KOMPEITO, an Atypical Arabidopsis Rhomboid-Related Gene, Is Required for Callose Accumulation and Pollen Wall Development.

Fertilization is a key event for sexually reproducing plants. Pollen-stigma adhesion, which is the first step in male-female interaction during fertilization, requires proper pollen wall patterning. Callose, which is a ß-1.3-glucan, is an essential polysaccharide that is required for pollen development and pollen wall formation. Mutations in CALLOSE SYNTHASE 5 (CalS5) disrupt male meiotic callose accumulation; however, how CalS5 activity and callose synthesis are regulated is not fully understood. In this paper, we report the isolation of a kompeito-1 (kom-1) mutant defective in pollen wall patterning and pollen-stigma adhesion in Arabidopsis thaliana. Callose was not accumulated in kom-1 meiocytes or microspores, which was very similar to the cals5 mutant. The KOM gene encoded a member of a subclass of Rhomboid serine protease proteins that lacked active site residues. KOM was localized to the Golgi apparatus, and both KOM and CalS5 genes were highly expressed in meiocytes. A 220 kDa CalS5 protein was detected in wild-type (Col-0) floral buds but was dramatically reduced in kom-1. These results suggested that KOM was required for CalS5 protein accumulation, leading to the regulation of meiocyte-specific callose accumulation and pollen wall formation.

Chitin-glucan supplementation improved postprandial metabolism and altered gut microbiota in subjects at cardiometabolic risk in a randomized trial.

Chitin-glucan (CG), an insoluble dietary fiber, has been shown to improve cardiometabolic disorders associated with obesity in mice. Its effects in healthy subjects has recently been studied, revealing its interaction with the gut microbiota. In this double-blind, randomized, cross-over, twice 3-week exploratory study, we investigated the impacts of CG on the cardiometabolic profile and gut microbiota composition and functions in 15 subjects at cardiometabolic risk. They consumed as a supplement 4.5 g of CG daily or maltodextrin as control. Before and after interventions, fasting and postprandial metabolic parameters and exhaled gases (hydrogen [H2] and methane [CH4]) were evaluated. Gut microbiota composition (16S rRNA gene sequencing analysis), fecal concentrations of bile acids, long- and short-chain fatty acids (LCFA, SCFA), zonulin, calprotectin and lipopolysaccharide binding protein (LBP) were analyzed. Compared to control, CG supplementation increased exhaled H2 following an enriched-fiber breakfast ingestion and decreased postprandial glycemia and triglyceridemia response to a standardized test meal challenge served at lunch. Of note, the decrease in postprandial glycemia was only observed in subjects with higher exhaled H2, assessed upon lactulose breath test performed at inclusion. CG decreased a family belonging to Actinobacteria phylum and increased 3 bacterial taxa: Erysipelotrichaceae UCG.003, Ruminococcaceae UCG.005 and Eubacterium ventriosum group. Fecal metabolites, inflammatory and intestinal permeability markers did not differ between groups. In conclusion, we showed that CG supplementation modified the gut microbiota composition and improved postprandial glycemic response, an early determinant of cardiometabolic risk. Our results also suggest breath H2 production as a non-invasive parameter of interest for predicting the effectiveness of dietary fiber intervention.

Dynamics of cell wall polysaccharides during the elongation growth of rye primary roots.

MAIN CONCLUSION: In cells of growing rye roots, xyloglucans and homogalacturonans demonstrate developmental stage specificity, while different xylans have tissue specificity. Mannans, arabinans and galactans are also detected within the protoplast. Mannans form films on sections of fresh material. The primary cell walls of plants represent supramolecular exocellular structures that are mainly composed of polysaccharides. Cell wall properties and architecture differ between species and across tissues within a species. We revised the distribution of cell wall polysaccharides and their dynamics during elongation growth and histogenesis in rye roots using nonfixed material and the spectrum of antibodies. Rye is a member of the Poaceae family and thus has so-called type II primary cell walls, which are supposed to be low in pectins and xyloglucans and instead have arabinoxylans and mixed-linkage glucans. However, rye cell walls at the earliest stages of cell development were enriched with the epitopes of xyloglucans and homogalacturonans. Mixed-linkage glucan, which is often considered an elongation growth-specific polysaccharide in plants with type II cell walls, did not display such dynamics in rye roots. The cessation of elongation growth and even the emergence of root hairs were not accompanied by the disappearance of mixed-linkage glucans from cell walls. The diversity of xylan motifs recognized by different antibodies was minimal in the meristem zone of rye roots, but this diversity increased and showed tissue specificity during root growth. Antibodies specific for xyloglucans, galactans, arabinans and mannans bound the cell content. When rye root cells were cut, the epitopes of xyloglucans, galactans and arabinans remained within the cell content, while mannans developed net-like or film-like structures on the surface of sections.

