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1.
Appl Environ Microbiol ; 88(6): e0218721, 2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35108084

RESUMO

Gum arabic is an arabinogalactan protein (AGP) that is effective as a prebiotic for the growth of bifidobacteria in the human intestine. We recently identified a key enzyme in the glycoside hydrolase (GH) family 39, 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase), for the assimilation of gum arabic AGP in Bifidobacterium longum subsp. longum. The enzyme released α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP and facilitated the action of other enzymes for degrading the AGP backbone and modified sugar. In this study, we identified an α-l-arabinofuranosidase (BlArafE; encoded by BLLJ_1850), a multidomain enzyme with both GH43_22 and GH43_34 catalytic domains, as a critical enzyme for the degradation of modified α-l-arabinofuranosides in gum arabic AGP. Site-directed mutagenesis approaches revealed that the α1,3/α1,4-Araf double-substituted gum arabic AGP side chain was initially degraded by the GH43_22 domain and subsequently cleaved by the GH43_34 domain to release α1,3-Araf and α1,4-Araf residues, respectively. Furthermore, we revealed that a tetrasaccharide, α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal, was a limited degradative oligosaccharide in the gum arabic AGP fermentation of B. longum subsp. longum JCM7052. The oligosaccharide was produced from gum arabic AGP by the cooperative action of the three cell surface-anchoring enzymes, GAfase, exo-ß1,3-galactanase (Bl1,3Gal), and BlArafE, on B. longum subsp. longum JCM7052. Furthermore, the tetrasaccharide was utilized by the commensal bacteria. IMPORTANCE Terminal galactose residues of the side chain of gum arabic arabinogalactan protein (AGP) are mainly substituted by α1,3/α1,4-linked Araf and ß1,6-linked α-l-Rhap-(1→4)-ß-d-GlcpA residues. This study found a multidomain BlArafE with GH43_22 and GH43_34 catalytic domains showing cooperative action for degrading α1,3/α1,4-linked Araf of the side chain of gum arabic AGP. In particular, the GH43_34 domain of BlArafE was a novel α-l-arabinofuranosidase for cleaving the α1,4-Araf linkage of terminal galactose. α-l-Rhap-(1→4)-ß-d-GlcpA-(1→6)-ß-d-Galp-(1→6)-d-Gal tetrasaccharide was released from gum arabic AGP by the cooperative action of GAfase, GH43_24 exo-ß-1,3-galactanase (Bl1,3Gal), and BlArafE and remained after B. longum subsp. longum JCM7052 culture. Furthermore, in vitro assimilation test of the remaining oligosaccharide using Bacteroides species revealed that cross-feeding may occur from bifidobacteria to other taxonomic groups in the gut.


Assuntos
Bifidobacterium longum , Bifidobacterium longum/metabolismo , Galactanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Goma Arábica , Humanos , Oligossacarídeos/química
2.
Sci Rep ; 12(1): 1089, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-35058551

RESUMO

In the present study, galactan exopolysaccharide (EPS) from Weissella confusa KR780676 was evaluated for its potential to alleviate oxidative stress using in vitro assays and in vivo studies in Saccharomyces cerevisiae (wild type) and its antioxidant (sod1∆, sod2∆, tsa1∆, cta2∆ and ctt1∆), anti-apoptotic (pep4∆ and fis1∆) and anti-aging (sod2∆, tsa1∆ and ctt1∆)) isogenic gene deletion mutants. Galactan exhibited strong DPPH and nitric oxide scavenging activity with an IC50 value of 450 and 138 µg/mL respectively. In the yeast mutant model, oxidative stress generated by H2O2 was extensively scavenged by galactan in the medium as confirmed using spot assays followed by fluorescencent DCF-DA staining and microscopic studies. Galactan treatment resulted in reduction in the ROS generated in the yeast mutant cells as demonstrated by decreased fluorescence intensity. Furthermore, galactan exhibited protection against oxidative damage through H2O2 -induced apoptosis inhibition in the yeast mutant strains (pep4∆ and fis1∆) leading to increased survival rate by neutralizing the oxidative stress. In the chronological life span assay, WT cells treated with galactan EPS showed 8% increase in viability whereas sod2∆ mutant showed 10-15% increase indicating pronounced anti-aging effects. Galactan from W. confusa KR780676 has immense potential to be used as a natural antioxidant for nutraceutical, pharmaceutical and food technological applications. As per our knowledge, this is the first report on in-depth assessment of in vivo antioxidant properties of a bacterial EPS in a yeast deletion model system.


