Taxonomic and functional stability overrules seasonality in polar benthic microbiomes.

Coastal shelf sediments are hot spots of organic matter mineralization. They receive up to 50% of primary production, which, in higher latitudes, is strongly seasonal. Polar and temperate benthic bacterial communities, however, show a stable composition based on comparative 16S rRNA gene sequencing despite different microbial activity levels. Here, we aimed to resolve this contradiction by identifying seasonal changes at the functional level, in particular with respect to algal polysaccharide degradation genes, by combining metagenomics, metatranscriptomics, and glycan analysis in sandy surface sediments from Isfjorden, Svalbard. Gene expressions of diverse carbohydrate-active enzymes changed between winter and spring. For example, ß-1,3-glucosidases (e.g. GH30, GH17, GH16) degrading laminarin, an energy storage molecule of algae, were elevated in spring, while enzymes related to α-glucan degradation were expressed in both seasons with maxima in winter (e.g. GH63, GH13_18, and GH15). Also, the expression of GH23 involved in peptidoglycan degradation was prevalent, which is in line with recycling of bacterial biomass. Sugar extractions from bulk sediments were low in concentrations during winter but higher in spring samples, with glucose constituting the largest fraction of measured monosaccharides (84% ± 14%). In porewater, glycan concentrations were ~18-fold higher than in overlying seawater (1107 ± 484 vs. 62 ± 101 µg C l-1) and were depleted in glucose. Our data indicate that microbial communities in sandy sediments digest and transform labile parts of photosynthesis-derived particulate organic matter and likely release more stable, glucose-depleted residual glycans of unknown structures, quantities, and residence times into the ocean, thus modulating the glycan composition of marine coastal waters.

Carbohydrates and carbohydrate degradation gene abundance and transcription in Atlantic waters of the Arctic.

Carbohydrates are chemically and structurally diverse, represent a substantial fraction of marine organic matter and are key substrates for heterotrophic microbes. Studies on carbohydrate utilisation by marine microbes have been centred on phytoplankton blooms in temperate regions, while far less is known from high-latitude waters and during later seasonal stages. Here, we combine glycan microarrays and analytical chromatography with metagenomics and metatranscriptomics to show the spatial heterogeneity in glycan distribution and potential carbohydrate utilisation by microbes in Atlantic waters of the Arctic. The composition and abundance of monomers and glycan structures in POM varied with location and depth. Complex fucose-containing sulfated polysaccharides, known to accumulate in the ocean, were consistently detected, while the more labile ß-1,3-glucan exhibited a patchy distribution. Through 'omics analysis, we identify variations in the abundance and transcription of carbohydrate degradation-related genes across samples at the community and population level. The populations contributing the most to transcription were taxonomically related to those known as primary responders and key carbohydrate degraders in temperate ecosystems, such as NS4 Marine Group and Formosa. The unique transcription profiles for these populations suggest distinct substrate utilisation potentials, with predicted glycan targets corresponding to those structurally identified in POM from the same sampling sites. By combining cutting-edge technologies and protocols, we provide insights into the carbohydrate component of the carbon cycle in the Arctic during late summer and present a high-quality dataset that will be of great value for future comparative analyses.

Dissolved storage glycans shaped the community composition of abundant bacterioplankton clades during a North Sea spring phytoplankton bloom.

