Cell Wall Carbohydrate Composition Research Articles

Study of fungal cell wall evolution through its monosaccharide composition: An insight into fungal species interacting with plants

Every fungal cell is encapsulated in a cell wall, essential for cell viability, morphogenesis, and pathogenesis. Most knowledge of the cell wall composition in fungi has focused on ascomycetes, especially human pathogens, but considerably less is known about early divergent fungal groups, such as species in the Zoopagomycota and Mucoromycota phyla. To shed light on evolutionary changes in the fungal cell wall, we studied the monosaccharide composition of the cell wall of 18 species including early diverging fungi and species in the Basidiomycota and Ascomycota phyla with a focus on those with pathogenic lifestyles and interactions with plants. Our data revealed that chitin is the most characteristic component of the fungal cell wall, and was found to be in a higher proportion in the early divergent groups. The Mucoromycota species possess few glucans, but instead have other monosaccharides such as fucose and glucuronic acid that are almost exclusively found in their cell walls. Additionally, we observed that hexoses (glucose, mannose and galactose) accumulate in much higher proportions in species belonging to Dikarya. Our data demonstrate a clear relationship between phylogenetic position and fungal cell wall carbohydrate composition and lay the foundation for a better understanding of their evolution and their role in plant interactions.

PSIII-1 Effect of Cannabidiol Extraction on the Cell Wall Carbohydrate Composition of Hemp Inflorescences

Abstract Since the 2018 Farm Bill, US growers have been allowed to cultivate hemp (Cannabis sativa L.), prompting interest in exploring the use of hemp products in ruminant diets. Extraction of cannabidiol (CBD) oil from hemp biomass generates spent inflorescent material that could be upcycled as a feedstuff, yet little is known about its fiber composition. A recent study measured in vitro true digestibility and fermentation parameters of multiple hemp cultivars, some intact and some CBD-extracted (Altman et al., 2022). Both categories of hemp inflorescence material had similar or greater total digestible nutrients (TDN) than an alfalfa cube control, supporting hemp’s potential as a feedstuff. However, comparisons between non-extracted and CBD-extracted samples could not be made, and the fiber content was only described as neutral detergent fiber (NDF). Fiber composition can significantly impact ruminal digestibility, and in-depth characterizations of cell wall carbohydrate composition can provide further insight into the digestibility of a specific plant-based feed. Therefore, in the present study, we examined the effect of CBD oil extraction on the cell wall carbohydrate composition of the inflorescent material of two hemp cultivars. Intact (Biomass) and CBD-extracted (Spent) inflorescent material from two hemp cultivars, Mary and Zelios, were analyzed. The scell wall monosaccharides of the samples were released via two complementary acid hydrolyses, methanolysis and Saeman (sulfuric acid) hydrolysis, and separated and quantified using high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Additionally, the carbohydrate linkage patterns were assessed by generating partially methylated alditol acetates (PMAAs). Student’s unpaired t-tests were run in GraphPad Prism, and no differences (P > 0.05) in monosaccharide concentrations based upon treatment (CBD extraction) or cultivar were found with either hydrolysis method except for the methanolysis glucose concentration between the biomass forms of the two cultivars where Mary Biomass possessed more glucose than Zelios Biomass (P <b0.001). In the Saeman hydrolysates, glucose was the dominant monosaccharide (38.75 ± 1.44 molar %), indicating a large amount of cellulose, supported by the presence of 1,4-Glcp in the linkage analysis. Linkage analysis also revealed the presence of xyloglucans and rhamnogalacturonan Type 1 pectins in all materials. The data showed no effect of CBD oil extraction on the monosaccharide composition, suggesting that the extraction process leaves the fiber components mostly intact, but there may be differences in glucose concentration (and consequently cellulose content) between cultivars of hemp. Future studies may benefit more from investigating the impact of hemp cultivar on fiber digestibility rather than impact of the CBD extraction process.

Cell Wall Carbohydrate Dynamics during the Differentiation of Infection Structures by the Apple Scab Fungus, Venturia inaequalis.

