Major Cell Wall Polysaccharides Research Articles

Do Cuticular Gaps Make It Possible to Study the Composition of the Cell Walls in the Glands of Drosophyllum lusitanicum?

Carnivorous plants can survive in poor habitats because they have the ability to attract, capture, and digest prey and absorb animal nutrients using modified organs that are equipped with glands. These glands have terminal cells with permeable cuticles. Cuticular discontinuities allow both secretion and endocytosis. In Drosophyllum lusitanicum, these emergences have glandular cells with cuticular discontinuities in the form of cuticular gaps. In this study, we determined whether these specific cuticular discontinuities were permeable enough to antibodies to show the occurrence of the cell wall polymers in the glands. Scanning transmission electron microscopy was used to show the structure of the cuticle. Fluorescence microscopy revealed the localization of the carbohydrate epitopes that are associated with the major cell wall polysaccharides and glycoproteins. We showed that Drosophyllum leaf epidermal cells have a continuous and well-developed cuticle, which helps the plant inhibit water loss and live in a dry environment. The cuticular gaps only partially allow us to study the composition of cell walls in the glands of Drosophyllum. We recoded arabinogalactan proteins, some homogalacturonans, and hemicelluloses. However, antibody penetration was only limited to the cell wall surface. The localization of the wall components in the cell wall ingrowths was missing. The use of enzymatic digestion improves the labeling of hemicelluloses in Drosophyllum glands.

The Localization of Cell Wall Components in the Quadrifids of Whole-Mount Immunolabeled Utricularia dichotoma Traps.

Utricularia (bladderworts) are carnivorous plants. They produce small hollow vesicles, which function as suction traps that work underwater and capture fine organisms. Inside the traps, there are numerous glandular trichomes (quadrifids), which take part in the secretion of digestive enzymes, the resorption of released nutrients, and likely the pumping out of water. Due to the extreme specialization of quadrifids, they are an interesting model for studying the cell walls. This aim of the study was to fill in the gap in the literature concerning the immunocytochemistry of quadrifids in the major cell wall polysaccharides and glycoproteins. To do this, the localization of the cell wall components in the quadrifids was performed using whole-mount immunolabeled Utricularia traps. It was observed that only parts (arms) of the terminal cells had enough discontinuous cuticle to be permeable to antibodies. There were different patterns of the cell wall components in the arms of the terminal cells of the quadrifids. The cell walls of the arms were especially rich in low-methyl-esterified homogalacturonan. Moreover, various arabinogalactan proteins also occurred. Cell walls in glandular cells of quadrifids were rich in low-methyl-esterified homogalacturonan; in contrast, in the aquatic carnivorous plant Aldrovanda vesiculosa, cell walls in the glandular cells of digestive glands were poor in low-methyl-esterified homogalacturonan. Arabinogalactan proteins were found in the cell walls of trap gland cells in all studied carnivorous plants: Utricularia, and members of Droseraceae and Drosophyllaceae.

Differences in the Occurrence of Cell Wall Components between Distinct Cell Types in Glands of Drosophyllum lusitanicum.

Carnivorous plants are mixotrophs that have developed the ability to lure, trap, and digest small organisms and utilize components of the digested bodies. Leaves of Drosophyllum lusitanicum have two kinds of glands (emergences): stalked mucilage glands and sessile digestive glands. The stalked mucilage glands perform the primary role in prey lure and trapping. Apart from their role in carnivory, they absorb water condensed from oceanic fog; thus, plants can survive in arid conditions. To better understand the function of carnivorous plant emergences, the molecular composition of their cell walls was investigated using immunocytochemical methods. In this research, Drosophyllum lusitanicum was used as a study system to determine whether cell wall immunocytochemistry differs between the mucilage and digestive glands of other carnivorous plant species. Light and electron microscopy were used to observe gland structure. Fluorescence microscopy revealed the localization of carbohydrate epitopes associated with the major cell wall polysaccharides and glycoproteins. The mucilage gland (emergence) consists of a glandular head, a connecting neck zone, and stalk. The gland head is formed by an outer and inner layer of glandular (secretory) cells and supported by a layer of endodermoid (barrier) cells. The endodermoid cells have contact with a core of spongy tracheids with spiral-shaped thickenings. Lateral tracheids are surrounded by epidermal and parenchymal neck cells. Different patterns of cell wall components were found in the various cell types of the glands. Cell walls of glandular cells generally are poor in both low and highly esterified homogalacturonans (HGs) but enriched with hemicelluloses. Cell walls of inner glandular cells are especially rich in arabinogalactan proteins (AGPs). The cell wall ingrowths in glandular cells are significantly enriched with hemicelluloses and AGPs. In the case of cell wall components, the glandular cells of Drosophyllum lusitanicum mucilage glands are similar to the glandular cells of the digestive glands of Aldrovanda vesiculosa and Dionaea muscipula.