BACKGROUND: Blooms of marine microalgae play a pivotal role in global carbon cycling. Such blooms entail successive blooms of specialized clades of planktonic bacteria that collectively remineralize gigatons of algal biomass on a global scale. This biomass is largely composed of distinct polysaccharides, and the microbial decomposition of these polysaccharides is therefore a process of prime importance. RESULTS: In 2020, we sampled a complete biphasic spring bloom in the German Bight over a 90-day period. Bacterioplankton metagenomes from 30 time points allowed reconstruction of 251 metagenome-assembled genomes (MAGs). Corresponding metatranscriptomes highlighted 50 particularly active MAGs of the most abundant clades, including many polysaccharide degraders. Saccharide measurements together with bacterial polysaccharide utilization loci (PUL) expression data identified ß-glucans (diatom laminarin) and α-glucans as the most prominent and actively metabolized dissolved polysaccharide substrates. Both substrates were consumed throughout the bloom, with α-glucan PUL expression peaking at the beginning of the second bloom phase shortly after a peak in flagellate and the nadir in bacterial total cell counts. CONCLUSIONS: We show that the amounts and composition of dissolved polysaccharides, in particular abundant storage polysaccharides, have a pronounced influence on the composition of abundant bacterioplankton members during phytoplankton blooms, some of which compete for similar polysaccharide niches. We hypothesize that besides the release of algal glycans, also recycling of bacterial glycans as a result of increased bacterial cell mortality can have a significant influence on bacterioplankton composition during phytoplankton blooms. Video Abstract.

Viral infection switches the balance between bacterial and eukaryotic recyclers of organic matter during coccolithophore blooms.

Algal blooms are hotspots of marine primary production and play central roles in microbial ecology and global elemental cycling. Upon demise of the bloom, organic carbon is partly respired and partly transferred to either higher trophic levels, bacterial biomass production or sinking. Viral infection can lead to bloom termination, but its impact on the fate of carbon remains largely unquantified. Here, we characterize the interplay between viral infection and the composition of a bloom-associated microbiome and consequently the evolving biogeochemical landscape, by conducting a large-scale mesocosm experiment where we monitor seven induced coccolithophore blooms. The blooms show different degrees of viral infection and reveal that only high levels of viral infection are followed by significant shifts in the composition of free-living bacterial and eukaryotic assemblages. Intriguingly, upon viral infection the biomass of eukaryotic heterotrophs (thraustochytrids) rivals that of bacteria as potential recyclers of organic matter. By combining modeling and quantification of active viral infection at a single-cell resolution, we estimate that viral infection causes a 2-4 fold increase in per-cell rates of extracellular carbon release in the form of acidic polysaccharides and particulate inorganic carbon, two major contributors to carbon sinking into the deep ocean. These results reveal the impact of viral infection on the fate of carbon through microbial recyclers of organic matter in large-scale coccolithophore blooms.

Marine bacteroidetes use a conserved enzymatic cascade to digest diatom ß-mannan.

The polysaccharide ß-mannan, which is common in terrestrial plants but unknown in microalgae, was recently detected during diatom blooms. We identified a ß-mannan polysaccharide utilization locus (PUL) in the genome of the marine flavobacterium Muricauda sp. MAR_2010_75. Proteomics showed ß-mannan induced translation of 22 proteins encoded within the PUL. Biochemical and structural analyses deduced the enzymatic cascade for ß-mannan utilization. A conserved GH26 ß-mannanase with endo-activity depolymerized the ß-mannan. Consistent with the biochemistry, X-ray crystallography showed the typical TIM-barrel fold of related enzymes found in terrestrial ß-mannan degraders. Structural and biochemical analyses of a second GH26 allowed the prediction of an exo-activity on shorter manno-gluco oligosaccharides. Further analysis demonstrated exo-α-1,6-galactosidase- and endo-ß-1,4-glucanase activity of the PUL-encoded GH27 and GH5_26, respectively, indicating the target substrate is a galactoglucomannan. Epitope deletion assays with mannanases as analytic tools indicate the presence of ß-mannan in the diatoms Coscinodiscus wailesii and Chaetoceros affinis. Mannanases from the PUL were active on diatom ß-mannan and polysaccharide extracts sampled during a microalgal bloom at the North Sea. Together these results demonstrate that marine microorganisms use a conserved enzymatic cascade to degrade ß-mannans of marine and terrestrial origin and that this metabolic pathway plays a role in marine carbon cycling.

Fucoid brown algae inject fucoidan carbon into the ocean.

