DOG1 (Delay of Germination1) is a gene regulating germination through abscisic acid (ABA)-mediated pathway. In this study, we have identified 21 DOG1 domain containing proteins in tomato (SlDOGs) and analyzed their phylogenetic relationship and collinearity with DOG genes in other crops and examined their expression in contrasting genotypes under polyethylene glycol (PEG)-induced moisture deficit stress. The motif analysis identified motif1 forms part of the DOG1 domain, and the conserved domain analysis confirmed the presence of the DOG1 domain in all the SlDOGs. The cis-acting elements associated with abiotic stress response such as ERE, AREB1, and MYC were found upstream of the SlDOGs and the corresponding transacting gene families showed differential expression under PEG-mediated stress. Furthermore, SlDOG5, SlDOG6, and SlDOG13 were upregulated under PEG stress, while SlDOG8 exhibited contrasting expression between the genotypes suggesting a potential role in stress tolerance. Overall, this study provides the first comprehensive identification and characterization of DOG1 domain containing proteins in tomato and highlights their possible involvement in drought tolerance.
This study investigated the development of Ligustrum sp. fruits through physical, chemical, physicochemical, proteomic, and metabolomic analyses. The ripening process was found to be accompanied by significant physicochemical changes and a complex dynamic of phenolic compounds, indicating metabolism physiological roles and potential nutritional and pharmacological applications. Proteomic analysis revealed 359 differentially expressed proteins throughout development, 47 of which were associated with metabolic functions such as energy metabolism, gene regulation, stress response, and cell wall remodeling. A considerable number of hypothetical proteins were also identified, highlighting the need for further functional studies. The results reveal multifaceted regulation and intense biochemical activity supporting fruit growth and maturation, contributing to a better understanding of the molecular mechanisms involved and emphasizing the biotechnological potential of the species for future functional and pharmacological applications.
The present study provides the first integrative genomic comparison of five Vigna species Vigna angularis, V. umbellata, V. mungo, V. radiata, and V. unguiculata to characterize their genomic architecture and diversity. It gives a valuable understanding to the evolutionary history and genetic diversity of these legumes that may have wide inference for crop improvement under breeding programs and ultimately contribute to the food and nutrition security. Protein and genomic fasta sequences were retrieved from the NCBI GenBank. A total of 8194 single-copy and 23,560 unique genes were identified. Vigna umbellata, V. angularis, V. radiata, and V. mungo exhibited closest relationship, while V. unguiculata showed a higher degree of genomic divergence. The study reported that 496 gene clusters were linked to response to salt stress, 406 for response to water-deficit stress, and 319 for response to oxidative stress, while 112 genes were identified for response to cold exhibiting the robustness of these legume species against abiotic stresses. The gene family expansion of the Vigna species indicated a significant increase in the gene families compared to their common ancestor. The finding unravels the evolutionary relationships and genome evolution of the five Vigna species. The study revealed the divergence of V. angularis, V. umbellata, V. mungo, and V. radiata from the common ancestor that have experienced evolutionary pressures favoring diversification and increased adaptability. Therefore, these Vigna species may be used to develop improved varieties of other Vigna species being cultivated for food purposes.
The synthesis of very-long-chain fatty acids (VLCFAs), regulation of membrane lipids, and responses to external stimuli all rely on 3-ketoacyl-CoA synthase (KCS). Nevertheless, the regulatory mechanisms of KCS genes in response to temperature during cotton fiber development remain poorly characterized. Herein, a comprehensive analysis of the KCS gene family was conducted across four Gossypium species, identifying 167 fatty acid elongase 1 (FAE1)-type and 44 elongation of very-long-chain fatty acids (ELO)-type KCS genes. Synteny analysis revealed that whole-genome and segmental duplications primarily contributed to the expansion of the KCS gene family. intragenomic paralogs underwent strong purifying selection, while intergenomic orthologs experienced pronounced positive selection following polyploidization. The subcellular localization of KCS proteins was highly conserved. Transcriptome analysis and RT-qPCR revealed that, at 4 days post-anthesis (DPA), high temperature significantly increased the expression of five FAE1-type and two ELO-type genes. Analysis of cis-elements in the promoters revealed numerous stress- and hormone-responsive motifs, suggesting a potential association between temperature and hormone signaling. Comparison of Gossypium hirsutum and Gossypium arboreum with contrasting fuzz phenotypes revealed differences in KCS gene expression. Except for GhKCS12, the expression levels of the other seven genes were positively correlated with the fuzz phenotype in G. hirsutum, while these genes were either barely expressed or showed no correlation with the fuzz phenotype in G. arboreum. The present study uncovered a KCS-VLCFA-ACO-ethylene-fuzz signaling cascade responsive to temperature. Importantly, the functional divergence of KCS genes between allopolyploid and diploid cotton provides insights into cotton adaptation to environmental conditions and epidermal cell differentiation and lays a solid foundation for cultivating temperature-adaptive cotton varieties with desirable fuzz traits.
