An electric molecular motor.

Macroscopic electric motors continue to have a large impact on almost every aspect of modern society. Consequently, the effort towards developing molecular motors1-3 that can be driven by electricity could not be more timely. Here we describe an electric molecular motor based on a [3]catenane4,5, in which two cyclobis(paraquat-p-phenylene)6 (CBPQT4+) rings are powered by electricity in solution to circumrotate unidirectionally around a 50-membered loop. The constitution of the loop ensures that both rings undergo highly (85%) unidirectional movement under the guidance of a flashing energy ratchet7,8, whereas the interactions between the two rings give rise to a two-dimensional potential energy surface (PES) similar to that shown by FOF1 ATP synthase9. The unidirectionality is powered by an oscillating10 voltage11,12 or external modulation of the redox potential13. Initially, we focused our attention on the homologous [2]catenane, only to find that the kinetic asymmetry was insufficient to support unidirectional movement of the sole ring. Accordingly, we incorporated a second CBPQT4+ ring to provide further symmetry breaking by interactions between the two mobile rings. This demonstration of electrically driven continual circumrotatory motion of two rings around a loop in a [3]catenane is free from the production of waste products and represents an important step towards surface-bound14 electric molecular motors.

Genome editing of a rice CDP-DAG synthase confers multipathogen resistance.

The discovery and application of genome editing introduced a new era of plant breeding by giving researchers efficient tools for the precise engineering of crop genomes1. Here we demonstrate the power of genome editing for engineering broad-spectrum disease resistance in rice (Oryza sativa). We first isolated a lesion mimic mutant (LMM) from a mutagenized rice population. We then demonstrated that a 29-base-pair deletion in a gene we named RESISTANCE TO BLAST1 (RBL1) caused broad-spectrum disease resistance and showed that this mutation caused an approximately 20-fold reduction in yield. RBL1 encodes a cytidine diphosphate diacylglycerol synthase that is required for phospholipid biosynthesis2. Mutation of RBL1 results in reduced levels of phosphatidylinositol and its derivative phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). In rice, PtdIns(4,5)P2 is enriched in cellular structures that are specifically associated with effector secretion and fungal infection, suggesting that it has a role as a disease-susceptibility factor3. By using targeted genome editing, we obtained an allele of RBL1, named RBL1Δ12, which confers broad-spectrum disease resistance but does not decrease yield in a model rice variety, as assessed in small-scale field trials. Our study has demonstrated the benefits of editing an LMM gene, a strategy relevant to diverse LMM genes and crops.

Electron-catalysed molecular recognition.

Molecular recognition1-4 and supramolecular assembly5-8 cover a broad spectrum9-11 of non-covalently orchestrated phenomena between molecules. Catalysis12 of such processes, however, unlike that for the formation of covalent bonds, is limited to approaches13-16 that rely on sophisticated catalyst design. Here we establish a simple and versatile strategy to facilitate molecular recognition by extending electron catalysis17, which is widely applied18-21 in synthetic covalent chemistry, into the realm of supramolecular non-covalent chemistry. As a proof of principle, we show that the formation of a trisradical complex22 between a macrocyclic host and a dumbbell-shaped guest-a molecular recognition process that is kinetically forbidden under ambient conditions-can be accelerated substantially on the addition of catalytic amounts of a chemical electron source. It is, therefore, electrochemically possible to control23 the molecular recognition temporally and produce a nearly arbitrary molar ratio between the substrates and complexes ranging between zero and the equilibrium value. Such kinetically stable supramolecular systems24 are difficult to obtain precisely by other means. The use of the electron as a catalyst in molecular recognition will inspire chemists and biologists to explore strategies that can be used to fine-tune non-covalent events, control assembly at different length scales25-27 and ultimately create new forms of complex matter28-30.

Latent feature extraction with a prior-based self-attention framework for spatial transcriptomics.

Rapid advances in spatial transcriptomics (ST) have revolutionized the interrogation of spatial heterogeneity and increase the demand for comprehensive methods to effectively characterize spatial domains. As a prerequisite for ST data analysis, spatial domain characterization is a crucial step for downstream analyses and biological implications. Here we propose a prior-based self-attention framework for spatial transcriptomics (PAST), a variational graph convolutional autoencoder for ST, which effectively integrates prior information via a Bayesian neural network, captures spatial patterns via a self-attention mechanism, and enables scalable application via a ripple walk sampler strategy. Through comprehensive experiments on data sets generated by different technologies, we show that PAST can effectively characterize spatial domains and facilitate various downstream analyses, including ST visualization, spatial trajectory inference and pseudotime analysis. Also, we highlight the advantages of PAST for multislice joint embedding and automatic annotation of spatial domains in newly sequenced ST data. Compared with existing methods, PAST is the first ST method that integrates reference data to analyze ST data. We anticipate that PAST will open up new avenues for researchers to decipher ST data with customized reference data, which expands the applicability of ST technology.

