INTRODUCTION:Plants have evolved complex defense systems to cope with herbivore attack, yet how these defense responses are coordinated across distinct cell types and time scales remains unclear. Understanding the cellular and spatial organization of such defenses is critical for elucidating the mechanisms underlying plant-insect interactions. OBJECTIVES:This study aimed to dissect the spatiotemporal regulation of tobacco leaf defense responses to herbivory by integrating time-series single-cell transcriptomics and spatial metabolomics. METHODS:We generated a high-resolution single-cell atlas of 28,318 tobacco leaf cells under simulated herbivory. Transcriptional dynamics, cell-cell communication networks, and spatial metabolite distributions were analyzed using co-expression, pseudotime, and ligand-receptor inference approaches. Functional validation of key regulators was conducted through qRT-PCR and VIGS assays. RESULTS:Our analyses revealed rapid transcriptional and metabolic remodeling following herbivore stress, with epidermal subpopulations serving as early signaling hubs. Spatial metabolomics confirmed epidermis-enriched accumulation of defense metabolites. Pseudotime and co-expression analyses identified epidermal subcluster 6 as an early-responsive population characterized by elevated WRKY81 expression. Silencing WRKY81 impaired defense activation, increasing herbivore feeding efficiency and metabolic assimilation. CONCLUSION:This study establishes the first single-cell-resolved dynamic defense network of tobacco leaves against herbivory. The findings uncover the central role of epidermal transcriptional reprogramming and identify WRKY81 as a critical regulator of early defense commitment, offering a mechanistic basis for the development of pest-resistant crops.

Tobacco waste represents a significant underutilized biomass with substantial potential for the extraction of high-value aromatic and bioactive metabolites. To evaluate the valorization of tobacco waste through S. cerevisiae M01 fermentation, this study systematically investigated the strain growth, genomic characteristics, and evolution of metabolic profiles. The result showed that the strain grew well in tobacco powder suspension and the physicochemical transformations occurred predominantly within the first 24h. Whole-genome analysis revealed a significant proportion of genes dedicated to amino acid, carbohydrate, and energy metabolism. HS-SPME-GC/MS analysis revealed that fermentation enhanced the floral, fruit, and sweet notes of tobacco extracts by increased the content of ketones, alcohols, and esters. Furthermore, metabolomics analysis identified 3408 differential non-volatile metabolites, primarily involving amino acid and derivatives, phenylpropanoids, and carbohydrates. The KEGG enrichment analysis revealed that the core pathways mainly associated with amino acid metabolism and nucleotide metabolism. Moreover, fermentation significantly increased the total phenols and flavonoids of tobacco extracts. Collectively, this study establishes a robust theoretical framework for the microbial fermentation of tobacco wastes, thereby contributing to its high-value utilization.

Plant synthetic biology is emerging as a transformative approach to reprogram plants into programmable, sustainable production systems. Unlike traditional genetic engineering, which relies on trial-and-error strategies, synthetic biology applies engineering principles to enable rational design of biological functions. Recent advances in CRISPR-based genome editing, modular DNA assembly, and synthetic gene circuits have significantly expanded the capacity for precise control of plant traits. However, progress in plants still lags behind microbial and mammalian systems due to intrinsic challenges, including complex multicellularity, context-dependent gene expression, and long Design-Build-Test-Learn (DBTL) cycles. This review provides a systematic overview of this field from an engineering perspective. We summarize the design principles and current progress of biological parts for plant synthetic biology, organized into sensor, processor, and actuator modules. We then examine major platforms for genetic circuit construction, including transcriptional regulation, recombinase-based systems, CRISPR/dCas tools, RNA-based regulation, and protein interaction networks, highlighting their respective strengths and limitations. Furthermore, we discuss the rapidly growing role of artificial intelligence (AI) in plant engineering. AI-driven approaches are enabling advances in part discovery, circuit design, and DBTL cycle acceleration. Finally, we outline key challenges, including the scarcity of standardized parts, limited orthogonality, and insufficient plant-specific datasets, and discuss future directions such as AI-integrated automation, virtual cell modeling, and translation from model plants to crops. Together, these developments are expected to shift plant synthetic biology from empirical practices toward predictive, scalable, and programmable engineering.