Brown algae annually convert gigatons of carbon dioxide into carbohydrates, including the complex extracellular matrix polysaccharide fucoidan. Due to its persistence in the environment, fucoidan is potentially a pathway for marine carbon sequestration. Rates of fucoidan secretion by brown algae remain unknown due to the challenge of identifying and quantifying complex polysaccharides in seawater. We adapted the techniques of anion exchange chromatography, enzyme-linked immunosorbent assay, and biocatalytic enzyme-based assay for detection and quantification of fucoidan. We found the brown alga Fucus vesiculosus at the Baltic Sea coast of south-west Finland to secrete 0.3% of their biomass as fucoidan per day. Dissolved fucoidan concentrations in seawater adjacent to algae reached up to 0.48 mg L-1. Fucoidan accumulated during incubations of F. vesiculosus, significantly more in light than in darkness. Maximum estimation by acid hydrolysis indicated fucoidan secretion at a rate of 28 to 40 mg C kg-1 h-1, accounting for 44 to 50% of all exuded dissolved organic carbon. Composed only of carbon, oxygen, hydrogen, and sulfur, fucoidan secretion does not consume nutrients enabling carbon sequestration independent of algal growth. Extrapolated over a year, the algae sequester more carbon into secreted fucoidan than their biomass. The global utility of fucoidan secretion is an alternative pathway for carbon dioxide removal by brown algae without the need to harvest or bury algal biomass.

Structures and functions of algal glycans shape their capacity to sequester carbon in the ocean.

Algae synthesise structurally complex glycans to build a protective barrier, the extracellular matrix. One function of matrix glycans is to slow down microorganisms that try to enzymatically enter living algae and degrade and convert their organic carbon back to carbon dioxide. We propose that matrix glycans lock up carbon in the ocean by controlling degradation of organic carbon by bacteria and other microbes not only while algae are alive, but also after death. Data revised in this review shows accumulation of algal glycans in the ocean underscoring the challenge bacteria and other microbes face to breach the glycan barrier with carbohydrate active enzymes. Briefly we also update on methods required to certify the uncertain magnitude and unknown molecular causes of glycan-controlled carbon sequestration in a changing ocean.

Biocatalytic quantification of α-glucan in marine particulate organic matter.

Marine algae drive the marine carbon cycle, converting carbon dioxide into organic material. A major component of this produced biomass is a variety of glycans. Marine α-glucans include a range of storage glycans from red and green algae, bacteria, fungi, and animals. Although these compounds are likely to account for a high amount of the carbon stored in the oceans they have not been quantified in marine samples so far. Here we present a method to extract and quantify α-glucans (and compare it with the ß-glucan laminarin) in particulate organic matter from algal cultures and environmental samples using sequential physicochemical extraction and enzymes as α-glucan-specific probes. This enzymatic assay is more specific and less susceptible to side reactions than chemical hydrolysis. Using HPAEC-PAD to detect the hydrolysis products allows for a glycan quantification in particulate marine samples down to a concentration of ≈2 µg/L. We measured glucans in three cultured microalgae as well as in marine particulate organic matter from the North Sea and western North Atlantic Ocean. While the ß-glucan laminarin from diatoms and brown algae is an essential component of marine carbon turnover, our results further indicate the significant contribution of starch-like α-glucans to marine particulate organic matter. Henceforth, the combination of glycan-linkage-specific enzymes and chromatographic hydrolysis product detection can provide a powerful tool in the exploration of marine glycans and their role in the global carbon cycle.

Structural Basis of Ligand Selectivity by a Bacterial Adhesin Lectin Involved in Multispecies Biofilm Formation.

