Isolated Cell Walls Research Articles

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, β-1,3- and β-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 β-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 β-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.

Nutritional uniformity of cell wall and lignin fractions in grass and red clover silage evaluated by the Lucas test with application to forage feed evaluation

The objective of this study was to investigate relationships between different cell wall (CW) fractions and in vivo digestibility of grass and red clover silages. The material consisted of 28 silages (6 primary growth grass, 16 regrowth grass and 6 red clover). Different fibre fractions predicted organic matter (OM) and neutral detergent fibre (aNDFom) digestibility reasonably well among the three forage types, but uniformly the digestibility predictions were not accurate enough for estimating the feeding value. Isolation of total CW did not improve digestibility predictions compared with aNDFom. The fraction insoluble in CW isolation, but soluble in aNDFom extraction was a uniform feed fraction with almost complete true digestibility. Isolation of CW fraction from faecal samples was not able to solubilize endogenous and metabolic components. True crude protein (CP) digestibility was higher than 0.90 when estimated by the Lucas test and by assuming that only N bound to neutral detergent fibre in faeces was of feed origin. Acid detergent lignin was more closely related to digestibility than lignin isolated from neutraldtergent fibre or CW (but all were inferior compared to indigestible NDF (iNDF) determined by 12-d ruminal in situ incubation. Lignin to iNDF ratio varied both within and between forage types. For evaluating nutritive value of forages actual measurements of iNDF (or undigested NDF) is recommended over detailed analysis of fibre and lignin fractions. Despite some CW fractions are solubilized in ND extractions, analysis of total CW is not a useful tool in ruminant nutrition research.

O-Linked Glycans of Candida albicans Interact with Specific GPCRs in the Coronary Endothelium and Inhibit the Cardiac Response to Agonists

Candida albicans is an opportunistic fungal pathogen that may cause invasive infections in immunocompromised patients, disseminating through the bloodstream to other organs. In the heart, the initial step prior to invasion is the adhesion of the fungus to endothelial cells. Being the fungal cell wall's outermost structure and the first to come in contact with host cells, it greatly modulates the interplay that later will derive in the colonization of the host tissue. In this work, we studied the functional contribution of N-linked and O-linked mannans of the cell wall of C. albicans to the interaction with the coronary endothelium. An isolated rat heart model was used to assess cardiac parameters related to vascular and inotropic effects in response to phenylephrine (Phe), acetylcholine (aCh) and angiotensin II (Ang II) when treatments consisting of: (1) live and heat-killed (HK) C. albicans wild-type yeasts; (2) live C. albicans pmr1Δ yeasts (displaying shorter N-linked and O-linked mannans); (3) live C. albicans without N-linked and O-linked mannans; and (4) isolated N-linked and O-linked mannans were administered to the heart. Our results showed that C. albicans WT alters heart coronary perfusion pressure (vascular effect) and left ventricular pressure (inotropic effect) parameters in response to Phe and Ang II but not aCh, and these effects can be reversed by mannose. Similar results were observed when isolated cell walls, live C. albicans without N-linked mannans or isolated O-linked mannans were perfused into the heart. In contrast, C. albicans HK, C. albicans pmr1Δ, C. albicans without O-linked mannans or isolated N-linked mannans were not able to alter the CPP and LVP in response to the same agonists. Taken together, our data suggest that C. albicans interaction occurs with specific receptors on coronary endothelium and that O-linked mannan contributes to a greater extent to this interaction. Further studies are necessary to elucidate why specific receptors preferentially interact with this fungal cell wall structure.

Functional differentiation of two autochthonous cohabiting strains of Pleurotus ostreatus and Cyclocybe aegerita from Serbia in lignin compound degradation

In nature, woody substrates are usually degraded by entire communities of microorganisms, which are nowadays jeopardised by anthropogenic influence, making it important to define the functional specificity of every species. Two strains of autochthonous fungi from Serbian lowland forests (Pleurotus ostreatus Ser1 and Cyclocybe aegerita Ser1) have been investigated for their ability to degrade lignin substrates [oak sawdust, oak isolated cell walls, and synthetic dehydrogenative polymer (DHP)]. Measuring the activities of the enzymes involved in lignin degradation was coupled with detecting the HPLC profile of the phenolics in the fungal growth media, and the lignin loss. While Pleurotus ostreatus Ser1 appeared highly effective within a very short time span, Cyclocybe aegerita Ser1 failed to degrade lignin. This situation was supported by very high enzyme activities and the low presence of phenolics in the media of Pleurotus ostreatus Ser1, compared to very low enzyme activity and the high presence of phenolics in the media with Cyclocybe aegerita Ser1.