Plant architecture is key to crop yield, with leaf angle being critical for high-density cultivation. Although TAC1 represents a promising regulator of leaf angle for breeding, its molecular mechanism remains poorly understood, particularly at single-nucleus resolution. Here, we performed single-nucleus RNA sequencing on NtTAC1 knockdown lines exhibiting reduced leaf angle. This analysis generated a transcriptional atlas comprising 20 distinct clusters corresponding to 14 cell types and identified the endodermis as a central regulatory hub. Weighted gene co-expression network analysis and trajectory inference revealed that the auxin transporter NtPIN3 acts as a key downstream effector of NtTAC1. The two genes are co-expressed in endodermal cells and promote their differentiation from meristematic cells. Spatial metabolomics further demonstrated that NtTAC1 suppression elevates auxin levels and alters its spatial distribution, resulting in asymmetric auxin accumulation preferentially in the abaxial region and consequent reduction in leaf angle. Silencing NtPIN3 recapitulated the NtTAC1 disruption phenotype, confirming that the NtTAC1-NtPIN3 axis regulates both auxin asymmetry and cell wall remodelling. Consistently, both knockdown lines exhibited enhanced lignin deposition, linking disrupted auxin flow to secondary wall thickening. Moreover, CRISPR/Cas9-mediated editing of SlTAC1 in tomato suppressed SlPIN3 expression, indicating evolutionary conservation of this module. Collectively, our findings uncover a cell-type-resolved mechanism underlying leaf angle regulation and provide a mechanistic framework for precision engineering of crop architecture adapted to high-density cultivation.

INTRODUCTION:Pseudomonas syringae pv. tabaci, a Gram-negative bacterial pathogen, causes devastating tobacco wildfire disease with global economic impacts. While its pathogenicity is well documented, the dynamic defense mechanisms of tobacco against infection remain poorly understood. OBJECTIVE:This study aimed to decipher phased defense mechanisms of tobacco against P. syringae infection through multi-omics integration, with emphasis on elucidating spatiotemporal coordination between transcriptional reprogramming and metabolic remodeling, and functionally validating critical regulatory modules. METHODS:Time-series transcriptomic and metabolomic profiling was integrated to reconstruct dynamic response patterns. Stage-specific regulatory modules were explored via TO-GCN and WGCNA, and the roles of WRKY6 and WRKY23 in disease resistance were validated by generating transgenic lines. RESULTS:Early infection (12-24 hpi) prioritized stress signaling and hormone pathway activation (salicylic acid/jasmonate), transitioning to cellular homeostasis regulation at late stages (48-60 hpi). WRKY, ERF, and NAC families orchestrated stage-specific gene expression. Notably, WRKY6 and WRKY23 functioned as negative regulators, with their silencing leading to a reduction in lesion area by 42-58% and pathogen load by 3.2-4.5 fold. Metabolomic analysis revealed sustained activation of phenylpropanoid metabolism, specifically regulating L-phenylalanine homeostasis and biosynthesis of its defense derivative xanthosine. Additionally, core modules involved in sphingolipid metabolism, light responses, and hormone cross-talk were also identified. CONCLUSION:We demonstrate that WRKY-mediated transcriptional reprogramming coordinates phytohormone signaling, sphingolipid dynamics, and light responses to spatiotemporally regulate secondary metabolite production. The identified WRKY6 and WRKY23 regulatory module establishes a molecular framework for engineering disease resistance, and the proposed two-phase defense model (early signaling → late metabolic remodeling) advances understanding of plant-pathogen interactions and offers targets for precision breeding.

