The combined treatment of 1-MCP and sucrose has been shown to delay postharvest senescence in leafy vegetables, but the underlying mechanisms remain unclear. In this study, pakchoi was treated with 1‑MCP, sucrose, or their combination and stored at 20 °C for 7 days. The combined treatment (MST) exhibited the strongest effect in comprehensively delaying leaf yellowing, reducing ROS accumulation, and suppressing the expression of chlorophyll degradation genes. MST also inhibited protease activity and downregulated the autophagy‑related gene BrATG7, while all treatments preserved RuBisCO activity relative to the control, with ST showing the strongest protective effect. Furthermore, MST effectively protected chloroplast ultrastructure. Correlation analysis revealed that BrATG7 expression was significantly associated with quality parameters and chlorophyll degradation. Overexpression of BrATG7 in pakchoi significantly reduced RuBisCO activity. Furthermore, the transcription factor BrNAC29 was shown to bind the BrATG7 promoter. These results suggest that the combined treatment delays senescence by simultaneously alleviating carbon starvation, suppressing ROS accumulation, and inhibiting the transcription factor BrNAC29. This, in turn, downregulates BrATG7, thereby limiting BrATG7-mediated autophagic degradation of chloroplast proteins. This study provides a novel mechanism for postharvest preservation and identifies BrATG7 as a potential molecular target.

Endogenous H2O2 participated in the ethylene-dependent ripening and senescence process of horticultural fruit as a secondary messenger; however, the molecular mechanism beneath such a phenomenon has not been fully clarified until recently. By a conjoint analysis of metabolite, enzyme activities, gene expression profiles in AsA-GSH cycle of 'Kolar' pear, PbrAPX14 might act as a negative factor in the ethylene-mediated H2O2 accumulation. PbrAPX14, located in cytosol, would reduce H2O2 in vitro and in vivo, inhibit ethylene production, and thus fruit ripening and senescence. After analysing the expression profiles of the differentially expressed transcription factors (TFs) followed by experimental validation, the nuclear PbrIDD2 could directly bind to the cis-acting element (core motif: TTTGTCG) in PbrAPX14 promoter, activate its expression and thus enhance the H2O2-scavenging capacity of fruit/calli, which was associated with the mitigated ethylene evolution and fruit ripening and senescence. Further study explored that the H2O2-mediated post-translational S-sulfenylation of Cys48 residue in PbrAPX14, which mitigated its function, existed in vitro and in vivo, and was upregulated by ethylene, facilitating endogenous H2O2 accumulation. Overall, our results implied that both transcriptional and post-translational regulation of PbrAPX14, which were (in)directly under the control of ethylene, functioned in pear ripening and senescence process via regulating endogenous H2O2 level.

The breakpoint and segment size of wheat-rye T5DL·5DS-2RS translocation and differentially expressed genes for enhancing grain hardness and pre-harvest sprouting resistance were determined. Wheat (Triticum aestivum L.) is increasingly challenged by pre-harvest sprouting and other various biotic and abiotic stresses due to increasing effects of climate change. Here, by utilizing oligonucleotide probe multiplex #6 FISH (fluorescence in situ hybridization), GISH (genomic in situ hybridization), molecular markers, RNA-seq, and phenotyping analyses, a wheat-rye (Secale cereale L.) segmental T5DL·5DS-2RS translocation was precisely characterized. This translocation chromosome has a 4.05-Mb deletion of 5DS harboring grain hardness genes Pina-D1, Pinb-D1 and soft-protein gene Gsp-D1 that is replaced by a 218-Mb rye 2RS segment carrying Secalin-2, a gene encoding a 75kγ-secalin subunit. This translocation line has markedly enhanced grain hardness and pre-harvest sprouting resistance, but showed small adverse effect on thousand-kernel weight and grain width. The translocation line has significantly changed gene expression profiles whereby down-regulation of genes Pina, Pinb, Gsp-1 contributed to enhanced grain hardness. Differentially expressed genes in the ABA (abscisic acid) and GAs (gibberellins) signaling pathways of both rye and wheat chromosomes appear to be key factors for PHS resistance.

