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1.
Int J Biol Macromol ; 268(Pt 1): 131619, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38692998

RESUMEN

The plant cell wall is a complex, heterogeneous structure primarily composed of cellulose, hemicelluloses, and lignin. Exploring the variations in these three macromolecules over time is crucial for understanding wood formation to enhance chemical processing and utilization. Here, we comprehensively analyzed the chemical composition of cell walls in the trunks of Pinus tabulaeformis using multiple techniques. In situ analysis showed that macromolecules accumulated gradually in the cell wall as the plant aged, and the distribution pattern of lignin was opposite that of polysaccharides, and both showed heterogenous distribution patterns. In addition, gel permeation chromatography (GPC) results revealed that the molecular weights of hemicelluloses decreased while that of lignin increased with age. Two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance (2D-HSQC NMR) analysis indicated that hemicelluloses mainly comprised galactoglucomannan and arabinoglucuronoxylan, and the lignin types were mainly comprised guaiacyl (G) and p-hydroxyphenyl (H) units with three main linkage types: ß-O-4, ß-ß, and ß-5. Furthermore, the C-O bond (ß-O-4) signals of lignin decreased while the C-C bonds (ß-ß and ß-5) signals increased over time. Taken together, these findings shed light on wood formation in P. tabulaeformis and lay the foundation for enhancing the processing and use of wood and timber products.


Asunto(s)
Pared Celular , Celulosa , Lignina , Pinus , Polisacáridos , Lignina/química , Pinus/química , Pared Celular/química , Polisacáridos/química , Celulosa/química , Peso Molecular , Árboles/química , Espectroscopía de Resonancia Magnética/métodos , Madera/química
2.
Carbohydr Polym ; 337: 122149, 2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-38710571

RESUMEN

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 ß-1,3-linked Glcp, both the α and ß-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.


Asunto(s)
Pared Celular , Quitina , Quitosano , Glucanos , Oryza , Enfermedades de las Plantas , Rhizoctonia , Rhizoctonia/efectos de los fármacos , Oryza/microbiología , Oryza/química , Pared Celular/química , Quitosano/química , Quitosano/farmacología , Quitina/química , Quitina/farmacología , Glucanos/química , Glucanos/farmacología , Enfermedades de las Plantas/microbiología , Resistencia a la Enfermedad , Especies Reactivas de Oxígeno/metabolismo
3.
Sci Rep ; 14(1): 11454, 2024 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-38769105

RESUMEN

This study focuses on pectin covalently linked in cell walls from two sources, apples and carrots, that was extracted using diluted alkali, and it describes changes in the rheological properties of diluted alkali-soluble pectin (DASP) due to enzymatic treatment. Given DASP's richness of rhamnogalacturonan I (RG-I), RG-I acetyl esterase (RGAE), rhamnogalacturonan endolyase (RGL), and arabinofuranosidase (ABF) were employed in various combinations for targeted degradation of RG-I pectin chains. Enzymatic degradations were followed by structural studies of pectin molecules using atomic force microscopy (AFM) as well as measurements of rheological and spectral properties. AFM imaging revealed a significant increase in the length of branched molecules after incubation with ABF, suggesting that arabinose side chains limit RG-I aggregation. Structural modifications were confirmed by changes in the intensity of bands in the pectin fingerprint and anomeric region on Fourier transform infrared spectra. ABF treatment led to a decrease in the stability of pectic gels, while the simultaneous use of ABF, RGAE, and RGL enzymes did not increase the degree of aggregation compared to the control sample. These findings suggest that the association of pectin chains within the DASP fraction may rely significantly on intermolecular interactions. Two mechanisms are proposed, which involve side chains as short-range attachment points or an extended linear homogalacturonan conformation favoring inter-chain interactions over self-association.


