Chemoenzymatic labeling strategies, relying on azide-modified sugar donors and subsequent Cu-based click chemistry to introduce enrichable handles, have greatly advanced the study of O-GlcNAcylation. However, multistep labeling and enrichment procedures often lead to substantial sample loss. While a recent one-step labeling approach using biotinylated UDP-GalNAc donors simplifies the procedures, the strong biotin-avidin interactions, as well as bulky tags, limited this strategy to protein-level identification rather than site-specific glycopeptide mapping. Herein, we developed an oxime-based reversible O-GlcNAc enrichment (ORO-GlcNAc) approach employing a ketone-functionalized UDP-GalNAc analogue (UDP-GalNLeV) for one-step chemoenzymatic labeling with >96% efficiency, eliminating intermediate reactions and minimizing handling-induced loss. Crucially, oxime-based capture using hydroxylamine-functionalized beads, followed by reversible methoxyamine-mediated release, overcomes the limitations of conventional ketone-based enrichment strategies and eliminates the need for azide-based chemistry. Using the ORO-GlcNAc strategy, we identified 4129 potential O-GlcNAcylation sites in HeLa cells, of which 64.3% were annotated in the O-GlcNAc Atlas, demonstrating the high coverage and enrichment performance of ORO-GlcNAc. Functional studies suggested a potential role of O-GlcNAcylation in the nucleocytoplasmic redistribution of the chromatin remodeler EP400 during stress-granule assembly. This platform thus provides a powerful and versatile tool for advancing the O-GlcNAcylation research.

The rapid degradation of the cell wall leads to firmness loss in fruit, resulting in a shorter shelf life and quality deterioration. Here, we evaluated the impact of preharvest foliar application of alginate oligosaccharides (AOS) on a perishable fruit kiwiberry (Actinidia arguta) over a 42 d storage period using physiological, molecular, and biochemical approaches. The selected plants were sprayed with the AOS solution 15 d before harvest, covering both the leaves and fruit surfaces. The preharvest AOS application significantly reduced decay incidence by about 60 %, maintained nearly twice the firmness, and 22 % less weight loss compared with control fruit after 42 d of storage. Transcriptome analysis reveals that AOS treatment downregulates the genes related to cell wall degradation. Moreover, biochemical analysis indicates that preharvest application suppressed the activation of cell wall degrading enzymes, reduced the solubilization of cell wall polysaccharides, and preserved their monosaccharide composition. These results reveal that preharvest application of AOS ensured the storability of kiwiberry by controlling cell wall degradation enzymes and gene expression. These findings highlight the significant potentiality of AOS preharvest treatments for delaying postharvest softening of kiwiberry.

Steviol glycosides (SGs) from Stevia rebaudiana are prized as noncaloric sweeteners, with rebaudioside M (Reb M)-a next-generation SG known for its sucrose-like sweetness and lack of off-tastes-standing out for its superior sensory profile. However, Reb M's limited natural abundance impedes its commercial production. Here, we report the identification of a glucosyltransferase, UGT76G4 that efficiently catalyzes the conversion of Reb D to Reb M with a strong preference for C19 glycosylation. Structural and functional analyses, including X-ray crystallography, molecular dynamics simulations, and mutagenesis, revealed key residues in UGT76G4 that dictate its regioselectivity, with residue 200 playing a pivotal role. Engineered UGT76G4 variants, including Q199I/ G200Y and H155S/Q199I/G200Y, enhanced Reb E and Reb D conversion efficiency by 1.46-fold and 23-fold, respectively, compared to UGT76G1. The engineered variants offer a promising pathway for increasing Reb M production, advancing biotechnological strategies for steviol glycoside biosynthesis and optimizing plant metabolic engineering approaches. Our findings deepen the understanding of SG biosynthesis and provide a basis for sustainable production of high-value sweeteners.