Blackening of Allium chinense at 70-80 °C enhanced total acidity and reducing sugars, producing a pleasant sweet-sour flavor. Total phenolic content increased from 10.20 to 94.54 mg GAE/g at 75 °C, with enhanced antioxidant capacity (DPPH 66.73%, ABTS 90.59%, FRAP 113.40 μg AAE/g). Non-targeted metabolomics revealed marked reduction of γ-glutamyl-S-allyl-L-cysteine (GSAC) and related γ-glutamyl dipeptides, responsible for the characteristic pungency of fresh A. chinense, alongside upregulation of terpenoids, alkaloids, and flavonoids (2- to 5-fold). Blackening at 75 °C yielded optimal flavor, color, texture, and bioactivity, with 5-HMF accumulation (166 μg/g) within safe limits. This study provides insights into physicochemical and metabolic changes during blackening, offering a foundation for optimizing functional blackened A. chinense production.

Ready-to-eat kiwifruit is increasingly favored by consumers, and fruit firmness is a key indicator of its edibility. Studies have shown that ethylene and its signaling pathways are involved in regulating fruit softening. However, in kiwifruit, research on the molecular mechanisms underlying ethylene-mediated postharvest softening remains limited, and the specific regulatory targets have yet to be fully elucidated. This study identified and characterized AcERF61, an ethylene response factor from 'Xuxiang' kiwifruit, and elucidated its regulatory role in fruit softening. AcERF61 was characterized as an ethylene-inducible nuclear protein with transcriptional activation activity. DNA affinity purification sequencing (DAP-seq) identified 2644 genome-wide binding sites for AcERF61, revealing three key target genes involved in cell wall degradation, including AcPME1 (pectin methylesterase), AcPL1 (pectin lyase), and AcCEL1 (cellulase). AcERF61 directly bound to a novel motif (GTGGTGGTGGTGGTGGTGGTGGG) in the promoter regions of these genes, as confirmed by yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter (DLR) assays. Transient overexpression of AcERF61 in kiwifruit significantly enhanced the expression of target genes (AcPME1, AcPL1, and AcCEL1), and caused cell wall loosening and degradation. Conversely, virus-induced gene silencing of AcERF61 suppressed target gene expression, and preserved cell wall integrity. These findings demonstrate that AcERF61 acts as a positive transcriptional regulator of kiwifruit softening by directly modulating the expression of genes involved in cell wall degradation, providing valuable insights for developing precision postharvest technologies and improving ready-to-eat kiwifruit quality.

Broccoli (Brassica oleracea var. italica) is highly nutritious, rich in vitamin C, glucosinolates, and minerals. However, its high postharvest respiratory rate leads to rapid quality deterioration, particularly chlorophyll degradation and yellowing under ambient conditions. In China, the lack of timely pre-cooling facilities exacerbates postharvest losses. Therefore, developing safe, effective and low-cost preservation methods for broccoli during transportation is of great practical importance. In this study, RNA sequencing was employed to analyze the effects of preharvest 1-methylcyclopropene (1-MCP) and postharvest pre-cooling combined with cold treatments on gene expression in broccoli. Transcriptome analysis revealed that both treatments significantly upregulated or maintained key genes involved in chlorophyll biosynthesis (e.g., Glutamyl-tRNA reductase (GluTR), porphobilinogen deaminase (PBGD), magnesium chelatase (MgCh)) and downregulated chlorophyll degradation-related genes (e.g., Chlorophyllase (CLH), pheophytinase (PPH), pheophorbide a oxygenase (PaO)), resulting in enhanced chlorophyll retention. Furthermore, chlorophyllide a oxygenase (CAO) was upregulated, while chlorophyll b reductase (CBR) was downregulated, suggesting modulation of the chlorophyll cycle. These findings elucidate the molecular mechanisms by which 1-MCP and pre-cooling combined with cold regulate chlorophyll metabolism, providing new insights into the gene regulatory network underlying the postharvest quality maintenance in broccoli.

