Architecture, Function, Regulation, and Evolution of α-Glucans Metabolic Enzymes in Prokaryotes.

Bacteria have acquired sophisticated mechanisms for assembling and disassembling polysaccharides of different chemistry. α-d-Glucose homopolysaccharides, so-called α-glucans, are the most widespread polymers in nature being key components of microorganisms. Glycogen functions as an intracellular energy storage while some bacteria also produce extracellular assorted α-glucans. The classical bacterial glycogen metabolic pathway comprises the action of ADP-glucose pyrophosphorylase and glycogen synthase, whereas extracellular α-glucans are mostly related to peripheral enzymes dependent on sucrose. An alternative pathway of glycogen biosynthesis, operating via a maltose 1-phosphate polymerizing enzyme, displays an essential wiring with the trehalose metabolism to interconvert disaccharides into polysaccharides. Furthermore, some bacteria show a connection of intracellular glycogen metabolism with the genesis of extracellular capsular α-glucans, revealing a relationship between the storage and structural function of these compounds. Altogether, the current picture shows that bacteria have evolved an intricate α-glucan metabolism that ultimately relies on the evolution of a specific enzymatic machinery. The structural landscape of these enzymes exposes a limited number of core catalytic folds handling many different chemical reactions. In this Review, we present a rationale to explain how the chemical diversity of α-glucans emerged from these systems, highlighting the underlying structural evolution of the enzymes driving α-glucan bacterial metabolism.

BnXTH1 regulates cadmium tolerance by modulating vacuolar compartmentalization and the cadmium binding capacity of cell walls in ramie (Boehmeria nivea).

Xyloglucan endotransglucosylase/hydrolases (XTH) are cell wall-modifying enzymes important in plant response to abiotic stress. However, the role of XTH in cadmium (Cd) tolerance in ramie remains largely unknown. Here, we identified and cloned BnXTH1, a member of the XTH family, in response to Cd stress in ramie. The BnXTH1 promoter (BnXTH1p) demonstrated that MeJA induces the response of BnXTH1p to Cd stress. Moreover, overexpressing BnXTH1 in Boehmeria nivea increased Cd tolerance by significantly increasing the Cd content in the cell wall and decreasing Cd inside ramie cells. Cadmium stress induced BnXTH1-expression and consequently increased xyloglucan endotransglucosylase (XET) activity, leading to high xyloglucan contents and increased hemicellulose contents in ramie. The elevated hemicellulose content increased Cd chelation onto the cell walls and reduced the level of intracellular Cd. Interestingly, overexpressing BnXTH1 significantly increased the content of Cd in vacuoles of ramie and vacuolar compartmentalization genes. Altogether, these results evidence that Cd stress induced MeJA accumulation in ramie, thus, activating BnXTH1 expression and increasing the content of xyloglucan to enhance the hemicellulose binding capacity and increase Cd chelation onto cell walls. BnXTH1 also enhances the vacuolar Cd compartmentalization and reduces the level of Cd entering the organelles and soluble solution.

Glucans and applications in drug delivery.

Glucan is a natural polysaccharide widely distributed in cereals and microorganisms that has various biological activities, including immunomodulatory, anti-infective, anti-inflammatory, and antitumor activities. In addition to wide applications in the broad fields of food, healthcare, and biomedicines, glucans hold promising potential as drug delivery carrier materials or ligands. Specifically, glucan microparticles or yeast cell wall particles are naturally enclosed vehicles with an interior cavity that can be exploited to carry and deliver drug payloads. The biological activities and targeting capacities of glucans depend largely on the recognition of glucan moieties by receptors such as dectin-1 and complement receptor 3, which are widely expressed on the cell membranes of mononuclear phagocytes, dendritic cells, neutrophils, and some lymphocytes. This review summarizes the chemical structures, sources, fundamental properties, extraction methods, and applications of these materials, with an emphasis on drug delivery. Glucans are utilized mainly as vaccine adjuvants, targeting ligands and as carrier materials for various drug entities. It is believed that glucans and glucan microparticles may be useful for the delivery of both small-molecule and macromolecular drugs, especially for potential treatment of immune-related diseases.