The lima bean (Phaseolus lunatus L.) is a legume of high agronomic and nutritional importance, particularly for smallholder farming in northeastern Brazil. The breeding program at the Federal University of Piauí (UFPI, Brazil) developed lines from biparental crosses, selecting traits of agronomic relevance for crop improvement, such as determinate growth habit, earliness, and yield. This study evaluated the genetic diversity and population structure of 34 ninth-generation lines using 3002 single-nucleotide polymorphism markers generated using genotyping-by-sequencing. Observed heterozygosity (HO) was lower than the expected heterozygosity (HE), with mean values of 0.01 and 0.19, respectively. Line H25-66 exhibited the highest number of private alleles and marked genetic divergence, whereas the lines from population H46 were highly homogeneous, with intermediate differentiation observed in the remaining populations. Multivariate analyses, including principal component analysis, Neighbor-joining clustering, heatmaps, and sparse non-negative matrix factorization ancestry plots, consistently revealed well-defined genetic groups. The consistent identification of highly homogeneous groups, such as population H46, together with strongly divergent materials, particularly line H25-66, demonstrates that the program harbored both stabilized genetic backgrounds suitable for cultivar development and contrasting sources of variation that can be strategically exploited in parental selection, enabling crosses between divergent genotypes to generate novel allele combinations. These findings provide valuable insights for guiding strategic breeding decisions and accelerating the development of improved lima bean cultivars adapted to Brazilian production systems.
Calcineurin B-like proteins (CBLs) are a class of plant-specific Ca2⁺ sensors that play critical roles in plant growth, development, and stress responses. CBL proteins contain four canonical EF-hand domains for Ca2⁺ binding, each consisting of an α-helix-loop-α-helix structure formed by 12 relatively conserved amino acids. At the transcriptional level, the promoter regions of CBL genes harbor cis-acting elements such as W-box, MBS, and G-BOX, which interact with upstream transcription factors (TFs) to activate or repress the expression of downstream genes. CBLs regulate stomatal movement through signaling pathways involving abscisic acid (ABA), respiratory burst oxidase homologs (RBOHs), and reactive oxygen species (ROS), thereby reducing water loss and enhancing plant adaptation to environmental stresses. Numerous studies have demonstrated that under adverse conditions such as salinity, drought, extreme temperatures, nutrient deficiency, and pathogen infection, CBLs rapidly respond to transient intracellular Ca2⁺ signals. CBLs form signaling modules with CBL-interacting protein kinases (CIPKs) to transduce Ca2⁺ signals and regulate downstream targets.. On the other hand, CBLs also interact with other proteins, including high-affinity K⁺ transporter 5, protein S-acyl transferase 10, and type 2C protein phosphatases, to positively or negatively regulate plant stress tolerance. Furthermore, CBLs are involved in the regulation of organ and tissue growth and development, promote sugar accumulation in fruits. CBLs also interact with the flowering time regulator GIGANTEA (GI) to modulate flowering time under salt stress conditions. This review systematically summarizes the discovery, protein structure, classification, regulatory networks, biological functions, and mechanisms underlying stress responses of plant CBLs, and provides perspectives for future research, aiming to provide theoretical foundations and genetic resources for the genetic improvement of crop stress tolerance and biological breeding.