Echinocandins - structure, mechanism of action and use in antifungal therapy.

With increasing number of immunocompromised patients as well as drug resistance in fungi, the risk of fatal fungal infections in humans increases as well. The action of echinocandins is based on the inhibition of ß-(1,3)-d-glucan synthesis that builds the fungal cell wall. Caspofungin, micafungin, anidulafungin and rezafungin are semi-synthetic cyclic lipopeptides. Their specific chemical structure possess a potential to obtain novel derivatives with better pharmacological properties resulting in more effective treatment, especially in infections caused by Candida and Aspergillus species. In this review we summarise information about echinocandins with closer look on their chemical structure, mechanism of action, drug resistance and usage in clinical practice. We also introduce actual trends in modification of this antifungals as well as new methods of their administration, and additional use in viral and bacterial infections.

Phloem iron remodels root development in response to ammonium as the major nitrogen source.

Plants use nitrate and ammonium as major nitrogen (N) sources, each affecting root development through different mechanisms. However, the exact signaling pathways involved in root development are poorly understood. Here, we show that, in Arabidopsis thaliana, either disruption of the cell wall-localized ferroxidase LPR2 or a decrease in iron supplementation efficiently alleviates the growth inhibition of primary roots in response to NH4+ as the N source. Further study revealed that, compared with nitrate, ammonium led to excess iron accumulation in the apoplast of phloem in an LPR2-dependent manner. Such an aberrant iron accumulation subsequently causes massive callose deposition in the phloem from a resulting burst of reactive oxygen species, which impairs the function of the phloem. Therefore, ammonium attenuates primary root development by insufficiently allocating sucrose to the growth zone. Our results link phloem iron to root morphology in response to environmental cues.

The graft framework: Quantitative changes in cell wall matrix polysaccharides throughout the tomato graft union formation.

Plant cell walls provide essential functions in cell recognition, differentiation, adhesion and wound responses. Therefore, it is tempting to hypothesize that cell walls play a key role in grafting, but to date there are no quantitative studies targeting on cell wall changes during grafting. The aim of this work was to investigate the dynamics of pectic and hemicellulosic polysaccharides at the graft junctions in tomato homografts throughout the first 12 days after grafting. Cell wall fractionation, combined with ATR-FTIR spectroscopy and gas-chromatography, evidenced a marked increase in pectin content and a decrease in the degree of methyl-esterification of homogalacturonan in scion and rootstock throughout grafting. Also, recovery of tightly-bound hemicelluloses decreased at late times after grafting suggesting an increase of cross-linked hemicelluloses along grafting. In addition, immuno-dot assays revealed an increase in xyloglucan and arabinogalactan proteins in the first days after grafting, pointing to a presumed role in tissue adhesion-cohesion.

Cooperative Kinetics of the Glucan Phosphatase Starch Excess4.

Glucan phosphatases are members of a functionally diverse family of dual-specificity phosphatase (DSP) enzymes. The plant glucan phosphatase Starch Excess4 (SEX4) binds and dephosphorylates glucans, contributing to processive starch degradation in the chloroplast at night. Little is known about the complex kinetics of SEX4 when acting on its complex physiologically relevant glucan substrate. Therefore, we explored the kinetics of SEX4 against both insoluble starch and soluble amylopectin glucan substrates. SEX4 displays robust activity and a unique sigmoidal kinetic response to amylopectin, characterized by a Hill coefficient of 2.77 ± 0.63, a signature feature of cooperativity. We investigated the basis for this positive kinetic cooperativity and determined that the SEX4 carbohydrate-binding module (CBM) dramatically influences the binding cooperativity and substrate transformation rates. These findings provide insights into a previously unknown but important regulatory role for SEX4 in reversible starch phosphorylation and further advances our understanding of atypical kinetic mechanisms.

Surface-enhanced Raman spectroscopic chemical imaging reveals distribution of pectin and its co-localization with xyloglucan inside onion epidermal cell wall.

The primary plant cell wall is a complex matrix composed of interconnected polysaccharides including cellulose, hemicellulose, and pectin. Changes of this dynamic polysaccharide system play a critical role during plant cell development and differentiation. A better understanding of cell wall architectures can provide insight into the plant cell development. In this study, a Raman spectroscopic imaging approach was developed to visualize the distribution of plant cell wall polysaccharides. In this approach, Surface-enhanced Raman scattering (SERS through self-assembled silver nanoparticles) was combined with Raman labels (4-Aminothiophenol. 4ATP) and targeted enzymatic hydrolysis to improve the sensitivity, specificity, and throughput of the Raman imaging technique, and to reveal the distribution of pectin and its co-localization with xyloglucan inside onion epidermal cell (OEC) wall. This technique significantly decreased the required spectral acquisition time. The resulted Raman spectra showed a high Raman signal. The resulted Raman images successfully revealed and characterized the pectin distribution and its co-localization pattern with xyloglucan in OEC wall.