Cofea: correlation-based feature selection for single-cell chromatin accessibility data.

Single-cell chromatin accessibility sequencing (scCAS) technologies have enabled characterizing the epigenomic heterogeneity of individual cells. However, the identification of features of scCAS data that are relevant to underlying biological processes remains a significant gap. Here, we introduce a novel method Cofea, to fill this gap. Through comprehensive experiments on 5 simulated and 54 real datasets, Cofea demonstrates its superiority in capturing cellular heterogeneity and facilitating downstream analysis. Applying this method to identification of cell type-specific peaks and candidate enhancers, as well as pathway enrichment analysis and partitioned heritability analysis, we illustrate the potential of Cofea to uncover functional biological process.

scPRAM accurately predicts single-cell gene expression perturbation response based on attention mechanism.

MOTIVATION: With the rapid advancement of single-cell sequencing technology, it becomes gradually possible to delve into the cellular responses to various external perturbations at the gene expression level. However, obtaining perturbed samples in certain scenarios may be considerably challenging, and the substantial costs associated with sequencing also curtail the feasibility of large-scale experimentation. A repertoire of methodologies has been employed for forecasting perturbative responses in single-cell gene expression. However, existing methods primarily focus on the average response of a specific cell type to perturbation, overlooking the single-cell specificity of perturbation responses and a more comprehensive prediction of the entire perturbation response distribution. RESULTS: Here, we present scPRAM, a method for predicting perturbation responses in single-cell gene expression based on attention mechanisms. Leveraging variational autoencoders and optimal transport, scPRAM aligns cell states before and after perturbation, followed by accurate prediction of gene expression responses to perturbations for unseen cell types through attention mechanisms. Experiments on multiple real perturbation datasets involving drug treatments and bacterial infections demonstrate that scPRAM attains heightened accuracy in perturbation prediction across cell types, species, and individuals, surpassing existing methodologies. Furthermore, scPRAM demonstrates outstanding capability in identifying differentially expressed genes under perturbation, capturing heterogeneity in perturbation responses across species, and maintaining stability in the presence of data noise and sample size variations. AVAILABILITY AND IMPLEMENTATION: https://github.com/jiang-q19/scPRAM and https://doi.org/10.5281/zenodo.10935038.

EpiCarousel: memory- and time-efficient identification of metacells for atlas-level single-cell chromatin accessibility data.

SUMMARY: Recent technical advancements in single-cell chromatin accessibility sequencing (scCAS) have brought new insights to the characterization of epigenetic heterogeneity. As single-cell genomics experiments scale up to hundreds of thousands of cells, the demand for computational resources for downstream analysis grows intractably large and exceeds the capabilities of most researchers. Here, we propose EpiCarousel, a tailored Python package based on lazy loading, parallel processing, and community detection for memory- and time-efficient identification of metacells, i.e. the emergence of homogenous cells, in large-scale scCAS data. Through comprehensive experiments on five datasets of various protocols, sample sizes, dimensions, number of cell types, and degrees of cell-type imbalance, EpiCarousel outperformed baseline methods in systematic evaluation of memory usage, computational time, and multiple downstream analyses including cell type identification. Moreover, EpiCarousel executes preprocessing and downstream cell clustering on the atlas-level dataset with 707 043 cells and 1 154 611 peaks within 2 h consuming <75 GB of RAM and provides superior performance for characterizing cell heterogeneity than state-of-the-art methods. AVAILABILITY AND IMPLEMENTATION: The EpiCarousel software is well-documented and freely available at https://github.com/biox-nku/epicarousel. It can be seamlessly interoperated with extensive scCAS analysis toolkits.

Post-Translational Modification ß-Hydroxybutyrylation Regulates Ustilaginoidea virens Virulence.