Blackgram (Vigna mungo L. Hepper) is an important pulse crop whose productivity is severely constrained by powdery mildew caused by Erysiphe polygoni. To investigate resistance mechanisms, 121 blackgram genotypes were evaluated under natural epiphytotic conditions during the 2023–2024 and 2024–2025 cropping seasons. Five genotypes consistently exhibited partial resistance. Pathometric descriptors, including final disease severity (FDS), coefficient of infection (CI), relative area under the disease progress curve (rAUDPC) and apparent infection rate (r), showed strong positive correlations, confirming their suitability for assessing partial resistance. Detailed disease progression studies on twelve contrasting genotypes during Rabi 2024–2025 revealed that moderately resistant genotypes (TU-40, MBG-1123 and TBG-138) exhibited prolonged latent periods, fewer lesions (≤ 0.54 per leaflet), smaller lesion diameters (≤ 1.24 mm), and reduced sporulation (< 22.04 × 104 conidia ml⁻1), whereas susceptible genotypes (PU-31 and TBG-129) showed rapid disease development and profuse sporulation (up to 55.66 × 104 conidia ml⁻1). Seventeen representative genotypes with contrasting disease reactions were further analysed using 20 resistance gene analogue (RGA) primers and 20 simple sequence repeat (SSR) markers. RGA primers generated polymorphic amplification profiles that differentiated resistant and susceptible genotypes, while the SSR marker VrCSSTS1 showed a preliminary putative association with powdery mildew resistance. These findings demonstrate that integrating field phenotyping with molecular marker analysis facilitates the identification of promising resistance sources. However, the identified marker–trait associations require validation across diverse germplasm, and the amplified RGA loci require sequencing to confirm their identity before deployment in marker-assisted breeding programmes.
Makhana (Euryale ferox Salisb.) is an economically and nutritionally important aquatic crop. In recent years, rapid progress in genomic and transcriptomic studies has advanced understanding of the molecular mechanisms underlying growth, development, stress tolerance, and secondary metabolite biosynthesis in this species. The availability of a high-quality reference genome and developmental stage-specific transcriptome datasets has facilitated the identification of key structural and regulatory genes involved in flavonoid, lignin, and starch biosynthesis, as well as in physiological and evolutionary adaptation to the aquatic environment. Despite these advances, molecular studies on E. ferox remain limited, and functional validation research is largely lacking. In addition, genetic diversity analysis has been performed using RAPD and SSR markers, which are useful for crop improvement programs. However, to date, no review in the public domain specifically focuses on molecular studies of E. ferox; therefore, this review presents a novel synthesis of current molecular knowledge and emerging research directions. Future prospects emphasize the integration of multi-omics approaches, genome-wide association studies, and modern gene-editing platforms such as CRISPR-Cas9 to improve metabolite production, enhance stress tolerance, increase yield, and develop improved genotypes or superior accessions. Such knowledge will help to accelerate genetic improvement strategies and promote sustainable utilization of E. ferox.
Light wheat is the immature fruit of wheat. It is a commonly used traditional Chinese medicine in clinic. It has different effects at different stages, especially in the treatment of hyperhidrosis. Five growth stages of Light wheat were analyzed by UPLC-MS/MS and RNA sequencing of gene expression. Metabolome and transcriptome analysis were used to explore the formation mechanism of Light wheat and MetMap analysis was used to analyze the pharmacodynamic material basis. Different metabolic product accumulation and gene expression patterns were observed, especially in the phenylpropane synthesis pathway and flavonoid and flavonol biosynthesis. The main findings include the following: the harvest period of main metabolites is stage C to stage D. The key regulatory genes for the formation of Light wheat, include module core genes and possible pharmacological substances. This study preliminarily explores the metabolic and transcriptional dynamics during Light wheat development; the critical harvesting period for the accumulation of pharmacologically active compounds in Light wheat has been preliminarily identified and provided an important theoretical basis for its pharmacodynamic material basis.
Parthenocarpy, the development of fruit in the absence of pollination or fertilization, is an important trait in cucurbit crops as it stabilizes yield under conditions unfavorable for pollination and produces seedless fruit preferred by consumers and processors. This review summarizes current knowledge of the genetic control, hormonal regulation, and breeding use of parthenocarpy in Cucurbitaceae. Inheritance ranges from simple to complex, and several quantitative trait loci (QTLs), including Parth2.1 and Parth6.1, have been mapped in cucumber. A small number of genes, such as CsWIP1, CsACO2, CsFUL1 and CsNPF1, have been confirmed by CRISPR/Cas9 or transgenic studies to influence fruit set, while a much larger group of genes, including members of the YUCCA, ARF, PIN and GA20-oxidase families, has been proposed mainly from transcriptomic and association studies and still requires functional confirmation. At the physiological level, parthenocarpy is controlled by an interconnected hormonal network: auxin initiates ovary growth, gibberellin and cytokinin promote cell division and expansion, and ethylene and abscisic acid restrain the response, with MADS-box transcription factors, microRNAs and the SPINDLY protein fine-tuning the overall output. Parthenocarpy can also be induced by stimulatory pollination, low temperature, short photoperiod, and ploidy manipulation, and can be fixed genetically through marker-assisted selection or CRISPR/Cas-based genome editing. Each of these approaches has practical benefits as well as clear limitations, including cost, genotype dependence, regulatory uncertainty, and the polygenic nature of the trait. The review concludes with emerging tools, such as base and prime editing, multi-parent mapping populations, and single-cell transcriptomics. It outlines the key questions that remain to be resolved for the development of stable, high-yielding parthenocarpic cultivars.
