Dataset Information

Multicolor 3D-dSTORM Reveals Native-State Ultrastructure of Polysaccharides' Network during Plant Cell Wall Assembly.

ABSTRACT: The plant cell wall, a form of the extracellular matrix, is a complex and dynamic network of polymers mediating a plethora of physiological functions. How polysaccharides assemble into a coherent and heterogeneous matrix remains mostly undefined. Further progress requires improved molecular-level visualization methods that would gain a deeper understanding of the cell wall nanoarchitecture. dSTORM, a type of super-resolution microscopy, permits quantitative nanoimaging of the cell wall. However, due to the lack of single-cell model systems and the requirement of tissue-level imaging, its use in plant science is almost absent. Here we overcome these limitations; we compare two methods to achieve three-dimensional dSTORM and identify optimal photoswitching dyes for tissue-level multicolor nanoscopy. Combining dSTORM with spatial statistics, we reveal and characterize the ultrastructure of three major polysaccharides, callose, mannan, and cellulose, in the plant cell wall precursor and provide evidence for cellulose structural re-organization related to callose content.

SUBMITTER: Peaucelle A

PROVIDER: S-EPMC7733027 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Multicolor 3D-dSTORM Reveals Native-State Ultrastructure of Polysaccharides' Network during Plant Cell Wall Assembly.

Peaucelle Alexis A Wightman Raymond R Haas Kalina Tamara KT

iScience 20201127 12

The plant cell wall, a form of the extracellular matrix, is a complex and dynamic network of polymers mediating a plethora of physiological functions. How polysaccharides assemble into a coherent and heterogeneous matrix remains mostly undefined. Further progress requires improved molecular-level visualization methods that would gain a deeper understanding of the cell wall nanoarchitecture. dSTORM, a type of super-resolution microscopy, permits quantitative nanoimaging of the cell wall. However, ...[more]

PMID: 33336161

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Project description:Symbiotic bacteria inhabiting the human gut have evolved under intense pressure to utilize complex carbohydrates, primarily plant cell wall glycans in our diets. These polysaccharides are not digested by human enzymes, but are processed to absorbable short chain fatty acids by gut bacteria. The Bacteroidetes, one of two dominant bacterial phyla in the adult gut, possess broad glycan-degrading abilities. These species use a series of membrane protein complexes, termed Sus-like systems, for catabolism of many complex carbohydrates. However, the role of these systems in degrading the chemically diverse repertoire of plant cell wall glycans remains unknown. Here we show that two closely related human gut Bacteroides, B. thetaiotaomicron and B. ovatus, are capable of utilizing nearly all of the major plant and host glycans, including rhamnogalacturonan II, a highly complex polymer thought to be recalcitrant to microbial degradation. Transcriptional profiling and gene inactivation experiments revealed the identity and specificity of the polysaccharide utilization loci (PULs) that encode individual Sus-like systems that target various plant polysaccharides. Comparative genomic analysis indicated that B. ovatus possesses several unique PULs that enable degradation of hemicellulosic polysaccharides, a phenotype absent from B. thetaiotaomicron. In contrast, the B. thetaiotaomicron genome has been shaped by increased numbers of PULs involved in metabolism of host mucin O-glycans, a phenotype that is undetectable in B. ovatus. Binding studies of the purified sensor domains of PUL-associated hybrid two-component systems in conjunction with transcriptional analyses demonstrate that complex oligosaccharides provide the regulatory cues that induce PUL activation and that each PUL is highly specific for a defined cell wall polymer. These results provide a view of how these species have diverged into different carbohydrate niches by evolving genes that target unique suites of available polysaccharides, a theme that likely applies to disparate bacteria from the gut and other habitats.

Project description:BackgroundClostridium cellulovorans is a mesophilic, cellulosome-producing bacterium containing 57 genomic cellulosomal enzyme-encoding genes. In addition to cellulosomal proteins, C. cellulovorans also secretes non-cellulosomal proteins to degrade plant cell wall polysaccharides. Unlike other cellulosome-producing Clostridium species, C. cellulovorans can metabolize all major plant cell wall polysaccharides (cellulose, hemicelluloses, and pectins). In this study, we performed a temporal proteome analysis of C. cellulovorans to reveal strategies underlying plant cell wall polysaccharide degradation.ResultsWe cultured C. cellulovorans with five different carbon sources (glucose, cellulose, xylan, galactomannan, and pectin) and performed proteome analysis on cellular and secreted proteins. In total, we identified 1895 cellular proteins and 875 secreted proteins. The identified unique carbohydrate-degrading enzymes corresponding to each carbon source were annotated to have specific activity against each carbon source. However, we identified pectate lyase as a unique enzyme in C. cellulovorans cultivated on xylan, which was not previously associated with xylan degradation. We performed k-means clustering analysis for elucidation of temporal changes of the cellular and secreted proteins in each carbon sources. We found that cellular proteins in most of the k-means clusters are involved in carbohydrate metabolism, amino acid metabolism, translation, or membrane transport. When xylan and pectin were used as the carbon sources, the most increasing k-means cluster contained proteins involved in the metabolism of cofactors and vitamins. In case of secreted proteins of C. cellulovorans cultured either on cellulose or xylan, galactomannan, and pectin, the clusters with the most increasing trend contained either 25 cellulosomal proteins and five non-cellulosomal proteins or 8-19 cellulosomal proteins and 9-16 non-cellulosomal proteins, respectively. These differences might reflect mechanisms for degrading cellulose of other carbon source. Co-abundance analysis of the secreted proteins revealed that proteases and protease inhibitors accumulated coordinately. This observation implies that the secreted protease inhibitors and proteases protect carbohydrate-degrading enzymes from an attack from the plant.ConclusionIn this study, we clarified, for the first time, the temporal proteome dynamics of cellular and secreted proteins in C. cellulovorans. This data will be valuable in understanding strategies employed by C. cellulovorans for degrading major plant cell wall polysaccharides.

Dataset Information

Multicolor 3D-dSTORM Reveals Native-State Ultrastructure of Polysaccharides' Network during Plant Cell Wall Assembly.

Publications

Multicolor 3D-dSTORM Reveals Native-State Ultrastructure of Polysaccharides' Network during Plant Cell Wall Assembly.

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