Asunto(s)
Pectinas , Reología , Pectinas/química , Pectinas/metabolismo , Microscopía de Fuerza Atómica , Álcalis/química , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/química , Solubilidad , Espectroscopía Infrarroja por Transformada de Fourier , Daucus carota/química , Polisacárido Liasas/metabolismo , Polisacárido Liasas/química , Pared Celular/química , Pared Celular/metabolismo
4.
Methods Mol Biol ; 2775: 329-347, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38758327

RESUMEN

The cell wall of the fungal pathogens Cryptococcus neoformans and C. gattii is critical for cell wall integrity and signaling external threats to the cell, allowing it to adapt and grow in a variety of changing environments. Chitin is a polysaccharide found in the cell walls of fungi that is considered to be essential for fungal survival. Chitosan is a polysaccharide derived from chitin via deacetylation that is also essential for cryptococcal cell wall integrity, fungal pathogenicity, and virulence. Cryptococcus has evolved mechanisms to regulate the amount of chitin and chitosan during growth under laboratory conditions or during mammalian infection. Therefore, levels of chitin and chitosan have been useful phenotypes to define mutant Cryptococcus strains. As a result, we have developed and/or refined various qualitative and quantitative methods for measuring chitin and chitosan. These techniques include those that use fluorescent probes that are known to bind to chitin (e.g., calcofluor white and wheat germ agglutinin), as well as those that preferentially bind to chitosan (e.g., eosin Y and cibacron brilliant red 3B-A). Techniques that enhance the localization and quantification of chitin and chitosan in the cell wall include (i) fluorescence microscopy, (ii) flow cytometry, (iii) and spectrofluorometry. We have also modified two highly selective biochemical methods to measure cellular chitin and chitosan content: the Morgan-Elson and the 3-methyl-2-benzothiazolone hydrazine hydrochloride (MBTH) assays, respectively.


Asunto(s)
Pared Celular , Quitina , Quitosano , Quitina/metabolismo , Quitina/química , Quitina/análisis , Quitosano/química , Quitosano/metabolismo , Pared Celular/metabolismo , Pared Celular/química , Cryptococcus neoformans/metabolismo , Colorantes Fluorescentes/química , Cryptococcus/metabolismo , Microscopía Fluorescente/métodos
5.
Artículo en Inglés | MEDLINE | ID: mdl-38752995

RESUMEN

A novel actinobacterium, strain ZYX-F-186T, was isolated from marine sediment sampled on Yongxing Island, Hainan Province, PR China. Based on the results of 16S rRNA gene sequence analysis, strain ZYX-F-186T belongs to the genus Phytohabitans, with high similarity to Phytohabitans kaempferiae KK1-3T (98.3 %), Phytohabitans rumicis K11-0047T (98.1 %), Phytohabitans flavus K09-0627T (98.1 %), Phytohabitans houttuyneae K11-0057T (97.9 %), Phytohabitans suffuscus K07-0523T (97.7 %), and Phytohabitans aurantiacus RD004123T (97.7 %). Phylogenetic analysis of 16S rRNA gene sequences showed that the strain formed a single subclade in the genus Phytohabitans. The novel isolate contained meso-diaminopimelic acid, d-glutamic acid, glycine, d-alanine, and l-lysine in the cell wall. The whole-cell sugars were xylose, arabinose, ribose, and rhamnose. The predominant menaquinones were MK-9(H8), MK-9(H6), and MK-9(H4). The characteristic phospholipids were phosphatidylethanolamine, phosphatidylinositol, phosphatidylmethylethanolamine, phosphatidylglycerol, and an unknown phospholipid. The major fatty acids (>5 %) were iso-C16 : 0, anteiso-C17 : 0, and iso-C18 : 0. Genome sequencing showed a DNA G+C content of 71.9 mol%. Low average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values demonstrated that strain ZYX-F-186T could be readily distinguished from its closely related species. Based on its phylogenetic, chemotaxonomic, and physiological characteristics, strain ZYX-F-186T represents a novel species of the genus Phytohabitans, for which the name Phytohabitans maris sp. nov. is proposed. The type strain is ZYX-F-186T (=CGMCC 4.8025T=CCTCC AA 2023025T=JCM 36507T).


Asunto(s)
Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano , Ácidos Grasos , Sedimentos Geológicos , Filogenia , ARN Ribosómico 16S , Análisis de Secuencia de ADN , Sedimentos Geológicos/microbiología , ARN Ribosómico 16S/genética , China , ADN Bacteriano/genética , Ácidos Grasos/química , Fosfolípidos , Vitamina K 2/análogos & derivados , Vitamina K 2/análisis , Vitamina K 2/química , Hibridación de Ácido Nucleico , Pared Celular/química
6.
Int J Biol Macromol ; 268(Pt 1): 131601, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38626833