Agronomically desirable homozygous genotypes for traits of interest are crucial for successful plant breeding programs. This study aimed to identify groundnut genotypes resistant to foliar diseases and high oleic acid content through natural field screening, biochemical analysis, and molecular validation for use in marker-assisted backcross breeding. Fifty-two groundnut genotypes were evaluated, including a resistant (ICG 15419) and susceptible (TMV 7) check for late leaf spot and rust. In field screening, ICG 8760, ICG 11088, ICG 12276, ICG 12625, and ICG 15419 were identified as resistant to both late leaf spot and rust. Biochemical assays showed that ICG 15419, ICG 8760, ICG 15398, and ICG 115 had elevated levels of phenols, proteins, chlorophyll, peroxidase, and polyphenol oxidase. Genotyping with 13 polymorphic SSR markers identified 35 alleles, with PIC values ranging from 0.50 to 0.66. Biometrical analysis revealed significant variation, high heritability, and strong positive correlations between key agronomic traits and pod yield. Principal component analysis identified six major principal components, which explained 72.22
Rising global temperatures and the increasing frequency of heat waves results in climate change and pose a major constraint on rice (Oryza sativa L.) productivity. Rice is highly sensitive to elevated temperatures during key developmental stages, including panicle initiation, meiosis, anthesis, and early grain filling. During these stages, heat stress reduces pollen viability, disrupts anther dehiscence, lowers spikelet fertility, and impairs grain filling. At the physiological level, heat stress accelerates respiration, destabilizes cellular membranes, suppresses photosynthesis, and alters assimilate partitioning, leading to significant yield penalties. Recent advances have deepened understanding of the genetic and regulatory bases of heat tolerance, highlighting the roles of quantitative trait loci, heat-responsive genes, transcription factors, phytohormone signaling pathways, and epigenetic regulation. In parallel, advances in phenotyping, including controlled-environment screening and high-throughput field-based platforms, have improved the precision of heat-adaptive trait evaluation. These developments are increasingly integrated into breeding strategies such as marker-assisted selection, genomic selection, and genome editing to improve heat tolerance alongside yield and grain quality. However, the expression of heat tolerance remains strongly stage-specific and is influenced by genetic background and genotype × environment interactions. This review synthesizes current knowledge on heat stress responses in rice and discusses their application in breeding programs. Future progress will depend on robust functional validation of key regulators, integration of multi-omics data with predictive breeding models, and broadening the genetic base of heat tolerance to sustain rice production under warming climates.
Table beet (Beta vulgaris var. rubra et lutea) is a flavonoid-rich root vegetable, but the coordinated regulation of flavonoid biosynthesis across varieties (red vs. yellow) and developmental stages (rosette vs. fleshy root enlargement) remains unclear. Here, we performed integrated LC-MS/MS-based metabolomic and RNA-seq transcriptomic analyses on 12 samples of red and yellow table beets at these two stages. A total of 374 flavonoid metabolites were detected. The relative abundance of detected flavonoid metabolites was highest in red beets at the fleshy root enlargement stage (RG3) and lowest in yellow beets at the rosette stage (YG2). Several characteristic flavonoids showed stage- and variety-associated accumulation patterns, including quercetin and rutin in RG3 and isoquercitrin and trifolin in YG3. Transcriptome sequencing identified 5503, 3253, 2661, and 6793 differentially expressed genes (DEGs) in RG3_vs_RG2, YG2_vs_RG2, YG3_vs_RG3, and YG3_vs_YG2, respectively, with enrichment in flavonoid biosynthesis-related pathways. Integrated analysis revealed strong correlations between flavonoid biosynthesis-related structural genes and metabolites, suggesting that CHS, CHI, F3H, CYP75B1, FLS, and LAR may contribute to stage- and variety-associated differences in flavonoid accumulation. We also identified MYB, bHLH, and WRKY family members as candidate regulators. These results provide a gene-metabolite framework for understanding flavonoid accumulation in table beet and offer candidate resources for high-flavonoid breeding.
