The cellulose synthase superfamily in fully sequenced plants and algae

Background  

The cellulose synthase superfamily has been classified into nine cellulose synthase-like (Csl) families and one cellulose synthase (CesA) family. The Csl families have been proposed to be involved in the synthesis of the backbones of hemicelluloses of plant cell walls. With 17 plant and algal genomes fully sequenced, we sought to conduct a genome-wide and systematic investigation of this superfamily through in-depth phylogenetic analyses.     

Taxonomic significance of cellulosic cell walls in the bangiales (Rhodophyta)

Cellulose has been characterized from isolated cell walls of the conchocelis phases of both Porphyra umbilicalis and P. leucostricta. Evidence for cellulose II (regenerated cellulose) in Schweitzer's reagent extracts was provided by X-ray powder analysis and paper chromatography of partial hydrolyzates. The presence of cellulose in the conchocelis phase of species of Porphyra provides evidence for the continuity of cell wall composition within the Rhodophyta. Adoption of a classification scheme incorporating consolidation of all red algal orders under the single class Rhodophyceae is proposed.     

BIOCHEMICAL AND MICROCHEMICAL STUDY OF THE CELL WALLS OF RHIZIDIOMYCES SP.

Fuller , Melvin S. (Brown U., Providence, R. I.) Biochemical and microchemical study of the cell walls of Rhizidiomyces sp. Amer. Jour. Bot. 47(10): 838–842. lllus. 1960.—The presence of chitin in the cell walls of the fungus Rhizidiomyces is demonstrated by qualitative analysis of enzymatic and hydrochloric-acid hydrolysates of partially cleaned cell walls. Qualitative examination of the enzymatic and acid hydrolysates did not, however, serve for the detection of cellulose present in the cell walls of Rhizidiomyces. With microchemical tests, both chitin and cellulose can be detected. These microchemical tests served to indicate the localization of the chitin and cellulose in the cell walls of mature plants before and during zoospore discharge.     

Hydrophilic domains of scaffolding protein CbpA promote glycosyl hydrolase activity and localization of cellulosomes to the cell surface of Clostridium cellulovorans

CbpA, the scaffolding protein of Clostridium cellulovorans cellulosomes, possesses one family 3 cellulose binding domain, nine cohesin domains, and four hydrophilic domains (HLDs). Among the three types of domains, the function of the HLDs is still unknown. We proposed previously that the HLDs of CbpA play a role in attaching the cellulosome to the cell surface, since they showed some homology to the surface layer homology domains of EngE. Several recombinant proteins with HLDs (rHLDs) and recombinant EngE (rEngE) were examined to determine their binding to the C. cellulovorans cell wall fraction. Tandemly linked rHLDs showed higher affinity for the cell wall than individual rHLDs showed. EngE was shown to have a higher affinity for cell walls than rHLDs have. C. cellulovorans native cellulosomes were found to have higher affinity for cell walls than rHLDs have. When immunoblot analysis was carried out with the native cellulosome fraction bound to cell wall fragments, the presence of EngE was also confirmed, suggesting that the mechanism anchoring CbpA to the C. cellulovorans cell surface was mediated through EngE and that the HLDs play a secondary role in the attachment of the cellulosome to the cell surface. During a study of the role of HLDs on cellulose degradation, the mini-cellulosome complexes with HLDs degraded cellulose more efficiently than complexes without HLDs degraded cellulose. The rHLDs also showed binding affinity for crystalline cellulose and carboxymethyl cellulose. These results suggest that the CbpA HLDs play a major role and a minor role in C. cellulovorans cellulosomes. The primary role increases cellulose degradation activity by binding the cellulosome complex to the cellulose substrate; secondarily, HLDs aid the binding of the CbpA/cellulosome to the C. cellulovorans cell surface.     