Scab, caused by the biotrophic fungal pathogen Venturia inaequalis, is the most economically important disease of apples. During infection, V. inaequalis colonizes the subcuticular host environment, where it develops specialized infection structures called runner hyphae and stromata. These structures are thought to be involved in nutrient acquisition and effector (virulence factor) delivery, but also give rise to conidia that further the infection cycle. Despite their importance, very little is known about how these structures are differentiated. Likewise, nothing is known about how these structures are protected from host defenses or recognition by the host immune system. To better understand these processes, we first performed a glycosidic linkage analysis of sporulating tubular hyphae from V. inaequalis developed in culture. This analysis revealed that the V. inaequalis cell wall is mostly composed of glucans (44%) and mannans (37%), whereas chitin represents a much smaller proportion (4%). Next, we used transcriptomics and confocal laser scanning microscopy to provide insights into the cell wall carbohydrate composition of runner hyphae and stromata. These analyses revealed that, during subcuticular host colonization, genes of V. inaequalis putatively associated with the biosynthesis of immunogenic carbohydrates, such as chitin and β-1,6-glucan, are downregulated relative to growth in culture, while on the surface of runner hyphae and stromata, chitin is deacetylated to the less-immunogenic carbohydrate chitosan. These changes are anticipated to enable the subcuticular differentiation of runner hyphae and stromata by V. inaequalis, as well as to protect these structures from host defenses and recognition by the host immune system. IMPORTANCE Plant-pathogenic fungi are a major threat to food security. Among these are subcuticular pathogens, which often cause latent asymptomatic infections, making them difficult to control. A key feature of these pathogens is their ability to differentiate specialized subcuticular infection structures that, to date, remain largely understudied. This is typified by Venturia inaequalis, which causes scab, the most economically important disease of apples. In this study, we show that, during subcuticular host colonization, V. inaequalis downregulates genes associated with the biosynthesis of two immunogenic cell wall carbohydrates, chitin and β-1,6-glucan, and coats its subcuticular infection structures with a less-immunogenic carbohydrate, chitosan. These changes are anticipated to enable host colonization by V. inaequalis and provide a foundation for understanding subcuticular host colonization by other plant-pathogenic fungi. Such an understanding is important, as it may inform the development of novel control strategies against subcuticular plant-pathogenic fungi.

Responses of leaf litter decomposability to nitrogen and phosphorus additions are associated with cell wall carbohydrate composition in a subtropical plantation

Responses of leaf litter decomposability to nitrogen and phosphorus additions are associated with cell wall carbohydrate composition in a subtropical plantation

Metabolic Potential, Ecology and Presence of Associated Bacteria Is Reflected in Genomic Diversity of Mucoromycotina.

Mucoromycotina are often considered mainly in pathogenic context but their biology remains understudied. We describe the genomes of six Mucoromycotina fungi representing distant saprotrophic lineages within the subphylum (i.e., Umbelopsidales and Mucorales). We selected two Umbelopsis isolates from soil (i.e., U. isabellina, U. vinacea), two soil-derived Mucor isolates (i.e., M. circinatus, M. plumbeus), and two Mucorales representatives with extended proteolytic activity (i.e., Thamnidium elegans and Mucor saturninus). We complement computational genome annotation with experimental characteristics of their digestive capabilities, cell wall carbohydrate composition, and extensive total lipid profiles. These traits inferred from genome composition, e.g., in terms of identified encoded enzymes, are in accordance with experimental results. Finally, we link the presence of associated bacteria with observed characteristics. Thamnidium elegans genome harbors an additional, complete genome of an associated bacterium classified to Paenibacillus sp. This fungus displays multiple altered traits compared to the remaining isolates, regardless of their evolutionary distance. For instance, it has expanded carbon assimilation capabilities, e.g., efficiently degrades carboxylic acids, and has a higher diacylglycerol:triacylglycerol ratio and skewed phospholipid composition which suggests a more rigid cellular membrane. The bacterium can complement the host enzymatic capabilities, alter the fungal metabolism, cell membrane composition but does not change the composition of the cell wall of the fungus. Comparison of early-diverging Umbelopsidales with evolutionary younger Mucorales points at several subtle differences particularly in their carbon source preferences and encoded carbohydrate repertoire. Nevertheless, all tested Mucoromycotina share features including the ability to produce 18:3 gamma-linoleic acid, use TAG as the storage lipid and have fucose as a cell wall component.