Lysine ß-hydroxybutyrylation (Kbhb) is an evolutionarily conserved and widespread post-translational modification that is associated with active gene transcription and cellular proliferation. However, its role in phytopathogenic fungi remains unknown. Here, we characterized Kbhb in the rice false smut fungus Ustilaginoidea virens. We identified 2204 Kbhb sites in 852 proteins, which are involved in diverse biological processes. The mitogen-activated protein kinase UvSlt2 is a Kbhb protein, and a strain harboring a point mutation at K72, the Kbhb site of this protein, had decreased UvSlt2 activity and reduced fungal virulence. Molecular dynamic simulations revealed that K72bhb increases the hydrophobic solvent-accessible surface area of UvSlt2, thereby affecting its binding to its substrates. The mutation of K298bhb in the septin UvCdc10 resulted in reduced virulence and altered the subcellular localization of this protein. Moreover, we confirmed that the NAD+-dependent histone deacetylases UvSirt2 and UvSirt5 are the major enzymes that remove Kbhb in U. virens. Collectively, our findings identify regulatory elements of the Kbhb pathway and reveal important roles for Kbhb in regulating protein localization and enzymatic activity. These findings provide insight into the regulation of virulence in phytopathogenic fungi via post-translational modifications.

Syntheses of three-dimensional catenanes under kinetic control.

Although catenanes comprising two ring-shaped components can be made in large quantities by templation, the preparation of three-dimensional (3D) catenanes with cage-shaped components is still in its infancy. Here, we report the design and syntheses of two 3D catenanes by a sequence of SN2 reactions in one pot. The resulting triply mechanically interlocked molecules were fully characterized in both the solution and solid states. Mechanistic studies have revealed that a suit[3]ane, which contains a threefold symmetric cage component as the suit and a tribromide component as the body, is formed at elevated temperatures. This suit[3]ane was identified as the key reactive intermediate for the selective formation of the two 3D catenanes which do not represent thermodynamic minima. We foresee a future in which this particular synthetic strategy guides the rational design and production of mechanically interlocked molecules under kinetic control.

Interface-Suppressed Nematicity and Enhanced Superconducting Pairing Strength of FeSe/NdFeO3 in the Low-Doping Regime.

The discovery of interfacial superconductivity in monolayer FeSe/oxides has spurred intensive research interest. Here we not only extend the FeSe/FeOx superconducting interface to FeSe/NdFeO3 but also establish robust interface-enhanced superconductivity at a very low doping level. Specifically, well-annealed FeSe/NdFeO3 exhibits a low doping level of 0.038-0.046 e-/Fe with a larger superconducting pairing gap without a nematic gap, indicating an enhancement of the enhanced superconducting pairing strength and suppression of nematicity by the FeSe/FeOx interface compared with those of thick FeSe films. These results improve our understanding of the roles of the oxide interface in the low-electron-doped regime.

Inferior Interfacial Superconductivity in 1 UC FeSe/SrVO3/SrTiO3 with Screened Interfacial Electron-Phonon Coupling.

Single-unit cell (1 UC) FeSe interfaced with TiOx or FeOx exhibits significantly enhanced superconductivity compared to that of bulk FeSe, with interfacial electron-phonon coupling (EPC) playing a crucial role. However, the reduced dimensionality in 1 UC FeSe, which may drive superconducting fluctuations, complicates our understanding of the enhancement mechanisms. We construct a new superconducting interface, 1 UC FeSe/SrVO3/SrTiO3. Here, the itinerant electrons of highly metallic SrVO3 films can screen all high-energy Fuchs-Kliewer phonons, including those of SrTiO3, making it the first FeSe/oxide system with screened interfacial EPC while maintaining the 1 UC FeSe thickness. Despite comparable doping levels, the heavily electron-doped 1 UC FeSe/SrVO3 exhibits a pairing temperature (Tg ∼ 48 K) lower than those of FeSe/SrTiO3 and FeSe/LaFeO3. Our findings disentangle the contributions of interfacial EPC from dimensionality in terms of enhancing Tg in FeSe/oxide interfaces, underscoring the critical importance of interfacial EPC. This FeSe/VOx interface also provides a platform for studying interfacial superconductivity.

Rbm46 inhibits reactive oxygen species in mouse embryonic stem cells through modulating BNIP3-mediated mitophagy.