RESUMEN

This study investigates the impact of water and salinity stress on Aloe vera, focusing on the role of Aloe vera polysaccharides in mitigating these stresses. Pectins and acemannan were the most affected polymers. Low soil moisture and high salinity (NaCl 80 mM) increased pectic substances, altering rhamnogalacturonan type I in Aloe vera gel. Aloe vera pectins maintained a consistent 60 % methyl-esterification regardless of conditions. Interestingly, acemannan content rose with salinity, particularly under low moisture, accompanied by 90 to 150 % acetylation increase. These changes improved the functionality of Aloe vera polysaccharides: pectins increased cell wall reinforcement and interactions, while highly acetylated acemannan retained water for sustained plant functions. This study highlights the crucial role of Aloe vera polysaccharides in enhancing plant resilience to water and salinity stress, leading to improved functional properties.


Asunto(s)
Aloe , Mananos , Pectinas , Aloe/química , Mananos/química , Pectinas/química , Agua/química , Pared Celular/química , Pared Celular/efectos de los fármacos , Salinidad , Polisacáridos/química , Polisacáridos/farmacología , Tolerancia a la Sal/efectos de los fármacos , Acetilación , Estrés Fisiológico/efectos de los fármacos
7.
Methods Mol Biol ; 2788: 81-95, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38656510

RESUMEN

Atomic force microscopy (AFM) has broken boundaries in the characterization of the supramolecular architecture of cell wall assemblies and single cell wall polysaccharides at the nanoscale level. Moreover, AFM provides an opportunity to evaluate the mechanical properties of cell wall material which is not possible with any other method. However, in the case of plant tissue, the critical step is a smart sample preparation that should not affect the polysaccharide structure or assembly and on the other hand should consider device limitations, especially scanner ranges. In this chapter, the protocols from the sample preparation, including isolation of cell wall material and extraction of cell wall polysaccharide fractions, through AFM imaging of polysaccharide assemblies and single molecules until an image analysis to obtain quantitative data characterizing the biopolymers are presented.


Asunto(s)
Pared Celular , Microscopía de Fuerza Atómica , Microscopía de Fuerza Atómica/métodos , Pared Celular/ultraestructura , Pared Celular/química , Polisacáridos/química , Polisacáridos/análisis
8.
ACS Nano ; 18(18): 11910-11920, 2024 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-38680054

RESUMEN

Personalized antitumor immunotherapy utilizing neoantigen vaccines holds great promise. However, the limited immunogenicity of existing recognized neoantigens and the inadequate stimulation of antitumor immune responses by conventional adjuvants pose significant challenges. To address these limitations, we developed a nanovaccine that combines a BCG bacterial cell wall skeleton (BCG-CWS) based nanoscale adjuvant (BCNA) with peptide neoantigens (M27 and M30). This integrated approach provides an efficient translational strategy for cancer immunotherapy. The BCNA nanovaccine, formulated with PLGA as an emulsifier, exhibits excellent biocompatibility and superior antigen presentation compared with conventional BCG-CWS adjuvants. Subcutaneous immunization with the BCNA-based nanovaccine effectively targets lymph nodes, eliciting robust innate and tumor-specific immune responses. Importantly, our findings demonstrate that BCNAs significantly enhance neoantigen immunogenicity while minimizing acute systemic toxicity. Furthermore, when combined with a mouse PD-L1 antibody, our strategy achieves complete tumor elimination in 60% of cases and prevents 25% of tumor growth in a melanoma mouse model. In conclusion, our BCNA-based nanovaccine represents a promising avenue for advancing personalized therapeutic neoantigen vaccines and holds significant implications for enhancing personalized immunotherapy and improving patient outcomes in the field of cancer treatment.


Asunto(s)
Adyuvantes Inmunológicos , Vacunas contra el Cáncer , Inmunoterapia , Animales , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Antígenos de Neoplasias/inmunología , Femenino , Humanos , Pared Celular/inmunología , Pared Celular/química , Mycobacterium bovis/inmunología , Nanopartículas/química , Vacuna BCG/inmunología , Línea Celular Tumoral
9.
J Agric Food Chem ; 72(18): 10206-10217, 2024 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-38597965