Switchgrass and Giant Miscanthus Biomass and Theoretical Ethanol Production from Reclaimed Mine Lands

Switchgrass (Panicum virgatum L.) and giant miscanthus (Miscanthus x giganteus Greef & Deuter ex Hodkinson & Renvoize) are productive on marginal lands in the eastern USA, but their productivity and composition have not been compared on mine lands. Our objectives were to compare biomass production, composition, and theoretical ethanol yield (TEY) and production (TEP) of these grasses on a reclaimed mined site. Following 25 years of herbaceous cover, vegetation was killed and plots of switchgrass cultivars Kanlow and BoMaster and miscanthus lines Illinois and MBX-002 were planted in five replications. Annual switchgrass and miscanthus yields averaged 5.8 and 8.9 Mg dry matter ha−1, respectively, during 2011 to 2015. Cell wall carbohydrate composition was analyzed via near-infrared reflectance spectroscopy with models based on switchgrass or mixed herbaceous samples including switchgrass and miscanthus. Concentrations were higher for glucan and lower for xylan in miscanthus than in switchgrass but TEY did not differ (453 and 450 L Mg−1, respectively). In response to biomass production, total ethanol production was greater for miscanthus than for switchgrass (5594 vs 3699 L ha−1), did not differ between Kanlow and BoMaster switchgrass (3880 and 3517 L ha−1, respectively), and was higher for MBX-002 than for Illinois miscanthus (6496 vs 4692 L ha−1). Relative to the mixed feedstocks model, the switchgrass model slightly underpredicted glucan and slightly overpredicted xylan concentrations. Estimated TEY was slightly lower from the switchgrass model but both models distinguished genotype, year, and interaction effects similarly. Biomass productivity and TEP were similar to those from agricultural sites with marginal soils.

Molecular and cellular responses of the pathogenic fungus Lomentospora prolificans to the antifungal drug voriconazole.

The filamentous fungus Lomentospora (Scedosporium) prolificans is an emerging opportunistic pathogen associated with fatal infections in patients with disturbed immune function. Unfortunately, conventional therapies are hardly of any use against this fungus due to its intrinsic resistance. Therefore, we performed an integrated study of the L. prolificans responses to the first option to treat these mycoses, namely voriconazole, with the aim of unveiling mechanisms involved in the resistance to this compound. To do that, we used a wide range of techniques, including fluorescence and electron microscopy to study morphological alterations, ion chromatography to measure changes in cell-wall carbohydrate composition, and proteomics-based techniques to identify the proteins differentially expressed under the presence of the drug. Significantly, we showed drastic changes occurring in cell shape after voriconazole exposure, L. prolificans hyphae being shorter and wider than under control conditions. Interestingly, we proved that the architecture and carbohydrate composition of the cell wall had been modified in the presence of the drug. Specifically, L. prolificans constructed a more complex organelle with a higher presence of glucans and mannans. In addition to this, we identified several differentially expressed proteins, including Srp1 and heat shock protein 70 (Hsp70), as the most overexpressed under voriconazole-induced stress conditions. The mechanisms described in this study, which may be directly related to L. prolificans antifungal resistance or tolerance, could be used as targets to improve existing therapies or to develop new ones in order to successfully eliminate these mycoses.

Microalgal biomass dewatering using forward osmosis membrane: Influence of microalgae species and carbohydrates composition

The potential application of forward osmosis (FO) in microalgae dewatering requires an improved understanding of the factors that control membrane fouling which can reduce dewatering performance in terms of water flux through membrane and algae recovery. The aim of this study was to elucidate the influence of algae cell wall carbohydrate composition on the FO dewatering performance using three types of draw solutions (sea salts, MgCl2 and CaCl2). Experimental results suggest that the interaction between microalgae and back diffused draw solutes plays a key role. Scenedesmus obliquus with fructose and abundant glucose and mannose in its cell wall showed strong response to the back diffusion of calcium ions which encouraged S. obliquus to produce more extracellular carbohydrates and formed a stable gel network between algal biomass and extracellular carbohydrates, leading to algae aggregation and severe loss in both water flux and algae biomass during FO dewatering with Ca2+-containing draw solution. Chlamydomonas reinhardtii without fructose but great galactose showed a similar response to the calcium back diffusion but to a lower extent. Both S. obliquus and C. reinhardtii did not cause obvious membrane fouling but dramatic algae biomass loss at the end of FO filtration with MgCl2 draw solution due to their interaction with back diffused Mg2+ ions which led to the deposition of algae flocs onto membrane surface and/or feed spacer. Chlorella vulgaris without fructose was the most suitable microalgae species to be dewatered by FO with algae recovery over 81% and negligible flux decline regardless of which draw solution was applied. The findings improve mechanical understanding of FO membrane fouling by microalgae; have significant implications for the algae species selection; and are critical for the development and optimization of FO dewatering processes.

