A refined polysaccharide component (ADP-0.1-1) was isolated and purified from Angelicae dahuricae radix. Structural analysis revealed that ADP-0.1-1 had a molecular weight of 1.32 × 107 Da and was primarily composed of galactose (Gal), arabinose (Ara), galacturonic acid (GalA), rhamnose (Rha), mannose (Man), and glucose (Glc). Methylation analysis, along with nuclear magnetic resonance (NMR) spectroscopy, indicated that the main chain of ADP-0.1-1 was →3,5)-α-Araf-(1→ and →4,6)-β-D-Galp-(1→, the side chains were →4)-β-D-Glcp-(1→, →4)-α-GalAp-(1→, →5)-α-Araf-(1→, and terminal units were α-Araf-(1→ residues. In an H2O2-induced oxidative stress model using RAW264.7 macrophages, ADP-0.1-1 not only reduced malondialdehyde (MDA) content but also enhanced the activities of key antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT). Furthermore, it up-regulated the mRNA expression of antioxidant-related genes such as HO-1, NQO1, SOD2, GPX1, and CAT. The mechanism underlying these effects is closely associated with the activation of the Nrf2/HO-1 signalling pathway.

Trypsin (Try) has been reported to possess specific superoxide anion-scavenging activity and to exert pronounced anti-senescence effects in Hylocereus undatus fruit; however, the mechanisms underlying its anti-senescence function remain unclear. This study identified TAP4 as the most bioactive peptide among Try autolysis peptides (TAPs). Single-cell transcriptomic analysis revealed that TAP4 alters pericarp cell differentiation trajectories during senescence, particularly in endocarp (EN) cells. It activates the transcription factor (TF) HuWRKY21-1, specifically inducing immune responses in inner pericarp cells and upregulating 44 immune-related genes, including HuIGP4 and HuLYK6. Downstream factors, such as HuFULL and ethylene response factors (ERFs), promote the biosynthesis of antioxidants, such as antheraxanthin and hyperin. Functional validation via virus-induced gene silencing (VIGS), callose deposition, disease resistance assays, and quantitative reverse-transcription PCR confirmed the roles of key TFs, including HuWRKY21-1, HuFULL, and HuERF30-1, in immune activation and resistance reconstruction. This work provides evidence for the potential of the EN as a defensive tissue in fruit and opens new avenues for understanding the mechanisms underlying the establishment of plant resistance.

The differentiation trajectories of 13 cell clusters in H. undatus during senescence have been revealed by single-cell transcriptome (scRNA-seq) analysis, and the five cell clusters in the exocarp have been found to induce resistance mainly. However, the precise localization and functional differentiation of these five cell clusters remain unclear. In this study, phenotypic changes in the exocarp were recorded, and the ROS production and flavonoid biosynthesis in the exocarp were elucidated. The trajectories of the cell clusters involved in exocarp function were further revealed in scRNA-seq profiles. Through integrated analysis of scRNA-seq and spatial RNA-seq data at resolutions of 0.5, 0.6, and 1.5, using algorithms SingleR, SciBet, CARD, and RCTD, the fine localization of these clusters from the outer to inner layers was achieved. RNA-FISH was used to validate the specific expression of marker genes HuSP2 and HuCEL2 in different locations of the exocarp. Finally, the differentiation trajectories of the five cell clusters were analyzed by selecting highly correlated cells with specific functions for each cluster. The core roles of HuPRP and HuCRRSP55 in the gene regulatory networks of exocarp clusters were revealed. A hypothesis that the five clusters in the exocarp participate in the induction of resistance was proposed.

Fruit senescence is a complex physiological process. Previous studies have found that the formation of early resistance to fruit senescence is mainly based on phenylpropanoid biosynthesis. However, the specific response mechanisms of phenylpropanoid biosynthesis within cells are not well understood. The results showed that during the senescence process of cells related to phenylpropanoid biosynthesis, the mature sample had significantly higher phenylpropanoid biosynthesis-related cell correlations compared to the senescent sample. In the pseudotime differentiation trajectory, cells related to phenylpropanoid biosynthesis were mainly distributed in the resistance branch. A pseudotime-related gene regulatory network (GRN) was constructed. And three hub genes HuSAG12, HuNAD1, and HuDC2.15 were identified. HuSAG12 was specifically expressed in the mesocarp, while the other two genes were specifically expressed in the exocarp. They were highly expressed during the late, middle, and early stages of senescence, respectively. In the HuSAG12 RNA-silenced lines, the silencing of SAG12 negatively regulated the expression of HuDC2.15 and accelerated the fruit senescence of Hylocereus undatus. In summary, HuSAG12 likely plays a crucial role in inducing early resistance during the senescence of Hylocereus undatus by regulating flavonoid biosynthesis. This study provides new insights into the response mechanisms of phenylpropanoid biosynthesis during the dynamic senescence of fruit cells.