Strawberry exhibits a very short shelf life due to its rapid softening and decay, resulting in postharvest loss. Our previous study showed that AOS, an eco-friendly nontoxic natural product, plays a role in preserving the freshness of strawberries by regulating ABA content and its signaling pathway. Considering the critical role that energy deficit plays in the senescence process of fruits, coupled with the potential influence of plant hormones on energy metabolism. In this study, the molecular mechanism of strawberry postharvest senescence in response to AOS treatment was further explored from the perspective of energy metabolism regulation. This was achieved through a two-layered approach, integrating the direct determination of energy-related substances with a comprehensive joint analysis of transcriptome and proteome data. AOS treatment significantly delayed the loss of ATP, ADP, and AMP contents compared to control fruit, implying that maintaining cell energy is a crucial factor in AOS postponing senescence in strawberry. Subsequently, the mechanisms were further revealed by transcriptome and proteome analysis. Overall, 255 DEGs were screened from the AOS group, mainly related to carbohydrate and energy metabolism, defense, plant hormone, and secondary metabolism. Additionally, A total of 227 proteins were identified that were differentially expressed in the AOS group, and the majority were related to carbohydrate and energy metabolism (85 proteins). Two omics data implied that AOS could effectively regulate the energy metabolism system to maintain the cellular energy level of harvested strawberry during storage, ultimately leading to longer shelf life. Therefore, our findings provide comprehensive information regarding the molecular mechanism underlying the postharvest storage of strawberry treated with AOS. From our results, it can be concluded that AOS postharvest treatment is very useful for keeping fruit quality and extending shelf life by maintaining a higher energy level.

Plant-derived oligosaccharides are promising ecofriendly elicitors of plant immunity. In this study, a β-1,4-mannanase (PpManA) from Paenibacillus polymyxa was characterized with broad pH stability, relatively low-temperature activity, and high specificity for konjac glucomannan (KGM), efficiently producing KGM oligosaccharides (KGMOS, DP 2-13). Treatment with KGMOS (10-50 mg/L) enhanced Arabidopsis resistance against Pseudomonas syringae pv tomato DC3000 (Pst DC3000), reducing bacterial colonization and disease symptoms while suppressing pathogen-responsive genes. KGMOS elevated H2O2 and NO levels and up-regulated SA- and JA-responsive genes. During infection, KGMOS activated both pathways, increasing hormone levels and defense gene expression. Genetic studies using SA-deficient (NahG) and JA-deficient (jar1) mutants showed attenuated resistance, with higher bacterial counts and severe symptoms versus those of wild-type plants under KGMOS treatment. These results demonstrate that KGMOS triggers immunomodulation via coordinated SA/JA signaling, offering a sustainable alternative to chemical pesticides for crop protection.

Phytopathogen cell wall polysaccharides have important physiological functions. In this study, we isolated and characterized the alkali-insoluble residue on the inner layers of the Rhizoctonia solani AG1 IA cell wall (RsCW-AIR). Through chemical composition and structural analysis, RsCW-AIR was mainly identified as a complex of chitin/chitosan and glucan (ChCsGC), with glucose and glucosamine were present in a molar ratio of 2.7:1.0. The predominant glycosidic bond linkage of glucan in ChCsGC was β-1,3-linked Glcp, both the α and β-polymorphic forms of chitin were presented in it by IR, XRD, and solid-state NMR, and the ChCsGC exhibited a degree of deacetylation measuring 67.08 %. RsCW-AIR pretreatment effectively reduced the incidence of rice sheath blight, and its induced resistance activity in rice was evaluated, such as inducing a reactive oxygen species (ROS) burst, leading to the accumulation of salicylic acid (SA) and the up-regulation of SA-related gene expression. The recognition of RsCW-AIR in rice is partially dependent on CERK1.

Alginate lyases, which degrade alginate into oligosaccharides, have broad applications in biorefinery, biomedical, and industrial fields. The Polysaccharide Lyase Family 7 (PL7) is particularly notable for its alginate lyase activity. In this study, two novel alginate lyases, PmAlg7A and PmAlg7B, from Pseudomonas mendocina E03 were cloned, heterologously expressed, and characterized. PmAlg7B exhibited limited activity toward alginate (0.10 U/mg-protein), while PmAlg7A demonstrated higher activity with a specific activity of 0.76 U/mg-protein. PmAlg7A was identified as an MG-specific alginate lyase, producing oligosaccharides with degrees of polymerization (Dp) ranging from 2 to 5. The enzyme exhibited optimal activity at a temperature of 30 °C and a pH of 8.0, with a Km of 7.94 ± 0.92 mg/ml and a kcat of 1.23 ± 0.06 s-1. Structural comparisons and amino acid sequence alignments indicated a potential role for residue 55 in loop B in modulating the activity of PmAlg7B, which was supported by mutagenesis experiments and molecular dynamics simulations. These findings enhance our understanding of the critical role of loop B in regulating substrate binding in PL7 alginate lyases.