Assuntos
Galactanos/isolamento & purificação , Galactanos/farmacologia , Weissella/metabolismo , Antioxidantes/farmacologia , Bactérias/efeitos dos fármacos , Galactanos/metabolismo , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Estresse Oxidativo/efeitos dos fármacos , Polissacarídeos Bacterianos/isolamento & purificação , Polissacarídeos Bacterianos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos
3.
Plant J ; 109(1): 164-181, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34726315

RESUMO

Arabinogalactan proteins (AGPs) are complex, hyperglycosylated plant cell wall proteins with little known about the biological roles of their glycan moieties in sexual reproduction. Here, we report that GLCAT14A, GLCAT14B, and GLCAT14C, three enzymes responsible for the addition of glucuronic acid residues to AGPs, function in pollen development, polytubey block, and normal embryo development in Arabidopsis. Using biochemical and immunolabeling techniques, we demonstrated that the loss of function of the GLCAT14A, GLCAT14B, and GLCAT14C genes resulted in disorganization of the reticulate structure of the exine wall, abnormal development of the intine layer, and collapse of pollen grains in glcat14a/b and glcat14a/b/c mutants. Synchronous development between locules within the same anther was also lost in some glcat14a/b/c stamens. In addition, we observed excessive attraction of pollen tubes targeting glcat14a/b/c ovules, indicating that the polytubey block mechanism was compromised. Monosaccharide composition analysis revealed significant reductions in all sugars in glcat14a/b and glcat14a/b/c mutants except for arabinose and galactose, while immunolabeling showed decreased amounts of AGP sugar epitopes recognized by glcat14a/b and glcat14a/b/c mutants compared with the wild type. This work demonstrates the important roles that AG glucuronidation plays in Arabidopsis sexual reproduction and reproductive development.


Assuntos
Arabidopsis/enzimologia , Galactanos/metabolismo , Mucoproteínas/metabolismo , Polissacarídeos/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Ácido Glucurônico/metabolismo , Mucoproteínas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/enzimologia , Pólen/genética , Pólen/fisiologia , Tubo Polínico/enzimologia , Tubo Polínico/genética , Tubo Polínico/fisiologia , Reprodução
4.
Plant J ; 109(1): 47-63, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34695268

RESUMO

Dwarfing rootstocks and dwarf cultivars are urgently needed for modern pear cultivation. However, germplasm resources for dwarfing pear are limited, and the underlying mechanisms remain unclear. We previously showed that dwarfism in pear is controlled by the single dominant gene PcDw (Dwarf). We report here that the expression of PcAGP7-1 (ARABINOGALACTAN PROTEIN 7-1), a key candidate gene for PcDw, is significantly higher in dwarf-type pear plants because of a mutation in an E-box in the promoter. Electrophoretic mobility shift assays and transient infiltration showed that the transcription factors PcBZR1 and PcBZR2 could directly bind to the E-box of the PcAGP7-1 promoter and repress transcription. Moreover, transgenic pear lines overexpressing PcAGP7-1 exhibited obvious dwarf phenotypes, whereas RNA interference pear lines for PcAGP7-1 were taller than controls. PcAGP7-1 overexpression also enhanced cell wall thickness, affected cell morphogenesis, and reduced brassinolide (BL) content, which inhibited BR signaling via a negative feedback loop, resulting in further dwarfing. Overall, we identified a dwarfing mechanism in perennial woody plants involving the BL-BZR/BES-AGP-BL regulatory module. Our findings provide insight into the molecular mechanism of plant dwarfism and suggest strategies for the molecular breeding of dwarf pear cultivars.


Assuntos
Brassinosteroides/metabolismo , Galactanos/metabolismo , Proteínas de Plantas/metabolismo , Pyrus/genética , Esteroides Heterocíclicos/metabolismo , Mucoproteínas/genética , Mucoproteínas/metabolismo , Mutação , Fenótipo , Filogenia , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Pyrus/química , Pyrus/crescimento & desenvolvimento , Pyrus/ultraestrutura , Tabaco/química , Tabaco/genética , Tabaco/crescimento & desenvolvimento , Tabaco/ultraestrutura
5.
BMC Plant Biol ; 21(1): 590, 2021 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-34903166

RESUMO

BACKGROUND: Arabinogalactan-proteins (AGPs) are structurally complex hydroxyproline-rich cell wall glycoproteins ubiquitous in the plant kingdom. AGPs biosynthesis involves a series of post-translational modifications including the addition of type II arabinogalactans to non-contiguous Hyp residues. To date, eight Hyp-galactosyltransferases (Hyp-GALTs; GALT2-GALT9) belonging to CAZy GT31, are known to catalyze the addition of the first galactose residues to AGP protein backbones and enable subsequent AGP glycosylation. The extent of genetic redundancy, however, remains to be elucidated for the Hyp-GALT gene family. RESULTS: To examine their gene redundancy and functions, we generated various multiple gene knock-outs, including a triple mutant (galt5 galt8 galt9), two quadruple mutants (galt2 galt5 galt7 galt8, galt2 galt5 galt7 galt9), and one quintuple mutant (galt2 galt5 galt7 galt8 galt9), and comprehensively examined their biochemical and physiological phenotypes. The key findings include: AGP precipitations with ß-Yariv reagent showed that GALT2, GALT5, GALT7, GALT8 and GALT9 act redundantly with respect to AGP glycosylation in cauline and rosette leaves, while the activity of GALT7, GALT8 and GALT9 dominate in the stem, silique and flowers. Monosaccharide composition analysis showed that galactose was decreased in the silique and root AGPs of the Hyp-GALT mutants. TEM analysis of 25789 quintuple mutant stems indicated cell wall defects coincident with the observed developmental and growth impairment in these Hyp-GALT mutants. Correlated with expression patterns, galt2, galt5, galt7, galt8, and galt9 display equal additive effects on insensitivity to ß-Yariv-induced growth inhibition, silique length, plant height, and pollen viability. Interestingly, galt7, galt8, and galt9 contributed more to primary root growth and root tip swelling under salt stress, whereas galt2 and galt5 played more important roles in seed morphology, germination defects and seed set. Pollen defects likely contributed to the reduced seed set in these mutants. CONCLUSION: Additive and pleiotropic effects of GALT2, GALT5, GALT7, GALT8 and GALT9 on vegetative and reproductive growth phenotypes were teased apart via generation of different combinations of Hyp-GALT knock-out mutants. Taken together, the generation of higher order Hyp-GALT mutants demonstrate the functional importance of AG polysaccharides decorating the AGPs with respect to various aspects of plant growth and development.