The Role of Isocitrate Lyase (ICL1) in the Metabolic Adaptation of Candida albicans Biofilms.

BackgroundA major characteristic of Candida biofilm cells that differentiates them from free-floating cells is their high tolerance to antifungal drugs. This high resistance is attributed to particular biofilm properties, including the accumulation of extrapolymeric substances, morphogenetic switching, and metabolic flexibility.ObjectivesThis study evaluated the roles of metabolic processes (in particular the glyoxylate cycle) on biofilm formation, antifungal drug resistance, morphology, and cell wall components.MethodsGrowth, adhesion, biofilm formation, and cell wall carbohydrate composition were quantified for isogenic Candida albicans ICL1/ICL1, ICL1/icl1, and icl1/icl1 strains. The morphology and topography of these strains were compared by light microscopy and scanning electron microscopy. FKS1 (glucan synthase), ERG11 (14-α-demethylase), and CDR2 (efflux pump) mRNA levels were quantified using qRT-PCR.ResultsThe ICL1/icl1 and icl1/icl1 strains formed similar biofilms and exhibited analogous drug-tolerance levels to the control ICL1/ICL1 strains. Furthermore, the drug sequestration ability of β-1, 3-glucan, a major carbohydrate component of the extracellular matrix, was not impaired. However, the inactivation of ICL1 did impair morphogenesis. ICL1 deletion also had a considerable effect on the expression of the FKS1, ERG11, and CDR2 genes. FKS1 and ERG11 were upregulated in ICL1/icl1 and icl1/icl1 cells throughout the biofilm developmental stages, and CDR2 was upregulated at the early phase. However, their expression was downregulated compared to the control ICL1/ICL1 strain.ConclusionsWe conclude that the glyoxylate cycle is not a specific determinant of biofilm drug resistance.

Spatially and temporally restricted expression of PtrMYB021 regulates secondary cell wall formation in Arabidopsis

Among the R2R3 MYB transcription factors that involve in the regulation of secondary cell wall formation in Arabidopsis, MYB46 alone is sufficient to induce the entire secondary cell wall biosynthesis program. PtrMYB021, the poplar homolog of MYB46, has been reported to regulate secondary cell wall formation when expressed in Arabidopsis. We report here that spatially and temporally restricted expression of PtrMYB021 is critical for its function in regulating secondary cell wall formation. By using quantitative RT-PCR, we found that PtrMYB021 was expressed primarily in xylem tissues. When expressed in Arabidopsis under the control of PtrCesA8, but not the 35S promoter, PtrMYB021 increased secondary cell wall thickness, which is likely caused by increased lignification as well as changes in cell wall carbohydrate composition. In consistent with this, elevated expression of lignin and cellulose biosynthetic genes were observed in the transgenic plants. When expressed in Arabidopsis protoplasts as fusion proteins to the Gal4 DNA binding domain, PtrMYB021 activated the reporter gene Gal4-GUS. In summary, our results suggest that PtrMYB021 is a transcriptional activator, and spatially and temporally restricted expression of PtrMYB021 in Arabidopsis regulates secondary cell wall formation by activating a subset of secondary cell wall biosynthesis genes.

Cell wall composition and digestibility alterations in Brachypodium distachyon achieved through reduced expression of the UDP-arabinopyranose mutase.