Although the advantages of physical field-assisted fermentation have been revealed in recent years, there is limited information on the effects of physical field treatments applied during the post-fermentation process on microbial metabolic regulation and fermentation characteristics. This study employed the screened Rhodotorula mucilaginosa and Lactobacillus plantarum, targeting high dehydrogenase and total protease activities, to perform the fermentation of lamb liver paste, followed by post-fermentation mediated individually by ultrasound (US), pulsed electric field (PEF), and their combined treatment (US-PEF). The results showed that US-PEF treatment significantly increased the activities of aldehyde dehydrogenase, alcohol dehydrogenase as well as intracellular and extracellular proteases, with a significant increase by inter-species interaction of screened strains. The degradation rates of off-flavor compounds were markedly improved, with trimethylamine, dimethylamine, and histamine reaching 73.10%, 42.66%, and 77.96%, respectively. Analysis of headspace volatile fingerprinting revealed that US-PEF assisted post-fermentation significantly reduced the levels of off-flavor aldehydes, including 2-methylpropanal, 2-methylbutanal, and 3-methylbutanal in lamb liver, while promoting the generation of aroma esters like methyl acetate and butyrolactone, thereby significantly improving the flavor characteristics of lamb liver. During the post-fermentation process, physical field stimulation generated cavitation and electromechanical coupling effects. These effects activated microbial metabolic activity and increased enzyme expression, driving the shift of volatile compounds and promoting the conversion of off-flavor aldehydes and ketones into acids and esters. This study provides a novel technical alternative to enhanced efficiencies in regulating flavor features of fermented products, particularly for livestock and poultry by-products with off-odor components.

Ascorbate (AsA) redox status participated in the scald development of Pyrus bretschneideri Rehd. fruit as a cellular redox sensor. By a conjoint analysis of metabolites, enzyme activities and gene expression profiles in AsA-GSH cycle of the chilled pear, PbrDHAR5 was characterized as the candidate gene involved in this process. PbrDHAR5, located in cytosol and nucleus, catalyzed DHA reduction into AsA in vitro and in vivo, elevating AsA redox status and thus fruit chilling tolerance; moreover, the catalytic Cys20 residue in PbrDHAR5 played critical role in this reaction. After analyzing the expression profiles of the differentially expressed TFs, PbrWRKY83 demonstrated higher correlation with PbrDHAR5 than others. PbrWRKY83, located in nucleus, could interact with the only two W-box elements in PbrDHAR5 promoter as monomer and then activate its expression, leading to the improvement of AsA redox status and thus fruit chilling tolerance. In a further study, we explored that the H2O2-mediated S-sulfenylation of Cys20 residue in PbrDHAR5 accumulated upon scald development, suppressed its activity, and thus caused the decrement of AsA redox status. Taken together, our results implied that the H2O2-mediated S-sulfenylation of PbrDHAR5 attenuates the role of PbrWRKY83-PbrDHAR5 module, which positively regulates AsA redox status during scald development in pear.

