参与木葡聚糖合成的糖基转移酶基因研究进展

半纤维素多糖木葡聚糖(XyG)存在于大多数植物的初生细胞壁中, 对细胞壁的结构组织和生长发育具有重要的调控作用。XyG在植物进化中存在结构的多样性。该文概述了参与XyG合成的糖基转移酶的最新研究进展, XyG合成需要多种糖基转移酶参与, 这些酶类很可能以蛋白酶复合体的形式存在并发挥作用, XyG的结构和组成的改变对植物的生长发育也产生影响。     

Poroelastic Mechanical Effects of Hemicelluloses on Cellulosic Hydrogels under Compression

Hemicelluloses exhibit a range of interactions with cellulose, the mechanical consequences of which in plant cell walls are incompletely understood. We report the mechanical properties of cell wall analogues based on cellulose hydrogels to elucidate the contribution of xyloglucan or arabinoxylan as examples of two hemicelluloses displaying different interactions with cellulose. We subjected the hydrogels to mechanical pressures to emulate the compressive stresses experienced by cell walls in planta. Our results revealed that the presence of either hemicellulose increased the resistance to compression at fast strain rates. However, at slow strain rates, only xyloglucan increased composite strength. This behaviour could be explained considering the microstructure and the flow of water through the composites confirming their poroelastic nature. In contrast, small deformation oscillatory rheology showed that only xyloglucan decreased the elastic moduli. These results provide evidence for contrasting roles of different hemicelluloses in plant cell wall mechanics and man-made cellulose-based composite materials.     

Pea Xyloglucan and Cellulose: VI. Xyloglucan-Cellulose Interactions in Vitro and in Vivo

Since xyloglucan is believed to bind to cellulose microfibrils in the primary cell walls of higher plants and, when isolated from the walls, can also bind to cellulose in vitro, the binding mechanism of xyloglucan to cellulose was further investigated using radioiodinated pea xyloglucan. A time course for the binding showed that the radioiodinated xyloglucan continued to be bound for at least 4 hours at 40°C. Binding was inhibited above pH 6. Binding capacity was shown to vary for celluloses of different origin and was directly related to the relative surface area of the microfibrils. The binding of xyloglucan to cellulose was very specific and was not affected by the presence of a 10-fold excess of (1→2)-β-glucan, (1→3)-β-glucan, (1→6)-β-glucan, (1→3, 1→4)-β-glucan, arabinogalactan, or pectin. When xyloglucan (0.1%) was added to a cellulose-forming culture of Acetobacter xylinum, cellulose ribbon structure was partially disrupted indicating an association of xyloglucan with cellulose at the time of synthesis. Such a result suggests that the small size of primary wall microfibrils in higher plants may well be due to the binding of xyloglucan to cellulose during synthesis which prevents fasciation of small fibrils into larger bundles. Fluorescent xyloglucan was used to stain pea cell wall ghosts prepared to contain only the native xyloglucan:cellulose network or only cellulose. Ghosts containing only cellulose showed strong fluorescence when prepared before or after elongation; as predicted, the presence of native xyloglucan in the ghosts repressed binding of added fluorescent xyloglucan. Such ghosts, prepared after elongation when the ratio of native xyloglucan:cellulose is substantially reduced, still showed only faint fluorescence, indicating that microfibrils continue to be coated with xyloglucan throughout the growth period.     

与木葡聚糖合成的糖基转移酶基因研究进展

半纤维素多糖木葡聚糖(XyG)存在于大多数植物的初生细胞壁中, 对细胞壁的结构组织和生长发育具有重要的调控作用。XyG在植物进化中存在结构的多样性。该文概述了参与XyG合成的糖基转移酶的最新研究进展, XyG合成需要多种糖基转移酶参与, 这些酶类很可能以蛋白酶复合体的形式存在并发挥作用, XyG的结构和组成的改变对植物的生长发育也产生影响。     

Structure of the Primary Cell Walls of Suspension-Cultured Rosa glauca Cells: II. Multiple Forms of Xyloglucans

Xyloglucans, characteristic hemicellulosic polysaccharides of plant primary walls, have been isolated from Rosa glauca suspension-cultured cells. The cell wall material was fractionated by two sequences of extraction based on solubilization of the hemicelluloses in alkaline and organic solvent systems, respectively. In both cases, only a part (about 50%) of the total xyloglucan could be extracted, the rest remaining tightly associated with cellulose and necessitating the use of acid to be solubilized. Purification of xyloglucans was effected by formation of a gel in appropriate mixtures of dimethyl sulfoxide and water. Further fractionation could be achieved on a cellulose column eluted with chaotropic solvents. This demonstrated the heterogeneity of xyloglucans in the primary cell walls. Analytical data show that all fractions are constituted with the same sugars: l-arabinose, l-fucose, d-galactose, d-xylose, and d-glucose, but their relative proportions differ, particularly the ratio of glucose to xylose which varies from 1.2 to 2 within the different xyloglucans. The structure of these hemicelluloses was established by methylation analysis and shown to consist of a (1 → 4)-linked glucan backbone which carries substituents on the O-6 of glucose. Here again, the multiple forms of xyloglucans was suggested by the various patterns of substitutions found on the different fractions. The configuration of the linkages were established by ¹³C nuclear magnetic resonance spectroscopy and shown to be β for the glucan backbone, α for the xylosyl and fucosyl substituents, and β for the galactosyl substituents. These configurations agree with the specific rotation of the xyloglucan.     