RESUMEN

Bamboo is a promising biomass resource. However, the complex multilayered structure and chemical composition of bamboo cell walls create a unique anti-depolymerization barrier, which increases the difficulty of separation and utilization of bamboo. In this study, the relationship between the connections of lignin-carbohydrate complexes (LCCs) within bamboo cell walls and their multilayered structural compositions was investigated. The chemical composition, structural properties, dissolution processes, and migration mechanisms of LCCs were analyzed. Alkali-stabilized LCC bonds were found to be predominantly characterized by phenyl glycoside (PhGlc) bonds along with numerous p-coumaric acid (PCA) linkage structures. As demonstrated by the NMR and CLSM results, the dissolution of the LCC during the alkaline pretreatment process was observed to migrate from the inner secondary wall (S-layer) of the bamboo fiber cell walls to the cell corner middle lamella (CCML) and compound middle lamella (CML), ultimately leading to its release from the bamboo. Furthermore, the presence of H-type lignin-FA-arabinoxylan linkage structures within the bamboo LCC was identified with their primary dissolution observed in the S-layer of the bamboo fiber cell walls. The study results provided a clear target for breaking down the anti-depolymerization barrier in bamboo, signifying a major advancement in achieving the comprehensive separation of bamboo components.


Asunto(s)
Carbohidratos , Pared Celular , Lignina , Lignina/química , Pared Celular/química , Carbohidratos/química , Álcalis/química , Sasa/química , Solubilidad , Poaceae/química , Xilanos/química , Espectroscopía de Resonancia Magnética
10.
Int J Biol Macromol ; 268(Pt 2): 131684, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38663695

RESUMEN

Cracking, warping, and decaying stemming from wood's poor dimensional stability and durability are the most annoying issues of natural wood. There is an urgent need to address these issues, of which, sustainable and green chemical treatments are favorably welcomed. Herein, we developed a facile method through the incorporation of environmentally friendly biopolymer lignin into wood cells for wood dimensional stability and durability enhancement. Enzymatic hydrolysis lignin (EHL) was dissolved into various solvents followed by impregnation and drying to incorporate lignin into wood cells. Impregnation treatment was developed to incorporate into wood to improve its dimensional stability, durability, and micromechanics. The anti-swelling efficiency reached up to 99.4 %, the moisture absorption decreased down to 0.55 %, the mass loss after brown rot decay decreased to 7.22 %, and the cell wall elasticity as well as hardness increased 8.7 % and 10.3 %, respectively. Analyses acquired from scanning electron microscopy, fluorescent microscopy, and Raman imaging revealed that the EHL was successfully colonized in cell lumen as well as in cell walls, thus improved wood dimensional stability and durability. Moreover, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy confirmed EHL interaction with the cell wall components, thus the wood mechanical property was not impaired significantly, whereas nanoindentation data indicated even slight mechanical enhancement on the cell walls. This facile approach can improve the wood properties in multiple aspects and remarkably enhance the outdoor performance of modified wood products. In addition, using lignin as a natural modifying agent to improve wood performance will have a great positive impact on the environment.


Asunto(s)
Lignina , Madera , Lignina/química , Madera/química , Pared Celular/química , Hidrólisis , Espectroscopía Infrarroja por Transformada de Fourier
11.
Food Chem ; 449: 139234, 2024 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-38608604

RESUMEN

Cuticle wax chemicals are cultivar-dependent and contribute to storage quality. Few research reported on wax analysis between melting flesh-type (MF; 'Jinhuami 25') and nonmelting flesh-type (NMF; 'Xizhoumi 17' and 'Chougua') Hami melons. Chemicals and crystal structures of Hami melon cuticular wax, cell wall metabolism related to fruit melting, and fruit physiology were analyzed to observe wax functions. Results showed that Hami melon cuticle wax predominantly consists of esters, alkanes, alcohols, aldehydes, and terpenoids. MF-type has a lower alkane/terpenoid ratio, concomitant to its higher weight loss and cuticle permeability. Micromorphology of wax crystals appears as numerous platelets with irregular crystals, and the transformation of wax structure in NMF Hami melon is delayed. Waxy components affect cell wall metabolism and physiological quality, which results in the pulp texture difference between MF-type and NMF-type during storage. Results provide a reference for the regulation of wax synthesis in both types of melons.