The burgeoning multi-omics data have provided deep insights into the regulatory mechanisms underlying plant growth and development. However, revealing the complete landscape of gene regulatory networks underpinning various developmental processes remains challenging. Here, a multi-omics integrative gene network of the pear fruit development process was constructed through integrating 3D genomic, transcriptomic, transcription factor (TF) binding, chromatin accessibility, protein structure, and proteomic data. This integrative network comprises over 45,678 elements interconnected by more than 3.15 million edges and exhibits great potential in predicting regulatory and interactive relationships involved in the formation of key fruit quality traits (e.g., sugar, stone cell). In particular, the integrative network was applied to predict interactors of PbrII5, an inhibitor of vacuolar sucrose hydrolysis, and the predicted interactors were further validated through molecular experiments. Moreover, the network showed good performance in automatically predicting fruit trait-related genes by leveraging machine learning models. Specifically, a set of sugar metabolism-related genes was newly predicted, and their functions were verified through overexpression in pear fruit. In addition, extensive regulatory network divergence was observed between duplicated genes, with neofunctionalization being the dominant evolutionary process reshaping network connections of duplicated genes. Lastly, a multi-omics network database, pearGRN (http://peargrn.njau.edu.cn), was developed to facilitate further research for resolving complex gene regulatory relationships. This study lays a strong foundation for revealing novel regulatory mechanisms underlying fruit development and quality formation.

To develop more effective preservation strategies for edible fungi, this study investigated the role of reactive oxygen species (ROS) in quality changes of shiitake mushrooms (Lentinula edodes cv. T2) during storage at 5 °C and −1 °C (near-freezing temperature, NFT), as well as the underlying metabolic mechanisms of ROS. Compared with storage at 5 °C, NFT storage significantly maintained mushroom quality, as evidenced by higher hardness, glutathione (GSH), ascorbic acid (AsA), and total phenolics contents, as well as lower pileus opening degree (PD) and browning index (BI). These changes were accompanied by reduced levels of O2•-, H2O2, and malondialdehyde (MDA). NFT storage suppressed NADPH oxidase (NOX) gene expression and reduced NOX activity, thereby decreasing ROS production. Meanwhile, NFT storage upregulated the expression of antioxidant-related genes, including LeSOD2, LeCAT1, LePOD1, LeAPX1, LeMDHAR1, LeDHAR1, LeGR1, and LeGPX1, resulting in increased activities of the corresponding enzymes and enhanced AsA-GSH cycle-related antioxidant activity in shiitake mushrooms during NFT storage. Correlation and cluster analyses revealed that O2•- and H2O2 levels in shiitake mushrooms showed a significant negative correlation with hardness and a significant positive correlation with PD and BI. Combined with gene silencing and dual-luciferase reporter assays, these results demonstrate that NFT storage maintains endogenous ROS homeostasis in shiitake mushrooms by initiating intrinsic transcriptional regulation, thereby reducing NOX gene expression and activity while upregulating antioxidant enzyme-related genes and their corresponding enzyme activities, thereby preserving mushroom quality. This study demonstrates the feasibility of non-freezing NFT storage of shiitake mushrooms at –1 °C. This nonchemical, low-cost physical technique extends the shelf life of shiitake mushrooms from 16 days (under conventional storage at 5 °C) to 32 days, thereby markedly reducing postharvest losses and facilitating long-distance transportation and off-season supply. These findings offer both a novel molecular framework for postharvest preservation and a practical low-temperature storage protocol for the edible mushroom cold-chain industry.

Ultrasound pretreatment combined with pineapple peel extract (PP) was investigated for its effect on the post-acidification behavior of Lactobacillus delbrueckii subsp. bulgaricus, a key factor influencing the quality and shelf stability of fermented dairy products. The combined treatment significantly altered cellular physiological status and reduced early-stage culturability, without causing persistent divergence in OD₆₀₀-based growth trends. The PPU condition was associated with a 29.77