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.     

Structure of Plant Cell Walls : XIX. Isolation and Characterization of Wall Polysaccharides from Suspension-Cultured Douglas Fir Cells

The partial purification and characterization of cell wall polysaccharides isolated from suspension-cultured Douglas fir (Pseudotsuga menziesii) cells are described. Extraction of isolated cell walls with 1.0 m LiCl solubilized pectic polysaccharides with glycosyl-linkage compositions similar to those of rhamnogalacturonans I and II, pectic polysaccharides isolated from walls of suspension-cultured sycamore cells. Treatment of LiCl-extracted Douglas fir walls with an endo-α-1,4-polygalacturonase released only small, additional amounts of pectic polysaccharide, which had a glycosyl-linkage composition similar to that of rhamnogalacturonan I. Xyloglucan oligosaccharides were released from the endo-α-1,4-polygalacturonase-treated walls by treatment with an endo-β-1,4-glucanase. These oligosaccharides included hepta- and nonasaccharides similar or identical to those released from sycamore cell walls by the same enzyme, and structurally related octa- and decasaccharides similar to those isolated from various angiosperms. Finally, additional xyloglucan and small amounts of xylan were extracted from the endo-β-1,4-glucanase-treated walls by 0.5 n NaOH. The xylan resembled that extracted by NaOH from dicot cell walls in that it contained 2,4- but not 3,4-linked xylosyl residues. In this study, a total of 15% of the cell wall was isolated as pectic material, 10% as xyloglucan, and less than 1% as xylan. The noncellulosic polysaccharides accounted for 26% of the cell walls, cellulose for 23%, protein for 34%, and ash for 5%, for a total of 88% of the cell wall. The cell walls of Douglas fir were more similar to dicot (sycamore) cell walls than to those of graminaceous monocots, because they had a predominance of xyloglucan over xylan as the principle hemicellulose and because they possessed relatively large amounts of rhamnogalacturonan-like pectic polysaccharides.     

木葡聚糖及其在植物抗逆过程中的功能研究进展

木葡聚糖(XyG)是一种存在于所有陆生植物细胞壁中的基质多糖, 是双子叶植物初生细胞壁中含量(20%-25%, w/w)最丰富的半纤维素成分。作为细胞壁的组分, XyG不仅与植物的生长发育密切相关, 还在植物抵抗各种生物和非生物逆境过程中发挥重要作用。XyG代谢相关基因主要通过改变植物细胞壁的组成以及对细胞壁进行重排进而改变细胞壁的弹性/硬度等特性, 影响植物的抗逆性。XyG及其寡糖也可能作为信号分子, 或与其它信号分子协同作用应对逆境胁迫。该文概述了XyG的结构与类型及参与XyG生物合成与降解的相关基因, 重点阐述XyG相关基因应答生物和非生物胁迫的作用机制。     

Hemicellulose biosynthesis

One major component of plant cell walls is a diverse group of polysaccharides, the hemicelluloses. Hemicelluloses constitute roughly one-third of the wall biomass and encompass the heteromannans, xyloglucan, heteroxylans, and mixed-linkage glucan. The fine structure of these polysaccharides, particularly their substitution, varies depending on the plant species and tissue type. The hemicelluloses are used in numerous industrial applications such as food additives as well as in medicinal applications. Their abundance in lignocellulosic feedstocks should not be overlooked, if the utilization of this renewable resource for fuels and other commodity chemicals becomes a reality. Fortunately, our understanding of the biosynthesis of the various hemicelluloses in the plant has increased enormously in recent years mainly through genetic approaches. Taking advantage of this knowledge has led to plant mutants with altered hemicellulosic structures demonstrating the importance of the hemicelluloses in plant growth and development. However, while we are on a solid trajectory in identifying all necessary genes/proteins involved in hemicellulose biosynthesis, future research is required to combine these single components and assemble them to gain a holistic mechanistic understanding of the biosynthesis of this important class of plant cell wall polysaccharides.     

A cross-polarization, magic-angle-spinning, 13C-nuclear-magnetic-resonance study of polysaccharides in sugar beet cell walls

Solid-state nuclear magnetic resonance relaxation experiments were used to study the rigidity and spatial proximity of polymers in sugar beet (Beta vulgaris) cell walls. Proton T_1ρ decay and cross-polarization patterns were consistent with the presence of rigid, crystalline cellulose microfibrils with a diameter of approximately 3 nm, mobile pectic galacturonans, and highly mobile arabinans. A direct-polarization, magic-angle-spinning spectrum recorded under conditions adapted to mobile polymers showed only the arabinans, which had a conformation similar to that of beet arabinans in solution. These cell walls contained very small amounts of hemicellulosic polymers such as xyloglucan, xylan, and mannan, and no arabinan or galacturonan fraction closely associated with cellulose microfibrils, as would be expected of hemicelluloses. Cellulose microfibrils in the beet cell walls were stable in the absence of any polysaccharide coating.     