Asunto(s)
Cucumis melo , Frutas , Ceras , Ceras/química , Frutas/química , Cucumis melo/química , Pared Celular/química
12.
Carbohydr Polym ; 334: 122024, 2024 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-38553223

RESUMEN

Upon tensile stress, the spiral cellulose fibrils in wood cell walls rotate like springs with decreasing microfibril angle (MFA), and the cellulose molecules elongate in the chain direction. Compression wood with high MFA and opposite wood with low MFA were comparatively studied by in-situ tensile tests combined with synchrotron radiation WAXS in the present study. FTIR spectroscopy revealed that compression wood had a higher lignin content and fewer acetyl groups. For both types of wood, the lattice spacing d004 increased and the MFA decreased gradually with the increase of tensile stress. At stresses beyond the yield point, cellulose lattice strain depended linearly on macroscopic stress, while the MFA depended linearly on macroscopic strain. The deformation mechanisms of compression wood and opposite wood are not essentially different but differ in their deformation behavior. Specifically, the contribution ratio of lattice strain and cellulose fibril reorientation to macroscopic strain was 0.25 and 0.53 for compression wood, and 0.40 and 0.33 for opposite wood, respectively. Due to the geometric effects of MFA, a greater contribution of cellulose fibril reorientation to the macroscopic deformation was detected in compression wood than in opposite wood.


Asunto(s)
Celulosa , Pinus , Celulosa/química , Madera/metabolismo , Microfibrillas/química , Lignina/metabolismo , Pared Celular/química
13.
Methods Mol Biol ; 2791: 57-70, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38532092

RESUMEN

Immunohistochemistry is a method that allows the detection of individual components of cell walls in an extremely precise way at the level of a single cell and wall domains. The cell wall antibodies detect specific epitopes of pectins, arabinogalactan proteins (AGP), hemicelluloses, and extensins. The presented method visualization of the selected pectic and AGP epitopes using antibodies directed to wall components is described. The method of the analysis of the chemical composition of the wall is present on the example of the shoot apical meristems of Fagopurum esculentum and Fagopyrum tataricum. Recommended protocols for immunostaining and examination on fluorescence microscopy level are presented.


Asunto(s)
Fagopyrum , Fagopyrum/química , Fagopyrum/metabolismo , Meristema/metabolismo , Pectinas/análisis , Inmunohistoquímica , Epítopos , Pared Celular/química
14.
Can J Microbiol ; 70(5): 190-198, 2024 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-38525892

RESUMEN

The cell envelope of the poly-extremophile bacterium Deinococcus radiodurans is renowned for its highly organized structure and unique functional characteristics. In this bacterium, a precise regularity characterizes not just the S-layer, but it also extends to the underlying cell envelope layers, resulting in a dense and tightly arranged configuration. This regularity is attributed to a minimum of three protein complexes located at the outer membrane level. Together, they constitute a recurring structural unit that extends across the cell envelope, effectively tiling the entirety of the cell body. Nevertheless, a comprehensive grasp of the vacant spaces within each layer and their functional roles remains limited. In this study, we delve into these aspects by integrating the state of the art with structural calculations. This approach provides crucial evidence supporting an evolutive pressure intricately linked to surface phenomena depending on the environmental conditions.


Asunto(s)
Membrana Celular , Deinococcus , Deinococcus/metabolismo , Deinococcus/química , Membrana Celular/metabolismo , Membrana Celular/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Pared Celular/química , Pared Celular/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/química
15.
Food Chem ; 448: 139062, 2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-38531297

RESUMEN

Avenanthramide-C (AVN-C) is the biomarker for oat with a variety of physiological functions, whereas its application is constrained by low stability and bioavailability. Avenanthramide-C is the biomarker for oat with a variety of physiological functions, whereas its application is constrained by low stability and bioavailability. This study evaluated the potential of yeast cell (YC) and yeast cell wall (YCW) capsules as delivery systems for stabilizing AVN-C. It was observed that these yeast capsules possessed the ellipsoidal morphology and intact structure without visual pores. Additionally, the YCW capsules exhibited higher encapsulation and loading capacity due to the large internal space. The interaction of yeast capsules with AVN-C involved the hydrophobic interactions and hydrogen bonding. Moreover, the loading of AVN-C induced high hydrophobicity inside the yeast capsules, which helped to protect AVN-C against degradation and release AVN-C in a slow and sustained manner in the simulated gastrointestinal tract. The YCW capsules have potential as controlled delivery system for AVN-C, which could be further used as a nutraceutical and added to functional foods.