Carbohydrate recognition by lectins governs critical host-microbe interactions. MpPA14 (Marinomonas primoryensis PA14 domain) lectin is a domain of a 1.5-MDa adhesin responsible for a symbiotic bacterium-diatom interaction in Antarctica. Here, we show that MpPA14 binds various monosaccharides, with l-fucose and N-acetylglucosamine being the strongest ligands (dissociation constant [Kd ], ∼150 µM). High-resolution structures of MpPA14 with 15 different sugars bound elucidated the molecular basis for the lectin's apparent binding promiscuity but underlying selectivity. MpPA14 mediates strong Ca2+-dependent interactions with the 3,4-diols of l-fucopyranose and glucopyranoses, and it binds other sugars via their specific minor isomers. Thus, MpPA14 only binds polysaccharides like branched glucans and fucoidans with these free end groups. Consistent with our findings, adhesion of MpPA14 to diatom cells was selectively blocked by l-fucose, but not by N-acetyl galactosamine. The MpPA14 lectin homolog present in a Vibrio cholerae adhesin was produced and was shown to have the same sugar binding preferences as MpPA14. The pathogen's lectin was unable to effectively bind the diatom in the presence of fucose, thus demonstrating the antiadhesion strategy of blocking infection via ligand-based antagonists.IMPORTANCE Bacterial adhesins are key virulence factors that are essential for the pathogen-host interaction and biofilm formation that cause most infections. Many of the adhesin-driven cell-cell interactions are mediated by lectins. Our study reveals for the first time the molecular basis underlying the binding selectivity of a common bacterial adhesin lectin from the marine bacterium Marinomonas primoryensis, homologs of which are found in both environmental and pathogenic species. The lectin-ligand interactions illustrated at the atomic level guided the identification of a ligand that serves as an inhibitor to block bacterium-host adhesion. With conventional bactericidal antibiotics losing their potency due to resistance, our work gives critical insight into an antiadhesion strategy to treat bacterial infections.

Diatom fucan polysaccharide precipitates carbon during algal blooms.

The formation of sinking particles in the ocean, which promote carbon sequestration into deeper water and sediments, involves algal polysaccharides acting as an adhesive, binding together molecules, cells and minerals. These as yet unidentified adhesive polysaccharides must resist degradation by bacterial enzymes or else they dissolve and particles disassemble before exporting carbon. Here, using monoclonal antibodies as analytical tools, we trace the abundance of 27 polysaccharide epitopes in dissolved and particulate organic matter during a series of diatom blooms in the North Sea, and discover a fucose-containing sulphated polysaccharide (FCSP) that resists enzymatic degradation, accumulates and aggregates. Previously only known as a macroalgal polysaccharide, we find FCSP to be secreted by several globally abundant diatom species including the genera Chaetoceros and Thalassiosira. These findings provide evidence for a novel polysaccharide candidate to contribute to carbon sequestration in the ocean.

Separating Golgi Proteins from Cis to Trans Reveals Underlying Properties of Cisternal Localization.

The order of enzymatic activity across Golgi cisternae is essential for complex molecule biosynthesis. However, an inability to separate Golgi cisternae has meant that the cisternal distribution of most resident proteins, and their underlying localization mechanisms, are unknown. Here, we exploit differences in surface charge of intact cisternae to perform separation of early to late Golgi subcompartments. We determine protein and glycan abundance profiles across the Golgi; over 390 resident proteins are identified, including 136 new additions, with over 180 cisternal assignments. These assignments provide a means to better understand the functional roles of Golgi proteins and how they operate sequentially. Protein and glycan distributions are validated in vivo using high-resolution microscopy. Results reveal distinct functional compartmentalization among resident Golgi proteins. Analysis of transmembrane proteins shows several sequence-based characteristics relating to pI, hydrophobicity, Ser abundance, and Phe bilayer asymmetry that change across the Golgi. Overall, our results suggest that a continuum of transmembrane features, rather than discrete rules, guide proteins to earlier or later locations within the Golgi stack.

Biphasic cellular adaptations and ecological implications of Alteromonas macleodii degrading a mixture of algal polysaccharides.

