajectories in Bacillus subtilis and capturing rapid network reorganization in Escherichia coli under antibiotic stress, revealing distinct sender-receiver subpopulations. Applied to human gut microbiomes, BACON unveiled diurnal fluctuations in cross-species signaling that transcend enterotype boundaries and uncovered conserved metabolic specialization in signal-responsive bacteria. In a clinical context, analysis of an ICU patient's gut microbiome revealed how Pseudomonas aeruginosa establishes a self-reinforcing communication circuit that upregulates virulence pathways. This work provides a unified framework for analyzing bacterial social interactions across diverse ecosystems. It opens new avenues for understanding microbial sociology, combating antimicrobial resistance, and engineering synthetic communities.

Cowpea cultivar B301 is resistant to the parasitic weed Striga gesnerioides races SG4 and SG3, developing a hypersensitive response (HR) at the site of parasite attachment on the host root. In contrast, race SG4z successfully parasitizes B301, rapidly making vascular connections following attachment, undergoing cotyledon expansion (CE), and completing its life cycle. Comparative transcriptomics identified a set of differentially expressed transcripts in the Striga haustorial secretome during incompatible host-parasite interactions that were annotated as candidate avirulence (Avr) factors. We cloned the full-length coding sequence of two candidate genes, SGall_094658.3 (SGCHE) encoding a carbohydrate esterase and SGall_060151.1 (SGFAD) encoding a flavin adenine dinucleotide-binding Berberine family protein. Both genes encoded proteins with N-terminal apoplast targeting signals. Full-length SGCHE and SGFAD and truncated versions lacking the apoplast signal peptide (SGCHE∆SP and SGFAD∆SP) were expressed in the roots of B301 composite plants. Functional analysis showed that the transgenic B301 roots expressing SGCHE, SGCHE∆SP, SGFAD, and SGFAD∆SP demonstrated enhanced resistance to SG4z parasitism as indicated by increased levels of HR and reduced levels of parasite CE. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) analysis showed that transgenic B301 roots expressing the various Avr candidate proteins (SGCHE, SGCHE∆SP, SGFAD, and SGFAD∆SP) exhibited upregulated levels of gene expression associated with innate immunity to Striga parasitism compared to non-transgenic roots prior to parasite attachment and significantly increased levels of expression when challenged by SG4z. These results offer new insights on the mechanisms by which parasitic plants trigger host defense responses and provide a possible foundation for developing effective strategies to combat parasitic weeds.

Plant glandular trichomes are specialised epidermal structures capable of synthesising, storing and secreting numerous varieties of secondary metabolites in different classes and are central to plant defence and the biosynthesis of high-value metabolites. In this study, we characterised the HAIRPLUS (HAP) gene family and uncovered its role as a conserved regulator of trichome development and metabolism in tobacco. Four homologues, NtHAP1a, NtHAP1b, NtHAP2a and NtHAP2b, were identified and functional studies using RNAi and CRISPR-Cas9 revealed that NtHAPs act as negative regulators of glandular trichome development. Suppression of NtHAPs resulted in increased trichome density and enlarged glandular heads, as well as enhanced accumulation of diterpenoids (e.g., neophytadiene) and increased nicotine levels. Additionally, NtHAP1 appeared to have a stronger effect on trichome density. This study establishes the NtHAP genes as key negative regulators of glandular trichome development in tobacco, expanding their functional scope from trichome morphogenesis to metabolic regulation and highlighting their evolutionary conservation across Solanaceae. These findings pave the way for both fundamental research into trichome biology and practical applications in metabolic engineering and crop improvement, such as pest resistance.