Reciprocal influence of ethylene and gibberellins on response-gene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A cortical band of fiber cells originate de novo in tendrils of redvine [Brunnichia ovata (Walt.) Shiners] when these convert from straight, supple young filaments to stiffened coiled structures in response to touch stimulation. We have analyzed the cell walls of these fibers by in situ localization techniques to determine their composition and possible role(s) in the coiling process. The fiber cell wall consists of a primary cell wall and two lignified secondary wall layers (S₁ and S₂) and a less lignified gelatinous (G) layer proximal to the plasmalemma. Compositionally, the fibers are sharply distinct from surrounding parenchyma as determined by antibody and affinity probes. The fiber cell walls are highly enriched in cellulose, callose and xylan but contain no homogalacturonan, either esterified or de-esterified. Rhamnogalacturonan-I (RG-I) epitopes are not detected in the S layers, although they are in both the gelatinous layer and primary wall, indicating a further restriction of RG-I in the fiber cells. Lignin is concentrated in the secondary wall layers of the fiber and the compound middle lamellae/primary cell wall but is absent from the gelatinous layer. Our observations indicate that these fibers play a central role in tendril function, not only in stabilizing its final shape after coiling but also generating the tensile strength responsible for the coiling. This theory is further substantiated by the absence of gelatinous layers in the fibers of the rare tendrils that fail to coil. These data indicate that gelatinous-type fibers are responsible for the coiling of redvine tendrils and a number of other tendrils and vines.     

Evidence for in vitro binding of pectin side chains to cellulose

Pectins of varying structures were tested for their ability to interact with cellulose in comparison to the well-known adsorption of xyloglucan. Our results reveal that sugar beet (Beta vulgaris) and potato (Solanum tuberosum) pectins, which are rich in neutral sugar side chains, can bind in vitro to cellulose. The extent of binding varies with respect to the nature and structure of the side chains. Additionally, branched arabinans (Br-Arabinans) or debranched arabinans (Deb-Arabinans; isolated from sugar beet) and galactans (isolated from potato) were shown bind to cellulose microfibrils. The adsorption of Br-Arabinan and galactan was lower than that of Deb-Arabinan. The maximum adsorption affinity of Deb-Arabinan to cellulose was comparable to that of xyloglucan. The study of sugar beet and potato alkali-treated cell walls supports the hypothesis of pectin-cellulose interaction. Natural composites enriched in arabinans or galactans and cellulose were recovered. The binding of pectins to cellulose microfibrils may be of considerable significance in the modeling of primary cell walls of plants as well as in the process of cell wall assembly.     

THE COMPOSITION AND PHYLOGENETIC SIGNIFICANCE OF THE MOUGEOTIA (CHAROPHYCEAE) CELL WALL1

The two-layered, fibrillar cell wall of Mougeotia C. Agardh sp. consisted of 63.6% non-cellulosic carbohydrates and 13.4% cellulose. The orientation of cellulose microfibrils in the native cell wall agrees with the multinet growth hypothesis, which has been employed to explain the shift in microfibril orientation from transverse (inner wall) toward axial (outer wall). Monosaccharide analysis of isolated cell walls revealed the presence of ten sugars with glucose, xylose and galactose most abundant. Methylation analysis of the acid-modified, 1 N NaOH insoluble residue fraction showed that it was composed almost exclusively of 4-linked glucose, confirming the presence of cellulose. The major hemicellulosic carbohydrate was semi-purified by DEAE Sephacel (Cl^?) anion-exchange chromatography of the hot 1 N NaOH soluble fraction. This hemicellulose was a xylan consisting of a 4-xylosyl backbone and 2,4-xylosyl branch points. The major hot water soluble neutral polysaccharide was identified as a 3-linked galactan. Mougeotia cell wall composition is similar to that of (Charophyceae) and has homologies with vascular plant cell walls. Our observations support transtructural evidence which suggests that members of the Charophyceae represent the phylogenetic line that gave rise to vascular plants. Therefore, the primary cell walls of vascular plants many have evolved directly from structures typical of the filamentous green algal cell walls found in the Charophyceae.     