Assuntos
Arabidopsis/genética , Galactanos/metabolismo , Galactosiltransferases/metabolismo , Mucoproteínas/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Flores/enzimologia , Flores/genética , Flores/fisiologia , Flores/ultraestrutura , Galactosiltransferases/genética , Pleiotropia Genética , Germinação , Glucosídeos/química , Glicosilação , Hidroxiprolina/metabolismo , Meristema/enzimologia , Meristema/genética , Meristema/fisiologia , Meristema/ultraestrutura , Mucoproteínas/genética , Mutação , Especificidade de Órgãos , Floroglucinol/análogos & derivados , Floroglucinol/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Caules de Planta/enzimologia , Caules de Planta/genética , Caules de Planta/fisiologia , Caules de Planta/ultraestrutura , Biossíntese de Proteínas , Estresse Salino , Sementes/enzimologia , Sementes/genética , Sementes/fisiologia , Sementes/ultraestrutura
6.
J Bacteriol ; 203(24): e0040321, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34606371

RESUMO

The alphaproteobacterium Sinorhizobium meliloti secretes two acidic exopolysaccharides (EPSs), succinoglycan (EPSI) and galactoglucan (EPSII), which differentially enable it to adapt to a changing environment. Succinoglycan is essential for invasion of plant hosts and, thus, for the formation of nitrogen-fixing root nodules. Galactoglucan is critical for population-based behaviors such as swarming and biofilm formation and can facilitate invasion in the absence of succinoglycan on some host plants. The biosynthesis of galactoglucan is not as completely understood as that of succinoglycan. We devised a pipeline to identify putative pyruvyltransferase and acetyltransferase genes, construct genomic deletions in strains engineered to produce either succinoglycan or galactoglucan, and analyze EPS from mutant bacterial strains. EPS samples were examined by 13C cross-polarization magic-angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). CPMAS NMR is uniquely suited to defining chemical composition in complex samples and enables the detection and quantification of distinct EPS functional groups. Galactoglucan was isolated from mutant strains with deletions in five candidate acyl/acetyltransferase genes (exoZ, exoH, SMb20810, SMb21188, and SMa1016) and a putative pyruvyltransferase (wgaE or SMb21322). Most samples were similar in composition to wild-type EPSII by CPMAS NMR analysis. However, galactoglucan produced from a strain lacking wgaE exhibited a significant reduction in pyruvylation. Pyruvylation was restored through the ectopic expression of plasmid-borne wgaE. Our work has thus identified WgaE as a galactoglucan pyruvyltransferase. This exemplifies how the systematic combination of genetic analyses and solid-state NMR detection is a rapid means to identify genes responsible for modification of rhizobial exopolysaccharides. IMPORTANCE Nitrogen-fixing bacteria are crucial for geochemical cycles and global nitrogen nutrition. Symbioses between legumes and rhizobial bacteria establish root nodules, where bacteria convert dinitrogen to ammonia for plant utilization. Secreted exopolysaccharides (EPSs) produced by Sinorhizobium meliloti (succinoglycan and galactoglucan) play important roles in soil and plant environments. The biosynthesis of galactoglucan is not as well characterized as that of succinoglycan. We employed solid-state nuclear magnetic resonance (NMR) to examine intact EPS from wild-type and mutant S. meliloti strains. NMR analysis of EPS isolated from a wgaE gene mutant revealed a novel pyruvyltransferase that modifies galactoglucan. Few EPS pyruvyltransferases have been characterized. Our work provides insight into the biosynthesis of an important S. meliloti EPS and expands the knowledge of enzymes that modify polysaccharides.


Assuntos
Proteínas de Bactérias/metabolismo , Polissacarídeos Bacterianos/metabolismo , Transferases/metabolismo , Proteínas de Bactérias/genética , Galactanos/química , Galactanos/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Glucanos/química , Glucanos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Mutação , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/genética , Sinorhizobium meliloti , Transferases/classificação , Transferases/genética
7.
Cells ; 10(8)2021 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-34440649

RESUMO

Arabinogalactan proteins (AGPs) are a class of heavily glycosylated proteins occurring as a structural element of the cell wall-plasma membrane continuum. The features of AGPs described earlier suggest that the proteins may be implicated in plant adaptation to stress conditions in important developmental phases during the plant reproduction process. In this paper, the microscopic and immunocytochemical studies conducted using specific antibodies (JIM13, JIM15, MAC207) recognizing the carbohydrate chains of AGPs showed significant changes in the AGP distribution in female and male reproductive structures during the first stages of Bellis perennis development. In typical conditions, AGPs are characterized by a specific persistent spatio-temporal pattern of distribution. AGP epitopes are visible in the cell walls of somatic cells and in the megasporocyte walls, megaspores, and embryo sac at every stage of formation. During development in stress conditions, the AGP localization is altered, and AGPs entirely disappear in the embryo sac wall. In the case of male development, AGPs are present in the tapetum, microsporocytes, and microspores in normal conditions. In response to development at lower temperature, AGPs are localized in the common wall of microspores and in mature pollen grains. Additionally, they are accumulated in remnants of tapetum cells.