The high-value carotenoid astaxanthin is biosynthesized through a dual-enzyme-catalyzed cascade and is getting increased attention for engineered biosynthesis in plants. When developing astaxanthin-producing tobacco by expressing 2A-peptide-linked CBFD (carotenoid β-ring-4-dehydrogenase) and HBFD (carotenoid 4-hydroxy-β-ring-4-dehydrogenase) from Adonis aestivalis, this work discovered an in-enzyme splicing site at the N-terminus of HBFD that has potentials for multiple protein expression in plant using monocistronic cassette. Based on this finding, we generated astaxanthin-producing tobacco plants expressing a directly fused protein of CBFD and HBFD with a monocistronic cassette. Further integrated IP (immunoprecipitation) and LC-MS/MS assays revealed the presence of an in-enzyme splicing site at the N-terminus of HBFD. Nevertheless, the obtained astaxanthin-producing tobacco plants exhibited a growth retardation as observed by previous researches. Subsequent studies revealed that the astaxanthin-producing caused growth retardation of tobacco was correlated with chloroplast disruption and chlorophyll reduction, and it could be alleviated by expressing a chlorophyll biosynthetic enzyme identified by proteomics. Additionally, crossing the astaxanthin-producing tobacco with a variety having higher chlorophyll content also alleviated the growth retardation caused by astaxanthin production, and improved the total astaxanthin yield per plant by at least threefold along with the biomass increase. This work provides novel approaches for expressing multiple proteins in tobacco and for engineering efficient astaxanthin-producing tobacco.

Buildings need practical ways to monitor indoor air quality (IAQ) beyond aggregate TVOC readings. We show that low-cost commercial VOC sensors, coupled with machine learning, can recover compound-specific information from plant-emitted terpenes, enabling practical, real-time bioindication in buildings. In an office testbed, we exposed sensors to 16 terpenes and trained random forest, support vector machine, and XGBoost models on time series features. The models detected "any terpene versus background" at 97%-100% accuracy, identified "plants versus background" at similar to 100%, and discriminated among individual compounds with accuracies up to 96%. Feature importance emphasized temporal dynamics (e.g., autocorrelation lags and entropy measures) rather than static peaks, highlighting the value of sequence information for commodity hardware. Complementary experiments with living basil plants showed reproducible VOC profiles and stress-induced bursts of similar to 70-100 ppb, confirming in situ feasibility. A placement analysis across 13 locations indicated that the HVAC return-air duct provides the most actionable, room-integrated signal for deployment, balancing accuracy and coverage. Together, these results establish a pathway from TVOC to compound-aware IAQ using sensors already common in smart buildings, with immediate applications to exposure triage and demand-controlled ventilation, and a foundation for plant-integrated environmental monitoring.

Salmonella, a gram-negative bacteria, is the leading cause of foodborne illness globally. Two serovars of Salmonella, S. enteritidis and S. typhimurium are responsible for the majority of human salmonellosis. Prolonged salmonellosis caused by Salmonella species leads to the development of colon cancer, which is 3rd most common cancer in the world. Porins in the outer membrane of Salmonella can be used to elicit immune response. The production of plant-based vaccine against salmonellosis and the subsequent colon cancer using outer membrane proteins can be helpful for the people of developing countries. In this study, OmpC protein from Salmonella enteritidis was subjected to various bioinformatics tools which exhibited OmpC vaccine construct to be sufficiently immunogenic, non-allergenic, non-toxic and non-homologous to human proteins. Docking analysis showed strong interaction of OmpC vaccine model with TLR-4. After in silico analysis, this vaccine construct was expressed in tobacco plants via Agrobacterium-mediated transformation. Gateway (R) cloning was used to clone OmpC gene. Transformation and integration of transgene within tobacco plants was confirmed through conventional PCR. qRT-PCR was done for expression analysis and copy number calculated was 2. The expressed OmpC protein accumulated up to 0.42% of total soluble protein. Immunization of mice with total soluble protein (TSP) and purified OmpC protein generated significant level of anti-OmpC antibodies. The vaccine candidate also demonstrated significant protective effect in mice upon challenging with Salmonella typhimurium. To the best of our knowledge, this is the first study reporting the expression of OmpC antigen in plants for potential use as vaccine against salmonellosis.

In the era of growing interest in healthy buildings and smart homes, the importance of sustainable, health conscious indoor environments is paramount. Smart tools, especially VOC sensors, are crucial for monitoring indoor air quality, yet interpreting signals from various VOC sources remains challenging. A promising approach involves understanding how indoor plants respond to environmental conditions. Plants produce terpenes, a type of VOC, when exposed to abiotic and biotic stressors - including pathogens, predators, light, and temperature - offering a novel pathway for monitoring indoor air quality. While prior work often relies on specialized laboratory sensors, our research leverages readily available commercial sensors to detect and classify plant emitted VOCs that signify changes in indoor conditions. We quantified the sensitivity of these sensors by measuring 16 terpenes in controlled experiments, then identified and tested the most promising terpenes in realistic environments. We also examined physics based models to map VOC responses but found them lacking for real world complexity. Consequently, we trained machine learning models to classify terpenes using commercial sensors and identified optimal sensor placement. To validate this approach, we analyzed emissions from a living basil plant, successfully detecting terpene output. Our findings establish a foundation for overcoming challenges in plant VOC detection, paving the way for advanced plant based sensors to enhance indoor environmental quality in future smart buildings.