DELLA proteins can participate in the biosynthesis pathway of flavonoids. It has been shown that trypsin can induce flavonoid synthesis, thereby enhancing the storage quality of Hylocereus undatus (H. undatus) fruit. However, whether trypsin induces flavonoid biosynthesis and improves fruit quality during storage by regulating the phenylpropanoid synthesis pathway through DELLA remains to be further elucidated. To investigate the molecular mechanism of trypsin-induced flavonoid synthesis in H. undatus, we conducted transcriptomic analysis and verified it through virus-induced gene silencing (VIGS). Analysis of transcription factors showed that the top five genes with the largest expression differences regulated by trypsin all belonged to the GRAS family. Further protein network interaction analysis identified HuGAI1 as a hub protein in the GRAS family. Trypsin treatment was able to extend the shelf life of fruit. However, after the expression of HuGAI1 was silenced, the storage quality of the fruit declined. GO and KEGG analysis after HuGAI1 silencing revealed that differentially expressed genes (DEGs) were mainly concentrated in metabolic pathways such as phenylpropanoid, flavonoid, and flavonol biosynthesis. Trypsin can upregulate the expression of HuGAI1. And HuGAI1, by participating in the phenylpropanoid biosynthesis pathway, regulates the biosynthesis of flavonoids and flavonols, leading to an increase in antioxidant flavonoid content and, consequently, enhancing fruit storage.

The phenomenon of microbial resistance is becoming increasingly serious. The efflux pump system plays a key role in bacterial resistance to antibiotics. The transcriptional profile of Escherichia coli treated with antibiotics revealed the pathways and response mechanisms of up- or down-regulation of efflux pump-related genes. In this study, 104 of all 2548 genes, efflux pump-related differentially significantly expressed genes were screened. The most significant spermidine transport in the Gene Ontology functional enrichment analysis was the most likely important pathway for the E. coli species to respond to antibiotics. The functional enrichment results of Kyoto Encyclopedia of Genes and Genomes showed that most of the antibiotic-induced transport proteins belong to the “ABC family efflux pumps”. The results of the protein-protein interaction network indicated that potC might act as a key gene for E. coli to respond to antibiotics. Finally, overexpressing strains of potC were constructed, and the survival rates of overexpressing strains of potC were improved by 18.9, 14.0, 43.3, and 31.7

Fruit senescence is a complex physiological process. Single-cell RNA sequencing (scRNA-seq) analysis revealed the differentiation trajectories of 13 cell clusters during the senescence of Hylocereus undatus(H. undatus). The mesocarp of the fruit contained four cell clusters, but their precise localization and functional division remained unclear. This work documented mesocarp phenotypic alterations and elucidated the time courses of mesocarp flavonoid biosynthesis and superoxide anion generation. Additionally, overall ROS changes were observed using fluorescence microscopy. By combining the single-cell atlas with spatial transcriptomics data at resolutions of 0.2 and 0.8, and applying four computational algorithms (SingleR, SciBet, CARD, and RCTD), we accurately mapped the spatial distribution of the four cell populations in the two layers of the mesocarp from outer to inner regions. Furthermore, we identified highly correlated cells with cell-specific functions, which allowed us to perform a detailed analysis of the differentiation trajectories of these four cell clusters. We proposed a hypothesis that these four clusters in the mesocarp participate in the senescence process. Finally, using SCODE, we uncovered the gene regulatory networks of the pericarp's highly correlated cell clusters during fruit senescence. Through single-cell technology, the functional division of the four cell clusters in the mesocarp—responsible for stress responses, signal transduction, material preparation, and cell differentiation trajectories—has been revealed. These findings provide insights from a single-cell dimension and a spatiotemporal perspective, enhancing the understanding of the dynamic process of plant senescence.