Assuntos
Asteraceae/metabolismo , Temperatura Baixa , Galactanos/metabolismo , Gametogênese Vegetal , Mucoproteínas/metabolismo , Óvulo Vegetal/metabolismo , Processamento de Proteína Pós-Traducional , Asteraceae/embriologia , Asteraceae/crescimento & desenvolvimento , Glicosilação , Imuno-Histoquímica , Microscopia Confocal , Óvulo Vegetal/embriologia , Óvulo Vegetal/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Fatores de Tempo
8.
Cells ; 10(8)2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34440704

RESUMO

Novel molecular pinball machines of the plasma membrane control cytosolic Ca2+ levels that regulate plant metabolism. The essential components involve: 1. an auxin-activated proton pump; 2. arabinogalactan glycoproteins (AGPs); 3. Ca2+ channels; 4. auxin-efflux "PIN" proteins. Typical pinball machines release pinballs that trigger various sound and visual effects. However, in plants, "proton pinballs" eject Ca2+ bound by paired glucuronic acid residues of numerous glycomodules in periplasmic AGP-Ca2+. Freed Ca2+ ions flow down the electrostatic gradient through open Ca2+ channels into the cytosol, thus activating numerous Ca2+-dependent activities. Clearly, cytosolic Ca2+ levels depend on the activity of the proton pump, the state of Ca2+ channels and the size of the periplasmic AGP-Ca2+ capacitor; proton pump activation is a major regulatory focal point tightly controlled by the supply of auxin. Auxin efflux carriers conveniently known as "PIN" proteins (null mutants are pin-shaped) pump auxin from cell to cell. Mechanosensitive Ca2+ channels and their activation by reactive oxygen species (ROS) are yet another factor regulating cytosolic Ca2+. Cell expansion also triggers proton pump/pinball activity by the mechanotransduction of wall stress via Hechtian adhesion, thus forming a Hechtian oscillator that underlies cycles of wall plasticity and oscillatory growth. Finally, the Ca2+ homeostasis of plants depends on cell surface external storage as a source of dynamic Ca2+, unlike the internal ER storage source of animals, where the added regulatory complexities ranging from vitamin D to parathormone contrast with the elegant simplicity of plant life. This paper summarizes a sixty-year Odyssey.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Membrana Celular/metabolismo , Mecanotransdução Celular , Proteínas de Membrana Transportadoras/metabolismo , Mucoproteínas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Plantas/metabolismo , Canais de Cálcio/metabolismo , Galactanos/metabolismo , Ácidos Indolacéticos/metabolismo , Desenvolvimento Vegetal , Proteínas de Plantas/metabolismo , Bombas de Próton/metabolismo , Espécies Reativas de Oxigênio , Estresse Mecânico
9.
Food Chem Toxicol ; 156: 112522, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34438010

RESUMO

BACKGROUND AND OBJECTIVES: Dietary fibers have beneficial effects on human health through the interaction with gut microbiota. Larch wood arabinogalactan (LA-AG) is one kind of complex soluble dietary fibers that may be utilized by human gut microbiota. METHODS AND RESULTS: In this study, the LA-AG degradation by gut microbiota were characterized by investigating the change of LA-AG, microbiota composition, and the production of short-chain fatty acids (SCFAs), lactic acid, succinic acid, as well as volatile organic metabolites. During the fermentation, pH decreased continuously, along with the organic acids (especially acetic acid and lactic acid) accumulating. LA-AG was degraded by gut microbiota then some beneficial metabolites were produced. In addition, LA-AG inhibited the proliferation of some gut microbiota (Unclassified_Enterobacteriaceae and Citrobacter) and the accumulation of some metabolites (Sulfide and indole) released by gut microbiota. CONCLUSION: LA-AG was partly fermentable fibers with prebiotic potential for human gut health.