Immunochemical Identification of the Main Cell Wall Polysaccharides of the Early Land Plant Marchantia polymorpha.

Plant primary cell walls are composite structures surrounding the protoplast and containing pectins, hemicelluloses, and cellulose polysaccharides, as well as proteins. Their composition changed during the evolution of the green lineage from algae to terrestrial plants, i.e., from an aquatic to a terrestrial environment. The constraints of life in terrestrial environments have generated new requirements for the organisms, necessitating adaptations, such as cell wall modifications. We have studied the cell wall polysaccharide composition of thalli of Marchantia polymorpha, a bryophyte belonging to one of the first land plant genera. Using a collection of specific antibodies raised against different cell wall polysaccharide epitopes, we were able to identify in polysaccharide-enriched fractions: pectins, including low-methylesterified homogalacturonans; rhamnogalacturonan I with arabinan side-chains; and hemicelluloses, such as xyloglucans with XXLG and XXXG modules, mannans, including galactomannans, and xylans. We could also show the even distribution of XXLG xyloglucans and galactomannans in the cell walls of thalli by immunocytochemistry. These results are discussed with regard to the cell wall proteome composition and in the context of the evolution of the green lineage. The cell wall polysaccharides of M. polymorpha illustrate the transition from the charophyte ancestors of terrestrial plants containing xyloglucans, xylans and mannans as hemicelluloses, and embryophytes which do not exhibit mannans as major primary cell wall polysaccharides.

Exploring the polysaccharide composition of plant cell walls in succulent aloes

Societal Impact StatementAloes are iconic succulent plants native to Africa, Madagascar and the Arabian Peninsula. The succulent leaf mesophyll of aloes has been used extensively as a herbal product for centuries, contributing to their overexploitation. Health benefits are attributed to their polysaccharide content. We present a comprehensive comparison of the polysaccharide composition of succulent tissues from 93 Aloe species. We found polysaccharide composition primarily related to leaf morphology in alignment with the broad range of Aloe species used medicinally. All aloes except Aloe ferox and Aloe vera are endangered raising concern about over‐harvesting of wild species.Summary Aloes are iconic succulent plants native to Africa, Madagascar and the Arabian Peninsula. All aloes except the commercially grown Aloe ferox and Aloe vera are protected according to the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Major factors contributing to their over‐exploitation are their ornamental value and medicinal use with more than 25% of Aloe species being utilised. The succulent inner leaf mesophyll of aloes is used in traditional medicine, with the healing effect ascribed to the properties of their structural polysaccharides. To explore the correlation between Aloe polysaccharide profiles and other biologically relevant traits across the genus, we (1) extracted polysaccharides and created profiles for nearly 100 representative species using carbohydrate microarrays and molecular probes. We targeted six major plant cell wall polysaccharide groups using 27 different molecular probes. We (2) tested for phylogenetic signal in the polysaccharide profiles and (3) assembled an exhaustive database from literature on the geographic region, level of endemism, altitude, habitat, habit, medicinal use and leaf morphology of the individual species of Aloe. In the absence of phylogenetic signal of polysaccharide profiles, multivariate linear modelling without phylogenetic correction was used and showed that polysaccharide composition primarily correlated with leaf morphology, highlighting the fundamental role of polysaccharides as the building blocks of plants. No correlations between polysaccharide composition of commercial and non‐commercial species were found. We found polysaccharide composition to primarily relate to leaf morphology emphasising the fundamental and structural role of polysaccharides.

Immunocytochemical Analysis of Bifid Trichomes in Aldrovanda vesiculosa L. Traps.