Cellular and extracellular localization of endocellulase in Trichoderma reesei

Abstract An endocellulase (1,4-β- d -glucan 4-glucanohydrolase, EC 3.2.1.4) was purified by preparative isoelectric focusing from culture fluids of Trichoderma reesei QM 9414 grown on cellulose. Its properties were studied by affinity titration curves and immunoelectrophoresis. FITC-labeled protein A-antibody was used to document its occurrence in cellulose and in fungal cell walls. Immunogold electron microscopy served to detect endocellulose sites within the outer exopolysaccharide layer of the fungal cell wall.     

Cellulose production by planktonic algae in lacustrine environments

Cellulose production by planktonic algae in a eutrophic pond and in an oligotrophic lake was estimated by comparing the amount of cellulose contained in intact algal cells with the amount of cellulose present in the water column. Cellulose contents of laboratory grown algal species representing the dominant cellulose producers were ranged from 2 to 39% of the total dry weight of cell mass, depending upon the species and stage of growth. The relative amounts of cellulose present in the water column ranged from 4 to 50% of the total dry weight of particulate matter.It was estimated that more than 30% of cellulose in the water column was actually contributed by the viable algal cells present during the algal bloom. Despite its algal origin, more cellulose was found in the water column than could be accounted for by the number of algal cells observed. The difference was due to the accumulation of cellulose from previous algal crops. This observation indicated that production of algal cellulose exceeded decomposition and little or no decomposition of cellulosic material took place in the water column.     

Overexpression of <Emphasis Type="Italic">PSP1</Emphasis> enhances growth of transgenic Arabidopsis plants under ambient air conditions

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.     

Pine xyloglucan. Occurrence, localization and interaction with cellulose

The occurrence, localization, and properties of xyloglucan in the cell walls of growing regions of Pinus pinaster hypocotyls have been studied. Xyloglucan was released from the cell wall with alkali solutions, the concentration increasing from 4 through 35%; KOH. In vitro experiments showed that xyloglucan and cellulose can interact, forming a macromolecular complex. Electron microscope observations showed that the cell wall material extracted with 35%; KOH, which contained some amount of xyloglucan, was enough to cover and join the cellulose microfibrils.     

Feasible mechanisms for algal digestion in the king angelfish

To determine the ability of the king angelfish Holacanthus passer to digest algae, three algal species were immersed in acidic conditions similar to that found in the stomach of fish. Only one of them was not susceptible to acidic lysis; two were affected after 40 and 60 min at pH 2·0. King angelfish have an expanded region of the intestine called here the hindgut chamber (HC) containing populations of micro-organisms. Some of these micro-organisms have the capacity to grow in cellulose, agar, and alginic acid; the main components of algal cell walls. Micro-organisms grew in carboxymethylcellulose cultures under aerobic and micro-aerobic conditions. The HC is highly vascularized, which could increase absorptive efficiency of material digested in it.     

Zinc distribution and speciation in roots of various genotypes of tobacco exposed to Zn

Cell walls of roots have a great reactivity towards metals, and may act as a barrier limiting the entry of metals, especially in non-hyperaccumulating species. The aim of this study was to determine the localization and speciation of Zn in roots of tobacco (Nicotiana tabacum) grown in Zn-contaminated substrates. Chemical extractions and EXAFS spectroscopy were applied on whole roots and on isolated cell walls of roots. Our results show that cell walls of roots exhibited a distribution of Zn affinity sites, from water-soluble to non-exchangeable Zn. In whole roots, Zn was bound with oxalate and other COOH/OH groups: the first species was probably intracellular while the second was attributed to Zn bound to the cell walls and, to a lesser extent, to intracellular organic acids. Moreover, Zn-phosphate was also identified, and this species was CuSO₄-extractable. It probably resulted from chemical precipitation in the apoplasm, and explained the steady increase in exchangeable root Zn observed in root of tobacco during the culture. This study shows the strength of combining EXAFS and chemical extractions for studying localization and speciation of metals in plants.     