Assuntos
Galactanos/metabolismo , Microbioma Gastrointestinal/fisiologia , Prebióticos , Bactérias/classificação , Bactérias/metabolismo , Fibras na Dieta , Fezes/microbiologia , Fermentação , Galactanos/química , Humanos , Larix/química
10.
Genes (Basel) ; 12(7)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206279

RESUMO

Guar (Cyamopsis tetragonoloba (L.) Taub.) is an annual legume crop native to India and Pakistan. Seeds of the plant serve as a source of galactomannan polysaccharide (guar gum) used in the food industry as a stabilizer (E412) and as a gelling agent in oil and gas fracturing fluids. There were several attempts to introduce this crop to countries of more northern latitudes. However, guar is a plant of a short photoperiod, therefore, its introduction, for example, to Russia is complicated by a long day length during the growing season. Breeding of new guar varieties insensitive to photoperiod slowed down due to the lack of information on functional molecular markers, which, in turn, requires information on guar genome. Modern breeding strategies, e.g., genomic predictions, benefit from integration of multi-omics approaches such as transcriptome, proteome and metabolome assays. Here we present an attempt to use transcriptome-metabolome integration to understand the genetic determination of flowering time variation among guar plants that differ in their photoperiod sensitivity. This study was performed on nine early- and six delayed-flowering guar varieties with the goal to find a connection between 63 metabolites and 1,067 differentially expressed transcripts using Shiny GAM approach. For the key biomarker of flowering in guar myo-inositol we also evaluated the KEGG biochemical pathway maps available for Arabidopsis thaliana. We found that the phosphatidylinositol signaling pathway is initiated in guar plants that are ready for flowering through the activation of the phospholipase C (PLC) gene, resulting in an exponential increase in the amount of myo-inositol in its free form observed on GC-MS chromatograms. The signaling pathway is performed by suppression of myo-inositol phosphate kinases (phosphorylation) and alternative overexpression of phosphatases (dephosphorylation). Our study suggests that metabolome and transcriptome information taken together, provide valuable information about biomarkers that can be used as a tool for marker-assisted breeding, metabolomics and functional genomics of this important legume crop.


Assuntos
Cyamopsis/genética , Redes e Vias Metabólicas/genética , Metaboloma/genética , Transcriptoma/genética , Biomarcadores/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Galactanos/genética , Galactanos/metabolismo , Perfilação da Expressão Gênica , Humanos , Mananas/genética , Mananas/metabolismo , Fotoperíodo , Desenvolvimento Vegetal/genética , Gomas Vegetais/genética , Gomas Vegetais/metabolismo
11.
Food Funct ; 12(17): 8181-8195, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34291785

RESUMO

This study aims to evaluate the colour texture, flow, viscoelastic, sensory, and simulated mastication properties, in the presence and absence of artificial saliva, of texture-modified Spanish sauce at different temperatures (25 °C, 37 °C and/or 55 °C). Sauce texture was modified using five hydrocolloids (modified starch (MS), guar gum (GG), tara gum (TG), sodium carboxymethylcellulose (CMC), and chia seed mucilage (CSM) as an alternative texturing agent), achieving two well-differentiated consistencies: honey-like and pudding-like. The MS, GG, TG and CSM sauces showed greater consistency, firmness, stiffness, and resistance to flow than the CMC samples. Furthermore, the internal structure of CMC sauces was the most affected by temperature changes. The addition of saliva decreased the apparent viscosity, consistency, and adhesiveness of the sauces. Among the samples studied, the GG and CSM texture-modified sauces would be suitable for dysphagic patients because of their good elasticity, relatively high resistance to deformation and structural stability, as well as better resistance to salivary α-amylase action. However, CSM sauces obtained the lowest sensory attribute scores. This work opens the door to the use of CSM as a texturing agent and demonstrates the importance of considering not only the hydrocolloid type and consistency level, but also the administration temperature of dysphagia-oriented products. Selecting a suitable texturing agent is of great importance for safe and easy swallowing by dysphagic patients.


Assuntos
Mastigação , Paladar , Adulto , Afasia/metabolismo , Feminino , Aditivos Alimentares/metabolismo , Qualidade dos Alimentos , Galactanos/metabolismo , Humanos , Masculino , Mananas/metabolismo , Pessoa de Meia-Idade , Gomas Vegetais/metabolismo , Reologia , Saliva/metabolismo , Viscosidade , Adulto Jovem
12.
Food Funct ; 12(17): 8017-8025, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34269784

RESUMO

The purpose of this study was to investigate the effect of starch-hydrocolloid (gum arabic, xanthan gum, and guar gum) complexes with heat-moisture treatment (HMT) on in vivo digestibility. In vivo digestibility experiments revealed that the body weight, liver weight, and fat index of mice in the intervention group were significantly reduced compared with those in the high-fat group. Glucose tolerance improved, and blood lipid levels, liver and adipose tissue morphology returned to normal. The results of mRNA expression levels showed that the intervention of corn starch-hydrocolloid complexes after HMT down-regulated the expression level of genes related to fat synthesis compared with the high-fat group, which could decrease lipid deposition and stabilize blood lipid levels. Results revealed that starch-xanthan gum complex (1 : 40 ratio) with HMT could markedly reduce the digestibility of starch. Overall, this study provides new ideas for the application of low-glycemic-index and functional foods.