Enzymatic browning in fresh-cut products represents a significant quality deterioration issue impacting commercial value and consumer acceptance. This study investigated 3-mercapto-2-butanone, a naturally occurring thiol compound with established food safety approval, as a novel anti-browning agent. Concentration-response experiments demonstrated that 3-mercapto-2-butanone at 50 mu L/L achieved optimal browning inhibition in fresh-cut potato shreds, maintaining excellent color after 4 days while also preventing browning in tobacco leaf pulp, confirming broad cross-species efficacy. Transcriptomic analysis revealed biphasic molecular responses: immediate modulation of phenylalanine metabolism and phenylpropanoid biosynthesis pathways, followed by sustained antioxidant enhancement and metabolic reorganization. Direct enzymatic assays confirmed potent polyphenol oxidase (PPO) inhibition, with complete activity suppression at the optimal concentration. Mechanistic studies demonstrated practical preservation of phenolic substrates while preventing quinone intermediate accumulation during enzymatic reactions. Molecular modeling revealed competitive binding at the PPO active site, with thiol groups forming stable copper coordination complexes that displaced natural substrates and prevented enzymatic oxidation. These findings establish 3-mercapto-2-butanone as a multi-target browning inhibitor operating through direct enzyme inhibition, transcriptional modulation of protective pathways, and competitive substrate displacement. The compound's established safety profile as a food flavoring agent, combined with superior efficacy compared to conventional inhibitors, positions it as a promising commercial alternative for fresh-cut produce preservation.

Tobacco (Nicotiana tabacum) is both a major industrial crop and a foundational model organism for plant biology. While alternative splicing significantly diversifies transcriptome complexity, tobacco isoform annotation remains incomplete, hampered by the limitations of previous short-read sequencing efforts. To address this gap, we generated a long-read transcriptome dataset across five tobacco tissues using the PacBio Sequel IIe platform, producing 198.4 million subreads. From these data, we successfully reconstructed 64,260 isoforms, including 35,013 novel transcripts. For the novel isoforms, we also performed systematic examinations of their structural features, biotypes and expression profiles. This dataset expands the tobacco isoform atlas and provides a valuable resource for genome annotation and regulatory studies.

Semi-Supervised Temporal Action Localization (SS-TAL) has attracted increasing attention for its ability to utilize unlabeled data. Most existing methods adopt pseudo labels to improve the utilization of unlabeled data. However, these methods neglect the unbalanced distribution of label noise in various samples and treat all pseudo labels equally. In this paper, we propose a novel framework called Mine the Pseudo Labels (MPL) to address this issue. Specifically, we first propose a method called pseudo-label confidence ranking which consists of three metrics to reliably measure the sample-wise quality of pseudo labels. Further, we design the pseudo-label adaptive correction module to refine the low confidence pseudo labels by estimating and rectifying the inherent bias of the temporal localization of the model. In addition, we introduce dual-level consistency learning with a Gaussian sampling scaling strategy, to train the model to learn scale-invariant and noise-unaware feature representation. Extensive experiments conducted on the benchmark datasets THUMOS14 and ActivityNet v1.3 demonstrate that our method outperforms previous state-of-the-art methods. Specifically, on THUMOS14, our method achieves an improvement of 5.1% and 3.5% in average mAP at 10% and 60% label rates, respectively. Our codes can be found in https://github.com/nohi191212/mpl.

The S-adenosyl-L-methionine (SAM)-dependent methyltransferase SABATH gene family is integral to regulating plant growth, development, and stress responses. Despite tobacco being an economically important crop and a widely used model organism, a systematic characterization of its SABATH gene family remains lacking. In this study, 48 SABATH genes were identified in tobacco and phylogenetically classified into three clades based on their methyltransferase functions. Comprehensive analyses of gene structure, motif composition, and evolutionary patterns revealed substantial divergence among these NtSABATH genes, with their evolution primarily driven by purifying selection. Additionally, NtSABATHs are predicted to be regulated by various miRNAs and multiple transcription factors associated with diverse stress responses. Expression profiling based on public RNA-seq datasets and qRT-PCR analyses demonstrated distinct tissue-specific expression patterns for NtSABATHs, indicating their crucial roles in plant growth, development, and responses to various biotic and abiotic stresses. Notably, we confirmed the crucial role of NtSABATH17 in enhancing tobacco’s resistance to black shank disease. This study provides a comprehensive characterization of the NtSABATH gene family through phylogenetic analysis and gene expression profiling, revealing their multifaceted roles in plant development and stress adaptation. These findings establish a solid foundation for future functional studies of NtSABATH genes.