Embryonic stem cells (ESCs) exhibit a metabolic preference for glycolysis over oxidative phosphorylation to meet their substantial adenosine triphosphate (ATP) demands during self-renewal. This metabolic choice inherently maintains low mitochondrial activity and minimal reactive oxygen species (ROS) generation. Nonetheless, the intricate molecular mechanisms governing the restraint of ROS production and the mitigation of cellular damage remain incompletely elucidated. In this study, we reveal the pivotal role of RNA-binding motif protein 46 (RBM46) in ESCs, acting as a direct post transcriptional regulator of ROS levels by modulating BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) mRNA expression. Rbm46 knockout lead to diminished mitochondrial autophagy, culminating in elevated ROS within ESCs, disrupting the delicate balance required for healthy self-renewal. These findings provide insights into a novel mechanism governing ROS regulation in ESCs.

Ustilaginoidea virens-secreted effector Uv1809 suppresses rice immunity by enhancing OsSRT2-mediated histone deacetylation.

Rice false smut caused by Ustilaginoidea virens is a devastating rice (Oryza sativa) disease worldwide. However, the molecular mechanisms underlying U. virens-rice interactions are largely unknown. In this study, we identified a secreted protein, Uv1809, as a key virulence factor. Heterologous expression of Uv1809 in rice enhanced susceptibility to rice false smut and bacterial blight. Host-induced gene silencing of Uv1809 in rice enhanced resistance to U. virens, suggesting that Uv1809 inhibits rice immunity and promotes infection by U. virens. Uv1809 suppresses rice immunity by targeting and enhancing rice histone deacetylase OsSRT2-mediated histone deacetylation, thereby reducing H4K5ac and H4K8ac levels and interfering with the transcriptional activation of defence genes. CRISPR-Cas9 edited ossrt2 mutants showed no adverse effects in terms of growth and yield but displayed broad-spectrum resistance to rice pathogens, revealing a potentially valuable genetic resource for breeding disease resistance. Our study provides insight into defence mechanisms against plant pathogens that inactivate plant immunity at the epigenetic level.

simCAS: an embedding-based method for simulating single-cell chromatin accessibility sequencing data.

MOTIVATION: Single-cell chromatin accessibility sequencing (scCAS) technology provides an epigenomic perspective to characterize gene regulatory mechanisms at single-cell resolution. With an increasing number of computational methods proposed for analyzing scCAS data, a powerful simulation framework is desirable for evaluation and validation of these methods. However, existing simulators generate synthetic data by sampling reads from real data or mimicking existing cell states, which is inadequate to provide credible ground-truth labels for method evaluation. RESULTS: We present simCAS, an embedding-based simulator, for generating high-fidelity scCAS data from both cell- and peak-wise embeddings. We demonstrate simCAS outperforms existing simulators in resembling real data and show that simCAS can generate cells of different states with user-defined cell populations and differentiation trajectories. Additionally, simCAS can simulate data from different batches and encode user-specified interactions of chromatin regions in the synthetic data, which provides ground-truth labels more than cell states. We systematically demonstrate that simCAS facilitates the benchmarking of four core tasks in downstream analysis: cell clustering, trajectory inference, data integration, and cis-regulatory interaction inference. We anticipate simCAS will be a reliable and flexible simulator for evaluating the ongoing computational methods applied on scCAS data. AVAILABILITY AND IMPLEMENTATION: simCAS is freely available at https://github.com/Chen-Li-17/simCAS.

Effects of Neolamarckia cadamba leaves extract on microbial community and antibiotic resistance genes in cecal contents and feces of broilers challenged with lipopolysaccharides.