Assuntos
Amido/metabolismo , Animais , Coloides/química , Coloides/metabolismo , Digestão , Manipulação de Alimentos , Galactanos/química , Galactanos/metabolismo , Índice Glicêmico , Goma Arábica/química , Goma Arábica/metabolismo , Temperatura Alta , Lipídeos/sangue , Masculino , Mananas/química , Mananas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Gomas Vegetais/química , Gomas Vegetais/metabolismo , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo , Amido/química
13.
Carbohydr Polym ; 267: 118219, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34119173

RESUMO

Polygonatum cyrtonema is a known tonic herb in Chinese Materia Medica, extensively consumed in China, but the structure and activity of its polysaccharide components remain to be clarified. Herein, two new polysaccharides (a fructan and a galactan) were purified from the dried and the processed P. cyrtonema rhizome, respectively. Structural analysis suggested that the fructan consisted of a (2 â†’ 6) linked ß-d-Fruf residues backbone with an internal α-d-Glcp residue and two (2 â†’ 1) linked ß-d-Fruf residues branches, and that the galactan was a (1 â†’ 4)-ß-d-galactan branched with a single ß-d-galactose at C-6 at about every nine residues in its main chain. The bioactive assay showed that the fructan and the galactan remarkably promoted growth of Bifidobacterium and Lactobacillus strains, indicating that they possess prebiotic activity. These findings may help expand the application of the polysaccharides from the tonic herb P. cyrtonema as functional ingredients in food products.


Assuntos
Frutanos/química , Frutanos/metabolismo , Galactanos/química , Galactanos/metabolismo , Polygonatum/química , Bifidobacterium/metabolismo , Sequência de Carboidratos , Frutanos/isolamento & purificação , Galactanos/isolamento & purificação , Lactobacillus/metabolismo , Peso Molecular , Prebióticos
14.
Gene ; 791: 145727, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34010707

RESUMO

Cluster bean (Guar) is the major source of industrial gum. Knowledge on the molecular events regulating galactomannan gum accumulation in guar will pave way for accelerated development of gummy guar genotypes. RNA Seq analysis in the immature seeds of contrasting cluster bean genotypes HGS 563 (gum type) and Pusa Navbahar (vegetable type) resulted in the generation of 19,855,490 and 21,488,472 quality reads. Data analysis identified 4938 differentially expressed genes between the gummy vs vegetable genotypes. A set of 2241 genes were up-regulated and 2587 genes were down-regulated in gummy guar. Significant up-regulation of genes involved in the biosynthesis of galactomannan and cell wall storage polysaccharides was observed in the gummy HGS 563. Genes involved in carotenoids, flavonoids, non mevalonic acid, terpenoids, and wax metabolism were also up-regulated in HGS 563. Mannose and galactose were the major nucleotide sugars in Pusa Navbahar and HGS 563 immature seeds. Immature seeds of HGS 563 showed high concentration of mannose and galactose accumulation compared to Pusa Navbahar. qRT-PCR analysis of selected genes confirmed the findings of transcriptome data.


Assuntos
Cyamopsis/genética , Cyamopsis/metabolismo , Galactanos/genética , Mananas/genética , Gomas Vegetais/genética , Cyamopsis/crescimento & desenvolvimento , Galactanos/metabolismo , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Mananas/metabolismo , Metaboloma/genética , Metabolômica/métodos , Gomas Vegetais/metabolismo , Polissacarídeos/metabolismo , Sementes/genética , Transcriptoma/genética
15.
BMC Plant Biol ; 21(1): 245, 2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34051740

RESUMO

BACKGROUND: Arabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone. Type II AGs are necessary for plant growth and critically important for the establishment of normal cellular functions. Despite the importance of type II AGs in plant development, our understanding of the underlying role of these glycans/sugar residues in mucilage formation and seed coat epidermal cell development is poorly understood and far from complete. One such sugar residue is the glucuronic acid residues of AGPs that are transferred onto AGP glycans by the action of ß-glucuronosyltransferase genes/enzymes. RESULTS: Here, we have characterized two ß-glucuronosyltransferase genes, GLCAT14A and GLCAT14C, that are involved in the transfer of ß-glucuronic acid (GlcA) to type II AGs. Using a reverse genetics approach, we observed that glcat14a-1 mutants displayed subtle alterations in mucilage pectin homogalacturonan (HG) compared to wild type (WT), while glcat14a-1glcat14c-1 mutants displayed much more severe mucilage phenotypes, including loss of adherent mucilage and significant alterations in cellulose ray formation and seed coat morphology. Monosaccharide composition analysis showed significant alterations in the sugar amounts of glcat14a-1glcat14c-1 mutants relative to WT in the adherent and non-adherent seed mucilage. Also, a reduction in total mucilage content was observed in glcat14a-1glcat14c-1 mutants relative to WT. In addition, glcat14a-1glcat14c-1 mutants showed defects in pectin formation, calcium content and the degree of pectin methyl-esterification (DM) as well as reductions in crystalline cellulose content and seed size. CONCLUSIONS: These results raise important questions regarding cell wall polymer interactions and organization during mucilage formation. We propose that the enzymatic activities of GLCAT14A and GLCAT14C play partially redundant roles and are required for the organization of the mucilage matrix and seed size in Arabidopsis thaliana. This work brings us a step closer towards identifying potential gene targets for engineering plant cell walls for industrial applications.