Reliable Approach for Pure Yeast Cell Wall Protein Isolation from Saccharomyces cerevisiae Yeast Cells.

Saccharomyces cerevisiae yeast is a fungus presenting a peripheral organelle called the cell wall. The cell wall protects the yeast cell from stress and provides means for communication with the surrounding environment. It has a complex molecular structure, composed of an internal part of cross-linked polysaccharides and an external part of mannoproteins. These latter are very interesting owing to their functional properties, dependent on their molecular features with massive mannosylations. Therefore, the molecular characterization of mannoproteins is a must relying on the optimal isolation and preparation of the cell wall fraction. Multiple methods are well reported for yeast cell wall isolation. The most applied one consists of yeast cell lysis by mechanical disruption. However, applying this classical approach to S288C yeast cells showed considerable contamination with noncell wall proteins, mainly comprising mitochondrial proteins. Herein, we tried to further purify the yeast cell wall preparation by two means: ultracentrifugation and Triton X-100 addition. While the first strategy showed limited outcomes in mitochondrial protein removal, the second strategy showed optimal results when Triton X-100 was added at 5%, allowing the identification of more mannoproteins and significantly enriching their amounts. This promising method could be reliably implemented on the lab scale for identification of mannoproteins and molecular characterization and industrial processes for “pure” cell wall isolation.

A Water Extract from Chlorella sorokiniana Cell Walls Stimulates Growth of Bone Marrow Cells and Splenocytes.

A water extract derived from the isolated cell walls of Chlorella sorokiniana (C. sorokiniana, Chlorella water extract, CWE) was analyzed for the presence of lipopolysaccharide (LPS)-related material via the Limulus amebocyte lysate (LAL) assay and evaluated for its growth stimulation effect on the bone marrow cells and splenocytes in vitro cell cultures. The extract contained low levels of LPS-related material, and a mass spectrum suggested that the extract contained many components, including a low level of a lipid A precursor, a compound known as lipid X, which is known to elicit a positive response in the LAL assay. Treatment with the CWE dose- and time-dependently stimulated the growth of mouse bone marrow cells (BMCs) and splenocytes (SPLs). Treatment with the CWE also increased specific BMC subpopulations, including antigen-presenting cells (CD19+ B cells, 33D1+ dendritic cells and CD68+ macrophages), and CD4+ and CD8+ T cells, but decreased the number of LY6G+ granulocytes. Treatment with the CWE also increased cytokine mRNA associated with T cell activation, including TNFα, IFNγ, and granzyme B in human lymphoblasts. The present study indicates that the cell wall fraction of C. sorokiniana contains an LPS-like material and suggests a candidate source for the bioactivity that stimulates growth of both innate and adaptive immune cells.

Calcium control of the hydraulic resistance in cells of Chara corallina.

The hydraulic resistance (the reciprocal of the hydraulic conductivity Lp) Lp-1 was measured in cells of Chara corallina by the method of transcellular osmosis. Treatment of cells with 100 mM KCl decreased Lp-1 significantly. Subsequent treatment of the cells with 70 mM CaCl2 recovered the decreased Lp-1 to the original value. To know whether K+ or Ca2+/Mg2+ acts on the cell wall and/or the membrane, the hydraulic resistances of the cell wall (Lpw-1) and that of the membrane (Lpm-1) were determined in one and the same cell. For this, a pair of cells (twin cells) were made from an internodal cell, one used for measurement of Lp-1 and the other used for the measurement of Lpw-1. From Lp-1 and Lpw-1, Lpm-1 was calculated. Both Lp-1 and Lpw-1 were decreased by K+, while Lpm-1 was not affected by K+. The same result was obtained with 5 mM EGTA. Lpw-1 was decreased more than it was by KCl but Lpm-1 remained constant after EGTA treatment. The recovery of the K+-decreased Lp-1 with Ca2+ can be explained exclusively by the recovery of Lpw-1 with Ca2+. The Ca2+ recovery of Lpw-1 was observed in the intact cell wall but not in the cell wall tube isolated from an internodal cell. The different response to Ca2+ between the intact cell wall and the isolated cell wall was discussed in relation to the tension in the cell wall which may be an important factor for the ionic regulation of hydraulic conductivity.