Cold and salinity stresses are major abiotic constraints limiting cucumber (Cucumis sativus L.) productivity worldwide. This study investigated the genetic basis of tolerance to cold and salinity stress using a diverse panel of 134 cucumber accessions. Plants were phenotyped under controlled stress conditions using standardized scoring indices, revealing broad and reproducible variation for both traits. Analysis of variance confirmed highly significant genotypic effects for cold tolerance and salinity stress survival (p < 0.001), demonstrating sufficient phenotypic resolution for association mapping. Genotyping-by-sequencing followed by stringent quality control yielded 18,383 high-confidence SNP markers distributed across the genome. Genome-wide association analyses were conducted using a multilocus mixed-model framework accounting for population structure and kinship, with defined significance thresholds and false discovery rate correction. Seven loci were significantly associated with cold stress tolerance, while three loci were associated with salinity stress tolerance, each explaining moderate proportions of phenotypic variance. Candidate genes underlying cold tolerance loci encode proteins involved in diverse biological functions, including transcriptional regulation, chromatin remodeling, signal transduction, protein turnover, and cellular homeostasis, indicating a multicomponent genetic basis of cold stress adaptation. In contrast, candidate genes associated with salinity tolerance were mainly related to membrane-associated processes and stress signaling pathways. Expression analysis using quantitative RT-PCR in contrasting tolerant and sensitive accessions revealed stress-responsive differential expression patterns for several candidate genes. Notably, cold and salinity tolerance were governed by largely distinct sets of genes, suggesting stress-specific genetic architectures. Overall, this study provides genomic insights into abiotic stress tolerance in cucumber and identifies candidate genes and molecular markers with potential utility for functional validation and breeding applications.
Nitrate transporter NRT1.1 plays a central role in nitrogen (N) assimilation in Arabidopsis thaliana (Arabidopsis), functioning as both a dual-affinity transporter and a nitrate sensor. However, the functional conservation of NRT1.1 orthologs in crops remains to be fully elucidated. Here, we characterized BrNPF6.6, a close homolog of NRT1.1 in Brassica rapa. Subcellular localization confirmed its plasma membrane localization, consistent with a role in nitrate transport. Expression analysis revealed tissue-specific responses to nitrate availability, with leaf BrNPF6.6 transcripts peaking under low N conditions. Functional complementation assays in the Arabidopsis chl1-5 mutant demonstrated that BrNPF6.6 restores nitrate accumulation, represses NRT2.1/2.4/2.5 expression under N-sufficient conditions, and partially complements the lateral root phenotype in chl1-5 mutant under low N conditions. These findings support that BrNPF6.6 is a close homolog of NRT1.1, highlighting the conservation of NRT1.1-mediated N assimilation functions.
The propagation of high-yield and high-quality hazelnut varieties through vegetative methods, such as micropropagation, is crucial for large-scale production. However, low adventitious root (AR) formation and regeneration remain major limitations in hazelnut micropropagation. AR formation is regulated by various environmental and internal factors, with the WUSCHEL-related homeobox (WOX) gene family playing a central role in its molecular control. Although WOX transcription factors (TFs) are known regulators of AR development in several plant species, this gene family has not yet been characterized in hazelnut. In this study, a genome-wide analysis identified WOX gene family members in hazelnut and examined their roles in AR development. Ten CaWOX genes were unevenly distributed across six chromosomes. Phylogenetic analysis grouped these proteins into three clades (WUS, intermediate, and ancient), consistent with other plant species. Conserved motif analysis revealed structural similarity among CaWOX proteins. The proteins ranged from 167 to 351 amino acids with molecular weights of 19.10–40.20 kDa, and three had isoelectric points below 7. All CaWOX proteins were predicted to localize in the nucleus. Promoter analysis revealed cis-acting regulatory elements associated with hormone signaling, development, and stress responses, indicating complex transcriptional regulation. CaWOX4 transcripts showed a statistically significant 1.82-fold increase on day 7 compared to day 1, whereas CaWOX13 transcripts showed a 1.36-fold increase that was not statistically significant. Overall, these results suggest that CaWOX4 and CaWOX13 may be associated with AR formation in hazelnut and represent candidate genes for future functional studies aimed at improving rooting efficiency in woody plants with recalcitrant regeneration systems.