The two-armed bifids (bifid trichomes) occur on the external (abaxial) trap surface, petiole, and stem of the aquatic carnivorous plant Aldrovanda vesiculosa (Droseracee). These trichomes play the role of mucilage trichomes. This study aimed to fill the gap in the literature concerning the immunocytochemistry of the bifid trichomes and compare them with digestive trichomes. Light and electron microscopy was used to show the trichome structure. Fluorescence microscopy revealed the localization of carbohydrate epitopes associated with the major cell wall polysaccharides and glycoproteins. The stalk cells and the basal cells of the trichomes were differentiated as endodermal cells. Cell wall ingrowths occurred in all cell types of the bifid trichomes. Trichome cells differed in the composition of their cell walls. The cell walls of the head cells and stalk cells were enriched with arabinogalactan proteins (AGPs); however, they were generally poor in both low- and highly-esterified homogalacturonans (HGs). The cell walls in the trichome cells were rich in hemicelluloses: xyloglucan and galactoxyloglucan. The cell wall ingrowths in the basal cells were significantly enriched with hemicelluloses. The presence of endodermal cells and transfer cells supports the idea that bifid trichomes actively transport solutes, which are polysaccharide in nature. The presence of AGPs (which are considered plant signaling molecules) in the cell walls in these trichome cells indicates the active and important role of these trichomes in plant function. Future research should focus on the question of how the molecular architecture of trap cell walls changes in cells during trap development and prey capture and digestion in A. vesiculosa and other carnivorous plants.

Cleavage of α-1,4-glycosidic linkages by the glycosylphosphatidylinositol-anchored α-amylase AgtA decreases the molecular weight of cell wall α-1,3-glucan in Aspergillus oryzae.

Aspergillus fungi contain α-1,3-glucan with a low proportion of α-1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored α-amylases are conserved in Aspergillus fungi. The GPI-anchored α-amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall α-1,3-glucan but not to degrade it in vivo. However, the detailed mechanism of cell wall α-1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by the Aspergillus oryzae agtA gene, an ortholog of the A. nidulans amyD gene. Similar to findings in A. nidulans, agtA overexpression in A. oryzae grown in submerged culture decreased the amount of cell wall α-1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced in Pichia pastoris and found that it degraded soluble starch, but not linear bacterial α-1,3-glucan. Furthermore, rAoAgtA cleaved 3-α-maltotetraosylglucose with a structure similar to the predicted boundary structure between the α-1,3-glucan main chain and a short spacer composed of α-1,4-linked glucose residues in cell wall α-1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the α-1,4-glycosidic bonds of 3-α-maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall α-1,3-glucan. Consistent with this hypothesis, heterologous overexpression of agtA in A. nidulans decreased the molecular weight of cell wall α-1,3-glucan. These in vitro and in vivo properties of AoAgtA suggest that GPI-anchored α-amylases can degrade the spacer α-1,4-glycosidic linkages in cell wall α-1,3-glucan before its insolubilization, and this spacer cleavage decreases the molecular weight of cell wall α-1,3-glucan in vivo.

Stellate Trichomes in Dionaea muscipula Ellis (Venus Flytrap) Traps, Structure and Functions

The digestive organs of carnivorous plants have external (abaxial) glands and trichomes, which perform various functions. Dionaea muscipula Ellis (the Venus flytrap) is a model carnivorous plant species whose traps are covered by external trichomes. The aim of the study was to fill in the gap regarding the structure of the stellate outer trichomes and their immunocytochemistry and to determine whether these data support the suggestions of other authors about the roles of these trichomes. Light and electron microscopy was used to show the trichomes' structure. Fluorescence microscopy was used to locate the carbohydrate epitopes that are associated with the major cell wall polysaccharides and glycoproteins. The endodermal cells and internal head cells of the trichomes were differentiated as transfer cells, and this supports the idea that stellate trichomes transport solutes and are not only tomentose-like trichomes. Trichome cells differ in the composition of their cell walls, e.g., the cell walls of the internal head cells are enriched with arabinogalactan proteins (AGPs). The cell walls of the outer head cells are poor in both low and highly homogalacturonans (HGs), but the immature trichomes are rich in the pectic polysaccharide (1-4)-β-D-galactan. In the immature traps, young stellate trichomes produce mucilage which may protect the trap surface, and in particular, the trap entrance. However, the role of these trichomes is different when the outer head cells collapse. In the internal head cells, a thick secondary wall cell was deposited, which together with the thick cell walls of the outer head cells played the role of a large apoplastic space. This may suggest that mature stellate trichomes might function as hydathodes, but this should be experimentally proven.