Tobacco (Nicotiana tabacum L.) is a major economic crop and a model for plant-pathogen interactions, yet the spatiotemporal dynamics of defense metabolism during infection remain poorly characterized. Here, we used MALDI-MSI-based spatial metabolomics to systematically profile tobacco leaves during Pseudomonas syringae infection. Multidimensional analysis of 1,399 annotated metabolites revealed distinct spatiotemporal regulation patterns. Temporally, early infection (12 h postinfection (hpi)) was characterized by increased organic acids and terpenoids, followed by a mid-stage shift toward phenolic acids and quinones (24 hpi) and a late-stage enrichment of alkaloids by 60 hpi. Spatially, constrained clustering produced anatomy-aligned segmentation maps and revealed cell type preferences across epidermal, mesophyll, and vascular regions, with directional redistribution of differentially expressed metabolites as infection progressed. Defense hormones, including salicylic acid (SA) and jasmonic acid (JA), preferentially accumulated in vascular bundles and varied dynamically over time. Functional validation through exogenous application of representative metabolites (eg calystegine C1 and L-phenylalanine) and hormones (JA and SA), together with genetic manipulation of JA-biosynthetic genes, confirmed their roles in reducing lesion development and suppressing bacterial proliferation. Notably, epidermal enrichment of alkaloids-especially nicotine-and amino acid derivatives showed a decrease-then-increase pattern consistent with early consumption and later replenishment; nicotine's defensive contribution was further supported using a low-nicotine mutant. Collectively, P. syringae infection orchestrates a coordinated, cell type-compartmentalized defense metabolic program in tobacco, providing a resource for mechanistic studies and metabolic engineering of disease resistance. This time- and tissue-resolved atlas links metabolite remodeling to hormone-associated signaling and chemical barrier formation during wildfire disease progression.

β-Glucosidases (BGLUs) are critical enzymes involved in plant stress responses and secondary metabolism. However, their genomic and functional roles in Nicotiana tabacum, a model species for specialized metabolism, remain poorly understood. In this study, we systematically identified 56 NtBGLU genes, which were classified into 10 distinct clades. Most of these genes exhibit strong conservation in gene structure, motif composition, and chromosomal distribution, with notable species-specific expansions in tobacco. Promoter analysis revealed abundant stress- and hormone-responsive elements, particularly for methyl jasmonate (MeJA) and abscisic acid (ABA), consistent with the known roles of BGLUs in stress signaling and metabolic regulation. Transcriptomic and qRT-PCR analyses revealed tissue-specific expression and differential responses to abiotic stresses and phytohormones. Among the identified genes, NtBGLU50 was selected for functional analysis due to its substantial upregulation under MeJA treatment. RNA interference (RNAi)-mediated knockdown of NtBGLU50 disrupted root development, altered phytohormone crosstalk, and impaired secondary metabolism, leading to reduced levels of nicotine and anabasine. These findings suggest that NtBGLU50 plays a role in integrating jasmonate (JA) signaling with glycoside metabolism and defense pathways, particularly in regulating nicotine biosynthesis. These results provide valuable insights into the functional roles of NtBGLU genes, particularly NtBGLU50, in mediating the integration of JA signaling with secondary metabolism, which may contribute to tobacco's stress response and alkaloid biosynthesis.