Nucleotide-activated sugars are essential substrates for plant cell-wall carbohydrate-polymer biosynthesis. The most prevalent grass cell wall (CW) sugars are glucose (Glc), xylose (Xyl), and arabinose (Ara). These sugars are biosynthetically related via the UDP–sugar interconversion pathway. We sought to target and generate UDP–sugar interconversion pathway transgenic Brachypodium distachyon lines resulting in CW carbohydrate composition changes with improved digestibility and normal plant stature. Both RNAi-mediated gene-suppression and constitutive gene-expression approaches were performed. CWs from 336 T0 transgenic plants with normal appearance were screened for complete carbohydrate composition. RNAi mutants of BdRGP1, a UDP-arabinopyranose mutase, resulted in large alterations in CW carbohydrate composition with significant decreases in CW Ara content but with minimal change in plant stature. Five independent RNAi-RGP1 T1 plant lines were used for in-depth analysis of plant CWs. Real-time PCR analysis indicated that gene expression levels for BdRGP1, BdRGP2, and BdRGP3 were reduced in RNAi-RGP1 plants to 15–20% of controls. CW Ara content was reduced by 23–51% of control levels. No alterations in CW Xyl and Glc content were observed. Corresponding decreases in CW ferulic acid (FA) and ferulic acid-dimers (FA-dimers) were observed. Additionally, CW p-coumarates (pCA) were decreased. We demonstrate the CW pCA decrease corresponds to Ara-coupled pCA. Xylanase-mediated digestibility of RNAi-RGP1 Brachypodium CWs resulted in a near twofold increase of released total carbohydrate. However, cellulolytic hydrolysis of CW material was inhibited in leaves of RNAi-RGP1 mutants. Our results indicate that targeted manipulation of UDP–sugar biosynthesis can result in biomass with substantially altered compositions and highlights the complex effect CW composition has on digestibility.

Elevated CO2 concentration impacts cell wall polysaccharide composition of green microalgae of the genus Chlorella.

The effect of CO2 concentration on the relative content of starch, lipid and cell wall carbohydrates in microalgal biomass was investigated for the four following Chlorella strains: C.vulgaris (UTEX 259), C.sorokiniana (UTEX 2805), C.minutissima (UTEX 2341) and C.variabilis (NC64A). Each strain had a different response to CO2 concentration. The starch content was higher in UTEX259 and NC64A cultured with 2% CO2 in the air supply than in cells cultured with ca. 0·04% CO2 (ambient air), while starch content was not affected for UTEX 2805 and UTEX 2341. The lipid content was higher in Chlorella minutissima UTEX 2341 cultured in 2% CO2 than in cells cultured in ambient air, but was unchanged for the other three strains. All four Chlorella strains tended to have a higher percentage of uronic acids and lower percentage of neutral sugars in their cell wall polysaccharide complement when grown with 2% CO2 supply. Although the percentage of neutral sugars in the cell walls varied with CO2 concentration, the relative proportions of different neutral sugar constituents remained constant for both CO2 conditions. The results demonstrate the importance of considering the effects of CO2 on the cell wall carbohydrate composition of microalgae. Microalgae have the potential to produce products that will reduce society's reliance on fossil fuels and address challenges related to food and feed production. An overlooked yet industrially relevant component of microalgae are their cell walls. Cell wall composition affects cell flocculation and the recovery of intracellular products. In this study, we show that increasing CO2 level results in greater cell wall polysaccharide and uronic acid content in the cell walls of three strains of microalgae. The results have implications on the management of systems for the capture of CO2 and production of fuels, chemicals and food from microalgae.

Effect of genotype on chemical composition, ruminal degradability and in vitro fermentation characteristics of maize residual plants

The objective of this study was to determine the changes to residual plant feeding value of early- and late-maturing maize varieties. The influence of the cell wall carbohydrate composition, in terms of neutral and acid detergent fibre (NDF and ADF) content, NDF and dry matter (DM) degradability, and in vitro organic matter digestibility and gas production on the feeding value of a range of maize genotypes, was measured. The different genotypes were allotted into two maturity groups (MG I--early to mid-early: S210-S240; MG II--mid-late to late: S 250-S280) and harvested at four different harvest dates (depending on the DM content of the kernels). The maize varieties of MG I had lower NDF and ADF contents and higher ruminal DM degradability, in vitro digestibility and gas production and thus a higher feeding value than MG II at the same stage of physiological maturity. A strong negative relationship between NDF content and the ruminal DM degradability (r = -0.81) was observed. The data indicate that the early-maturing varieties permit a larger flexibility in harvesting due to a longer period of starch inclusion into the kernel whilst simultaneously maintaining a good supply of rumen-available fibre. Conclusively, the higher feeding value of the early-maturing varieties, based on lower NDF and high DM digestibility, permits more flexibility in the harvesting period over the later-maturing varieties.