Algal polysaccharides are an important bacterial nutrient source and central component of marine food webs. However, cellular and ecological aspects concerning the bacterial degradation of polysaccharide mixtures, as presumably abundant in natural habitats, are poorly understood. Here, we contextualize marine polysaccharide mixtures and their bacterial utilization in several ways using the model bacterium Alteromonas macleodii 83-1, which can degrade multiple algal polysaccharides and contributes to polysaccharide degradation in the oceans. Transcriptomic, proteomic and exometabolomic profiling revealed cellular adaptations of A. macleodii 83-1 when degrading a mix of laminarin, alginate and pectin. Strain 83-1 exhibited substrate prioritization driven by catabolite repression, with initial laminarin utilization followed by simultaneous alginate/pectin utilization. This biphasic phenotype coincided with pronounced shifts in gene expression, protein abundance and metabolite secretion, mainly involving CAZymes/polysaccharide utilization loci but also other functional traits. Distinct temporal changes in exometabolome composition, including the alginate/pectin-specific secretion of pyrroloquinoline quinone, suggest that substrate-dependent adaptations influence chemical interactions within the community. The ecological relevance of cellular adaptations was underlined by molecular evidence that common marine macroalgae, in particular Saccharina and Fucus, release mixtures of alginate and pectin-like rhamnogalacturonan. Moreover, CAZyme microdiversity and the genomic predisposition towards polysaccharide mixtures among Alteromonas spp. suggest polysaccharide-related traits as an ecophysiological factor, potentially relating to distinct 'carbohydrate utilization types' with different ecological strategies. Considering the substantial primary productivity of algae on global scales, these insights contribute to the understanding of bacteria-algae interactions and the remineralization of chemically diverse polysaccharide pools, a key step in marine carbon cycling.

Molecular recognition of the beta-glucans laminarin and pustulan by a SusD-like glycan-binding protein of a marine Bacteroidetes.

Marine bacteria catabolize carbohydrate polymers of algae, which synthesize these structurally diverse molecules in ocean surface waters. Although algal glycans are an abundant carbon and energy source in the ocean, the molecular details that enable specific recognition between algal glycans and bacterial degraders remain largely unknown. Here we characterized a surface protein, GMSusD from the planktonic Bacteroidetes-Gramella sp. MAR_2010_102 that thrives during algal blooms. Our biochemical and structural analyses show that GMSusD binds glucose polysaccharides such as branched laminarin and linear pustulan. The 1.8 Å crystal structure of GMSusD indicates that three tryptophan residues form the putative glycan-binding site. Mutagenesis studies confirmed that these residues are crucial for laminarin recognition. We queried metagenomes of global surface water datasets for the occurrence of SusD-like proteins and found sequences with the three structurally conserved residues in different locations in the ocean. The molecular selectivity of GMSusD underscores that specific interactions are required for laminarin recognition. In conclusion, our findings provide insight into the molecular details of ß-glucan binding by GMSusD and our bioinformatic analysis reveals that this molecular interaction may contribute to glucan cycling in the surface ocean.

Carbohydrate Microarray Technology Applied to High-Throughput Mapping of Plant Cell Wall Glycans Using Comprehensive Microarray Polymer Profiling (CoMPP).

Cell walls are an important feature of plant cells and a major component of the plant glycome. They have both structural and physiological functions and are critical for plant growth and development. The diversity and complexity of these structures demand advanced high-throughput techniques to answer questions about their structure, functions and roles in both fundamental and applied scientific fields. Microarray technology provides both the high-throughput and the feasibility aspects required to meet that demand. In this chapter, some of the most recent microarray-based techniques relating to plant cell walls are described together with an overview of related contemporary techniques applied to carbohydrate microarrays and their general potential in glycoscience. A detailed experimental procedure for high-throughput mapping of plant cell wall glycans using the comprehensive microarray polymer profiling (CoMPP) technique is included in the chapter and provides a good example of both the robust and high-throughput nature of microarrays as well as their applicability to plant glycomics.

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition.

The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens The data identified six previously unidentified CBM families that targeted ß-glucans, ß-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize ß-glucans and ß-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

Cell wall composition profiling of parasitic giant dodder (Cuscuta reflexa) and its hosts: a priori differences and induced changes.