Chilling exposure of tomato, especially at early ripening stage, would impact flavor quality in the red fruit; nevertheless, the molecular mechanism beneath such phenomenon has not been fully clarified until recently. In this study, ethylene evolution and thus ripening process of 'FL 47' tomato was suppressed after 5 degrees C exposure at mature green stage for 4 d. And the suppressed ethylene abundance was associated with the downregulated gene (SlACO1, SlACS2/4, SlETR3/4/7, SlEIN2, SlEIL1, SlEBF1/2, and SlERF1) expression profiles, enzyme (ACO and ACS) activities, and ACC production in its biosynthesis & signaling transduction pathways. Additionally, the formation of TSS, glucose, fructose, and 13 key volatiles were suppressed by low-temperature treatment, while sucrose, TA, malate, citrate, and MeSA were enhanced, causing the negative impact on flavor profile. And the these quality changes were associated with the alternations of substrate (linoleic and linolenic acids, phytoene, phytofluene, trans-lycopene, gamma-carotenoid, beta-carotenoid, (iso)leucine, phenylalanine, salicylate (SA)) production, enzyme (SPS, A/NI, VI, PEPCK, IDH, NADP-ME, NADP-MDH, PEPC, LOX, HPL, PAL) activities, and gene (SlSPSA2/B/C, SlSUS4/6, SlA/NI1, SlVI, SlVIF, SlIDH3, SlNADP-MDH, SlPEPC3, SlNADP-ME2, SlPEPCK1, SlLOXC, Sl13-HPL, SlPSY1, SlCCD1A/B, SlBCAT1, SlAADC1A/B, SlAADC2, SlPAR1/2, SlICS, SlPAL4, SlSAMT1) expression profiles in their metabolic pathways. Further study explored that ethylene and SAs played an antagonistic role in fruit ripening and then flavor metabolism. Taken together, our study implied that chilling exposure at mature green stage impacted flavor quality in the red fruit via regulating the crosstalk between ethylene and SAs metabolism.

The functional integrity of a mammalian chromosome is shaped by its long-term, co-evolution with species-specific nuclear environment. How chromosomes co-adapt with their native environment to define 3D architecture and transcriptional activity remain poorly understood, largely due to a lack of experimental models capable of systematically dissecting this co-evolution relationship. Here, we report a cross-species chromosome substitution (CROSS) method, a robust genomic engineering method that enables the stable, scarless replacement of host chromosomes with evolutionarily divergent orthologs. By integrating microcell-mediated chromosome transfer with CRISPR/Cas9, we imported the intact 158-Mb rat X chromosome into mouse embryonic stem cells, and subsequently achieved targeted substitution of its endogenous mouse counterpart, maintaining stability and integrity. Using this model, we found that rat-specific LINE1 and RatSatRep2 repeats failed to adequately recruit host SETDB1 in mouse cells, leading to localized erosion of H3K9me3 heterochromatin. This further triggered 3D structural remodeling, characterized by the de novo formation of topologically associating domain (TAD) boundaries that aberrantly activated adjacent genes—including Rhox5 , the master regulator of the Rhox cluster—impairing cellular differentiation. Our method provides a powerful chromosome engineering platform for dissecting how genomic sequences and epigenetic mechanisms cooperate in regulating chromosome architecture and function, and for evaluating the structural and functional fidelity of large-scale synthetic or heterologous DNA across species.

There exists a pressing demand to establish a rapid, sensitive, and cost-effective method for identifying single nucleotide polymorphisms (SNPs), which are intricately linked to an individual's pathogen response, phenotypic variation, and gene functionality. In this study, we devised a novel strategy that harnesses the power of loop-mediated isothermal amplification (LAMP) in conjunction with a universal molecular beacon (uMB) to achieve visual differentiation of SNPs. Moreover, a delicately engineered stem-loop primer complex (SPCX) was designed to orchestrate the conformational change of uMB. Results highlighted that the LP region, situated at the 3 ' end of SPCX, can be effectively utilized to ensure target specificity. However, the hybridization between SPCX and the dumbbell-shaped intermediate of LAMP was undermined by the introduced SNPs. Notably, visual detection was precisely achieved by positioning SNPs at the third from the 3 ' end of the complementary sequence for the LP region. Method validation was successfully conducted on the authentication of bigeye tuna, wherein the SPCX (BE-a6y4-3-1) with a 30 bp LP region and a 3 bp Cp region was selected. With this optimized SPCX, the minimum amount of genomic DNA required for detection reached as low as 0.1 pg. Therefore, our strategy embraces a one-step, closed-tube approach, demonstrating the potential in addressing the challenges associated with allele discrimination and signal visualization.