ObjectiveTo investigate the mechanism of biofilm-mediated resistance to berberine in Escherichia coli.MethodsThe resistance of berberine against E. coli was induced by 1/2 MIC (minimum inhibitory concentration). Biofilm formation was detected by crystal violet staining. The mRNA level was detected by RT-qPCR, and the gene csgD was determined. the csgD-overexpressed strain was constructed. We measured the MIC of berberine against E. coli, as well as biofilm formation and the expression of mRNA.ResultsThe MIC after berberine induction was more than 32 times than the MIC before induction. the biofilm was significantly increased at 24, 48 and 72 hours (p<0.01) after berberine induction. In addition, the amount of biofilm production at 24, 48 and 72 hours was 1.3, 1.51 and 1.98 times after berberine induction than that before induction, respectively. The expression of csgD gene was significantly increased (p=0.016) after induction compared with that before induction. the MIC of csgD-overexpressed strain was about 5.8 times that before induction. The expression of csgD gene was significantly increased (p=0.016), which was 5.8 times higher than that before induction. The MIC of csgD-overexpressed strain was 100 μg/mL. Biofilm formation in csgD-overexpressed strain was 2.9 times higher than that of the control. The expression of biofilm-related genes, bcsA, luxS and csgD, was 45, 22.5 and 1628 times higher than that of the control, respectively.ConclusionBerberine might increase biofilm formation by inducing the expression of csgD gene, which might result in drug resistance in E. coli.

Fruit is highly susceptible to postharvest quality deterioration, and changes at the cellular level in the vascular bundle, a key hub for substance transport within the fruit, have a significant impact on the senescence process. Single-cell transcriptome sequencing (scRNA-seq) enables the precise analysis of gene expression dynamics in vascular cells across different stages of postharvest freshness and senescence. Utilizing scRNA-seq, this study developed a cellular atlas of the Hylocereus undatus (H. undatus) pericarp. Among the 13 cell clusters identified in the pericarp, vascular cells were predominantly localized in cluster 10, and the cell number within this cluster showed a significant decline with senescence. At a resolution of 0.2, two subpopulations of vascular cells were subdivided and identified, and their dynamics during the senescence process were analysed. In addition, through pseudo-timing trajectory analysis, two senescence hub regulators, HuCEP and HuSCPL48, were screened out by the pericarp vascular senescence gene regulatory network constructed by SCODE based on connectivity. In particular, genes related to mass transport, such as HuAAE3, and related to energy metabolism, such as HuNAD7, were significantly upregulated. At the same time, in the later stages of senescence, genes related to specific subsets of vascular cell activation protein degradation such as HuATG8CL and oxidative stress such as HuPMA4 were significantly up-regulated, leading to a stress response to fruit physiological decline. This study explored the dynamic changes in gene expression during vascular cell senescence.

Fruit senescence is a complex physiological process. In this study, we employed single-cell transcriptomics combined with phenotypic and functional analyses to investigate the role of endocarp cell clusters in postharvest senescence of Hylocereus undatus. Morphological changes of the endocarp were recorded, the temporal dynamics of superoxide anion production and flavonoid biosynthesis were characterized, and overall ROS accumulation was visualized by fluorescence microscopy. Single-cell RNA sequencing identified three endocarp-specific cell clusters (ScC4, ScC11, and ScC12), which were respectively enriched in antioxidant defense, secondary metabolism, and hormone signaling pathways. Pseudotime trajectory analysis revealed that ScC4 acted as the initiating stress-responsive cells, ScC11 mediated flavonoid biosynthesis and ROS scavenging, while ScC12 functioned as late-stage signaling cells associated with abscisic acid and ethylene pathways. Gene regulatory network inference further identified HuGIP2 as a central hub gene in ScC11. Virus-induced gene silencing demonstrated that suppression of HuGIP2 reduced flavonoid accumulation, increased ROS levels, enhanced lipoxygenase (LOX) activity, and accelerated senescence. Collectively, this study delineates the differentiation trajectories of endocarp cell populations during fruit senescence and proposes a mechanistic hypothesis whereby flavonoid-mediated ROS scavenging and LOX inhibition play pivotal roles in delaying senescence. These findings provide single-cell-level insights and spatiotemporal perspectives into the dynamic process of plant senescence.