Lectin-microarray technique for glycomic profiling of fungal cell surfaces.

Lectin microarrays are rows of lectins with different carbohydrate-binding specificities spotted on surfaces of glass slides. Lectin microarray technique enables glycomic analyses of carbohydrate composition of fungal cell walls. We will describe an application of the technique in analyzing cell surface glycome of yeast-form fungal cells in the living state. The analysis reveals genus- and species-dependent complex cell surface carbohydrate structures of fungi, and enabled us, therefore, to suggest that cell walls of yeast cells, which have been considered to have relatively simple structures, actually have a more complex structure containing galactose and fucose. This shows that the technique can be used to find new insights into the study of phylogenetic relations and into the classification of cells in the fungal kingdom based on cell wall glycome.

Chitinases CtcB and CfcI modify the cell wall in sporulating aerial mycelium of Aspergillus niger

Sporulation is an essential part of the life cycle of the industrially important filamentous fungus Aspergillus niger. The formation of conidiophores, spore-bearing structures, requires remodelling of the fungal cell wall, as demonstrated by the differences in carbohydrate composition of cell walls of vegetative mycelium and spores. Glycoside hydrolases that are involved in this process have so far remained unidentified. Using transcriptome analysis, we have identified genes encoding putative cell-wall-modifying proteins with enhanced expression in sporulating aerial mycelium compared to vegetative mycelium. Among the most strongly induced genes were those encoding a protein consisting of a putative chitin binding module (CBM14) and the chitinolytic enzymes NagA, CfcI and CtcB. Reporter studies showed that the N-acetyl-β-hexosaminidase gene nagA was expressed both in vegetative hyphae and in aerial structures (aerial hyphae, conidiophores and conidia) upon starvation. In contrast, promoter activities of the chitinase genes ctcB and cfcI were specifically localized in the conidiophores and conidia. CtcB is an endo-chitinase and CfcI releases monomers from chitin oligosaccharides: together these enzymes have the potential to degrade chitin of the fungal cell wall. Inactivation of both the cfcI and ctcB genes affected neither radial growth rate, nor formation and germination of spores. The amount of chitin in the spore walls of a ΔcfcIΔctcB double deletion strain, however, was significantly increased compared with the wild-type, thus indicating that CfcI and CtcB indeed modify the A. niger cell walls during sporulation. These novel insights in the sporulation process in aspergilli are of strong scientific relevance, and also may aid industrial strain engineering.

Gross karyotypic and phenotypic alterations among different progenies of the Candida glabrata CBS138/ATCC2001 reference strain.

Genomic plasticity is a mechanism for adaptation to environmental cues such as host responses and antifungal drug pressure in many fungi including the human pathogenic yeast Candida glabrata. In this study we evaluated the phenotypic and genotypic stability of the world-wide used C. glabrata reference strain CBS138/ATCC2001 under laboratory conditions. A set of ten lineages of this wild type strain and genetically modified progenies were obtained from different scientific laboratories, and analyzed for genotypic and phenotypic alterations. Even though the derivates were indistinguishable by multi locus sequence typing, different phenotypic groups that correlated with specific karyotypic changes were observed. In addition, modifications in the adherence capacity to plastic surface emerged that were shown to correlate with quantitative changes in adhesin gene expression rather than subtelomeric gene loss or differences in the number of macrosatellite repeats within adhesin genes. These results confirm the genomic plasticity of C. glabrata and show that chromosomal aberrations and functional adaptations may occur not only during infection and under antimicrobial therapy, but also under laboratory conditions without extreme selective pressures. These alterations can significantly affect phenotypic properties such as cell surface attributes including adhesion and the cell wall carbohydrate composition and therefore, if unnoticed, may adulterate the outcome of genetic studies.

The impact of cell wall carbohydrate composition on the chitosan flocculation of Chlorella

The carbohydrate composition of the algal cell wall was investigated for its role in cell flocculation. Cultures of Chlorella variabilis NC64A, which were found to have different levels of neutral sugar, uronic acid and amino sugar in the cell wall when cultured in different nitrogen sources and concentrations, were subjected to flocculation with chitosan at dosages of 0–69.6mg/l and pH values of 5.5, 7 and 8.5. In addition, flocculations of another three strains of Chlorella, which have different levels of cell wall components, were tested. Flocculation improved for all strains at pH 8.5 suggesting that inter molecular forces such as hydrogen bonding might be more important than charge neutralization in the flocculation of Chlorella. Total carbohydrate content in the cell wall was the most significant factor positively affecting the flocculation efficiency of C. variabilis NC64A cells with different cell wall compositions and the other Chlorella strains. The results presented in this study suggest that chitosan flocculation can be improved by optimizing the cell culture conditions to achieve higher cell wall polysaccharide content or selecting an algal strain with higher cell wall polysaccharide content.