ABSTRACTPesticides play a crucial role in safeguarding crops against pathogens and pests, yet the limited bioavailability of conventional formulations leads to excessive usage, causing environmental and health concerns. In this study, we developed a novel intelligent response‐controlled release nano‐pesticide, Vali@mPEG‐TK‐COS, by coupling chitosan oligosaccharide (COS), a plant immunity regulator, and methoxy polyethylene glycols (mPEG) at either end of thioketal (TK), a material responsive to reactive oxygen species (ROS), and incorporating validamycin (Vali) for controlled release. Vali@mPEG‐TK‐COS exhibits ROS‐responsive behaviour, releasing Vali and COS in response to plant ROS bursts, exhibiting dual capabilities in pathogen control and plant immunity induction during pathogenic attacks. Plant disease experiments confirmed the superior efficacy of Vali@mPEG‐TK‐COS against Rhizoctonia solani, surpassing the control effects of Vali or COS alone. This study presents a sophisticated ROS‐responsive controlled‐release nano‐pesticide with dual fungicidal and plant immunomodulatory properties, offering an advanced strategy to enhance the efficiency of existing pesticides and promote sustainable crop production by reducing overall pesticide usage.

The objective of this study was to investigate the impact of thermophilic bacteria on crude fiber content, carbohydrate-active enzyme (CAZyme) genes, and associated microbial communities during Chinese medicine residues composting. The study examines changes over 15 days of composting with (T) and without (CK) thermophilic microbial agents. Results show that the group T compost temperature reached a maximum of 71.0 °C and remained above 70 °C for 2 days, while the group CK maximum temperature was only 60.9 °C. On Day 15, the seed germination index (GI) of group T reached 98.7%, while the group CK GI was only 56.7%. After composting, the degradation rates of cellulose, hemicellulose, and lignin in group T increased by 5.1, 22.5, and 18.5%, respectively, compared to those in group CK. Thermophilic microbial agents changed the microbial communities related to CAZymes, increasing unclassified_o_Myxococcales and Sphaerobacter abundance and reducing Acinetobacter and Sphingobacterium abundance. Thermophilic microbial agents also increased the abundance of the GT4, GT2_Glycos_transf_2, and AA3 gene families. These results show that thermophilic microbial agents can increase composting temperature, accelerate compost maturation, and promote crude fiber degradation. Therefore, they have broad application potential.

Steviol glycosides (SGs) are a variety of important natural sweeteners. They are 200-350 times sweeter than sucrose without calories. Currently, their production is still mainly dependent on extraction from Stevia rebaudiana Bertoni (stevia). Oligosaccharides are environmentally friendly elicitors that promote plant growth and accumulation of secondary metabolites. In the present study, different concentrations of chitosan oligo-saccharides (COS) and alginate oligosaccharides (AOS) were applied to stevia to explore their effect on growth and SGs biosynthesis. It was found that both COS and AOS promoted biomass production by increasing the leaf number and photosynthetic efficiency, which may be related to the decreased content of abscisic acid. The content of SGs was significantly increased after 50 mg/L AOS treatment, which not only increased the contents of stevioside (STV) and rebaudioside A (Reb A) significantly, but some important minority glucosides, like Reb E, Reb D, and Reb M. The increased SGs contents were the combined effect of the higher expression of SGs biosynthesis related genes, including KAH, UGT74G1, UGT85C2, and UGT91D2. The geometry changes of stem induced by COS and AOS may help to increase the lodging resistance of stevia. Thus, COS and AOS can be used in the field planting of stevia to increase the yield of SGs for industrial purposes.