Saccharomyces cerevisiae CellWall Remodeling in the Absence of Knr4 and Kre6 Revealed by Nano-FourierTransform Infrared Spectroscopy.

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.

Structure-function analysis of the cyclic ß-1,2-glucan synthase from Agrobacterium tumefaciens.

The synthesis of complex sugars is a key aspect of microbial biology. Cyclic ß-1,2-glucan (CßG) is a circular polysaccharide critical for host interactions of many bacteria, including major pathogens of humans (Brucella) and plants (Agrobacterium). CßG is produced by the cyclic glucan synthase (Cgs), a multi-domain membrane protein. So far, its structure as well as the mechanism underlining the synthesis have not been clarified. Here we use cryo-electron microscopy (cryo-EM) and functional approaches to study Cgs from A. tumefaciens. We determine the structure of this complex protein machinery and clarify key aspects of CßG synthesis, revealing a distinct mechanism that uses a tyrosine-linked oligosaccharide intermediate in cycles of polymerization and processing of the glucan chain. Our research opens possibilities for combating pathogens that rely on polysaccharide virulence factors and may lead to synthetic biology approaches for producing complex cyclic sugars.

ß-1,3-Glucan recognition by Acanthamoeba castellanii as a putative mechanism of amoeba-fungal interactions.

In this study, we conducted an in-depth analysis to characterize potential Acanthamoeba castellanii (Ac) proteins capable of recognizing fungal ß-1,3-glucans. Ac specifically anchors curdlan or laminarin, indicating the presence of surface ß-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to Ac was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, Histoplasma capsulatum (Hc) G217B, possessing a ß-1,3-glucan outer layer, showed significant adhesion to Ac compared to a Hc G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble ß-1,3-glucan substantially inhibited this adhesion, indicating the involvement of ß-1,3-glucan recognition. Biotinylated ß-1,3-glucan-binding proteins from Ac exhibited higher binding to Hc G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the ß-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identified several Ac proteins capable of binding ß-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identified proteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinity to ß-1,3-glucans. These findings underscore the complexity of binding via ß-1,3-glucan and suggest the existence of alternative fungal recognition pathways in Ac.IMPORTANCEAcanthamoeba castellanii (Ac) and macrophages both exhibit the remarkable ability to phagocytose various extracellular microorganisms in their respective environments. While substantial knowledge exists on this phenomenon for macrophages, the understanding of Ac's phagocytic mechanisms remains elusive. Recently, our group identified mannose-binding receptors on the surface of Ac that exhibit the capacity to bind/recognize fungi. However, the process was not entirely inhibited by soluble mannose, suggesting the possibility of other interactions. Herein, we describe the mechanism of ß-1,3-glucan binding by A. castellanii and its role in fungal phagocytosis and survival within trophozoites, also using macrophages as a model for comparison, as they possess a well-established mechanism involving the Dectin-1 receptor for ß-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.

A CO2 sensing module modulates ß-1,3-glucan exposure in Candida albicans.