Immunocytochemical Analysis of the Wall Ingrowths in the Digestive Gland Transfer Cells in Aldrovanda vesiculosa L. (Droseraceae).

Carnivorous plants are unique due to their ability to attract small animals or protozoa, retain them in specialized traps, digest them, and absorb nutrients from the dissolved prey material; however, to this end, these plants need a special secretion-digestive system (glands). A common trait of the digestive glands of carnivorous plants is the presence of transfer cells. Using the aquatic carnivorous species Aldrovanda vesiculosa, we showed carnivorous plants as a model for studies of wall ingrowths/transfer cells. We addressed the following questions: Is the cell wall ingrowth composition the same between carnivorous plant glands and other plant system models? Is there a difference in the cell wall ingrowth composition between various types of gland cells (glandular versus endodermoid cells)? Fluorescence microscopy and immunogold electron microscopy were employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins. The cell wall ingrowths were enriched with arabinogalactan proteins (AGPs) localized with the JIM8, JIM13, and JIM14 epitopes. Both methylesterified and de-esterified homogalacturonans (HGs) were absent or weakly present in the wall ingrowths in transfer cells (stalk cells and head cells of the gland). Both the cell walls and the cell wall ingrowths in the transfer cells were rich in hemicelluloses: xyloglucan (LM15) and galactoxyloglucan (LM25). There were differences in the composition between the cell wall ingrowths and the primary cell walls in A. vesiculosa secretory gland cells in the case of the absence or inaccessibility of pectins (JIM5, LM19, JIM7, LM5, LM6 epitopes); thus, the wall ingrowths are specific cell wall microdomains. Even in the same organ (gland), transfer cells may differ in the composition of the cell wall ingrowths (glandular versus endodermoid cells). We found both similarities and differences in the composition of the cell wall ingrowths between the A. vesiculosa transfer cells and transfer cells of other plant species.

Candida albicans phosphate transport, facilitating nucleotide sugar biosynthesis, contributes to cell wall stability.

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin signaling, oxidative stress resistance and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild type cells recovering from phosphate starvation. Non-phosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar,GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. Our model is that low substrate concentrations of beta-D-glucan- and chitin synthases diminish enzymatic reaction rates and potentiate pharmacologic inhibitors to decrease the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-D-glucans or chitin. Hence inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.

Liberation of eicosapentaenoic acid and degradation of the major cell wall polysaccharide porphyran by fermentation of nori, the dried thalli of Pyropia yezoensis, with koji

In this study, dried sheets of nori, shredded and processed thalli of the red alga Pyropia yezoensis, were fermented with either barley, rice or soybean koji. High-performance liquid chromatographic analyses of the lipid extracts of the fermented products indicated that the fermentation of nori with all kinds of tested koji released free fatty acids, including the eicosapentaenoic acid, from ester lipids. We found that approximately half of the eicosapentaenoic acid in nori had been released as the free fatty acid at up to 4 weeks of fermentation at 30 °C and more than 65% at 8 to12 weeks in the fermented products with barley and rice koji. We also demonstrated the degradation of porphyran, a major cell wall polysaccharide of nori, by gel chromatography on Sephacryl S-300 HR of hot water extracts of the fermented products of nori with barley koji. Approximately two-third of porphyran had been degraded to porphyran oligosaccharides up to 6 weeks of fermentation. Fermentation of nori with koji may bring out the potential health-promoting functions of nori.