Rhizoctonia solani (R. solani) is an important pathogenic fungus that causes symptoms of sheath blight, and the polysaccharide-rich cell wall plays a major role in plant-pathogen interactions. However, the composition and structure of its cell wall polysaccharides are insufficiently understood, and its specific function in plant-pathogen interactions is unknown, which makes effective control of sheath blight difficult at present. Herein, five cell wall polysaccharides (WF-1, WF-2, CAF-1, HAF-1 and HAF 2-1) were sequentially extracted by boiling water, cold and hot alkali from R. solani AG1 IA. They were heteropolysaccharides containing mainly glucose, mannose and galactose and less fucose, with molecular weights above 1100 kDa. These five polysaccharides mainly composed of →4)-Glcp-(1→, →6)-Glcp-(1→, →4,6)-Glcp-(1→, →3,4)-Glcp-(1→, and Manp-(1→. Several polysaccharides, except WF-1, showed different induced resistance degrees on rice plant, with HAF 2-1 having the most significant effect. Further analysis using NMR confirmed that the backbone of HAF 2-1 mainly consisted of →4)-α-D-Glcp-(1→ and →6)-α-D-Glcp-(1→ with branches of →4,6)-D-Glcp-(1→. HAF 2-1 enhance the resistance of rice against R. solani through salicylic acid (SA)-mediated immune signaling pathway. This work improves our knowledge of the cell wall polysaccharides in plant pathogens and facilitates the study of pathogenic mechanisms and effective disease control.

Sheath blight, caused by Rhizoctonia solani (R. solani), is one of the most serious diseases of rice. Extracellular polysaccharides (EPS) are complex polysaccharides secreted by microbes that have a pivotal role in the plant-microbe interaction. At present, many studies have been carried out on R. solani, but it is not very clear whether the EPS is secreted by R. solani exists. Therefore, we isolated and extracted the EPS from R. solani, two kinds of EPS (EW-I and ES-I) were obtained by DEAE-cellulose 52 and Sephacryl S-300HR column further purification, and their structures were characterized by FT-IR, GC-MS, and NMR analysis. The results showed that EW-I and ES-I had similar monosaccharide composition but different molar ratio, they were composed of fucose, arabinose, galactose, glucose, and mannose with a ratio of 7.49: 27.72: 2.98: 6.66: 55.15 and 3.81: 12.98: 6.15: 10.83: 66.23, and their backbone may be composed of →2)-α-Manp-(1→ residues, beside ES-I was highly branched compared to EW-I. The exogenous application of EW-I and ES-I had no effect on the growth of R. solani AG1 IA itself, but their pretreatment of rice induced plant defense through activation of the salicylic acid pathway, resulting in enhanced resistance to sheath blight.

As an essential enzyme in the uridine diphosphate (UDP)-GlcNAc biosynthesis pathway, the significant role of N-acetylglucosamine phosphate mutase (AGM) remains unknown in plants. In the present study, a functional plant AGM (AtAGM) was identified from Arabidopsis thaliana. AtAGM catalyzes the isomerization of GlcNAc-1-P and GlcNAc-6-P, and has broad catalytic activity on different phosphohexoses. UDP-GlcNAc contents were significantly decreased in AtAGM T-DNA insertional mutants, which caused temperature-dependent growth defects in seedlings and vigorous growth in adult plants. Further analysis revealed that protein O-GlcNAcylation but not N-glycosylation was dramatically impaired in Atagm mutants due to UDP-GlcNAc shortage. Combined with the results from O-GlcNAcylation or N-glycosylation deficient mutants, and O-GlcNAcase inhibitor all suggested that protein O-GlcNAcylation impairment mainly leads to the phenotypic variations of Atagm plants. In conclusion, based on the essential role in UDP-GlcNAc biosynthesis, AtAGM is important for plant growth mainly via protein O-GlcNAcylation-level regulation.

ε-Poly-L-lysine (ε-PL) is a widely used antibacterial peptide polymerized of 25–35 L-lysine residues. The antibacterial effect of ε-PL is closely related to the polymerization degree. However, the mechanism of ε-PL degradation in S. albulus remains unclear. This study utilized the integrative plasmid pSET152-based CRISPRi system to transcriptionally repress the ε-PL degrading enzyme (pldII). The expression of pldII is regulated by changing the recognition site of dCas9. Through the ε-PL bacteriostatic experiments of repression strains, it was found that the repression of pldII improves the antibacterial effect of the ε-PL product. The consecutive MALDI-TOF-MS results confirmed that the molecular weight distribution of the ε-PL was changed after repression. The repression strain S1 showed a particular peak with a polymerization degree of 44, and other repression strains also generated ε-PL with a polymerization degree of over 40. Furthermore, the homology modeling and substrate docking of pldII, a typical endo-type metallopeptidase, were performed to resolve the degradation mechanism of ε-PL in S. albulus. The hydrolysis of ε-PL within pldII, initiated from the N-terminus by two amino acid-binding residues, Thr194 and Glu281, led to varying levels of polymerization of ε-PL.