Microbial species capable of co-existing with healthy individuals, such as the commensal fungus Candida albicans, exploit multifarious strategies to evade our immune defenses. These strategies include the masking of immunoinflammatory pathogen-associated molecular patterns (PAMPs) at their cell surface. We reported previously that C. albicans actively reduces the exposure of the proinflammatory PAMP, ß-1,3-glucan, at its cell surface in response to host-related signals such as lactate and hypoxia. Here, we show that clinical isolates of C. albicans display phenotypic variability with respect to their lactate- and hypoxia-induced ß-1,3-glucan masking. We have exploited this variability to identify responsive and non-responsive clinical isolates. We then performed RNA sequencing on these isolates to reveal genes whose expression patterns suggested potential association with lactate- or hypoxia-induced ß-1,3-glucan masking. The deletion of two such genes attenuated masking: PHO84 and NCE103. We examined NCE103-related signaling further because NCE103 has been shown previously to encode carbonic anhydrase, which promotes adenylyl cyclase-protein kinase A (PKA) signaling at low CO2 levels. We show that while CO2 does not trigger ß-1,3-glucan masking in C. albicans, the Sch9-Rca1-Nce103 signaling module strongly influences ß-1,3-glucan exposure in response to hypoxia and lactate. In addition to identifying a new regulatory module that controls PAMP exposure in C. albicans, our data imply that this module is important for PKA signaling in response to environmental inputs other than CO2.IMPORTANCEOur innate immune defenses have evolved to protect us against microbial infection in part via receptor-mediated detection of "pathogen-associated molecular patterns" (PAMPs) expressed by invading microbes, which then triggers their immune clearance. Despite this surveillance, many microbial species are able to colonize healthy, immune-competent individuals, without causing infection. To do so, these microbes must evade immunity. The commensal fungus Candida albicans exploits a variety of strategies to evade immunity, one of which involves reducing the exposure of a proinflammatory PAMP (ß-1,3-glucan) at its cell surface. Most of the ß-1,3-glucan is located in the inner layer of the C. albicans cell wall, hidden by an outer layer of mannan fibrils. Nevertheless, some ß-1,3-glucan can become exposed at the fungal cell surface. However, in response to certain specific host signals, such as lactate or hypoxia, C. albicans activates an anticipatory protective response that decreases ß-1,3-glucan exposure, thereby reducing the susceptibility of the fungus to impending innate immune attack. Here, we exploited the natural phenotypic variability of C. albicans clinical isolates to identify strains that do not display the response to ß-1,3-glucan masking signals observed for the reference isolate, SC5314. Then, using genome-wide transcriptional profiling, we compared these non-responsive isolates with responsive controls to identify genes potentially involved in ß-1,3-glucan masking. Mutational analysis of these genes revealed that a sensing module that was previously associated with CO2 sensing also modulates ß-1,3-glucan exposure in response to hypoxia and lactate in this major fungal pathogen of humans.

The Ruminococcus bromii amylosome protein Sas6 binds single and double helical α-glucan structures in starch.

Resistant starch is a prebiotic accessed by gut bacteria with specialized amylases and starch-binding proteins. The human gut symbiont Ruminococcus bromii expresses Sas6 (Starch Adherence System member 6), which consists of two starch-specific carbohydrate-binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). Here, we present the crystal structures of Sas6 and of RbCBM74 bound with a double helical dimer of maltodecaose. The RbCBM74 starch-binding groove complements the double helical α-glucan geometry of amylopectin, suggesting that this module selects this feature in starch granules. Isothermal titration calorimetry and native mass spectrometry demonstrate that RbCBM74 recognizes longer single and double helical α-glucans, while RbCBM26 binds short maltooligosaccharides. Bioinformatic analysis supports the conservation of the amylopectin-targeting platform in CBM74s from resistant-starch degrading bacteria. Our results suggest that RbCBM74 and RbCBM26 within Sas6 recognize discrete aspects of the starch granule, providing molecular insight into how this structure is accommodated by gut bacteria.

Enzyme immobilization on α-1,3-glucan: development of flow reactor with fusion protein of α-1,3-glucan binding domains and histamine dehydrogenase.