Cowpea (Vigna unguiculata (L.) Walp.) is the most important grain legume in sub-Saharan Africa where it provides a significant source of human nutrition and animal fodder to millions of low-input farmers. While adapted to a wide range of soils and generally considered climate resilient due to its drought and heat tolerance, cowpea yields are suboptimal throughout its main growing region because of numerous abiotic and biotic stress factors and low soil fertility. Among the major limitations is low available soil phosphorus (P) and, therefore, improving cowpea cultivars for growth under limiting P conditions would be of great importance and benefit to local low-input farmers. We grew a multi-parent advanced generation inter-cross (MAGIC) population of cowpea consisting of 305 F8 recombinant inbred lines (RILs) generated from eight genetically diverse founder parents and phenotyped them for root system, shoot system, and total biomass production under normal and low phosphate conditions in an ebb and flow hydroponic system. We then performed a genome wide association study (GWAS) to identify genes associated with phosphorus use efficiency (PUE). Two SNPs were identified that are significantly associated with shoot system growth under phosphate stress: one located on chromosome Vu09 and the other on Vu10. A third SNP was identified on Vu03 that is associated with enhanced root production under phosphate stress. Characterizing the underlying genetic basis for PUE can assist in the improvement of cowpea for better productivity across Africa.

While most cowpea cultivars are susceptible to parasitism by the root parasitic weed Striga gesnerioides (Willd.) Vatke, cultivar B301 is resistant to all Striga races except for SG4z. Resistance to Striga parasitism is manifested by the elicitation of a hypersensitive response (HR) at the site of parasite attachment on the host root followed by rapid death of the attached parasite. We isolated a papain-like cysteine protease (PLCP) designated SGCP1 that is highly expressed in the haustoria of S. gesnerioides race SG3 at the time of parasite attachment to the host root. SGCP1 contains an apoplast-targeting signal peptide, a Cathepsin pro-peptide inhibitory domain, a papain family cysteine protease domain, and a granulin domain. Full-length SGCP1 and a variant lacking the signal peptide (SGCP∆SP) were expressed in the roots of composite B301 plants. Expression of SGCP1 and SGCP∆SP resulted in activation of host innate immune responses exemplified by increased frequency of HR and decreased levels of parasite cotyledon expansion (CE), indicative of successful host parasitism, in transgenic compared to wild-type B301 roots parasitized by SG4z. These data indicate that SGCP1 functions as an avirulence factor capable of activating host innate immunity and furthers our understanding of how compatible and incompatible host–parasite interactions are controlled.

The human body is inhabited by trillions of microorganisms that play a crucial role in health and diseases. Our understanding of the species and functional composition of the human gut microbiome is rapidly expanding, but it is still mainly based on taxonomic profiles or gene abundance measurements. As such, little is known about the species-function heterogeneity and dynamic activities in human microecosystem niches. By applying a novel gut-specific single-microbe ribonucleic acid (RNA) sequencing and analytical framework on three healthy donors with distinct enterotypes, we created a comprehensive transcriptional landscape of the human gut microbiome and dissected functional specialization in 38,922 single microbes across 198 species. We investigated the functional redundancy and complementarity involved in short-chain fatty acids related central carbon metabolism and studied the heterogeneity and covariation of single-microbe metabolic capacity. Comparing the human gut microbiome at different times throughout the day, we were able to map diurnal dynamic activities of the gut microbiome and discovered its association with sub-population functional heterogeneous. Remarkably, using single-microbe RNA sequencing, we systematically dissected the metabolic function heterogeneity of Megamonas funiformis, a keystone species in Asian populations. Together with in vitro and in vivo experimental validations, we proved M. funiformis can effectively improve mineral absorption through exogenous phytic acid degradation, which could potentially serve as a probiotic that reduces malnutrition caused by deficiency of mineral elements. Our results indicated that species-function heterogeneity widely exists and plays important roles in the human gut microbiome, and through single-microbe RNA sequencing, we have been able to capture the transcriptional activity variances and identify keystone species with specialized metabolic functions of possible biological and clinical importance.