Inflammation underlies a wide variety of physiological and pathological processes, the Lipopolysaccharide (LPS)-induced inflammation model is widely recognized as a classical inflammatory paradigm, while Transforming growth factor-β (TGF-β) serves as a potent immunosuppressant capable of inhibiting immune responses and mitigating inflammation. However, its in vivo instability and the high cost associated with purification have imposed limitations on its clinical application. Therefore, we propose a therapeutic strategy for genetically modifying extracellular vesicles (HEVs) derived from HEK-293 T cells to incorporate TGF-β which holds potential for mitigating LPS-induced inflammation. In this study, we engineered a TGF-β lentivirus that specific incorporates TGF-β into HEVs and efficiently produces highly expressed TGF-β HEVs (HEVTs) through infection of HEK293 cells. Our data demonstrated that, compared to the LPS group, HEVTs internalized by immune cells significantly regulated pro-inflammatory cytokine expression in RAW 264.7 cells, such as IL-1β (p < 01), TNF-α (p < 001). Moreover, HEVTs were found to effectively reach the lesion area, compared to the LPS group, resulting in a remarkable inhibition in the activation of macrophages (p < 0.0001), dendritic cells (p < 0.0001), and neutrophils (p < 0.0001) in the peripheral immune system as well as microglia in the central nervous system of LPS-induced inflammation model mice. The utilization of this endogenous loading technique may present a promising strategy for the protein-based pharmacotherapy of inflammatory disorders.

BackgroundUntil recently, the mechanism underlying methyl jasmonate (MeJA)-mediated suppression of ethylene metabolism and its impact on quality formation in ripening tomatoes has not been fully clarified. This study aimed to investigate how exogenous MeJA application at the breaker stage affects endogenous jasmonates (JAs), ethylene production, metabolic pathways, and flavor profiles in 'FL 47' tomatoes.MethodsExogenous MeJA was applied to red 'FL 47' tomatoes at the breaker stage. We measured endogenous JAs (especially JA-Ile), ethylene production, enzyme activities (LOX, AOC, AOS, OPR, ACO, ACS), mRNA abundances of related genes (SlLOXD, SlAOC, SlAOS, SlOPR3, SlACO1, SlACS2, SlACS4, SlMYC2, SlMED25, SlETR3, SlETR4, SlETR7, SlEIN2, SlEIL1, SlEBF1, SlEBF2, SlERF1, SlLOXC, SlPSY1, SlCCD1A, SlCCD1B, SlBCAT1), production of sugars, organic acids, and 21 volatiles, as well as substrate contents (phytoene, phytofluene, trans-lycopene, γ-carotenoid, β-carotenoid, linoleic and linolenic acid) in MeJA-treated and control fruits.ResultsMeJA treatment suppressed endogenous JAs (JA-Ile by 13%) and ethylene production by 33%. Enzyme activities in metabolic pathways were reduced to over 87% of control levels, and mRNA abundances of the aforementioned genes decreased by 17-30%. Production of sugars, organic acids, and volatiles was altered, leading to changes in flavor profile. Specifically, six key ethylene-regulated volatiles (geranyl acetone, 1-penten-3-one, 6-methyl-5-hepten-2-one, 2-methyl butanal, 3-methyl butanal, 3-methyl butanol) were reduced, concomitant with 19-29% lower mRNA abundances of biosynthetic genes (SlLOXC, SlPSY1, SlCCD1A, SlCCD1B, SlBCAT1) and substrate contents over 74% of control levels.DiscussionBy considering the positive relationship between endogenous JA-Ile and ethylene levels in ripening fruit, our results imply that MeJA-mediated changes in aroma profile in red 'FL 47' tomatoes may result from mitigated endogenous JAs (especially JA-Ile) and ethylene biosynthesis and signaling transduction processes. These findings enhance understanding of hormone interactions in fruit quality formation and suggest potential applications for improving post-harvest tomato flavor.