α-1,3-Glucanase Agl-KA from Bacillus circulans KA-304 consists of a discoidin domain (DS1), a carbohydrate binding module family 6 (CBM6), a threonine-proline-rich-linker (TP linker), a discoidin domain (DS2), an uncharacterized domain, and a catalytic domain. The binding of DS1, CBM6, and DS2 to α-1,3-glucan can be improved in the presence of two of these three domains. In this study, DS1, CBM6, and TP linker were genetically fused to histamine dehydrogenase (HmDH) from Nocardioides simplex NBRC 12069. The fusion enzyme, AGBDs-HmDH, was expressed in Escherichia coli Rosetta 2 (DE3) and purified from the cell-free extract. AGBDs-HmDH bound to 1% micro-particle of α-1,3-glucan (diameter: less than 1 µm) and 7.5% coarse-particle of α-1,3-glucan (less than 200 µm) at about 97 % and 70% of the initial amounts of the enzyme, respectively. A reactor for flow injection analysis filled with AGBDs-HmDH immobilized on the coarse-particle of α-1,3-glucan was successfully applied to determine histamine. A linear calibration curve was observed in the range for about 0.1 to 3.0 mM histamine. These findings suggest that the combination of α-1,3-glucan and α-1,3-glucan binding domains is a candidate for novel enzyme immobilization.

Transcriptional expression of PHR2 is positively controlled by the calcium signaling transcription factor Crz1 through its binding motif in the promoter.

IMPORTANCE: The fungal cell wall consists of glucans, mannoproteins, and chitin and is essential for cell viability, morphogenesis, and pathogenesis. The enzymes of the GH72 family are responsible for ß-(1,3)-glucan elongation and branching, which is crucial for the formation of the glucan-chitin polymer at the bud neck of yeast cells. In the human fungal pathogen Candida albicans, there are five GH72 enzyme-encoding genes: PHR1, PHR2, PHR3, PGA4, and PGA5. It is known that expression of PHR1 and PHR2 is controlled by the pH-responsive Rim101 pathway through the transcription factor Rim101. In this study, we have demonstrated that the transcription expression of PHR2 is also controlled by the transcription factor Crz1 through its binding motif in the promoter. Therefore, we have uncovered a dual-control mechanism by which PHR2 expression is negatively regulated via CaRim101 through the pH-responsive pathway and positively modulated by CaCrz1 through the calcium/calcineurin signaling pathway.

Magnetic Yeast Glucan Particles for Antibody-Free Separation of Viable Macrophages from Drosophila melanogaster.

Currently available methods for cell separation are generally based on fluorescent labeling using either endogenously expressed fluorescent markers or the binding of antibodies or antibody mimetics to surface antigenic epitopes. However, such modification of the target cells represents potential contamination by non-native proteins, which may affect further cell response and be outright undesirable in applications, such as cell expansion for diagnostic or therapeutic applications, including immunotherapy. We present a label- and antibody-free method for separating macrophages from living Drosophila based on their ability to preferentially phagocytose whole yeast glucan particles (GPs). Using a novel deswelling entrapment approach based on spray drying, we have successfully fabricated yeast glucan particles with the previously unachievable content of magnetic iron oxide nanoparticles while retaining their surface features responsible for phagocytosis. We demonstrate that magnetic yeast glucan particles enable macrophage separation at comparable yields to fluorescence-activated cell sorting without compromising their viability or affecting their normal function and gene expression. The use of magnetic yeast glucan particles is broadly applicable to situations where viable macrophages separated from living organisms are subsequently used for analyses, such as gene expression, metabolomics, proteomics, single-cell transcriptomics, or enzymatic activity analysis.

Genome-wide identification and functional analysis of the cellulose synthase-like gene superfamily in common oat (Avena sativa L.).