A new carbohydrate-active oligosaccharide dehydratase is involved in the degradation of ulvan

Marine algae catalyze half of all global photosynthetic production of carbohydrates. Owing to their fast growth rates, Ulva spp. rapidly produce substantial amounts of carbohydrate-rich biomass and represent an emerging renewable energy and carbon resource. Their major cell wall polysaccharide is the anionic carbohydrate ulvan. Here, we describe a new enzymatic degradation pathway of the marine bacterium Formosa agariphila for ulvan oligosaccharides involving unsaturated uronic acid at the nonreducing end linked to rhamnose-3-sulfate and glucuronic or iduronic acid (Δ-Rha3S-GlcA/IdoA-Rha3S). Notably, we discovered a new dehydratase (P29_PDnc) acting on the nonreducing end of ulvan oligosaccharides, i.e., GlcA/IdoA-Rha3S, forming the aforementioned unsaturated uronic acid residue. This residue represents the substrate for GH105 glycoside hydrolases, which complements the enzymatic degradation pathway including one ulvan lyase, one multimodular sulfatase, three glycoside hydrolases, and the dehydratase P29_PDnc, the latter being described for the first time. Our research thus shows that the oligosaccharide dehydratase is involved in the degradation of carboxylated polysaccharides into monosaccharides.

Genome-wide transcriptome profiling and characterization of mannuronan C5-epimerases in Saccharina japonica

Alginate is a linear polysaccharide comprised of 1,4-linked-β-D-mannuronic acid (M) and α-L-guluronic acid (G). As the major cell wall polysaccharide in brown algae, alginate is widely used in the food, pharmaceutical, and chemical industries. Mannuronan C5-epimerases (MC5Es) catalyze the specific conversion of M to its C-5 epimer G, playing a key role in the modification of alginate. In this study, 143 MC5E gene sequences from Saccharina japonica (SjMC5Es) were identified based on the genome and transcriptome data. Transcriptomics analysis was conducted, and the results showed that the SjMC5Es varied in their spatial and temporal expression patterns. Most SjMC5Es exhibited higher expression in the basal blades of the algae at earlier developmental stages than at later adult stages, and the transcription levels of the SjMC5Es showed a clear trend of gradual increase or decrease from the basal to the distal blades. In addition, the β-D-mannuronic acid/α-L-guluronic acid (M/G) ratio of alginate from all samples was measured, and the M/G ratios were associated with the transcription levels of the SjMC5E genes. Finally, the highly expressed SjMC5E2 was characterized, soluble SjMC5E2 was heterologously expressed, and its mannuronan C5-epimerase activity was confirmed by 1H-nuclear magnetic resonance analysis. Our results provide a global view of the SjMC5Es in S. japonica and lay a foundation for further applications of MC5Es in improving the physiochemical properties of alginate.

RNAi suppression of xylan synthase genes in wheat starchy endosperm.

The xylan backbone of arabinoxylan (AX), the major cell wall polysaccharide in the wheat starchy endosperm, is synthesised by xylan synthase which is a complex of three subunits encoded by the GT43_1, GT43_2 and GT47_2 genes. RNAi knock-down of either GT43_1 or all three genes (triple lines) resulted in decreased AX measured by digestion with endoxylanase (to 33 and 34.9% of the controls) and by monosaccharide analysis (to 45.9% and 47.4% of the controls) with greater effects on the amount of water-extractable AX (to 20.6 and 19.9% of the controls). Both sets of RNAi lines also had greater decreases in the amounts of substituted oligosaccharides released by digestion of AX with endoxylanase than in fragments derived only from the xylan backbone. Although the GT43_1 and triple lines had similar effects on AX they did differ in their contents of soluble sugars (increased in triple only) and on grain size (decreased in triple only). Both sets of transgenic lines had decreased grain hardness, indicating effects on cell wall mechanics. These results, and previously published studies of RNAi suppression of GT43_2 and GT47_2 and of a triple mutant of GT43_2, are consistent with the model of xylan synthase comprising three subunits one of which (GT47_2) is responsible for catalysis with the other two subunits being required for correct functioning but indicate that separate xylan synthase complexes may be responsible for the synthesis of populations of AX which differ in their structure and solubility.

Engineering of a new Escherichia coli strain efficiently metabolizing cellobiose with promising perspectives for plant biomass-based application design

The necessity to decrease our fossil energy dependence requests bioprocesses based on biomass degradation. Cellobiose is the main product released by cellulases when acting on the major plant cell wall polysaccharide constituent, the cellulose. Escherichia coli, one of the most common model organisms for the academy and the industry, is unable to metabolize this disaccharide. In this context, the remodeling of E. coli to catabolize cellobiose should thus constitute an important progress for the design of such applications. Here, we developed a robust E. coli strain able to metabolize cellobiose by integration of a small set of modifications in its genome. Contrary to previous studies that use adaptative evolution to achieve some growth on this sugar by reactivating E. coli cryptic operons coding for cellobiose metabolism, we identified easily insertable modifications impacting the cellobiose import (expression of a gene coding a truncated variant of the maltoporin LamB, modification of the expression of lacY encoding the lactose permease) and its intracellular degradation (genomic insertion of a gene encoding either a cytosolic β-glucosidase or a cellobiose phosphorylase). Taken together, our results provide an easily transferable set of mutations that confers to E. coli an efficient growth phenotype on cellobiose (doubling time of 2.2 ​h in aerobiosis) without any prior adaptation.