Mugwort is derived from the dried leaves of Artemisia argyi Lévl. et Vant. (Asteraceae). Flavonoids represent a major class of bioactive constituents in A. argyi leaves and contribute significantly to its pharmacological activities. However, the regulatory mechanisms of flavonoid biosynthesis in A. argyi remain unclear. In this study, one R2R3-MYB family transcription factor, AaMYB57, was cloned from A. argyi leaves. The open reading frame of AaMYB57 was 993 bp, encoding 330 amino acids. The predicted relative molecular weight was 37,720.08, the theoretical isoelectric point was 8.44, the total number of atoms was 5224, the aliphatic index was 65.91, the instability index was 50.68, and the hydrophilicity coefficient was -0.840, suggesting that the AaMYB57 protein is a hydrophilic protein. AaMYB57 was localized in the nucleus. Sequence alignment results indicated that AaMYB57 had the highest homology with AanMYB1 from A. annua. The plant expression vector pBWA(V)HS-AaMYB57-Glosgfp was constructed using the double enzyme digestion method, and overexpression lines of the AaMYB57 in tobacco were obtained through Agrobacterium-mediated transformation. Compared with wild-type tobacco, the total flavonoid and rutin contents were higher in the AaMYB57 overexpression lines. Meanwhile, qRT-PCR analysis showed that the expression levels of genes involved in the flavonoid synthesis pathway, such as 4CL, CHI, and F3H, were upregulated in the overexpression lines. It is preliminarily speculated that AaMYB57 is involved in regulating the expression of enzyme genes in the flavonoid biosynthesis pathway, thereby affecting flavonoid synthesis in A. argyi leaves. This study establishes a foundation for further understanding the molecular mechanisms underlying flavonoid biosynthesis in A. argyi.
Rice serves as a primary dietary source of calories for over half of the global population. Bacterial blight (BB), caused by the pathogen Xanthomonas oryzae pv. oryzae (Xoo), poses a significant challenge to rice cultivation worldwide, including Bangladesh. This study aimed to utilize a marker-assisted backcross breeding strategy to stack three BB resistance (R) genes (Xa4, xa13, and Xa21) into the genetic background of a susceptible mega rice variety BRRI dhan28. Because BRRI dhan28 inherently possesses the Xa4 gene, which is ineffective when present alone against the prevalent BB isolates in Bangladesh. Donor parent IRBB60 was utilized as the source to introgress the xa13 and Xa21 genes, thereby achieving the three-gene stack. Backcrossing, selfing, and foreground selection were conducted across the generations (F1 to BC3F7) to develop advanced resistant lines. Genotypic and phenotypic data analysis of 400 BC3F2 individuals revealed that BB resistance followed the simple Mendelian inheritance. Among the best selected 40 fixed BILs (backcross inbred lines) in the BC3F7 generation, 33 possessed all the candidate R genes, while four lines contained only Xa4 and Xa21, and three lines consisted of xa13 with Xa4 genes. Disease scoring for BB ranged from 0 to 5 for these BILs, in contrast to 7 to 9 for the recipient parent. Marker-trait association analysis displayed a negative correlation between specific molecular markers and BB disease. The used molecular markers demonstrated significant potential in marker-assisted selection breeding. Furthermore, multi-location trials of the best eight BILs recorded that these lines possess broad-spectrum resistance to BB disease with superior agronomic performance. These BILs could serve as a promising candidate for developing novel rice cultivars resistance to bacterial blight or may be used as potential genetic stock for future rice breeding programs.
JA-Ile has been recognized as a crucial signaling molecule in plant defense responses against insect herbivory. The oxidative metabolism catalyzed by cytochrome P450 monooxygenase CYP94B3 is considered one of the major pathways regulating JA-Ile catabolism. In this study, four CYP94B3 homologs were identified in cultivated tobacco (Nicotiana tabacum), two of which were found to be mutated, suggesting possible functional redundancy among these genes. To determine whether mutation of CYP94B3 could enhance insect resistance in tobacco, we generated knockout lines using the CRISPR-Cas9 system. The results showed that homozygous CYP94B3 knockout lines exhibited significantly enhanced insect resistance. In addition, the JA-Ile content was elevated in the knockout lines, and the levels of insect resistance-related secondary metabolites, including TPI and nicotine, were also significantly increased, which may account for the enhanced insect resistance. Taken together, these findings provide a basis for the genetic improvement of insect resistance in plants through targeted manipulation of the JA-Ile metabolic pathway.