Hemicelluloses constitute approximately one-third of the plant cell wall and can be used as a dietary fiber and food additive, and as raw materials for biofuels. Although genes involved in hemicelluloses synthesis have been investigated in some model plants, no comprehensive analysis has been conducted in common oat at present. In this study, we identified and systematically analyzed the cellulose synthase-like gene (Csl) family members in common oat and investigated them using various bioinformatics tools. The results showed that there are 76 members of the oat Csl gene family distributed on 17 chromosomes, and phylogenetic analysis indicated that the 76 Csl genes belong to the CslA, CslC, CslD, CslE, CslF, CslH, and CslJ subfamilies. A total of 14 classes of cis-acting elements were identified in the promoter regions, including hormone response, light response, cell development, and defense stress elements. The collinearity analysis identified 28 pairs of segmentally duplicated genes, most of which were found on chromosomes 2D and 6A. Expression pattern analysis showed that oat Csl genes display strong tissue-specific expression; of the 76 Csl genes, 33 were significantly up-regulated in stems and 30 were up-regulated in immature seeds. The expression of most members of the AsCsl gene family is repressed by abiotic stress, while the expression of some members is up-regulated by light. Immunoelectron microscopy shows that the product of AsCsl61, a member of CslF subfamily, mediates (1,3; 1,4)-ß-D-glucan synthesis in transgenic Arabidopsis. These findings provide a fundamental understanding of the structural, functional, and evolutionary features of the oat Csl genes and may contribute to our general understanding of hemicellulose biosynthesis. Moreover, this information will be helpful in designing experiments for genetic manipulation of mixed-linkage glucan (MLG) synthesis with the goal of quality improvement in oat.

Transcriptome analysis of the effects of different carbon dioxide concentrations on paramylon accumulation in Euglena gracilis Z.

Appropriate concentration of carbon dioxide (CO2) will promote algae growth and metabolism. Building upon this finding, the present study investigated the impact of different CO2 concentrations (5% and 20%) on the carbon sequestration capacity of E. gracilis through aeration culturing, employing a combination of physiological analyses and transcriptome analysis. The results demonstrated that under 5% CO2 concentration, the cell density of E. gracilis was 1.79 times higher than that achieved in an air culture condition, and the paramylon content of E. gracilis was found to be 6.18 times higher than that of the air group. Based on transcriptome analysis, the carbon metabolism of E. gracilis was discussed. Significant up-regulation expression of genes associated with carbon synthesis was validated by an increase in paramylon content. This study revealed that under 5% CO2 conditions, E. gracilis exhibited elevated growth rate and enhanced photosynthetic carbon assimilation efficiency.

A role for ß-1,6- and ß-1,3-glucans in kinetochore function in Saccharomyces cerevisiae.

Chromosome segregation is crucial for the faithful inheritance of DNA to the daughter cells after DNA replication. For this, the kinetochore, a megadalton protein complex, assembles on centromeric chromatin containing the histone H3 variant CENP-A, and provides a physical connection to the microtubules. Here, we report an unanticipated role for enzymes required for ß-1,6- and ß-1,3-glucan biosynthesis in regulating kinetochore function in Saccharomyces cerevisiae. These carbohydrates are the major constituents of the yeast cell wall. We found that the deletion of KRE6, which encodes a glycosylhydrolase/ transglycosidase required for ß-1,6-glucan synthesis, suppressed the centromeric defect of mutations in components of the kinetochore, foremost the NDC80 components Spc24, Spc25, the MIND component Nsl1, and Okp1, a constitutive centromere-associated network protein. Similarly, the absence of Fks1, a ß-1,3-glucan synthase, and Kre11/Trs65, a TRAPPII component, suppressed a mutation in SPC25. Genetic analysis indicates that the reduction of intracellular ß-1,6- and ß-1,3-glucans, rather than the cell wall glucan content, regulates kinetochore function. Furthermore, we found a physical interaction between Kre6 and CENP-A/Cse4 in yeast, suggesting a potential function for Kre6 in glycosylating CENP-A/Cse4 or another kinetochore protein. This work shows a moonlighting function for selected cell wall synthesis proteins in regulating kinetochore assembly, which may provide a mechanism to connect the nutritional status of the cell to cell-cycle progression and chromosome segregation.

Schizophyllan from Schizophyllum commune BRM 060008: Potential application as an inhibitor of lipase.