L-serine is an industrially valuable amino acid that is widely used in the food, cosmetics and pharmaceutical industries. In this study, transcriptome sequencing technology was applied to analyze the changes in gene expression levels during the synthesis of L-serine in Escherichia coli fermentation. The optimal carbon–nitrogen ratio for L-serine synthesis in E. coli was determined by setting five carbon–nitrogen ratios for shake flask fermentation. Transcriptome sequencing was performed on E. coli fermented in five carbon–nitrogen ratio medium in which a total of 791 differentially expressed genes (DEGs) were identified in the CZ4_vs_CZ1 group, including 212 upregulated genes and 579 downregulated genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of these DEGs showed that the effect of an altered carbon–nitrogen ratio on the fermentability of E. coli was mainly focused on metabolic pathways such as GABAergic synapse and the two-component system (TCS) in which the genes playing key roles were mainly gadB, gadA, glsA, glnA, narH and narJ. In summary, these potential key metabolic pathways and key genes were proposed to provide valuable information for improving glucose conversion during E. coli fermentation.

Antibiotic resistance caused by antibiotic resistance genes (ARGs) threatens human health. ARGs in animal manure can be degraded by composting. This study explored the changes in ARGs and microbial communities during co-composting of pig manure and tea stalks with (T) thermophilic microbial agents, including Geobacillus toebii ZF1 and Geobacillus sp. ZF2 for 15 days in a 4 L vacuum flask. Composting without thermophilic microbial agents served as control (CK). The results showed that the compost temperature of group T peaked at 72.1 °C and maintained above 70 °C for 4 days. The maximum temperature of group CK was 64.0 °C. The seed germination index showed that group T had reached maturity on day 9, while group CK reached maturity on day 15. The dominant bacteria in group CK were Bacillus. In group T, the dominant bacteria changed from Bacillus and Corynebacterium to Geobacillus. Compared with that of group CK, group T increased the removal of cmx, baeS and TaeA by 24.6%, 18.6% and 12.1%, respectively. Group T promoted and inhibited the removal of arlR and novA, respectively (p < 0.05), while group CK showed the opposite effect. Network analysis suggested that group T effectively inactivated Dietzia, Clostridium and Corynebacterium (p < 0.05) and promoted the removal of cmx and baeS. These results showed that thermophilic microbial agents could accelerate the maturation of pig manure and tea stalks, change microbial communities and promote the removal of ARGs. It is of great significance to reduce the spread of ARGs and, in turn, human health risks.

The esterase PTCL1-EstA from Paenarthrobacter aurescens TC1 was expressed in Escherichia coli and characterized. An 1152 bp open reading frame encoding a 383 amino acid polypeptide was successfully expressed, the C-terminally His6-tagged PTCL1-EstA enzyme was purified, and the predicted molecular mass of the purified PTCL1-EstA was 40.6 kDa. The EstA family serine hydrolase PTCL1-EstA belongs to the esterase family VIII, contains esterase-labeled S-C-S-K sequences, and homologous class C beta-lactamase sequences. PTCL1-EstA favored p-nitrophenyl esters with C2-C6 chain lengths, but it was also able to hydrolyze long-chain p-nitrophenyl esters. Homology modelling and substrate docking predicted that Ser59 was an active site residue in PTCL1-EstA, as well as Tyr148, Ala325, and Asp323, which are critical in catalyzing the enzymatic reaction of p-nitrophenyl esters. PTCL1-EstA reached the highest specific activity against p-nitrophenyl butyrate (C4) at pH 7.0 and 45 °C but revealed better thermal stability at 40 °C and maintained high relative enzymatic activity and stability at pH 5.0–9.0. Fermentation medium optimization for PTCL1-EstA increased the enzyme activity to 510.76 U/mL, tapping the potential of PTCL1-EstA for industrial production.