PURPOSE:Peripheral neuropathies are commonly occurring conditions that are chronic and debilitating for patients. Established nonsurgical treatments have yielded mixed and patient-dependent results. Although cannabinoids have demonstrated efficacy as a treatment for central neuropathic pain, the therapeutic potential of cannabis-based medications for the management of peripheral neuropathic pain caused by nerve injury, trauma, and other noncompressive etiologies has yet to be definitively established. This study aims to determine whether cannabinoids are a potentially effective treatment for pain and symptoms associated with peripheral neuropathy. METHODS:A systematic search was conducted by two independent reviewers across PubMed, Cochrane, Ovid Medline, and CINAHL to identify studies in accordance with the predetermined inclusion/exclusion criteria. Information regarding study design, medication, dosage, effect on neuropathic pain, and other related outcomes was extracted. Meta-analysis of pain scores was performed for seven studies, and descriptive statistics were used to summarize other study findings as appropriate. RESULTS:Of the 927 studies identified, 14 randomized controlled trials were included. Thirteen of 14 studies (79%) observed a statistically significant decrease in neuropathic pain score following treatment with a cannabinoid. Meta-analysis yielded a mean difference of -0.67 [-0.89, -0.45]) on a 0-10 scale compared with placebo. Improvements in secondary outcomes such as sleep, sensory symptoms, and quality of life were observed. CONCLUSIONS:Our analysis of the literature shows that cannabis-based medicines may be effective in treating the pain and symptoms of peripheral neuropathy. These findings suggest the applicability of cannabis-based medicines for peripheral neuropathy. TYPE OF STUDY/LEVEL OF EVIDENCE:Therapeutic IV.

Uraria picta is a woody herb found throughout Asia, Africa and Australia. It has been long known to possess significant ethnomedicinal value. It is a key ingredient of more than a hundred Ayurvedic formulations and an important component of many patents in countries like India, China, Japan and USA. U. picta is commercially important and in high demand in India and Western African countries. As a consequence, U. picta has now been classified as a rare, endangered and threatened (RET) species in India. Biochemical analysis of different plant parts of U. picta and its tissue culture has shown that it is a valuable source of several bioactive phytochemicals among which Rhoifolin is the most recognized. In this work, relevant details on ethnobotany, bioactive compounds, pharmacology, toxicology, tissue culture and commercial applicability U. picta were retrieved from Google Scholar, Scopus, SpringerLink, Pubmed and Science Direct from 1950 to 2022. The seeds have a good balance of essential amino acids and the proportion of these amino acids is almost on par with that of nutritionally important common legumes and cereals. Given its status as an endangered species a number of reports have now appeared on methods of micropropagation of the plant as a means for conservation. The plant possesses several medicinal properties viz., anticancer, anti-inflammatory, antidiabetic, antimicrobial, etc. These studies mainly focus on the plant extract and not on the individual metabolites. Also, there is a dearth of reports on the mechanism of action of phytochemicals at the molecular levels. Thus, we provide perspective useful for bridging the gap that remains in the scientific validation of traditional uses of the plant through proper objectives and experimentation to explore novel therapeutic leads from U. picta with industrial viability. We also recommend the use of metabolic engineering and plant tissue culture (using elicitors and precursors) as approaches to increase the production of important bioactive molecules from this plant.

Loquat (Eriobotrya japonica) is an economically important subtropical fruit crop in China. Field surveys conducted in different loquat orchards located in Chongqing, Sichuan, and Fujian provinces between 2017 and 2020 resulted in a collection of 56 Alternaria-like isolates from trees exhibiting symptoms of loquat leaf spot. Multigene phylogenetic analyses using seven gene regions, namely, ITS, gapdh, RPB2, tef1, Alt a 1, endoPG, and OPA10-2, showed that all the isolates belonged to the genus Alternaria, and supporting morphological analysis identified them as members of species A. alternata, A. gaisen, and A. chongqingensis sp. nov. In vitro and in vivo pathogenicity tests showed all the identified species to be pathogenic and able to cause leaf spot disease on loquat. Moreover, comprehensive phylogenetic analyses employing all combinations of the above seven gene sequences revealed the capability of Alt a 1-tef1-endoPG to provide a well-resolved gene tree for Alternaria spp. at the species level. This study adds to the current knowledge on an unknown species (A. chongqingensis sp. nov.) and is the first report of A. gaisen in loquat worldwide.