Effect of Fungal Endophyte Epichloë bromicola Infection on Cd Tolerance in Wild Barley (Hordeum brevisubulatum).

Hydroponic Hordeum brevisubulatum (wild barley) was used as material in the greenhouse to study the effects of endophyte infection on plant growth, Cd absorption and transport, subcellular distribution, and Cd chemical forms under CdCl2 stress. Endophytic fungi respond positively to chlorophyll content and photosynthetic efficiency under Cd stress. The order of Cd absorption in different parts of the plant was: roots > stems > leaves. Endophyte infection increased the plant’s absorption and transport of Cd while causing a significant difference in the stem, which was associated with the distribution density of endophyte hyphae. The proportion of organelle Cd in endophyte-infected wild barley was significantly higher, which facilitated more Cd transport to aboveground. Cd stress showed a slight effect on the chemical forms of Cd in leaves. The proportion of phosphate, oxalate, and residual Cd increased in the stem. Cd existed in the form of inorganic salt, organic acid, pectin, and protein in roots. Endophyte infection reduced the Cd content of the more toxic chemical forms to protect the normal progress of plant physiological functions. Therefore, the isolation of cell walls and vacuoles is a key mechanism for plant Cd tolerance and detoxification. As endophyte infections have more ability to absorb Cd in plants, H. brevisubulatum–Epichloë bromicola symbionts can improve heavy metal contaminated soil and water.

Structural and biophysical properties of whole leaf and root tissue and isolated cell walls of common green bean and tomato seedlings grown in an aquaponics system relative to soil-grown counterparts

Structural and biophysical properties of whole leaf and root tissue and isolated cell walls of common green bean and tomato seedlings grown in an aquaponics system relative to soil-grown counterparts

Intact cells: "Nutritional capsules" in plant foods.

Macronutrients of pulses or cereals are stored in the cotyledon or endosperm cells with protection from intact cell walls. However, pulses and cereals are generally processed into fine particles during food production. For example, after milling, the macronutrients enclosed in the intact cells are released and are easily accessible to digestive enzymes in the gastrointestinal tract, leading to high metabolic responses. Therefore, studies on the health effects of intact cells and developing an alternative ingredient with a higher proportion of intact cells are areas of emerging interest. In this review, we highlighted the smallest unit of whole grain, an individual cell, as "nutritional capsules" and elucidated the structure-function of the nutritional capsules, followed by isolation techniques, as a potential novel functional ingredient and food. The polysaccharides' monomeric composition, secondary structure, and interactions determine the cell wall properties including the cell detachment during isolation and isolated cell properties. The intact cellular structure is retained after mild food processing and digestion, thereby, contributing to a lower extent/rate of digestion of entrapped macronutrients. Furthermore, the excursed intact capsules in the colonic environment modulate the population and diversity of microbiota, favouring the increased production of the short-chain fatty acids (SCFAs). The structural schematic model of Type-I and Type-II cells is developed together with the schematics of the cell wall isolation process. The review provides a critical summary of the recent trends in intact plant cells as a functional-nutritional food. It paves the way for the industrial production of intact cells as a novel food ingredient.

Differential Response of Two Tomato Genotypes, Wild Type cv. Ailsa Craig and Its ABA-Deficient Mutant flacca to Short-Termed Drought Cycles.