Senescence is the last critical stage of the fruit life cycle, which directly affects the maintenance of fruit quality and post harvest lifespan. The pericarp consists of various cell types, making it challenging to elucidate their roles in fruit senescence. In this study, an expression atlas of H. undatus pericarp was constructed using scRNA-seq profiles, encompassing data from thousands of individual cells. Five different algorithms, including deconvolution (SingleR and SciBet), supervised learning (RCTD and CARD), and multi-modal cross-analysis (MIA), were employed to assign 17 transcriptionally distinct cell clusters identified in single cells to four different cell types: exocarp, mesocarp, endocarp, and vascular bundles. Pseudotime trajectory analysis revealed a clear spatiotemporal transition from ROS-induced stress and disease resistance to senescence during the fruit ripening process, moving from the exocarp to the mesocarp. Previously unknown early response genes related to senescence and resistance were identified, providing novel tools for fruit senescence prediction based on mesocarp and enhancement of fruit resistance based on exocarp.

The senescence of fruit is a complex physiological process, with various cell types within the pericarp, making it highly challenging to elucidate their individual roles in fruit senescence. In this study, a single-cell expression atlas of the pericarp of pitaya (Hylocereus undatus) is constructed, revealing exocarp and mesocarp cells undergoing the most significant changes during the fruit senescence process. Pseudotime analysis establishes cellular differentiation and gene expression trajectories during senescence. Early-stage oxidative stress imbalance is followed by the activation of resistance in exocarp cells, subsequently senescence-associated proteins accumulate in the mesocarp cells at late-stage senescence. The central role of the early response factor HuCMB1 is unveiled in the senescence regulatory network. This study provides a spatiotemporal perspective for a deeper understanding of the dynamic senescence process in plants. Fruit senescence is a complex physiological process. Here, the authors construct a single-cell expression atlas of pitaya pericarp pitaya to provide a spatiotemporal perspective of the dynamic process of plant senescence.

The NTM1-like (NTL) proteins constitute a subfamily of the NAM, ATAF and CUC (NAC) transcription factor family that regulates diverse biological processes including stress response in plants. Ammopiptanthus mon-golicus is a desert shrub with very high tolerance to harsh environments. Here, an NTL-like gene, AmNTL1, was cloned from this species and its function in abiotic stress tolerance was analyzed. AmNTL1 encodes a canonical NTL protein. The full-length protein was primarily located at the plasma membrane and had trans-activation activity in yeast. The expression of AmNTL1 was differentially induced by cold, salt, drought, and exogenous abscisic acid (ABA) in laboratory-cultured A. mongolicus seedlings. The expression was also induced by the cold winter and spring weather in leaves of the A. mongolicus shrubs naturally growing in the wild and was at moderate levels in lateral roots, young leaves and flower buds of the shrubs. The trans-genic Arabidopsis lines constitutively expressing AmNTL1 showed enhanced tolerance to freezing, chilling, salinity, and osmotic stress as well as chilling-and salt-induced oxidative stresses. The transgenic lines also displayed ABA-sensitive phenotypes at the seed germination stage. These results suggest that AmNTL1 may play an important role in plants coping with abiotic stresses, especially cold injury, through the ABA-depen-dent signaling pathway.(c) 2023 SAAB. Published by Elsevier B.V. All rights reserved.

The environmental pollution caused by antibiotics is becoming more and more serious. Outer membrane proteins (OMPs) play key roles in the resistance of bacteria to antibiotics. Transcriptomic profiles of Esherichia coli treated with different types of antibiotics revealed the pathways and response mechanisms of OMP-related genes up- or down-regulated following antibiotics treatment. Among all the 4497 genes, 73 OMPs were screened. In gentamicin- or ampicillin-treated groups, 51 or 23 genes were differentially expressed, respectively. The results of protein-protein interaction networks showed that ompA , chiP , phoE , and cusC were overlapped hub genes in both groups. Considering the scores of nodes calculated by cytoHubba, OmpA was the key one among the OMPs through the calculation of Cytoscape. Finally, the knockout and overexpression mutants and complementation strain of ompA were constructed. The results of drug resistance to 4 types of antibiotics analysis confirmed the role of ompA in the resistance of E. coli to antibiotics. This work provides direct evidence for the role of outer membrane proteins, especially OmpA, in drug resistance of E. coli to different types of antibiotics. The findings in this study can lead to new strategies for improving the effectiveness of antibiotics.