Cannabis sativa (hemp) is a fiber crop that has historically been used for manufacturing textiles. Recently, limitations on cultivating industrial hemp in the United States have been lifted, as hemp has been reclassified as an agricultural commodity with the potential to serve as a bioenergy crop. This work used liquid nitrogen (LN2) to cryo-mill hemp (CMH) before NaOH chemical pretreatment. Pretreatments were conducted at 10% (w/v) solids loading with NaOH loadings ranging from 0 to 0.4 g of NaOH/g of dry biomass. Increasing NaOH chemical loading removed up to 80% percent of the original hemicellulose, but delignification leveled off around 20% even at the highest NaOH loadings. Moderate NaOH pretreatment conditions (0.1 to 0.2 g of NaOH/g of dry biomass) provided optimal conditions for sugar recovery following enzymatic hydrolysis with glucan yields approaching 90% and xylan yields at 80%. Pretreated CMH hydrolysate was fermented by Paenibacillus polymyxa to produce 2,3-butanediol (2,3-BDO). Under anoxic conditions, P. polymyxa generated 19 g/L of 2,3-BDO without conversion into acetoin. Aerobic fermentations produced 2,3-BDO titers near 13 g/L after 24 h, however, the resulting 2,3-BDO was converted to acetoin reaching around 9 g/L after 144 h.

Plant species can accumulate secondary metabolites in optically pure form or, occasionally, as enantiomeric mixtures. Interestingly, enantiomers of the same molecule can confer different biological activities. In tobacco (Nicotiana tabacum L.), the pyridine alkaloids nicotine, nornicotine, anatabine, and anabasine naturally exist as scalemic mixtures of (R)- or (S)-enantiomers, with the (S)-isoforms predominating. The mechanisms by which tobacco alkaloid enantiomers accumulate remain largely unknown. Experiments were carried out involving tobacco genotypes possessing induced deleterious mutations in three genes coding for nicotine demethylase (NND) enzymes and three genes coding for Berberine Bridge Like (BBL) enzymes that act near the end of the nicotine, anatabine, and anabasine biosynthetic pathways. Data indicate that (R)-nicotine is naturally produced at appreciable levels but is observed in only small amounts due to preferential demethylation by NND enzymes. Data further suggest that BBL-a and BBL-b are preferentially involved in the biosynthesis of (S)-alkaloid enantiomers, while BBL-c is preferentially involved in the biosynthesis of (R)-enantiomers. Gene duplication followed by genetic divergence thus played a role in the evolution of scalemic alkaloid accumulation in tobacco. Through a combination of mutation breeding and transgene overexpression, tobacco genotypes were generated in which the predominant alkaloid enantiomers were reversed from the (S)- to the (R)-isoforms. These results shed light on the genetic control of alkaloid accumulation in N. tabacum and on mechanisms of scalemic mixture formation of secondary metabolites in plants.

Striga gesnerioides is one of the major biotic constraints to cowpea (Vigna unguiculata [L.] Walp) production throughout West Africa. The best way to control and limit the spread of this weed remains genetic control, but recent works have shown that all cowpea varieties commonly grown in Togo are susceptible to S. gesnerioides. This study aims to investigate the resistance response of cowpea germplasm from Togo to S. gesnerioides. A field test was performed under natural infestation followed by a pots test under artificial infestation. Genotyping was carried out with three molecular markers SSR1, 61RM2 and C42-2B, known for their linkage to S. gesnerioides resistance genes. The results have revealed three cowpea accessions, TG20_66, TG20_82 and TG20_108, phenotypically resistant to S. gesnerioides. All three resistant accessions have shown the RSG3-301 resistance gene presence. Potential resistance gene sources to S. gesnerioides exist among cowpea accessions from Togo.