Two tomato genotypes with constitutively different ABA level, flacca mutant and wild type of Ailsa Craig cv. (WT), were subjected to three repeated drought cycles, with the aim to reveal the role of the abscisic acid (ABA) threshold in developing drought tolerance. Differential responses to drought of two genotypes were obtained: more pronounced stomatal closure, ABA biosynthesis and proline accumulation in WT compared to the mutant were compensated by dry weight accumulation accompanied by transient redox disbalance in flacca. Fourier-transform infrared (FTIR) spectra analysis of isolated cell wall material and morphological parameter measurements on tomato leaves indicated changes in dry weight accumulation and carbon re-allocation to cell wall constituents in flacca, but not in WT. A higher proportion of cellulose, pectin and lignin in isolated cell walls from flacca leaves further increased with repeated drought cycles. Different ABA-dependent stomatal closure between drought cycles implies that acquisition of stomatal sensitivity may be a part of stress memory mechanism developed under given conditions. The regulatory role of ABA in the cell wall restructuring and growth regulation under low leaf potential was discussed with emphasis on the beneficial effects of drought priming in developing differential defense strategies against drought.

Effect of varying the concentration of sulphuric acid and ammonium hydroxide on the release of cellulose from isolated cell wall component of corn cob

The scarcity and high price associated with fossil fuel has urged countries to research resources for alternative energy sources. Biofuels like bioethanol produced from lignocellulosic biomass (corn cob) were considered potential alternative. Cellulose composition from isolated cell wall material of corn cobs was investigated under two different pre-treatments using H2SO4 and NH4OH at varying concentrations of 5%, 10%, 20%, 30% and 40%. Cell wall not treated acted as control. Colorimetric anthrone-assay followed by absorbance reading at 625nm revealed that glucose is present in reasonable amount in corn cob. The analysis of variance (ANOVA) indicated significant differences among pre-treated compared to untreated (Control) corn cob samples at p≤0.05. Acid pre-treatment showed better glucose yield compared to alkali pre-treatment with results revealing 20% (19.37µg/ml) H2SO4 to be the optimal concentration producing highest glucose yield. The study reveals the potential of corn cob as a lignocellulosic feed stock for biofuel production.

Characterization of cadmium accumulation in the cell walls of leaves in a low-cadmium rice line and strengthening by foliar silicon application

Cadmium (Cd) remobilization in leaves is affected by whether Cd is stored in nonlabile subcellular compartments, which might be regulated by silicon (Si) application. However, the underlying mechanism is still far from being completely understood. In this research, the Cd distribution pattern in leaves and a Cd-binding characterization in the cell wall of the low-Cd rice line YaHui2816 were investigated through one hydroponic experiment with 10 μM Cd in solutions. Foliar Si application was further adopted to explore its influence on the Cd accumulation in the cell walls of leaves in YaHui2816. Most of the Cd (69.4%) was distributed in the cell walls of YaHui2816 leaves, whereas the isolated cell walls of leaves from YaHui2816 exhibited a lower capacity for Cd chemisorption than the contrasting line C268A, which was resulted from its fewer relative peak areas of functional groups in the cell wall, such as carboxyl CO and OH stretching. Foliar Si application significantly increased the Cd concentration in leaves and various cell wall fractions (pectin, hemicellulose 1 and residue) by 191% and 137–160%, respectively. RNA-seq analysis revealed that foliar Si application depressed the expression of the metal transporters OsZIP7 and OsZIP8, up-regulated the expression of genes participating in the glutathione metabolism and the cellulose synthesis. Overall, the influence of foliar Si application on Cd-accumulation in the cell wall of leaves in a low-Cd rice line was demonstrated in this research, which inspires further avenues to ensure the food safety of rice grains.

Evidence of intermolecular associations of β-glucan and high-molar mass xylan in a hot water extract of raw oat groat

High molar mass (HM) β-glucan present in oat products can reduce the risk of diet-related diseases, mainly owing to significantly increased digesta viscosity in human small intestine. To verify a research hypothesis that arabinoxylan (AX) present in oat water extract is associated with HM-β-glucan and thus may influence its functionality, multi-detection HPSEC coupled with enzymatic hydrolysis, sugar and 1H NMR analyses were performed. Isolated cell wall polysaccharide fraction comprised branched AX (arabinose-to-xylose ratio, Ara/Xyl ~ 0.8) and arabinogalactan-protein (AG-P). Nevertheless, it contained (10%) unbranched HM-xylan subfraction (weight-average molar mass, Mw ~ 3153 kg/mol), which was aggregated with β-glucan (Mw ~ 1029–1589 kg/mol) through its HM cellulose-like region. Of the two low molar mass-AXs (LM-AXs) isolated, the first having C(O)-3-monosubstituted and C(O)-2,3-disubstituted β-d-xylopyranosyl residues was covalently interlinked to HM-xylan. The second highly feruloylated, containing C(O)-3- and C(O)-2-monosubstituted units, was tightly bound to AG-P with terminal and 5-linked α-L-arabinofuranosyl residues, and non-covalently associated with LM-β-glucan subfraction.