Deep learning is revolutionizing enzyme engineering through efficient residue redesign. Leveraging deep learning for enzyme engineering, we redesigned a pectinase using ProteinMPNN guided by multiple sequence alignment. Our top-performing variant, DS-5, incorporated 72 mutations and achieved an 8.9-fold increase in catalytic activity compared to the wild-type. DS-5 also displayed significantly improved thermostability, with an optimal temperature increasing by 10°C, and robust performance over a wide pH range (7.0-11.0). Structural and molecular dynamics analyses revealed the source of this enhancement: a remodeled surface electrostatic potential due to the increase of five positively charged residues, forming an extended positive groove that potentially improves substrate binding affinity. This rationally designed enzyme demonstrated superior performance in applied settings, including apple juice clarification and tobacco degradation. Furthermore, treating tobacco leaves with DS-5 substantially improved their sensory profile by elevating the concentration of desirable flavor compounds like sucrose and lactones. Our study provides a framework for deep learning-guided engineering of highly efficient enzymes, directly linking catalytic improvements to enhanced end-product quality for industrial applications.

Nicotiana benthamiana has become a leading transient expression host for plant molecular farming, supporting the production of recombinant proteins, vaccine antigens, therapeutic antibodies and specialised metabolites. Here, we review the trajectory of N. benthamiana from a model for plant–virus interactions to a programmable synthetic-biology chassis, reframing it as a plant biofactory whose engineering value is rooted in its evolutionary origin, allopolyploid genome architecture, attenuated antiviral silencing, accession-level variation and tolerance of heterologous pathway reconstruction. We link these biological features to the engineering toolkit they have enabled, including genome editing, glycoengineering and combinatorial pathway reconstruction. We survey applications ranging from antibodies, vaccines and therapeutic proteins to complex plant natural products, distinguishing experimental proofs of concept from preclinical, clinical and approved products. We further compare N. benthamiana with other plant and plant-derived chassis, clarifying where it is uniquely powerful and where alternative systems remain competitive or complementary. Drawing lessons from selected plant-made biopharmaceutical programmes, we critically examine why strong proof-of-concept performance has not always translated into sustained commercial success. Finally, we discuss how genome-informed domestication, defined regulatory parts, controlled glycosylation, subcellular and protease engineering, and scalable production systems may help transform N. benthamiana from an empirical expression host into a rationally designed plant biofactory chassis.

IntroductionPlant-associated microbiota critically modulates host growth and environmental adaptation, yet assembly mechanisms, niche differentiation, and ecological strategies of bacterial communities inhabiting tobacco microhabitats remain poorly elucidated across geographical gradients.MethodsHere, we systematically characterized bacterial microbiome assembly across five tobacco-associated niches (bulk soil, rhizosphere soil, root, stem, and leaf) from seven typical tobacco-planting regions using 16S rRNA amplicon sequencing, genome annotation, and niche breadth analysis. The independent and interactive effects of geographical location and host compartment on community structure, and further compared genomic traits, functional profiles, and life-history strategies between specialist and generalist bacterial populations were quantified.ResultsThe results revealed a deterministic soil–plant continuum stratification of bacterial communities and diversity, with progressively simplified communities and decreasing alpha diversity from bulk soil to above-ground tissues, accompanied by progressive dominance of Proteobacteria. Geographical factors predominantly structured soil microbial communities via divergent edaphic properties, while host filtering acted as a universal dominant driver shaping endophytic microbiome assembly. Niche differentiation analysis demonstrated that niche-specialized bacterial ASVs overwhelmingly dominated all microhabitats and geographical sites, whereas generalist taxa only constituted auxiliary populations. Although specialist and generalist microbes exhibited highly conserved core genomic architectures and overall functional repertoires, they displayed distinct niche-specific functional divergence in metabolic pathways, stress resistance, and secondary metabolism across host compartments. Life-history strategy analysis further revealed that Y-strategist represented the core adaptive bacterial population, especially enriched in above-ground tobacco tissues.DiscussionOur study establishes a hierarchical dual-filtering assembly model for tobacco microbiota, clarifies the ecological differentiation and functional adaptation of specialist and generalist bacteria, and provides fundamental insights into the assembly rules and adaptive mechanisms of crop-associated microbiomes for future microbial resource utilization and agricultural microbiome regulation.