Per- and polyfluoroalkyl substances (PFAS) are a class of recalcitrant, highly toxic contaminants, with limited remediation options. Phytoremediation – removal of contaminants using plants – is an inexpensive, community-friendly strategy for reducing PFAS concentrations and exposures. This project is a collaboration between the Mi'kmaq Nation, Upland Grassroots, and researchers at several institutions who conducted phytoremediation field trials using hemp to remove PFAS from soil at the former Loring Air Force base, which has now been returned to the Mi’kmaq Nation. PFAS were analyzed in paired hemp and soil samples using targeted and non-targeted analytical approaches. Additionally, we used hydrothermal liquefaction (HTL) to degrade PFAS in the harvested hemp tissue. We identified 28 PFAS in soil and found hemp uptake of 10 of these PFAS. Consistent with previous studies, hemp exhibited greater bioconcentration for carboxylic acids compared to sulfonic acids, and for shorter-chain compounds compared to longer-chain. In total, approximately 1.4 mg of PFAS was removed from the soil via uptake into hemp stems and leaves, with an approximate maximum of 2% PFAS removed from soil in the most successful area. Degradation of PFAS by HTL was nearly 100% for carboxylic acids, but a portion of sulfonic acids remained. HTL also decreased precursor PFAS and extractable organic fluorine. In conclusion, while hemp phytoremediation does not currently offer a comprehensive solution for PFAS-contaminated soil, this project has effectively reduced PFAS levels at the Loring site and underscores the importance of involving community members in research aimed at remediating their lands.

This manuscript reviews two decades of projects funded by the Kirkhouse Trust (KT), a charity registered in the UK. KT was established to improve the productivity of legume crops important in African countries and in India. KT’s requirements for support are: (1) the research must be conducted by national scientists in their home institution, either a publicly funded agricultural research institute or a university; (2) the projects need to include a molecular biology component, which to date has mostly comprised the use of molecular markers for the selection of one or more target traits in a crop improvement programme; (3) the projects funded are included in consortia, to foster the creation of scientific communities and the sharing of knowledge and breeding resources. This account relates to the key achievements and challenges, reflects on the lessons learned and outlines future research priorities.

WRKY transcription factors play a pivotal role in regulating stress signaling pathways, including those associated with salt stress response. The present work characterized the effects of two WRKY genes from Vigna unguiculata, namely VuWRKY21 and VuWRKY87, on enhancing plant salinity tolerance. Under salt stress conditions, Arabidopsis lines expressing VuWRKY21 or VuWRKY87 showed elevated expression of genes participating in saline stress response pathways and reduced oxidative stress induced by reactive oxygen species (ROS). Among the salt-responsive genes in Arabidopsis, AtP5CS1, AtNHX1, AtRD29A, AtSOS3, AtSOS2, and AtSOS1 exhibited modulated expression levels after stress imposition. Furthermore, compared to wild-type plants, at most evaluated times, transgenic lines, on average, presented lower H2O2 content while displaying higher content of SOD (EC: 1.15.1.1) and CAT (EC: 1.11.1.6) at early stages of salt stress. These findings suggest that the expression of both VuWRKY genes in Arabidopsis, particularly VuWRKY21, activated genes involved in salinity tolerance.

Terpenes and terpenoids contribute aroma and flavor that influence consumer preferences in selecting plant-based products. Computational identification of biosynthetic gene clusters (BGCs) in plants can pave the way for future biosynthetic genetic engineering. Using integrative genomic, transcriptomic, and metabolic pathway annotation analyses, 35 BGCs were identified in tobacco with high confidence. Among the 35 BGCs identified, 7 were classified as terpene biosynthesis-related BGCs. Two BGCs found on C13 and C14 chromosomes belonged to terpene and saccharide-terpene biosynthetic classes that were only 93 Mb and 189 Kb apart, respectively. Other clusters have lengths ranging from 120 Kb (Cluster 9) to 1.6 Mb (Cluster 18). Each cluster contained five (Cluster 21) to twenty genes (Cluster 32), and the number of terpene synthase genes present in the clusters also varied from one (Clusters 18 and 21) to eight (Cluster 32). Gene expression profiling using diurnal and topping transcriptome datasets identified co-expressing genes within modules and varying levels of expression among modules as represented by the normalized enrichment score measured in each module. The positions pinpointed from these computational analyses will allow for the more efficient modifications of specific genes and BGCs for the development of tobacco-based products with improved aroma and flavor.