Trypsin has a new activity of scavenging superoxide anion and generating hydrogen peroxide. Trypsin can significantly improve the storage quality of C. sativus. To illustrate the mechanism of trypsin-induced resistance in fruits and vegetables, an integrated analysis of widely targeted metabolomics and transcriptomics was carried out. Transcriptomic results showed that 1068 genes highly related to phenylpropanoid biosynthesis gathered in the brown module were obtained by WGCNA. In KEGG analysis, differentially expressed genes (DEGs) were also highly enriched in EIP (Environmental Information Processing) pathways “Plant hormone signal transduction (map04075)” and “MAPK signaling pathway-plant (map04016)”. Next, 87 genes were identified as the leading edge by GSEA analysis. So far, CsMYC2 was highlighted as a key transcription factor that regulates phenylpropanoid biosynthesis identified by GSEA and WGCNA. Furthermore, the major route of biosynthesis of phenylpropanoid compounds including coumarins, lignins, chlorogenic acid, flavonoids, and derivatives regulated by trypsin was also illustrated by both transcriptomic and metabolomic data. Results of O2PLS showed that CsMYC2 was positively correlated with Rosmarinic acid-3-O-glucoside, Epigallocatechin, Quercetin-3-O-sophoroside (Baimaside), and so on. Correlation between CsMYC2, phenylpropanoid related genes, and metabolites in C. sativus was illustrated by co-expression networks. Roles of CsMYC2 were further checked in C. sativus by VIGS. The results of this study might give new insight into the exploration of the postharvest resistance mechanism of C. sativus induced by trypsin and provide useful information for the subsequent mining of resistance genes in C. sativus.

Abstract Background The Mitogen-Activated Protein Kinase (MAPK) cascade pathway extensively participates in the physiological regulation processes of plants. Trypsin, as an inducer of resistance, significantly enhanced the storage quality of Cucumis sativus (C. sativus). However, the mechanism by which trypsin regulated the synthesis of phenylpropanoid compounds through the MAPK pathway to influence fruit resistance remained to be elucidated. Methods To investigate the molecular mechanism underlying trypsin-induced C. sativus resistance, we conducted a combined transcriptomic and widely targeted metabolomic analysis, validated through Virus-Induced Gene Silencing (VIGS). Results Transcriptomic results revealed that a total of 83 differentially expressed genes (DEGs)were enriched in the MAPK pathway, with 48 genes significantly downregulated and 35 genes significantly upregulated. GSEA analysis further identified the WRKY33 transcription factor from the leading edge subset. Bioinformatics analysis indicated that CsWRKY33 shared high homology with WRKY22 in Arabidopsis. The combined analysis of transcriptomics and widely targeted metabolomics demonstrated significant upregulation in the synthesis of compounds such as vanillin, dihydrocharcone-4'-O-glucoside, and 2-hydroxy-3-phenylpropanoic acid. Co-expression network analysis showed that these key metabolites were negatively regulated by CsWRKY33. VIGS results showed that silencing CsWRKY33 enhanced fruit resistance and extended storage time. Conclusion This study revealed that trypsin could downregulate the expression of CsWRKY33 and promote the synthesis of compounds with high antioxidant and antibacterial activity, such as vanillin, dihydrocharcone-4'-O-glucoside, and 2-hydroxy-3-phenylpropanoic acid. This enhancement led to increasing fruit disease resistance and delayed senescence and decay.