Assuntos
Arabidopsis/enzimologia , Galactanos/metabolismo , Glucuronosiltransferase/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cálcio/metabolismo , Parede Celular/metabolismo , Celulose/metabolismo , Esterificação , Glucuronosiltransferase/genética , Monossacarídeos/metabolismo , Pectinas/metabolismo , Fenótipo , Polissacarídeos/metabolismo , Sementes/enzimologia , Sementes/genética
16.
Appl Environ Microbiol ; 87(14): e0271920, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33990299

RESUMO

The cellulolytic insect symbiont bacterium Streptomyces sp. strain SirexAA-E secretes a suite of carbohydrate-active enzymes (CAZymes), which are involved in the degradation of various polysaccharides in the plant cell wall, in response to the available carbon sources. Here, we examined a poorly understood response of this bacterium to mannan, one of the major plant cell wall components. SirexAA-E grew well on mannose, carboxymethyl cellulose (CMC), and locust bean gum (LBG) as sole carbon sources in the culture medium. The secreted proteins from each culture supernatant were tested for their polysaccharide-degrading ability, and the composition of secreted CAZymes in each sample was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that mannose, LBG, and CMC induced the secretion of mannan and cellulose-degrading enzymes. Interestingly, two α-1,2-mannosidases were abundantly secreted during growth on mannose and LBG. Using genomic analysis, we found a unique 12-bp palindromic sequence motif at 4 locations in the SirexAA-E genome, two of which were found upstream of the above-mentioned α-1,2-mannosidase genes, along with a newly identified mannose and mannobiose-responsive transcriptional regulator, SsManR. Furthermore, the previously reported cellobiose-responsive repressor, SsCebR, was determined to also use mannobiose as an effector ligand. To test whether mannobiose induces the sets of genes under the control of the two regulators, SirexAA-E was grown on mannobiose, and the secretome composition was analyzed. As hypothesized, the composition of the mannobiose secretome combined sets of CAZymes found in both LBG and CMC secretomes, and thus they are likely under the regulation of both SsManR and SsCebR. IMPORTANCE Streptomyces sp. SirexAA-E, a microbial symbiont of biomass-harvesting insects, secretes a suite of polysaccharide-degrading enzymes dependent on the available carbon sources. However, the response of this bacterium to mannan has not been documented. In this study, we investigated the response of this bacterium to mannose, mannobiose, and galactomannan (LBG). By combining biochemical, proteomic, and genomic approaches, we discovered a novel mannose and mannobiose responsive transcriptional regulator, SsManR, which selectively regulates three α-1,2-mannosidase-coding genes. We also demonstrated that the previously described cellobiose responsive regulator, SsCebR, could use mannobiose as an effector ligand. Overall, our findings suggest that the Streptomyces sp. SirexAA-E responds to mannose and mannooligosaccharides through two different transcriptional repressors that regulate the secretion of the plant cell wall-degrading enzymes to extract carbon sources in the host environment.


Assuntos
Proteínas de Bactérias/metabolismo , Mananas/metabolismo , Manose/metabolismo , Streptomyces/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/genética , Carboximetilcelulose Sódica/metabolismo , Galactanos/metabolismo , Galactose/análogos & derivados , Insetos/microbiologia , Manosidases/genética , Manosidases/metabolismo , Gomas Vegetais/metabolismo , Streptomyces/crescimento & desenvolvimento , Fatores de Transcrição/genética
17.
Mar Drugs ; 19(5)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946151

RESUMO

Sulfated galactans (SG) isolated from red alga Gracilaria fisheri have been reported to inhibit the growth of cholangiocarcinoma (CCA) cells, which was similar to the epidermal growth factor receptor (EGFR)-targeted drug, cetuximab. Herein, we studied the anti-cancer potency of SG compared to cetuximab. Biological studies demonstrated SG and cetuximab had similar inhibition mechanisms in CCA cells by down-regulating EGFR/ERK pathway, and the combined treatment induced a greater inhibition effect. The molecular docking study revealed that SG binds to the dimerization domain of EGFR, and this was confirmed by dimerization assay, which showed that SG inhibited ligand-induced EGFR dimer formation. Synchrotron FTIR microspectroscopy was employed to examine alterations in cellular macromolecules after drug treatment. The SR-FTIR-MS elicited similar spectral signatures of SG and cetuximab, pointing towards the bands of RNA/DNA, lipids, and amide I vibrations, which were inconsistent with the changes of signaling proteins in CCA cells after drug treatment. Thus, this study demonstrates the underlined anti-cancer mechanism of SG by interfering with EGFR dimerization. In addition, we reveal that FTIR signature spectra offer a useful tool for screening anti-cancer drugs' effect.


Assuntos
Antineoplásicos/farmacologia , Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Galactanos/farmacologia , Simulação de Acoplamento Molecular , Espectroscopia de Infravermelho com Transformada de Fourier , Compostos de Enxofre/farmacologia , Antineoplásicos/metabolismo , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cetuximab/farmacologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Galactanos/metabolismo , Humanos , Microespectrofotometria , Ligação Proteica , Multimerização Proteica , Transdução de Sinais , Compostos de Enxofre/metabolismo , Síncrotrons
18.
Int J Mol Sci ; 22(8)2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33920014