Polyamine oxidase in oat leaves: a cell wall-localized enzyme

The localization and activity of polyamine oxidase (PAO; EC 1.5.3.3), was investigated in leaves and protoplasts of oat seedlings. Activity of the enzyme is highest with spermine as substrate; spermidine is also oxidized, but putrescine and cadaverine are unaffected by the enzyme. Protoplasts isolated following digestion of leaves with cellulase in hypertonic osmoticum showed no PAO activity, and about 80% of the total leaf PAO activity could be accounted for in the cell wall debris. Histochemical localization experiments showed intense PAO activity in guard cells and in vascular elements whose walls are not digested by cellulase. When protoplasts were cultured in a medium suitable for regeneration of cell wall, PAO activity could be detected as the cellulose wall developed. Thus, PAO appears to be localized in cell walls.     

Roles of Cellulose and Xyloglucan in Determining the Mechanical Properties of Primary Plant Cell Walls

The primary cell walls of growing and fleshy plant tissue mostly share a common set of molecular components, cellulose, xyloglucan (XyG), and pectin, that are required for both inherent strength and the ability to respond to cell expansion during growth. To probe molecular mechanisms underlying material properties, cell walls and analog composites from Acetobacter xylinus have been measured under small deformation and uniaxial extension conditions as a function of molecular composition. Small deformation oscillatory rheology shows a common frequency response for homogenized native cell walls, their sequential extraction residues, and bacterial cellulose alone. This behavior is characteristic of structuring via entanglement of cellulosic rods and is more important than cross-linking with XyG in determining shear moduli. Compared with cellulose alone, composites with XyG have lower stiffness and greater extensibility in uniaxial tension, despite being highly cross-linked at the molecular level. It is proposed that this is due to domains of cross-linked cellulose behaving as mechanical elements, whereas cellulose alone behaves as a mat of individual fibrils. The implication from this work is that XyG/cellulose networks provide a balance of extensibility and strength required by primary cell walls, which is not achievable with cellulose alone.     

Understanding the biological rationale for the diversity of cellulose-directed carbohydrate-binding modules in prokaryotic enzymes

Plant cell walls are degraded by glycoside hydrolases that often contain noncatalytic carbohydrate-binding modules (CBMs), which potentiate degradation. There are currently 11 sequence-based cellulose-directed CBM families; however, the biological significance of the structural diversity displayed by these protein modules is uncertain. Here we interrogate the capacity of eight cellulose-binding CBMs to bind to cell walls. These modules target crystalline cellulose (type A) and are located in families 1, 2a, 3a, and 10 (CBM1, CBM2a, CBM3a, and CBM10, respectively); internal regions of amorphous cellulose (type B; CBM4-1, CBM17, CBM28); and the ends of cellulose chains (type C; CBM9-2). Type A CBMs bound particularly effectively to secondary cell walls, although they also recognized primary cell walls. Type A CBM2a and CBM10, derived from the same enzyme, displayed differential binding to cell walls depending upon cell type, tissue, and taxon of origin. Type B CBMs and the type C CBM displayed much weaker binding to cell walls than type A CBMs. CBM17 bound more extensively to cell walls than CBM4-1, even though these type B modules display similar binding to amorphous cellulose in vitro. The thickened primary cell walls of celery collenchyma showed significant binding by some type B modules, indicating that in these walls the cellulose chains do not form highly ordered crystalline structures. Pectate lyase treatment of sections resulted in an increased binding of cellulose-directed CBMs, demonstrating that decloaking cellulose microfibrils of pectic polymers can increase CBM access. The differential recognition of cell walls of diverse origin provides a biological rationale for the diversity of cellulose-directed CBMs that occur in cell wall hydrolases and conversely reveals the variety of cellulose microstructures in primary and secondary cell walls.     

Enhanced cellulose degradation using cellulase-nanosphere complexes

Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production.     