The plant microbiome plays a pivotal role in host adaptation and disease suppression, yet niche-specific microbial responses to biotic stress, particularly within distinct plant compartments, remain poorly understood. Here, we revealed that bacterial wilt disease (BWD) induced pronounced niche-specific microbiome alterations in tobacco, with the rhizoplane (RP) emerging as a critical hub for beneficial microbial recruitment and defense coordination. Utilizing 16S and ITS amplicon sequencing across 6 distinct plant niches, we observed significantly enhanced bacterial diversity and a striking enrichment of potentially beneficial microbes in the RP under BWD stress. Eight potent antagonistic bacterial strains were isolated from this key niche, with Stenotrophomonas sp. ASV61 and Chryseobacterium sp. ASV172 demonstrating robust in vitro biocontrol potential and confirming in vivo plant resistance and growth promotion. We further elucidated the superior biocontrol mechanisms of Chryseobacterium sp. ASV172, attributing its superior efficacy to enhanced colonization and flexirubin-mediated antagonism. Crucially, plant transcriptomic profiling unveiled that these beneficial microbes engaged in a signaling dialogue with host plants, dynamically modulating defense hormone pathways. While Ralstonia alone manipulated host defenses by sustaining salicylic acid responses, antagonistic strains re-directed the plant toward robust jasmonic acid signaling, thereby restoring a more effective defense posture. Collectively, our findings underscore the disproportionate importance of the RP over the rhizosphere in assembling a resilient microbiome against soil-borne diseases, paving the way for targeted RP microbiome engineering strategies for sustainable disease management.

Background Light and calcium signaling regulate gibberellic acid (GA) pathways, but their coordination mechanism remains unclear. Purpose To investigate how PhyB integrates light and calcium signals to regulate GA signaling in rice. Methods phyB and voz2 mutants were analyzed for phenotypes and transcriptomic changes. Protein interactions among PhyB, VOZ2, CIPK31, and SLR1 were examined, and comparative transcriptome analysis identified co-regulated genes. Results Both phyB and voz2 mutants showed semi-dwarf phenotypes. PhyB regulated GA biosynthesis, catabolism, and signaling genes. VOZ2, a PhyB-interacting transcription factor, co-regulated 62.9% of differentially expressed genes with PhyB and directly modulated GA biosynthetic genes (GA20ox3, GA3ox1, GA3ox2), activating biosynthesis while repressing catabolism. CIPK31 interacted with both PhyB and VOZ2 and stabilized SLR1, a GA signaling repressor. These components form a competitive regulatory module integrating light and calcium signaling. Conclusions PhyB-CIPK31-VOZ2 module integrates light and calcium signals to regulate GA signaling and plant growth in rice.

BRI1-EMS-SUPPRESSOR 1 (BES1) is a plant-specific transcription factor mediating brassinosteroid (BR) signaling and regulating cell proliferation, differentiation, morphogenesis, and stress responses. Tobacco (Nicotiana tabacum) is an important model plant and crop, but the BES1 family in tobacco remains poorly understood. In this study, we identified 17 BES1 genes and analyzed their tissue-specific expression and responses to hormones and abiotic stresses. NtBES1–17 was down-regulated by BR and up-regulated by brassinazole, indicating a key role in BR signaling, while NtBES1–1 and NtBES1–6 responded strongly to stress. Subcellular localization analysis showed that NtBES1–1 was localized in the nucleus, while NtBES1–6 and NtBES1–17 exhibited nucleo-cytoplasmic localization. These three proteins were capable of forming heterodimers and interacting with BIN2-like kinases. Overexpression of NtBES1–17 increased leaf and branch numbers, delayed flowering and senescence, and produced larger seeds, whereas knockout lines displayed opposite phenotypes. Hormone quantification and transcriptome analysis revealed increased feedback inhibition of BR biosynthesis and an antagonistic interaction with the jasmonic acid (JA) signaling pathway in the overexpression lines, while KEGG analysis showed that genes associated with ribosome, carbon fixation, and amino acid biosynthesis were widely down-regulated in the NtBES1–17/-11 knockout lines. Collectively, this study provides a systematic characterization of the BES1 family in tobacco and highlights NtBES1–17 as a central regulator of growth, development, and yield traits, offering molecular targets for crop improvement and stress resilience.