Asunto(s)
Avena , Cápsulas , Pared Celular , Saccharomyces cerevisiae , ortoaminobenzoatos , Avena/química , ortoaminobenzoatos/química , Cápsulas/química , Pared Celular/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Biomarcadores , Interacciones Hidrofóbicas e Hidrofílicas
16.
J Agric Food Chem ; 72(11): 6028-6039, 2024 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-38457781

RESUMEN

The fungal cell wall, primarily comprising a glucan-chitin matrix and cell wall proteins (CWPs), serves as a key mediator for fungal interactions with the environment and plays a pivotal role in virulence. In this study, we employed a comprehensive proteomics approach to analyze the CWPs in the plant pathogenic fungus Fusarium graminearum. Our methodology successfully extracted and identified 1373 CWPs, highlighting their complex linkages, including noncovalent bonds, disulfide bridges, alkali-sensitive linkages, and glycosylphosphatidylinositol (GPI) anchors. A significant subset of these proteins, enriched in Gene Ontology terms, suggest multifunctional roles of CWPs. Through the integration of transcriptomic and proteomic data, we observed differential expression patterns of CWPs across developmental stages. Specifically, we focused on two genes, Fca7 and Cpd1, which were upregulated in planta, and confirmed their localization predominantly outside the plasma membrane, primarily in the cell wall and periplasmic space. The disruption of FCA7 reduced virulence on wheat, aligning with previous findings and underscoring its significance. Overall, our findings offer a comprehensive proteomic profile of CWPs in F. graminearum, laying the groundwork for a deeper understanding of their roles in the development and interactions with host plants.


Asunto(s)
Proteínas Fúngicas , Fusarium , Proteínas Fúngicas/metabolismo , Proteómica , Pared Celular/química , Fusarium/genética , Fusarium/metabolismo , Enfermedades de las Plantas
17.
J Biol Chem ; 300(3): 105734, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38336294

RESUMEN

Numerous putative glycosyltransferases (GTs) have been identified using bioinformatic approaches. However, demonstrating the activity of these GTs remains a challenge. Here, we describe the development of a rapid in vitro GT-array screening platform for activity of GTs. GT-arrays are generated by cell-free in vitro protein synthesis and binding using microplates precoated with a N-terminal Halo- or a C-terminal GST-tagged GT-encoding plasmid DNA and a capture antibody. These arrays are then used for screening of transferase activities and the reactions are monitored by a luminescence GLO assay. The products formed by these reactions can be analyzed directly from the microplates by mass spectrometry. Using this platform, a total of 280 assays were performed to screen 22 putative fucosyltransferases (FUTs) from family GT37 (seven from Arabidopsis and 15 from rice) for activity toward five acceptors: non-fucosylated tamarind xyloglucan (TXyG), arabinotriose (Ara3), non-fucosylated rhamnogalacturonan I (RG-I), and RG-II from the mur1-1 Arabidopsis mutant, and the celery RG-II monomer lacking Arap and MeFuc of chain B and l-Gal of chain A. Our screen showed that AtFUT2, AtFUT5, and AtFUT10 have activity toward RG-I, while AtFUT8 was active on RG-II. Five rice OsFUTs have XyG-FUT activity and four rice OsFUTs have activity toward Ara3. None of the putative OsFUTs were active on the RG-I and RG-II. However, promiscuity toward acceptors was observed for several FUTs. These findings extend our knowledge of cell wall polysaccharide fucosylation in plants. We believe that in vitro GT-array platform provides a valuable tool for cell wall biochemistry and other research fields.


Asunto(s)
Pruebas de Enzimas , Fucosiltransferasas , Glicosiltransferasas , Proteínas de Plantas , Apium/enzimología , Apium/genética , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/metabolismo , Pared Celular/química , Pared Celular/enzimología , Pared Celular/metabolismo , Pruebas de Enzimas/instrumentación , Pruebas de Enzimas/métodos , Fucosiltransferasas/análisis , Fucosiltransferasas/clasificación , Fucosiltransferasas/metabolismo , Glicosiltransferasas/análisis , Glicosiltransferasas/metabolismo , Espectrometría de Masas , Oryza/enzimología , Proteínas de Plantas/análisis , Proteínas de Plantas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo
18.
Int J Biol Macromol ; 262(Pt 2): 130121, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38350588