Trypsin can significantly improve the storage quality of Cucumis sativus (C. sativus) due to its novel superoxide scavenging activity. Based on the results of physiological indices, an integrated analysis of widely targeted metabolomics and transcriptomics was carried out, gene function was further confirmed by VIGS, a potential molecular mechanism of resistance improvement of C. sativus due to flavonoid biosynthesis induced by trypsin was proposed and discussed. Transcriptomic results showed that 1,068 genes highly related to flavonoid biosynthesis gathered in the brown module were obtained by the advanced method WGCNA. An isomeric compound of dihydrochalcone, trilobatin, was likely to be the key metabolite regulated by trypsin. Results of O2PLS and co-expression network illustrated that CsUGT91C1 acted as the hub gene which regulated the biosynthesis of flavonoids, especially trilobatin, in C. sativus. CsUGT91C1 also played as a hub node in the Protein-Protein Interactions network of WGCNA screened genes (WSGs). GO and KEGG analyses of WSGs enriched the transferase activities, especially UDP-glycosyltransferase activity and KEGG pathways of "Phenylpropanoid biosynthesis (map00940) " and "Flavonoid biosynthesis (map00941) ". Results of webtools of the PlantCARE database identified the G-box region of CsUGT91C1, which can bind to the transcription factor CsMYC2. The expression and biological effects of CsUGT91C1 were significantly impeded by the silencing of CsMYC2 through VIGS. The results of this study might give new insight into the exploration of the postharvest resistance mechanism of C. sativus induced by trypsin and provide useful information for the subsequent mining of resistance genes in C. sativus.

To analyze the mechanism of the effect of trypsin on the preservation of Hylocereus undatus, the transcriptomic and widely targeted metabolomic profiles of H. undatus after trypsin treatment were evaluated. Among 477 genes related to lipid metabolism, 32 genes had significant expression differences. GO analysis results showed that the main enriched GO functions include pectinesterase and asparagine esterase activities, and so on. The KEGG metabolic pathway with the highest enrichment rate was fatty acid elongation. The protein-protein interaction (PPI) network analysis results showed that the PPI network of lipid metabolism is a complex biological network of scale-free cells. KCS1, QRT1, and ACC1 acted as hubs to regulate a large number of other proteins and amplify the regulatory role of trypsin to achieve a preservation effect. In addition, three unsaturated fatty acids were upregulated, while eight saturated fatty acids were downregulated. PRACTICAL APPLICATIONS: The postharvest storage of fresh fruits and vegetables brings about bottlenecks to fresh fruits and vegetables. There was also an increasing need for biopreservation techniques. Trypsin could significantly enhance the antioxidant capacity of fruits and vegetables, as a preserver for the storage of fruits and vegetables, which was convenient to operate and more economical. The regulation mechanism of trypsin on lipid metabolism in fruits and vegetables during storage of H. undatus is studied in this paper. The application of trypsin would provide a new strategy for quality control of fruit and vegetable storage.

Trypsin can significantly improve the storage quality of Hylocereus undatus (H. undatus). To verify the hub WRKY gene of H. undatus in trypsin preservation, joint analysis of transcriptome and protein-protein interaction (PPI) network was carried out, and virus-induced gene silencing (VIGS) was conducted. In the transcriptome of H. undatus, GO directed acyclic graph (DAG) showed that the GO terms of 55 WRKY genes were mainly enriched in sequence-specific DNA binding, DNA binding transcription factor activity, and so on. The GO enrichment chord diagram showed that HuWRKY40 was significantly up-regulated in the enriched top10 GO terms. KEGG enrichment analysis showed that 55 WRKY genes were mainly enriched in plant-pathogen interaction and MAPK pathway. The results of PPI network showed that HuWRKY40 was a hub protein of WRKY transcription factors (TFs) family regulated by trypsin, which was consistent with the results of transcriptome analysis. Bioinformatics analysis showed that HuWRKY40 of H. undatus had the highest homology with Beta vulgaris L. and Spinacia oleracea L. The function of the core regulatory protein HuWRKY40 was further clarified by VIGS technology. The results of VIGS showed that there was a big difference between the phenotype of the pTRV2-HuWRKY40 group and that of the control group. Finally, it was confirmed that HuWRKY40 accelerated the synthesis of flavonoids and improved the fruit quality during the storage of H. undatus. This study found that trypsin may regulate HuWRKY40 activity through the MAPK cascade pathway, affect the participation of flavonoid synthesis, and then delay fruit corruption. Practical applications With attention of people to the safety and freshness of fruits and vegetables, biological preservation technology has become one of the hotspots in the field of preservation in recent years. Trypsin can significantly improve the antioxidant capacity of fruits and vegetables. As a new biological preservative, it is convenient to operate and economical. In the current work, the mechanism of trypsin on the WRKY TFs during H. undatus storage was investigated. The application of trypsin would provide a new strategy for the storage quality control of fruits and vegetables.