Qingke ß-glucan synergizes with a ß-glucan-utilizing Lactobacillus strain to relieve capsaicin-induced gastrointestinal injury in mice.

Capsaicin (CAP) is the main pungent component in capsicum fruits. Eating too much CAP leads to gastrointestinal injury. Previously, Qingke ß-glucan combined with ß-glucan-utilizing Lactobacillus plantarum S58 (LP.S58) ameliorated high fat-diet-induced obesity, but their effects on CAP-induced gastrointestinal injury have not been investigated. Our results showed that Qingke ß-glucan reduced the CAP-induced gastrointestinal injury in Kunming mice. The serum levels of inflammatory cytokines and gastrointestinal hormones, and the localized inflammation and the expression of EGF, EGFR, VEGF, and ZO-1 in the gastrointestinal tissues in CAP-treated mice were partly restored by Qingke ß-glucan. The CAP-induced increase in the abundances of proinflammatory intestinal bacteria was also reduced by Qingke ß-glucan. More importantly, we found that these beneficial effects of Qingke ß-glucan were markedly enhanced by ß-glucan-utilizing LP.S58 supplementation. Our study indicated that Qingke ß-glucan coupled with ß-glucan-utilizing LP.S58 relieved CAP-induced gastrointestinal injury.

Analysis of sticky generative cell mutants reveals that suppression of callose deposition in the generative cell is necessary for generative cell internalization and differentiation in Arabidopsis.

In flowering plants, double fertilization between male and female gametophytes, which are separated by distance, largely depends on the unique pattern of the male gametophyte (pollen): two non-motile sperm cells suspended within a tube-producing vegetative cell. A morphological screen to elucidate the genetic control governing the strategic patterning of pollen has led to the isolation of a sticky generative cell (sgc) mutant that dehisces abnormal pollen with the generative cell immobilized at the pollen wall. Analyses revealed that the sgc mutation is specifically detrimental to pollen development, causing ectopic callose deposition that impedes the timely internalization and differentiation of the generative cell. We found that the SGC gene encodes the highly conserved domain of unknown function 707 (DUF707) gene that is broadly expressed but is germline specific during pollen development. Additionally, transgenic plants co-expressing fluorescently fused SGC protein and known organelle markers showed that SGC localizes in the endoplasmic reticulum, Golgi apparatus and vacuoles in pollen. A yeast two-hybrid screen with an SGC bait identified a thaumatin-like protein that we named GCTLP1, some homologs of which bind and/or digest ß-1,3-glucans, the main constituent of callose. GCTLP1 is expressed in a germline-specific manner and colocalizes with SGC during pollen development, indicating that GCTLP1 is a putative SGC interactor. Collectively, our results show that SGC suppresses callose deposition in the nascent generative cell, thereby allowing the generative cell to fully internalize into the vegetative cell and correctly differentiate as the germline progenitor, with the potential involvement of the GCTLP1 protein, during pollen development in Arabidopsis.

Oligogalacturonide-accelerated healing of mechanical wounding in tomato fruit requires calcium-dependent systemic acquired resistance.

Mechanical wounding causes significant economic losses of fresh produce due to accelerated senescence and spoilage as well as loss of nutritional value. Here, pre-application of oligogalacturonides (OGs) enzymatically hydrolyzed from apple pectin effectively reduced the healing times of mechanical wounds from>24 h in mock groups to 12 h, and the Botrytis cinerea infection rate was reduced from 37.5% to 12.5%. OGs accordingly increased callose deposition; SlPR1, SlPAL and SlHCT gene expression; and phenylalanine ammonia-lyase (PAL) activity around the wounds. Inhibition of Ca2+ signaling using the inhibitor Ruthenium Red markedly inhibited OG accelerated healing of mechanical wounding on fruit. SlPG2, SlEXP1, and SlCEL2 mRNAs accumulation was reduced in OG-elicited tomato fruit compared to water-treated fruit with subsequent retardation of the fruit softening during ripening. These results indicated that apple pectin OGs accelerate wound healing and inhibit fruit softening by activating calcium signaling in tomato fruits during postharvest storage.

Supplementation with beta-1,3-glucan improves productivity, immunity and antioxidative status in transition Holstein cows.