Glycoprotein Hypersecretion Alters the Cell Wall in Trichoderma reesei Strains Expressing the Saccharomyces cerevisiae Dolichylphosphate Mannose Synthase Gene

Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increased amount of N-acetylglucosamine, suggesting an increase in chitin content. Calcofluor white staining followed by fluorescence microscopy indicated changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble beta-(1,6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants. We also discuss the relationships between the observed changes in the cell wall, increased protein glycosylation, and the greater secretory capacity of T. reesei strains expressing the yeast DPM1 gene.

Interactions between MUR10/CesA7-dependent secondary cellulose biosynthesis and primary cell wall structure.

Primary cell walls are deposited and remodeled during cell division and expansion. Secondary cell walls are deposited in specialized cells after the expansion phase. It is presently unknown whether and how these processes are interrelated. The Arabidopsis (Arabidopsis thaliana) MUR10 gene is required for normal primary cell wall carbohydrate composition in mature leaves as well as for normal plant growth, hypocotyl strength, and fertility. The overall sugar composition of young mur10 seedlings is not significantly altered; however, the relative proportion of pectin side chains is shifted toward an increase in 1 --> 5-alpha-arabinan relative to 1 --> 4-beta-galactan. mur10 seedlings display reduced fucogalactosylation of tightly cell wall-bound xyloglucan. Expression levels of genes encoding either nucleotide sugar interconversion enzymes or glycosyl transferases, known to be involved in primary and secondary cell wall biosynthesis, are generally unaffected; however, the CesA7 transcript is specifically suppressed in the mur10-1 allele. The MUR10 locus is identical with the CesA7 gene, which encodes a cellulose catalytic subunit previously thought to be specifically involved in secondary cell wall formation. The xylem vessels in young mur10 hypocotyls are collapsed and their birefringence is lost. Moreover, a fucogalactosylated xyloglucan epitope is reduced and a 1 --> 5-alpha-arabinan epitope increased in every cell type in mur10 hypocotyls, including cells that do not deposit secondary walls. mur10 also displays altered distribution of an arabinogalactan-protein epitope previously associated with xylem differentiation and secondary wall thickening. This work indicates the existence of a mechanism that senses secondary cell wall integrity and controls biosynthesis or structural remodeling of primary cell walls and cellular differentiation.

Estimation of phylogenetic relationships within the Ascomycota on the basis of 18S rDNA sequences and chemotaxonomy

Small subunit rRNA gene sequences (18S rDNA), cell wall carbohydrate composition and ubiquinone components were analysed within a larger number of ascomycetous yeasts and dimorphic fungi to validate their congruence in predicting phylogenetic relationships. The glucose-mannose pattern distinguishes the Hemiascomycetes from the Euascomycetes and the Protomycetes which are characterised with the glucose-mannose-galactose-rhamnose-(fucose) profile. The glucose-mannose-galactose pattern was found in the cell walls of all the three classes. Different coenzyme Q component (CoQ5 to CoQ10) were found within the representatives of the Hemiascomycetes. Whereas CoQ9, CoQ10 and CoQ10H2 predominate within the Euascomycetes, CoQ9 and CoQ10 characterise the Protomycetes. Chemotaxonomic studies coupled with additional molecular and co-evolution studies support the idea that the Hemiascomycetes occupy a basal position in the phylogeny of Ascomycota. These results are not in line with the phylogenetic studies based on the sequences of 18S rRNA encoding gene. The maximum parsimony analysis indicated that Hemiascomycetes and Protomycetes might represent sister groups, opposing to the earlier reported results, where the Archiascomycetes (Protomycetes) or the Hemiascomycetes had been considered to be the most primitive ascomycetous fungi. Instead of the class Archiascomycetes, the term Protomycetes was introduced reflecting much better the properties of the whole class.