In vitro ability of nonviable cells of lactic acid bacteria strains in combination with sorbitan monostearate to bind to aflatoxin M1 in skimmed milk

This study aimed to evaluate the in vitro ability of heat-killed (HK) and acid-killed (AK) cells from three commercial strains of lactic acid bacteria, alone or in combination with sorbitan monostearate (SM), to bind to aflatoxin M1 (AFM1) in skimmed milk at concentrations of 0.05, 0.2 and 0.5 ng mL−1. The stability of the AFM1-bacterial cell bonds and the cell wall and exopolysaccharides (EPS) contribution to AFM1 decontamination were also investigated. Compared with HK cells, AK cells exhibited higher binding capacities in milk containing the highest AFM1 level, with maximum removal of 81.4, 56.8, and 50.8% by Lactococcus lactis ssp. cremoris, Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus), and L. lactis ssp. lactis at 1010 cells mL−1, respectively. The combination of HK cells with SM increased the AFM1 binding capacity, although no effect was found regarding AK cells. SM treatment also enhanced the stability of the AFM1 bond formed with both HK and AK cells. Cell wall isolates and EPS were the major components involved in the AFM1 binding process. Combining SM with HK cells is an interesting approach to reduce AFM1 levels in milk, although further studies are needed to optimize the adsorbing process.

Genetic variability of cooking time in dry beans (Phaseolus vulgaris L.) related to seed coat thickness and the cotyledon cell wall

Dry beans are an affordable, nutritious food that often require long cooking times. Storage time and conditions, growing environment, and genotype influence cooking times. Little is known about factors underlying genetic variation for cooking time. Using fast and slow cooking genotypes from four different seed types (brown, cranberry, red mottled, yellow), the objectives of this study were to (1) characterize genetic variability for cooking time across multiple soaking time points; (2) determine the roles of seed coat and cotyledon cell wall physical traits in genetic variability for cooking time; and (3) identify seed coat and cotyledon cell wall compositional differences associated with genetic variability for cooking time. Genotypes were evaluated for cooking time on unsoaked beans and beans soaked for 3, 6, 12, 18, and 24 h. Cooking times were sharply reduced after 3 h of soaking and plateaued after 6 h of soaking. Interestingly, the genotypes in each pair that cooked faster when soaked did not necessarily cook faster when unsoaked. Greater seed coat percentage, cotyledon cell wall thickness, total and insoluble whole seed dietary fiber, and insoluble cotyledon cell wall isolate were genotypic factors associated with longer cooking times of soaked beans. Thicker seed coat macrosclereid- and osteosclereid-layers were genotypic factors associated with longer cooking times of unsoaked beans. These findings suggest that cotyledon cell wall thickness and composition have a significant role in genetic variability for cooking time of soaked beans and seed coat layer thickness relates to the genetic variability for cooking time of unsoaked beans.

Intranasal Bifidobacterium longum protects against viral-induced lung inflammation and injury in a murine model of lethal influenza infection.