Dairy cows undergo dramatic physiological changes during the transition from late pregnancy to early lactation, which make them vulnerable to metabolic stress and immune dysfunction. The objective of this study was to evaluate the effects of a commercial beta-1,3-glucan product (Aleta™, containing 50% beta-1,3-glucan) on productivity, immunity and antioxidative status in transition cows. Fifty-four multiparous Holstein cows received a control diet or a diet supplemented with 5 or 10 g of beta-1,3-glucan per cow per day from 21 days before expected calving to 21 days after parturition. Blood samples were collected at day -21, 1, and 21 relative to calving. Colostrum and milk were collected at day 1 and 21 after calving, respectively. Data showed that supplementation with beta-1,3-glucan had no effect on milk composition, but increased milk production. Beta-1,3-glucan treatment also improved the milk quality, as shown by reduced milk somatic cell count and increased immunoglobulin levels in colostrum. Notably, beta-1,3-glucan markedly reduced serum levels of pro-inflammatory cytokines and C-reactive protein, while elevated serum immunoglobulin levels, indicating its immunity enhancement in transition cows. Moreover, beta-1,3-glucan addition reduced the serum malondialdehyde level and enhanced the activities of serum superoxide dismutase and catalase, which enhanced the antioxidative capacity in transition cows. In summary, supplementation with beta-1,3-glucan improves productivity, immunity and antioxidative status in transition dairy cows.

Aucklandia lappa Causes Membrane Permeation of Candida albicans.

Candida albicans is a major fungal pathogen in humans. In our previous study, we reported that an ethanol extract from Aucklandia lappa weakens C. albicans cell wall by inhibiting synthesis or assembly of both (1,3)-ß-D-glucan polymers and chitin. In the current study, we found that the extract is involved in permeabilization of C. albicans cell membranes. While uptake of ethidium bromide (EtBr) was 3.0% in control cells, it increased to 7.4% for 30 min in the presence of the A. lappa ethanol extract at its minimal inhibitory concentration (MIC), 0.78 mg/ml, compared to uptake by heat-killed cells. Besides, leakage of DNA and proteins was observed in A. lappa-treated C. albicans cells. The increased uptake of EtBr and leakage of cellular materials suggest that A. lappa ethanol extract induced functional changes in C. albicans cell membranes. Incorporation of diphenylhexatriene (DPH) into membranes in the A. lappa-treated C. albicans cells at its MIC decreased to 84.8%, after 60 min of incubation, compared with that of the controls, indicate that there was a change in membrane dynamics. Moreover, the anticandidal effect of the A. lappa ethanol extract was enhanced at a growth temperature of 40°C compared to that at 35°C. The above data suggest that the antifungal activity of the A. lappa ethanol extract against C. albicans is associated with synergistic action of membrane permeabilization due to changes in membrane dynamics and cell wall damage caused by reduced formation of (1,3)-ß-D-glucan and chitin.

Metabolite profiling reveals the interaction of chitin-glucan with the gut microbiota.

Dietary fibers are considered beneficial nutrients for health. Current data suggest that their interaction with the gut microbiota largely contributes to their physiological effects. In this context, chitin-glucan (CG) improves metabolic disorders associated with obesity in mice, but its effect on gut microbiota has never been evaluated in humans. This study explores the effect of a 3-week intervention with CG supplementation in healthy individuals on gut microbiota composition and bacterial metabolites. CG was given to healthy volunteers (n = 15) for three weeks as a supplement (4.5 g/day). Food diary, visual analog and Bristol stool form scales and a "quality of life" survey were analyzed. Among gut microbiota-derived metabolites, bile acids (BA), long- and short-chain fatty acids (LCFA, SCFA) profiling were assessed in stool samples. The gut microbiota (primary outcome) was analyzed by Illumina sequencing. A 3-week supplementation with CG is well tolerated in healthy humans. CG induces specific changes in the gut microbiota composition, with Eubacterium, Dorea and Roseburia genera showing the strongest regulation. In addition, CG increased bacterial metabolites in feces including butyric, iso-valeric, caproic and vaccenic acids. No major changes were observed for the fecal BA profile following CG intervention. In summary, our work reveals new potential bacterial genera and gut microbiota-derived metabolites characterizing the interaction between an insoluble dietary fiber -CG- and the gut microbiota.

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin.