Crops are the primary source of food for humans, providing essential nutrients such as carbohydrates and proteins. With economic development and rising living standards, demand for high-quality crops has increased, encompassing not only high and stable yields but also enhanced nutritional profiles, flavor, taste, and functional components. These quality traits are typically controlled by complex polygenic networks and often involve trade-offs among yield, nutrition, and processing performance. Traditional breeding methods such as crossbreeding and phenotypic selection are limited by low efficiency, long breeding cycles, and difficulties in the coordinated improvement of multiple traits. The CRISPR/Cas9 system, characterized by its programmability, simplicity, and high editing efficiency, offers a novel strategy for precise trait improvement and the breeding of high-quality crop varieties. Through targeted modification of key genes, CRISPR/Cas9 can effectively regulate the synthesis and accumulation of nutrients such as starch, proteins, lipids, and vitamins, thereby enabling the targeted optimization of crop quality traits. This review systematically summarizes recent applications of CRISPR/Cas9 in enhancing nutritional components, improving eating quality, and reducing harmful substances in crops. In addition, current challenges and future prospects are discussed to provide theoretical guidance and practical support for the precise and efficient application of gene-editing technologies in crop quality improvement.

Plant peptides represent a promising class of signaling molecules with considerable potential as sustainable agricultural inputs, due to their high specificity, low toxicity, and biocompatibility. However, their widespread application remains hampered by high synthesis costs and the inability of conventional production systems to support proper folding and essential post-translational modifications. To address these limitations, we established an efficient plant-based expression system in tobacco for synthesizing bioactive peptides, using the key signaling peptide Rapid Alkalization Factor 32 (RALF32) as a proof of concept. We identified the CsVMV promoter as the most effective driver of peptide expression in tobacco and demonstrated that incorporating the Ref 4 sequence into the 3′ UTR significantly increased mRNA stability. Fusion with a GFP tag further enhanced peptide accumulation, enabling the establishment of a robust expression system that facilitates peptide extraction from the apoplastic fluid. Biological activity assays confirmed that RALF32 produced in this system is fully functional. Our study provides a potential platform for peptide production, opening new avenues for the widespread application of peptide-based agrochemicals.

Jasmonic acid (JA) is a critical phytohormone that mediates plant defense mechanisms against herbivores and pathogens. Within the JA biosynthesis pathway, allene oxide synthase (AOS), a rate-limiting enzyme, plays a pivotal role in plant responses to biotic and abiotic stresses. However, the specific involvement of AOS in Nicotiana tabacum (tobacco) during stress conditions has yet to be explored. In this study, we identified 12 AOS genes distributed across eight chromosomes in tobacco, encoding proteins that range from 300 to 550 amino acids and contain up to 12 conserved motifs. Cis-acting element analysis revealed the presence of elements responsive to light, stress, and hormones in the promoters of these genes. Quantitative RT-PCR analysis demonstrated that several NtAOS genes were responsive to a variety of abiotic and hormonal stresses, with NtAOS7 showing significant upregulation under both abiotic stress and methyl jasmonate (MeJA) treatment. To explore its functional role, NtAOS7 knockout and overexpression lines were generated via gene editing. Phenotypic analysis revealed that NtAOS7 knockout mutants exhibited increased plant height, whereas overexpression of NtAOS7 led to reduced plant height and delayed flowering. Hormonal profiling further demonstrated that JA levels were significantly reduced in the NtAOS7 knockout lines, while overexpression of NtAOS7 resulted in elevated JA levels, indicating that NtAOS7 plays an important role in endogenous JA biosynthesis. Stress tolerance assays showed that NtAOS7 enhances plant resistance to cold and salt stresses. This study provides novel insights into the role of AOS in tobacco, setting the stage for future research into its involvement in stress adaptation and hormone signaling. Our findings establish NtAOS7 as a key regulatory player in JA biosynthesis, coordinating plant growth, developmental timing, and abiotic stress tolerance in tobacco. These insights contribute to the broader understanding of JA’s role in plant adaptation and open avenues for future biotechnological applications aimed at improving stress resilience in crops.