Plants control the properties and actuation of their organs through the orientation of cellulose fibrils in their cell walls

Plants use the orientation of cellulose microfibrils to create cell walls with anisotropic properties related to specific functions. This enables organisms to control the shape and size of cells during growth, to adjust the mechanical performance of tissues, and to perform bending movements of organs. We review the key function of cellulose orientation in defining structural-functional relationships in cell walls from a biomechanics perspective, and illustrate this by examples mainly from our own work. First, primary cell-wall expansion largely depends on the organization of cellulose microfibrils in newly deposited tissue and model calculations allow an estimate of how their passive re-orientation may influence the growth of cells. Moreover, mechanical properties of secondary cell walls depend to a large extent on the orientation of cellulose fibrils and we discuss strategies whereby plants utilize this interrelationship for adaptation. Lastly, we address the question of how plants regulate complex organ movements by designing appropriate supramolecular architectures at the level of the cell wall. Several examples, from trees to grasses, show that the cellulose architecture in the cell wall may be used to direct the swelling or shrinking of cell walls and thereby generate internal growth stress or movement of organs.     

Changes in pectin structure and localization during the growth of unadapted and NaCl-adapted tobacco cells

Tobacco cells adapted to grow in high concentrations of NaCl exhibit a drastically altered growth physiology that results in cells whose fully expanded volume is only one-fifth to one-eighth those of unadapted cells. Comparison between NaCl-adapted and unadapted tobacco cells provides an opportunity to evaluate current concepts of the structural and mechanical determinants of cell wall expansion. Both biochemical studies of pectic polymers and the ultrastructural localization of pectic epitopes at three specific phases of cell culture, maximal cell division, maximal elongation, and stationary phase are reported here. One-half of the galactosyluronic acid units in wall polymers of NaCl-adapted cells are esterified throughout the culture period, while wall polymers of unadapted cells show a rise in esterified polygalacturonic acid from 50 to 80% during elongation and then a decrease to 70% at stationary phase. Methyl esters account for only a proportion of the total esterified polygalacturonic acid at any stage in both unadapted and NaCl-adapted cell walls. Using monoclonal antibodies, we show differences in the localization of relatively methyl-esterified and unesterified pectic epitopes at different stages of growth and corroborate the chemical determinations. Fourier transform infrared (FTIR) microspectroscopy of representative walls of both NaCl-adapted and unadapted cells confirms, at the single cell wall level, that results obtained from chemical analysis of bulk samples are applicable to the entire cell population. FTIR microspectroscopy also reveals an increase in wall protein in the walls of adapted cells. Images obtained by the fast-freeze, deep-etch, rotary-shadowed replica technique show clearly different cell wall architectures in NaCl-adapted compared with unadapted cells; walls of elongating unadapted cells contain long, thin fibres that show a net orientation with respect to the long axis of the cell, whereas walls of adapted cells have thicker, flatter bundles of fibres with no clear net orientation. Polarized FTIR microspectroscopy indicates that, in unadapted tobacco cells during elongation, pectin molecules may be oriented within the wall in a similar manner to cellulose. Possible ways in which pectin structure and conformation may affect the behaviour of the cellulose-xyloglucan network are discussed.     

Polysaccharide-degrading Enzymes are Unable to Attack Plant Cell Walls without Prior Action by a "Wall-modifying Enzyme"

A study of the degradation of plant cell walls by the mixture of enzymes present in Pectinol R-10 is described. A “wall-modifying enzyme” has been purified from this mixture by a combination of diethylaminoethyl cellulose, Bio Gel P-100, and carboxymethyl cellulose chromatography. Treatment of cell walls with the “wall-modifying enzyme” is shown to be a necessary prerequisite to wall degradation catalyzed by a mixture of polysaccharide-degrading enzymes prepared from Pectinol R-10 or by an α-galactosidase secreted by the pathogenic fungus Colletotrichum lindemuthianum. The action of the “wall-modifying enzyme” on cell walls is shown to result in both a release of water-soluble, 70% ethanol-insoluble polymers and an alteration of the residual cell wall. A purified preparation of the “wall-modifying enzyme” is unable to degrade a wide variety of polysaccharide, glycoside, and peptide substrates. However, the purified preparation of wall-modifying enzyme has a limited ability to degrade polygalacturonic acid. The fact that polygalacturonic acid inhibits the ability of the “wall-modifying enzyme” to affect cell walls suggests that the “wall-modifying enzyme” may be responsible for the limited polygalacturonic acid-degrading activity present in the purified preparation. The importance of a wall-modifying enzyme in developmental processes and in pathogenesis is discussed.