We report on the homo- and hetero-transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488-labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta-galacturonide [α(1-4)GalAp]5 acceptor - the homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [α(1-4)GalAp]5 acceptor substrates at the -4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed-localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero-transglycosylation reaction with the xyloglucan/[α(1-4)GalAp]5 substrate pair, while wild-type TmXET6.3 lacked this activity. Expression data obtained by real-time quantitative polymerase chain reaction of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co-expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots and a basal Angiosperm. The discovered hetero-transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[α(1-4)GalAp]5 substrate pair is discussed against the background of roles of xyloglucan-pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and re-modelling, and affect the structural features of walls.

Integrated transcriptomic and metabolomic analyses reveal the effects of callose deposition and multihormone signal transduction pathways on the tea plant-Colletotrichum camelliae interaction.

Colletotrichum infects diverse hosts, including tea plants, and can lead to crop failure. Numerous studies have reported that biological processes are involved in the resistance of tea plants to Colletotrichum spp. However, the molecular and biochemical responses in the host during this interaction are unclear. Cuttings of the tea cultivar Longjing 43 (LJ43) were inoculated with a conidial suspension of Colletotrichum camelliae, and water-sprayed cuttings were used as controls. In total, 10,592 differentially expressed genes (DEGs) were identified from the transcriptomic data of the tea plants and were significantly enriched in callose deposition and the biosynthesis of various phytohormones. Subsequently, 3,555 mass spectra peaks were obtained by LC-MS detection in the negative ion mode, and 27, 18 and 81 differentially expressed metabolites (DEMs) were identified in the tea leaves at 12 hpi, 24 hpi and 72 hpi, respectively. The metabolomic analysis also revealed that the levels of the precursors and intermediate products of jasmonic acid (JA) and indole-3-acetate (IAA) biosynthesis were significantly increased during the interaction, especially when the symptoms became apparent. In conclusion, we suggest that callose deposition and various phytohormone signaling systems play important roles in the tea plant-C. camelliae interaction.

Characterization of two family AA9 LPMOs from Aspergillus tamarii with distinct activities on xyloglucan reveals structural differences linked to cleavage specificity.

Aspergillus tamarii grows abundantly in naturally composting waste fibers of the textile industry and has a great potential in biomass decomposition. Amongst the key (hemi)cellulose-active enzymes in the secretomes of biomass-degrading fungi are the lytic polysaccharide monooxygenases (LPMOs). By catalyzing oxidative cleavage of glycoside bonds, LPMOs promote the activity of other lignocellulose-degrading enzymes. Here, we analyzed the catalytic potential of two of the seven AA9-type LPMOs that were detected in recently published transcriptome data for A. tamarii, namely AtAA9A and AtAA9B. Analysis of products generated from cellulose revealed that AtAA9A is a C4-oxidizing enzyme, whereas AtAA9B yielded a mixture of C1- and C4-oxidized products. AtAA9A was also active on cellopentaose and cellohexaose. Both enzymes also cleaved the ß-(1→4)-glucan backbone of tamarind xyloglucan, but with different cleavage patterns. AtAA9A cleaved the xyloglucan backbone only next to unsubstituted glucosyl units, whereas AtAA9B yielded product profiles indicating that it can cleave the xyloglucan backbone irrespective of substitutions. Building on these new results and on the expanding catalog of xyloglucan- and oligosaccharide-active AA9 LPMOs, we discuss possible structural properties that could underlie the observed functional differences. The results corroborate evidence that filamentous fungi have evolved AA9 LPMOs with distinct substrate specificities and regioselectivities, which likely have complementary functions during biomass degradation.

Elongating maize root: zone-specific combinations of polysaccharides from type I and type II primary cell walls.

The dynamics of cell wall polysaccharides may modulate the cell wall mechanics and thus control the expansion growth of plant cells. The unique composition of type II primary cell wall characteristic of grasses suggests that they employ specific mechanisms for cell enlargement. We characterized the transcriptomes in five zones along maize root, clustered the expression of genes for numerous glycosyltransferases and performed extensive immunohistochemical analysis to relate the changes in cell wall polysaccharides to critical stages of cell development in Poaceae. Specific patterns of cell wall formation differentiate the initiation, realization and cessation of elongation growth. Cell walls of meristem and early elongation zone represent a mixture of type I and type II specific polysaccharides. Xyloglucans and homogalacturonans are synthesized there actively together with mixed-linkage glucans and glucuronoarabinoxylans. Rhamnogalacturonans-I with the side-chains of branched 1,4-galactan and arabinan persisted in cell walls throughout the development. Thus, the machinery to generate the type I primary cell wall constituents is completely established and operates. The expression of glycosyltransferases responsible for mixed-linkage glucan and glucuronoarabinoxylan synthesis peaks at active or late elongation. These findings widen the number of jigsaw pieces which should be put together to solve the puzzle of grass cell growth.