The effects of foliar-applied manganese on major cell wall polysaccharide fractions and soluble sugar composition of cucumber leaves

Increasing evidence shows that micronutrients can induce changes in cell wall (CW) properties in plants. A detailed knowledge of these changes will contribute to understand the beneficial and detrimental impacts of trace elements on plant development and defence. In the present work, the influence of foliar spray of manganese (Mn) on the composition of CW polysaccharides and soluble sugars was investigated in cucumber leaves using spectrophotometric and chromatographic techniques. Cucumber seedlings were hydroponically cultured under controlled situations for 21 days. At the two-leaf stage, the above facade of cucumber leaves was fertilized with MnSO4 solutions at various dosages (1.8, 4.5 and 7.2 mM). Results showed that the foliar Mn spray decreased the galacturonic acid (GalA) content, while it increased the degree-of-methyl-esterification (DM) of pectin. The amounts of fucose, arabinose and galactose diminished upon Mn application. The contents of rhamnose and xylose decreased or did not change by Mn treatment. The glucose level enhanced at low concentration of Mn (1.8 mM), but it decreased at medium and high concentrations of Mn (4.5 and 7.2 mM). The increase of mannose content was significant only at medium concentration of Mn. Overall, the amount of total hemicellulose increased at 1.8 mM concentration of Mn, while a decrease was observed at 4.5 and 7.2 mM concentrations of Mn. The cellulose content decreased at low and high concentrations of Mn, although it remained unchanged at medium concentration of Mn. Generally, the total contents of CW polysaccharides increased at low concentration of Mn, but they decreased at the higher concentrations of Mn. Such changes can be important in plant resistance to environmental stresses via reduction of permeability against fungal pathogens and decrease of water loss from tissues under stresses such as drought and salinity.

Rearrangement of the Cellulose-Enriched Cell Wall in Flax Phloem Fibers over the Course of the Gravitropic Reaction

The plant cell wall is a complex structure consisting of a polysaccharide network. The rearrangements of the cell wall during the various physiological reactions of plants, however, are still not fully characterized. Profound changes in cell wall organization are detected by microscopy in the phloem fibers of flax (Linum usitatissimum) during the restoration of the vertical position of the inclined stems. To characterize the underlying biochemical and structural changes in the major cell wall polysaccharides, we compared the fiber cell walls of non-inclined and gravistimulated plants by focusing mainly on differences in non-cellulosic polysaccharides and the fine cellulose structure. Biochemical analysis revealed a slight increase in the content of pectins in the fiber cell walls of gravistimulated plants as well as an increase in accessibility for labeling non-cellulosic polysaccharides. The presence of galactosylated xyloglucan in the gelatinous cell wall layer of flax fibers was demonstrated, and its labeling was more pronounced in the gravistimulated plants. Using solid state NMR, an increase in the crystallinity of the cellulose in gravistimulated plants, along with a decrease in cellulose mobility, was demonstrated. Thus, gravistimulation may affect the rearrangement of the cell wall, which can enable restoration in a vertical position of the plant stem.