BackgroundProphylactic strategies are urgently needed for prevention of severe inflammatory responses to respiratory viral infections. Bacterial-host interactions may modify the immune response to viral infections.MethodsWe examined the contribution of Intranasal administration of two different Bifidobacterium longum strains or its isolated cell wall in controlling viral induced inflammation using a murine model of influenza infection. We monitored mortality and morbidity over a 10-day period and viral load, differential broncho alveolar lavage (BAL) fluid inflammatory cell counts, Lung tissue histology, BAL and serum cytokines, markers of vascular damage and cell death were quantified.FindingsIntranasal administration of Bifidobacterium longum35624® or its isolated cell wall prior to virus inoculation significantly reduced viral load within the lungs and significantly improved survival. Reduced viral load was associated with reduced lung injury as suggested by cell death and vascular leakage markers, a shift from neutrophil to macrophage recruitment, reduced inflammatory cytokine levels (including IL-6), reduced type 1 and 2 interferon levels, but increased levels of interferon-λ and surfactant protein D. These protective effects were maintained when the bifidobacterial cell wall preparation was administered 24 h after viral inoculation. The protective effects were also observed for the Bifidobacterium longumPB-VIR™ strain.InterpretationExposure to these bifidobacterial strains protect against the inflammatory sequelae and damage associated with uncontrolled viral replication within the lung.FundingThis work has been funded, in part, by a research grant from GlaxoSmithKline, PrecisionBiotics Group Ltd., Swiss National Science Foundation grants (project numbers CRSII3_154488, 310030_144219, 310030_127356 and 310030_144219) and Christine Kühne – Center for Allergy Research and Education (CK-CARE).

The effects of ionizing radiation on the structure and antioxidative and metal-binding capacity of the cell wall of microalga Chlorella sorokiniana

The impact of ionizing radiation on microorganisms such as microalgae is a topic of increasing importance for understanding the dynamics of aquatic ecosystems in response to environmental radiation, and for the development of efficient approaches for bioremediation of mining and nuclear power plants wastewaters. Currently, nothing is known about the effects of ionizing radiation on the microalgal cell wall, which represents the first line of defence against chemical and physical environmental stresses. Using various microscopy, spectroscopy and biochemical techniques we show that the unicellular alga Chlorella sorokiniana elicits a fast response to ionizing radiation. Within one day after irradiation with doses of 1–5 Gy, the fibrilar layer of the cell wall became thicker, the fraction of uronic acids was higher, and the capacity to remove the main reactive product of water radiolysis increased. In addition, the isolated cell wall fraction showed significant binding capacity for Cu2+, Mn2+, and Cr3+. The irradiation further increased the binding capacity for Cu2+, which appears to be mainly bound to glucosamine moieties within a chitosan-like polymer in the outer rigid layer of the wall. These results imply that the cell wall represents a dynamic structure that is involved in the protective response of microalgae to ionizing radiation. It appears that microalgae may exhibit a significant control of metal mobility in aquatic ecosystems via biosorption by the cell wall matrix.

Innovation in Wood Preservation.

The wood preservation industry has depended on toxicity as a mechanism of effectiveness against decay fungi to extend the life of wood used in adverse conditions. An alternative to toxicity, however, is to study and understand the mechanism of fungal attack and stop it before it can start. Knowing that fungi need moisture for colonization, a new approach to wood preservation is to lower the cell wall moisture content below that needed for fungal attack. Acetylation chemistry is known to reduce the moisture content in the cell wall, and it was used to study moisture levels in the bulk cell wall and in the isolated cell wall polymers. Resistance to brown-rot was determined using a 12-week soil block test with Gloeophyllum trabeum. Weight loss was measured and an analysis of what was lost was determined.

BdGT43B2 functions in xylan biosynthesis and is essential for seedling survival in Brachypodium distachyon.

Xylan is the predominant hemicellulose in the primary cell walls of grasses, but its synthesis and interactions with other wall polysaccharides are complex and incompletely understood. To probe xylan biosynthesis, we generated CRISPR/Cas9 knockout and amiRNA knockdown lines of BdGT43B2, an ortholog of the wheat TaGT43‐4 xylan synthase scaffolding protein in the IRX14 clade, in Brachypodium distachyon. Knockout of BdGT43B2 caused stunting and premature death in Brachypodium seedlings. Immunofluorescence labeling of xylans was greatly reduced in homozygous knockout BdGT43B2 mutants, whereas cellulose labeling was unchanged or slightly increased. Biochemical analysis showed reductions in digestible xylan in knockout mutant walls, and cell size was smaller in knockout leaves. BdGT43B2 knockdown plants appeared morphologically normal as adults, but showed slight reductions in seedling growth and small decreases in xylose content in isolated cell walls. Immunofluorescence labeling of xylan and cellulose staining was both reduced in BdGT43B2 knockdown plants. Together, these data indicate that BdGT43B2 functions in the synthesis of a form of xylan that is required for seedling growth and survival in Brachypodium distachyon.