RESUMEN

This study identified a rhamnose-containing cell wall polysaccharide (RhaCWP) in an alkaline extract prepared to analyze intracellular polysaccharides (IPS) from Streptococcus mutans biofilm. IPS was an 1,4-α-D-glucan with branchpoints introduced by 1,6-α-glucan while RhaCWP presented 1,2-α-L-and 1,3-α-L rhamnose backbone and side chains connected by 1,2-α-D-glucans, as identified by nuclear magnetic resonance (NMR) spectroscopy and methylation analyses. The MW of IPS and RhaCWP was 11,298 Da, as determined by diffusion-ordered NMR spectroscopy. Therefore, this study analyzed the chemical structure of RhaCWP and IPS from biofilm in a single fraction prepared via a convenient hot-alkali extraction method. This method could be a feasible approach to obtain such molecules and improve the comprehension of the structure-function relationships in polymers from S. mutans in future studies.


Asunto(s)
Ramnosa , Streptococcus mutans , Ramnosa/análisis , Polisacáridos/análisis , Glucanos/química , Pared Celular/química
19.
Appl Spectrosc ; 78(4): 355-364, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38378014

RESUMEN

The cell wall integrity (CWI) signaling pathway regulates yeast cell wall biosynthesis, cell division, and responses to external stress. The cell wall, comprised of a dense network of chitin, ß-1,3- and ß-1,6- glucans, and mannoproteins, is very thin, <100 nm. Alterations in cell wall composition may activate the CWI pathway. Saccharomyces cerevisiae, a model yeast, was used to study the role of individual wall components in altering the structure and biophysical properties of the yeast cell wall. Near-field Fourier transform infrared spectroscopy (nano-FT-IR) was used for the first direct, spectrochemical identification of cell wall composition in a background (wild-type) strain and two deletion mutants from the yeast knock-out collection: kre6Δ and knr4Δ. Killer toxin resistant 6 (Kre6) is an integral membrane protein required for biosynthesis of ß-1,6-glucan, while Knr4 is a cell signaling protein involved in the control of cell wall biosynthesis, in particular, biosynthesis and deposition of chitin. Complementary spectral data were obtained with far-field (FF)-FT-IR, in transmission, and with attenuated total reflectance (ATR) spectromicroscopy with 3-10 µm wavelength-dependent spatial resolution. The FF-FT-IR spectra of cells and spectra of isolated cell wall components showed that components of the cell body dominated transmission spectra and were still evident in ATR spectra. In contrast, the nano-FT-IR at ∼25 nm spatial resolution could be used to characterize the yeast wall chemical structure. Our results show that the ß-1,6-glucan content is decreased in kre6Δ, while all glucan content is decreased in the knr4Δ cell wall. The latter may be thinner than in wild type, since not only are mannan and chitin detectable by nano-FT-IR, but also lipid membranes and protein, indicative of cell interior.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , beta-Glucanos , beta-Glucanos/análisis , Pared Celular/química , Quitina/análisis , Quitina/metabolismo , Glucanos/análisis , Glucanos/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier
20.
J Agric Food Chem ; 72(8): 4195-4206, 2024 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-38354398

RESUMEN

The increase of polysaccharides in the dark tea pile process is thought to be connected to the cell wall polysaccharides' breakdown. However, the relationship between tea polysaccharides (TPSs) and tea cell wall polysaccharides has not been further explored. In this study, the structural changes in the cell wall polysaccharides [e.g., cellulose, hemicellulose (HC), and pectin] in Liupao tea were characterized before and after traditional fermentation and tank fermentation. Additionally, the degradation mechanism of tea cell wall polysaccharides during fermentation was assessed. The results showed that cellulose crystallinity decreased by 11.9-49.6% after fermentation. The molar ratio of monosaccharides, such as arabinose, rhamnose, and glucose in HC, was significantly reduced, and the molecular weight decreased. The esterification degree and linearity of water-soluble pectin (WSP) were reduced. TPS content increases during pile fermentation, which may be due to HC degradation and the increase in WSP caused by cell wall structure damage. Microorganisms were shown to be closely associated with the degradation of cell wall polysaccharides during fermentation according to correlation analyses. Traditional fermentation had a greater effect on the cellulose structure, while tank fermentation had a more noticeable impact on HC and WSP.


Asunto(s)
Camellia sinensis , Polisacáridos , Fermentación , Polisacáridos/química , Camellia sinensis/química , Pectinas/química , Celulosa/metabolismo , Agua/metabolismo , Pared Celular/química , Té/química , China
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