The effects of Neolamarckia cadamba leaves extract (NCLE), with effective ingredients of flavonoids, on antibiotic resistance genes (ARGs) and relevant microorganisms in cecal contents and feces of broilers treated with or without lipopolysaccharide stimulation (LPS) were investigated. LPS stimulation increased (P < 0.05) the relative abundance of ARGs and mobile genetic elements (MGEs), such as tet(W/N/W), APH(3')-IIIa, ErmB, tet (44), ANT (6)-Ia, tet(O), tet (32), Vang_ACT_CHL, myrA, ANT (6)-Ib, IncQ1, tniB, and rep2 in cecal contents. However, the difference disappeared (P > 0.05) when NCLE was added at the same time. These differential ARGs and MGEs were mainly correlated (P < 0.01) with Clostridiales bacterium, Lachnospiraceae bacterium, and Candidatus Woodwardibium gallinarum. These species increased in LPS-stimulated broilers and decreased when NCLE was applied at the same time. In feces, LPS stimulation decreased (P < 0.05) the relative abundance of tet(Q), adeF, ErmF, Mef(En2), OXA-347, tet (40), npmA, tmrB, CfxA3, and ISCrsp1, while the LPS + NCLE treated group showed no significant effect (P > 0.05) on these ARGs. These differential ARGs and MGEs in feces were mainly correlated (P < 0.01) with Clostridiales bacterium, Pseudoflavonifractor sp. An184, Flavonifractor sp. An10, Ruminococcaceae bacterium, etc. These species increased in LPS-stimulated broilers and increased when NCLE was applied at the same time. In conclusion, LPS stimulation and NCLE influenced microbial communities and associated ARGs in both cecal contents and feces of broilers. NCLE alleviated the change of ARGs and MGEs in LPS-induced broilers by maintaining the microbial balance.IMPORTANCEAntibiotics showed a positive effect on gut health regulation and growth performance improvement in livestock breeding, but the antimicrobial resistance threat and environment pollution problem are increasingly severe with antibiotics abuse. As alternatives, plant extract containing bioactive substances are increasingly used to improve immunity and promote productivity. However, little is known about their effects on diversity and abundance of ARGs. Here, we investigated the effects of NCLE, with effective ingredients of flavonoids, on ARGs and relevant microorganisms in cecal contents and feces of broilers treated with or without lipopolysaccharide stimulation. We found that NCLE reduced the abundance of ARGs in cecal contents of lipopolysaccharide-induced broilers by maintaining the microbial balance. This study provides a comprehensive view of cecal and fecal microbial community, ARGs, and MGEs of broiler following LPS stimulation and NCLE treatment. It might be used to understand and control ARGs dissemination in livestock production.

Prediction of heterosis in the recent rapeseed (Brassica napus) polyploid by pairing parental nucleotide sequences.

The utilization of heterosis is a successful strategy in increasing yield for many crops. However, it consumes tremendous manpower to test the combining ability of the parents in fields. Here, we applied the genomic-selection (GS) strategy and developed models that significantly increase the predictability of heterosis by introducing the concept of a regional parental genetic-similarity index (PGSI) and reducing dimension in the calculation matrix in a machine-learning approach. Overall, PGSI negatively affected grain yield and several other traits but positively influenced the thousand-seed weight of the hybrids. It was found that the C subgenome of rapeseed had a greater impact on heterosis than the A subgenome. We drew maps with overviews of quantitative-trait loci that were responsible for the heterosis (h-QTLs) of various agronomic traits. Identifications and annotations of genes underlying high impacting h-QTLs were provided. Using models that we elaborated, combining abilities between an Ogu-CMS-pool member and a potential restorer can be simulated in silico, sidestepping laborious work, such as testing crosses in fields. The achievements here provide a case of heterosis prediction in polyploid genomes with relatively large genome sizes.

Potential Value of Probiotics on Lipid Profiles in Hyperlipidemia and Healthy Participants: Systematic Review and Meta-Analysis.

Objective: Many randomized controlled trials (RCTs) have reported the effect of probiotics on reducing plasma lipids with inconsistent results. An explicit systematic review and meta-analysis were conducted in this study to evaluate the effect of probiotics on the lipid profile of healthy and hyperlipidemia participants. Methods: A comprehensive literature search of RCTs was conducted using PubMed, Embase, World Health Organization (WHO) Global Index Medicus, WHO clinical trial registry, and Clinicaltrials.gov. Inclusion criteria included RCTs comparing the use of any strain of a specified probiotic with the placebo control group. The change in lipid profiles was analyzed. Results: The probiotics can decrease the total cholesterol (TC) level in hyperlipidemia participants but not healthy persons (MD = -0.43, 95% CI -0.60 - -0.25, P < .01; MD = -0.09, 95% CI -0.26 - 0.08, P > .05). Probiotics did not reduce high-density lipoprotein cholesterol (HDL-C) in patients with hyperlipidemia or healthy people (MD = -0.01, 95% CI -0.09 - 0.07, P > .05; MD = 0.02, 95% CI -0.04 - 0.09, P > .05). Furthermore, probiotics can reduce the low-density lipoprotein cholesterol (LDL-C) level both in hyperlipidemia and healthy persons (MD = -0.34, 95% CI -0.43 - -0.26, P < .01; MD = -0.15, 95% CI -0.28 - -0.02, P < .05). Lastly, the effect of probiotics on reducing triglyceride (TG) levels was significant in hyperlipidemia persons but not in the healthy population (MD = -0.20, 95% CI -0.37 - -0.04, P < .01; MD = -0.01, 95% CI -0.02 - 0.04, P > .05). Conclusions: Through our analysis, the effect of probiotics on lowering plasma lipid was more obvious in hyperlipidemia participants than healthy population. However, further studies are required to confirm the findings due to pronounced clinical heterogeneity.