RESUMO

Galectins represent ß-galactoside-binding proteins with numerous functions. Due to their role in tumor progression, human galectins-1, -3 and -7 (Gal-1, -3 and -7) are potential targets for cancer therapy. As plant derived glycans might act as galectin inhibitors, we prepared galactans by partial degradation of plant arabinogalactan-proteins. Besides commercially purchased galectins, we produced Gal-1 and -7 in a cell free system and tested binding capacities of the galectins to the galactans by biolayer-interferometry. Results for commercial and cell-free expressed galectins were comparable confirming functionality of the cell-free produced galectins. Our results revealed that galactans from Echinacea purpurea bind to Gal-1 and -7 with KD values of 1-2 µM and to Gal-3 slightly stronger with KD values between 0.36 and 0.70 µM depending on the sensor type. Galactans from the seagrass Zostera marina with higher branching of the galactan and higher content of uronic acids showed stronger binding to Gal-3 (0.08-0.28 µM) compared to galactan from Echinacea. The results contribute to knowledge on interactions between plant polysaccharides and galectins. Arabinogalactan-proteins have been identified as a new source for production of galactans with possible capability to act as galectin inhibitors.


Assuntos
Galectina 1/genética , Galectina 3/genética , Galectinas/genética , Sistema Livre de Células , Galactanos/química , Galactanos/metabolismo , Galectina 1/química , Galectina 3/química , Galectinas/química , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Ligação Proteica , Zosteraceae/química
19.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33879617

RESUMO

Mycobacterium tuberculosis, one of the deadliest pathogens in human history, is distinguished by a unique, multilayered cell wall, which offers the bacterium a high level of protection from the attacks of the host immune system. The primary structure of the cell wall core, composed of covalently linked peptidoglycan, branched heteropolysaccharide arabinogalactan, and mycolic acids, is well known, and numerous enzymes involved in the biosynthesis of its components are characterized. The cell wall biogenesis takes place at both cytoplasmic and periplasmic faces of the plasma membrane, and only recently some of the specific transport systems translocating the metabolic intermediates between these two compartments have been characterized [M. Jackson, C. M. Stevens, L. Zhang, H. I. Zgurskaya, M. Niederweis, Chem. Rev., 10.1021/acs.chemrev.0c00869 (2020)]. In this work, we use CRISPR interference methodology in Mycobacterium smegmatis to functionally characterize an ATP-binding cassette (ABC) transporter involved in the translocation of galactan precursors across the plasma membrane. We show that genetic knockdown of the transmembrane subunit of the transporter results in severe morphological changes and the accumulation of an aberrantly long galactan precursor. Based on similarities with structures and functions of specific O-antigen ABC transporters of gram-negative bacteria [C. Whitfield, D. M. Williams, S. D. Kelly, J. Biol. Chem. 295, 10593-10609 (2020)], we propose a model for coupled synthesis and export of the galactan polymer precursor in mycobacteria.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Galactanos/metabolismo , Lipopolissacarídeos/metabolismo , Mycobacterium smegmatis/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Modelos Moleculares , Mycobacterium smegmatis/genética
20.
Appl Environ Microbiol ; 87(10)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33674431

RESUMO

Gum arabic arabinogalactan (AG) protein (AGP) is a unique dietary fiber that is degraded and assimilated by only specific strains of Bifidobacterium longum subsp. longum Here, we identified a novel 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052 and classified it into glycoside hydrolase family 39 (GH39). GAfase released α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP and ß-l-Arap-(1→3)-l-Ara from larch AGP, and the α-d-Galp-(1→3)-l-Ara release activity was found to be 594-fold higher than that of ß-l-Arap-(1→3)-l-Ara. The GAfase gene was part of a gene cluster that included genes encoding a GH36 α-galactosidase candidate and ABC transporters for the assimilation of the released α-d-Galp-(1→3)-l-Ara in B. longum Notably, when α-d-Galp-(1→3)-l-Ara was removed from gum arabic AGP, it was assimilated by both B. longum JCM7052 and the nonassimilative B. longum JCM1217, suggesting that the removal of α-d-Galp-(1→3)-l-Ara from gum arabic AGP by GAfase permitted the cooperative action with type II AG degradative enzymes in B. longum The present study provides new insight into the mechanism of gum arabic AGP degradation in B. longum IMPORTANCE Bifidobacteria harbor numerous carbohydrate-active enzymes that degrade several dietary fibers in the gastrointestinal tract. B. longum JCM7052 is known to exhibit the ability to assimilate gum arabic AGP, but the key enzyme involved in the degradation of gum arabic AGP remains unidentified. Here, we cloned and characterized a GH39 3-O-α-d-galactosyl-α-l-arabinofuranosidase (GAfase) from B. longum JCM7052. The enzyme was responsible for the release of α-d-Galp-(1→3)-l-Ara and ß-l-Arap-(1→3)-l-Ara from gum arabic AGP. The presence of a gene cluster including the GAfase gene is specifically observed in gum arabic AGP assimilative strains. However, GAfase carrier strains may affect GAfase noncarrier strains that express other type II AG degradative enzymes. These findings provide insights into the bifidogenic effect of gum arabic AGP.


Assuntos
Proteínas de Bactérias/genética , Bifidobacterium/enzimologia , Glicosídeo Hidrolases/genética , Proteínas de Bactérias/metabolismo , Bifidobacterium/genética , Galactanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Goma Arábica
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