Host plant penetration is the gateway to survival for holoparasitic Cuscuta and requires host cell wall degradation. Compositional differences of cell walls may explain why some hosts are amenable to such degradation while others can resist infection. Antibody-based techniques for comprehensive profiling of cell wall epitopes and cell wall-modifying enzymes were applied to several susceptible hosts and a resistant host of Cuscuta reflexa and to the parasite itself. Infected tissue of Pelargonium zonale contained high concentrations of de-esterified homogalacturonans in the cell walls, particularly adjacent to the parasite's haustoria. High pectinolytic activity in haustorial extracts and high expression levels of pectate lyase genes suggest that the parasite contributes directly to wall remodeling. Mannan and xylan concentrations were low in P. zonale and in five susceptible tomato introgression lines, but high in the resistant Solanum lycopersicum cv M82, and in C. reflexa itself. Knowledge of the composition of resistant host cell walls and the parasite's own cell walls is useful in developing strategies to prevent infection by parasitic plants.

A new versatile microarray-based method for high throughput screening of carbohydrate-active enzymes.

Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing together with associated bioinformatics tools have identified vast numbers of putative carbohydrate-degrading and -modifying enzymes including glycoside hydrolases and lytic polysaccharide monooxygenases. However, there is a paucity of methods for rapidly screening the activities of these enzymes. By combining the multiplexing capacity of carbohydrate microarrays with the specificity of molecular probes, we have developed a sensitive, high throughput, and versatile semiquantitative enzyme screening technique that requires low amounts of enzyme and substrate. The method can be used to assess the activities of single enzymes, enzyme mixtures, and crude culture broths against single substrates, substrate mixtures, and biomass samples. Moreover, we show that the technique can be used to analyze both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified uncharacterized enzymes and by screening enzyme activities from fungal culture broths.

Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1.

Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K M was 1.81 mg/mL and k cat was 1.82 × 10(7) U M(-1). The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn(2+) addition caused regain of the activity, but Zn(2+)addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1' at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing.

Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation.

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are known to carry out oxidative cleavage of glycoside bonds in chitin and cellulose, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. Because biomass-degrading microorganisms tend to produce a plethora of LPMOs, and considering the complexity and copolymeric nature of the plant cell wall, it has been speculated that some LPMOs may act on other substrates, in particular the hemicelluloses that tether to cellulose microfibrils. We demonstrate that an LPMO from Neurospora crassa, NcLPMO9C, indeed degrades various hemicelluloses, in particular xyloglucan. This activity was discovered using a glycan microarray-based screening method for detection of substrate specificities of carbohydrate-active enzymes, and further explored using defined oligomeric hemicelluloses, isolated polymeric hemicelluloses and cell walls. Products generated by NcLPMO9C were analyzed using high performance anion exchange chromatography and multidimensional mass spectrometry. We show that NcLPMO9C generates oxidized products from a variety of substrates and that its product profile differs from those of hydrolytic enzymes acting on the same substrates. The enzyme particularly acts on the glucose backbone of xyloglucan, accepting various substitutions (xylose, galactose) in almost all positions. Because the attachment of xyloglucan to cellulose hampers depolymerization of the latter, it is possible that the beneficial effect of the LPMOs that are present in current commercial cellulase mixtures in part is due to hitherto undetected LPMO activities on recalcitrant hemicellulose structures.

Carbohydrate microarrays in plant science.

Almost all plant cells are surrounded by glycan-rich cell walls, which form much of the plant body and collectively are the largest source of biomass on earth. Plants use polysaccharides for support, defense, signaling, cell adhesion, and as energy storage, and many plant glycans are also important industrially and nutritionally. Understanding the biological roles of plant glycans and the effective exploitation of their useful properties requires a detailed understanding of their structures, occurrence, and molecular interactions. Microarray technology has revolutionized the massively high-throughput analysis of nucleotides, proteins, and increasingly carbohydrates. Using microarrays, the abundance of and interactions between hundreds and thousands of molecules can be assessed simultaneously using very small amounts of analytes. Here we show that carbohydrate microarrays are multifunctional tools for plant research and can be used to map glycan populations across large numbers of samples to screen antibodies, carbohydrate binding proteins, and carbohydrate binding modules and to investigate enzyme activities.