ß-D-glucan has significant implications in regulating lipid metabolism and preventing diseases associated with lipid accumulation. Schizophyllan (SPG) from Schizophyllum commune fungus is a commercially important ß-glucan with applications in the health food industry, pharmacy, and cosmetics. However, SPG was obtained by submerged culture of the wood-rotting and filamentous fungus S. commune BRM 060008, which may have been isolated from the Cerrado Biome of Brazil. In this study, to confirm that the polysaccharide produced by BRM 060008 strain fermentation was indeed (1→3)(1→6)-ß-D-glucan, it was purified and characterized using Fourier transform infrared spectroscopy, thermogravimetric analysis, high-performance size exclusion chromatography, nuclear magnetic resonance, and methylation analysis. The polysaccharide produced was identified as the ß-D-glucan expected with a high molecular weight (1.093 × 106 g/mol) and the thermogravimetric analysis indicated a maximum degradation temperature of ~324 °C and a 60 % residual weight, lower than commercial SPG. The molecular structure and thermal properties of the ß-D-glucan were similar to the commercial sample. Additionally, the in vitro pancreatic lipase inhibitory activity was evaluated, investigating anti-obesity and anti-lipidemic properties. The results showed unprecedented lipase inhibition activity to SPG prepared using the S. commune strain BRM 060008, making it promising for food and pharmaceutical applications.

Functional Roles of N-Terminal Domains in Pullulanase from Human Gut Lactobacillus acidophilus.

Pullulanases are multidomain α-glucan debranching enzymes with one or more N-terminal domains (NTDs) including carbohydrate-binding modules (CBMs) and domains of unknown function (DUFs). To elucidate the roles of NTDs in Lactobacillus acidophilus NCFM pullulanase (LaPul), two truncated variants, Δ41-LaPul (lacking CBM41) and Δ(41+DUFs)-LaPul (lacking CBM41 and two DUFs), were produced recombinantly. LaPul recognized 1.3- and 2.2-fold more enzyme attack-sites on starch granules compared to Δ41-LaPul and Δ(41+DUFs)-LaPul, respectively, as measured by interfacial kinetics. Δ41-LaPul displayed markedly lower affinity for starch granules and ß-cyclodextrin (10- and >21-fold, respectively) in comparison to LaPul, showing substrate binding mainly stems from CBM41. Δ(41+DUFs)-LaPul exhibited a 12 °C lower melting temperature than LaPul and Δ41-LaPul, indicating that the DUFs are critical for LaPul stability. Notably, Δ41-LaPul exhibited a 14-fold higher turnover number (kcat) and 9-fold higher Michaelis constant (KM) compared to LaPul, while Δ(41+DUFs)-LaPul's values were close to those of LaPul, possibly due to the exposure of aromatic by truncation.

Structural adaptation of fungal cell wall in hypersaline environment.

Halophilic fungi thrive in hypersaline habitats and face a range of extreme conditions. These fungal species have gained considerable attention due to their potential applications in harsh industrial processes, such as bioremediation and fermentation under unfavorable conditions of hypersalinity, low water activity, and extreme pH. However, the role of the cell wall in surviving these environmental conditions remains unclear. Here we employ solid-state NMR spectroscopy to compare the cell wall architecture of Aspergillus sydowii across salinity gradients. Analyses of intact cells reveal that A. sydowii cell walls contain a rigid core comprising chitin, ß-glucan, and chitosan, shielded by a surface shell composed of galactomannan and galactosaminogalactan. When exposed to hypersaline conditions, A. sydowii enhances chitin biosynthesis and incorporates α-glucan to create thick, stiff, and hydrophobic cell walls. Such structural rearrangements enable the fungus to adapt to both hypersaline and salt-deprived conditions, providing a robust mechanism for withstanding external stress. These molecular principles can aid in the optimization of halophilic strains for biotechnology applications.

Plant structural and storage glucans trigger distinct transcriptional responses that modulate the motility of Xanthomonas pathogens.