Molecular beacon (MB) has demonstrated great potential for target-specific detection during loop-mediated isothermal amplification (LAMP), which, however, is constrained by the designing complexity and the great cost of target-dedicated probe screening. The present study aimed to establish a universal MB (uMB), and develop a novel sequence-specific LAMP assay based on the uMB probe for end-point visual inspection. Specifically, a novel uMB and a stem-loop primer complex (SPCX) were successfully designed, which maintained relatively high stability, avoiding unfolding for non-target specimen at the reaction temperature. Instead, SPCX could get open in the presence of target DNA, and afterwards rearranged to serve as the complementary analyte for uMB. The target-probe binding would trigger the conformational change of uMB, thus realizing end-point visual inspection. Under the optimal conditions, the novel uMB-LAMP assay can specifically detect mislabeled escolar and Salmonella contamination within 30 min in a close-tube format, with the detection limit of 1 pg DNA and 2 x 101 CFU/mL, respectively. When the target was changed, only the LAMP primers need to be re-designed and partial sequence of SPCX at the both ends needs to be substituted, demonstrating remarkable versatility and adaptability.

High hydrostatic pressure (HHP) modulates noncovalent interactions of myofibrillar proteins (MP) at low temperature and influences microstructure-quality-function coupling for tailored meat quality and product development. Nevertheless, multiscale structural responses to pressure gradients remain incompletely defined. This review summarizes recent evidence on HHP-induced MP conformational transitions, aggregation-reorganization behaviors, and functional outcomes and discusses their qualitative/quantitative relationship with improvements in meat quality attributes. Comparative analyses across meats highlight differences in domain flexibility, hydrophobic exposure, and cross-linking propensity, underscoring the need for a cross-species structure-quality-function predictive model. At the processing level, combining HHP with fermentation, curing, and aging processes can accelerate diffusion and throughput, reduce salt inputs, and optimize the texture and flavor. Future work should prioritize cross-species, multiparameter predictive models, in situ time-resolved characterization, and carbon-footprint assessment frameworks. In summary, HHP provides a controllable molecular-level pathway to develop low-salt, clean-label, high-quality meat products while supporting mechanistic innovation and industrial application in nonthermal processing.

Citrate is critical to the flavor of horticultural fruit and governed by ACO. However, the specific ACO and its upstream regulators involved in citrate metabolism during pear (Pyrus spp.) fruit development remained uncharacterized. This study identified and characterized six PbrACOs from the Pyrus bretschneideri Rehd. genome. Comprehensive analyses of citrate levels, cyt/mitACO activities, and PbrACOs expression profiles in the pericarp and cortex tissues of developing 'Yali' and 'Dangshansuli' fruits revealed PbrACO2 as a candidate gene. Subsequently, PbrACO2 was confirmed as a mitochondrial aconitase catalyzing citrate-to-isocitrate conversion in vitro and in vivo. Analysis of differentially expressed transcription factors (TFs) and cis-acting elements in the PbrACO2 promoter identified nuclear PbrMYB3 and PbrMYB65, derived from whole genome duplication/segmental duplication, as candidate upstream regulators. These MYB TFs, without direct relationship, bound, as monomers, to the same two MYB-binding sites in the PbrACO2 promoter to activate its transcription, thereby promoting citrate isomerization in pear and tomato. Further investigation revealed that PbrMYB3 and PbrMYB65 are transcriptionally regulated by PbrNAC34a. Given their tissue-dependent expression profiles, the PbrNAC34a-PbrMYB3/65-PbrACO2 cascade partially accounts for citrate differences between pear fruit pericarp and cortex tissues. These findings enhance understanding of citrate accumulation in Rosaceae fruit and provide genetic resources for pear breeding.