Improving Image Segmentation with Contextual and Structural Similarity.

Deep learning models for medical image segmentation are usually trained with voxel-wise losses, e.g., cross-entropy loss, focusing on unary supervision without considering inter-voxel relationships. This oversight potentially leads to semantically inconsistent predictions. Here, we propose a contextual similarity loss (CSL) and a structural similarity loss (SSL) to explicitly and efficiently incorporate inter-voxel relationships for improved performance. The CSL promotes consistency in predicted object categories for each image sub-region compared to ground truth. The SSL enforces compatibility between the predictions of voxel pairs by computing pair-wise distances between them, ensuring that voxels of the same class are close together whereas those from different classes are separated by a wide margin in the distribution space. The effectiveness of the CSL and SSL is evaluated using a clinical cone-beam computed tomography (CBCT) dataset of patients with various craniomaxillofacial (CMF) deformities and a public pancreas dataset. Experimental results show that the CSL and SSL outperform state-of-the-art regional loss functions in preserving segmentation semantics.

Aromatic Ketones as Mild Presodiating Reagents toward Cathodes for High-Performance Sodium-Ion Batteries.

Chemical presodiation (CP) is an effective strategy to enhance energy density of sodium ion batteries. However, the sodiation reagents reported so far are basically polycyclic aromatic hydrocarbons (PAHs) wth low reductive potential (~0.1 V vs. Na+ /Na), which could easily cause over-sodiation and structural deterioration of the presodiated cathodes. In this work, Aromatic ketones (AKs) are rationally designed as mild presodiating reagents by introducing a carbonyl group (C=O) into PAHs to balance the conjugated and inductive effect. As the representatives, two compounds 9-Fluorenoneb (9-FN) and Benzophenone (BP) manifest favorable equilibrium potential of 1.55 V and 1.07 V (vs. Na+ /Na), respectively. Note that 9-FN demonstrates versatile presodiating capability toward multiple Na uptake hosts (tunneled Na0.44 MnO2 , layered Na0.67 Ni0.33 Mn0.67 O2 , polyanionic Na4 Fe2.91 (PO4 )2 P2 O7 , Na3 V2 (PO4 )3 and Na3 V2 (PO4 )2 F3 ), enabling greatly improved initial charging capacity of the cathode to balance the irrevisible capacity of the anode. Our results indicate that the Aromatic ketones are competitive presodiating cathodic reagents for high-performance sodium-ion batteries, and will inspire more studies and application attempts in the future.

Chromosome-level assembly of the Neolamarckia cadamba genome provides insights into the evolution of cadambine biosynthesis.

Neolamarckia cadamba (Roxb.), a close relative of Coffea canephora and Ophiorrhiza pumila, is an important traditional medicine in Southeast Asia. Three major glycosidic monoterpenoid indole alkaloids (MIAs), cadambine and its derivatives 3ß-isodihydrocadambine and 3ß-dihydrocadambine, accumulate in the bark and leaves, and exhibit antimalarial, antiproliferative, antioxidant, anticancer and anti-inflammatory activities. Here, we report a chromosome-scale N. cadamba genome, with 744.5 Mb assembled into 22 pseudochromosomes with contig N50 and scaffold N50 of 824.14 Kb and 29.20 Mb, respectively. Comparative genomic analysis of N. cadamba with Co. canephora revealed that N. cadamba underwent a relatively recent whole-genome duplication (WGD) event after diverging from Co. canephora, which contributed to the evolution of the MIA biosynthetic pathway. We determined the key intermediates of the cadambine biosynthetic pathway and further showed that NcSTR1 catalyzed the synthesis of strictosidine in N. cadamba. A new component, epoxystrictosidine (C27H34N2O10, m/z 547.2285), was identified in the cadambine biosynthetic pathway. Combining genome-wide association study (GWAS), population analysis, multi-omics analysis and metabolic gene cluster prediction, this study will shed light on the evolution of MIA biosynthetic pathway genes. This N. cadamba reference sequence will accelerate the understanding of the evolutionary history of specific metabolic pathways and facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding in plants.