IMPORTANCE: Pathogenic Xanthomonas bacteria can affect a variety of economically relevant crops causing losses in productivity, limiting commercialization and requiring phytosanitary measures. These plant pathogens exhibit high level of host and tissue specificity through multiple molecular strategies including several secretion systems, effector proteins, and a broad repertoire of carbohydrate-active enzymes (CAZymes). Many of these CAZymes act on the plant cell wall and storage carbohydrates, such as cellulose and starch, releasing products used as nutrients and modulators of transcriptional responses to support host colonization by mechanisms yet poorly understood. Here, we reveal that structural and storage ß-glucans from the plant cell function as spatial markers, providing distinct chemical stimuli that modulate the transition between higher and lower motility states in Xanthomonas citri, a key virulence trait for many bacterial pathogens.

Spatiotemporal deposition of cell wall polysaccharides in oat endosperm during grain development.

Oat (Avena sativa) is a cereal crop whose grains are rich in (1,3;1,4)-ß-D-glucan (mixed-linkage glucan or MLG), a soluble dietary fiber. In our study, we analyzed oat endosperm development in 2 Canadian varieties with differing MLG content and nutritional value. We confirmed that oat undergoes a nuclear type of endosperm development but with a shorter cellularization phase than barley (Hordeum vulgare). Callose and cellulose were the first polysaccharides to be detected in the early anticlinal cell walls at 11 days postemergence (DPE) of the panicle. Other polysaccharides such as heteromannan and homogalacturonan were deposited early in cellularization around 12 DPE after the first periclinal walls are laid down. In contrast to barley, heteroxylan deposition coincided with completion of cellularization and was detected from 14 DPE but was only detectable after demasking. Notably, MLG was the last polysaccharide to be laid down at 18 DPE within the differentiation phase, rather than during cellularization. In addition, differences in the spatiotemporal patterning of MLG were also observed between the 2 varieties. The lower MLG-containing cultivar AC Morgan (3.5% w/w groats) was marked by the presence of a discontinuous pattern of MLG labeling, while labeling in the same walls in CDC Morrison (5.6% w/w groats) was mostly even and continuous. RNA-sequencing analysis revealed higher transcript levels of multiple MLG biosynthetic cellulose synthase-like F (CSLF) and CSLH genes during grain development in CDC Morrison compared with AC Morgan that likely contributes to the increased abundance of MLG at maturity in CDC Morrison. CDC Morrison was also observed to have smaller endosperm cells with thicker walls than AC Morgan from cellularization onwards, suggesting the processes controlling cell size and shape are established early in development. This study has highlighted that the molecular processes influencing MLG content and deposition are more complex than previously imagined.

The LIKE SEX FOUR 1-malate dehydrogenase complex functions as a scaffold to recruit ß-amylase to promote starch degradation.

In plant leaves, starch is composed of glucan polymers that accumulate in chloroplasts as the products of photosynthesis during the day; starch is mobilized at night to continuously provide sugars to sustain plant growth and development. Efficient starch degradation requires the involvement of several enzymes, including ß-amylase and glucan phosphatase. However, how these enzymes cooperate remains largely unclear. Here, we show that the glucan phosphatase LIKE SEX FOUR 1 (LSF1) interacts with plastid NAD-dependent malate dehydrogenase (MDH) to recruit ß-amylase (BAM1), thus reconstituting the BAM1-LSF1-MDH complex. The starch hydrolysis activity of BAM1 drastically increased in the presence of LSF1-MDH in vitro. We determined the structure of the BAM1-LSF1-MDH complex by a combination of cryo-electron microscopy, crosslinking mass spectrometry, and molecular docking. The starch-binding domain of the dual-specificity phosphatase and carbohydrate-binding module of LSF1 was docked in proximity to BAM1, thus facilitating BAM1 access to and hydrolysis of the polyglucans of starch, thus revealing the molecular mechanism by which the LSF1-MDH complex improves the starch degradation activity of BAM1. Moreover, LSF1 is phosphatase inactive, and the enzymatic activity of MDH was dispensable for starch degradation, suggesting nonenzymatic scaffold functions for LSF1-MDH in starch degradation. These findings provide important insights into the precise regulation of starch degradation.