Temporal proteome dynamics of Clostridium cellulovorans cultured with major plant cell wall polysaccharides

BackgroundClostridium cellulovorans is a mesophilic, cellulosome-producing bacterium containing 57 genomic cellulosomal enzyme-encoding genes. In addition to cellulosomal proteins, C. cellulovorans also secretes non-cellulosomal proteins to degrade plant cell wall polysaccharides. Unlike other cellulosome-producing Clostridium species, C. cellulovorans can metabolize all major plant cell wall polysaccharides (cellulose, hemicelluloses, and pectins). In this study, we performed a temporal proteome analysis of C. cellulovorans to reveal strategies underlying plant cell wall polysaccharide degradation.ResultsWe cultured C. cellulovorans with five different carbon sources (glucose, cellulose, xylan, galactomannan, and pectin) and performed proteome analysis on cellular and secreted proteins. In total, we identified 1895 cellular proteins and 875 secreted proteins. The identified unique carbohydrate-degrading enzymes corresponding to each carbon source were annotated to have specific activity against each carbon source. However, we identified pectate lyase as a unique enzyme in C. cellulovorans cultivated on xylan, which was not previously associated with xylan degradation. We performed k-means clustering analysis for elucidation of temporal changes of the cellular and secreted proteins in each carbon sources. We found that cellular proteins in most of the k-means clusters are involved in carbohydrate metabolism, amino acid metabolism, translation, or membrane transport. When xylan and pectin were used as the carbon sources, the most increasing k-means cluster contained proteins involved in the metabolism of cofactors and vitamins. In case of secreted proteins of C. cellulovorans cultured either on cellulose or xylan, galactomannan, and pectin, the clusters with the most increasing trend contained either 25 cellulosomal proteins and five non-cellulosomal proteins or 8–19 cellulosomal proteins and 9–16 non-cellulosomal proteins, respectively. These differences might reflect mechanisms for degrading cellulose of other carbon source. Co-abundance analysis of the secreted proteins revealed that proteases and protease inhibitors accumulated coordinately. This observation implies that the secreted protease inhibitors and proteases protect carbohydrate-degrading enzymes from an attack from the plant.ConclusionIn this study, we clarified, for the first time, the temporal proteome dynamics of cellular and secreted proteins in C. cellulovorans. This data will be valuable in understanding strategies employed by C. cellulovorans for degrading major plant cell wall polysaccharides.

Engineer multi-functional cellulase/xylanase/β-glucosidase with improved efficacy to degrade rice straw

Plant lignocellulosic biomass can be enzymatically degraded and converted into biofuel. We have studied a bifunctional cellulase/xylanase from thermophilic Clostridium thermocellum (CtCel5E), which can digest two major plant cell wall polysaccharides, cellulose (a polymer of glucose) and xylan (a major hemicellulose composed of xylose), into disaccharides cellobiose and xylobiose, respectively. In the study presented here, the bifunctional CtCel5E was fused with a β-glucosidase from Clostridium cellulovorans (CcBglA) to convert cellulose into glucose with improved activities toward carboxymethyl cellulose (CMC), regenerated amorphous cellulose (RAC), and alkali-treated rice straw as compared to the mixture of two individual enzymes by eliminating accumulation of cellobiose intermediate. The engineered CtCel5E-CcBglA was secreted from yeast to degrade alkali-treated rice straw into glucose that sustained cell growth. Our study provides a strategy of consolidating enzymes for more efficient biomass processing in vitro and by yeast cells.

Mechanistic studies on the biosorption of Pb(II) by Pseudomonas aeruginosa.

The biosorption of Pb(II) ions from aqueous solution has been studied using both the intact and thermolyzed cells of Pseudomonas aeruginosa. Further, the role of the major cell wall components, namely DNA, protein, polysaccharide, and lipid, in Pb(II) binding has been assessed using an enzymatic treatment method. The Pb(II) bioremediation capability of P. aeruginosa cells has been investigated by varying the parameters of pH, time of interaction, amount of biomass, and concentration of Pb(II). The complete bioremoval of Pb(II) using intact cells has been achieved for an initial Pb(II) concentration of 12.4 mg L-1 at pH 6.2 and temperature 29 ± 1 °C. The biosorption isotherm follows Langmuirian behavior with a Gibbs free energy of -30.7 kJ mol-1, indicative of chemisorption. The biosorption kinetics is consistent with a pseudo-second-order model. The possible Pb(II) binding mechanisms of P. aeruginosa cells are discussed based on characterization using zeta potential measurements, Fourier transform infra-red spectroscopy, and energy dispersive X-ray spectroscopy. The results confirm that among the major cell wall components studied, polysaccharide shows the highest contribution towards Pb(II) binding, followed by DNA, lipid, and protein. Similar studies using thermolyzed cells show higher Pb(II) uptake compared to the intact cells both before and after enzymatic treatment.