In silico prediction of proteins related to xyloglucan fucosyltransferases in Solanaceae genomes

Two independent studies have shown that the cell wall of pollen tubes from tobacco and tomato species contained fucosylated xyloglucan (XyG). These findings are intriguing as many reports have shown that XyG of somatic cells of these species is not fucosylated but instead is arabinosylated. In order to produce fucosylated XyG, plants must express a functional galactoside α-2-fucosyltransferase. Here, using a bioinformatics approach, we show that several candidate genes coding for XyG fucosyltransferases are present in the genome of coffee and several Solanaceae species including tomato, tobacco, potato, eggplant and pepper. BLAST and protein alignments with the 2 well-characterized XyG fucosyltransferases from Arabidopsis thaliana and Pisum sativum revealed that at least 6 proteins from different Solanaceae species and from coffee displayed the 3 conserved motifs required for XyG fucosyltransferase activity.     

A Galacturonic Acid–Containing Xyloglucan Is Involved in Arabidopsis Root Hair Tip Growth

Root hairs provide a model system to study plant cell growth, yet little is known about the polysaccharide compositions of their walls or the role of these polysaccharides in wall expansion. We report that Arabidopsis thaliana root hair walls contain a previously unidentified xyloglucan that is composed of both neutral and galacturonic acid–containing subunits, the latter containing the β-d-galactosyluronic acid-(1→2)-α-d-xylosyl-(1→ and/or α-l-fucosyl-(1→2)-β-d-galactosyluronic acid-(1→2)-α-d-xylosyl-(1→) side chains. Arabidopsis mutants lacking root hairs have no acidic xyloglucan. A loss-of-function mutation in At1g63450, a root hair–specific gene encoding a family GT47 glycosyltransferase, results in the synthesis of xyloglucan that lacks galacturonic acid. The root hairs of this mutant are shorter than those of the wild type. This mutant phenotype and the absence of galacturonic acid in the root xyloglucan are complemented by At1g63450. The leaf and stem cell walls of wild-type Arabidopsis contain no acidic xyloglucan. However, overexpression of At1g63450 led to the synthesis of galacturonic acid–containing xyloglucan in these tissues. We propose that At1g63450 encodes XYLOGLUCAN-SPECIFIC GALACTURONOSYLTRANSFERASE1, which catalyzes the formation of the galactosyluronic acid-(1→2)-α-d-xylopyranosyl linkage and that the acidic xyloglucan is present only in root hair cell walls. The role of the acidic xyloglucan in root hair tip growth is discussed.     

Pea Xyloglucan and Cellulose : III. Metabolism during Lateral Expansion of Pea Epicotyl Cells

Lateral expansion of the third internodes of pea epicotyls was evoked by treatment with either 2,4-dichlorophenoxyacetic acid (2,4-D) or ethylene gas. During growth, 2,4-D enhanced and ethylene inhibited the deposition of xyloglucan and cellulose in the cell wall, with the result that the wall framework (ghost) from ethylene-treated swollen tissue was much thinner than that from 2,4-D-treated. The level of activity of xyloglucan synthase, alkali-insoluble β-glucan synthases, and endo-1,4-β-glucanases were all enhanced by 2,4-D treatment but not by ethylene. Both 2,4-D and ethylene treatments led to increased osmotic potential in the swelling tissues. Accordingly, swelling after 2,4-D treatment was accompanied by xyloglucan degradation, concomitant with substantial net synthesis, but swollen tissue as a result of ethylene treatment was characterized by walls whose integrity was weakened by relatively low levels of newly deposited polysaccharides rather than by the degradation.     

Plant protein inhibitors of cell wall degrading enzymes

Plant cell walls, which consist mainly of polysaccharides (i.e. cellulose, hemicelluloses and pectins), play an important role in defending plants against pathogens. Most phytopathogenic microorganisms secrete an array of cell wall degrading enzymes (CWDEs) capable of depolymerizing the polysaccharides in the plant host wall. In response, plants have evolved a diverse battery of defence responses including protein inhibitors of these enzymes. These include inhibitors of pectin degrading enzymes such as polygalacturonases, pectinmethyl esterases and pectin lyases, and hemicellulose degrading enzymes such as endoxylanases and xyloglucan endoglucanases. The discovery of these plant inhibitors and the recent resolution of their three-dimensional structures, free or in complex with their target enzymes, provide new lines of evidence regarding their function and evolution in plant-pathogen interactions.     

Biosynthesis of plant cell wall polysaccharides - a complex process

Cellulose, a major component of plant cell walls, is made by dynamic complexes that move within the plasma membrane while depositing cellulose directly into the wall. On the other hand, matrix polysaccharides are made in the Golgi and delivered to the wall via secretory vesicles. Several Golgi proteins that are involved in glucomannan and xyloglucan biosynthesis have been identified, including some glycan synthases that show sequence similarity to the cellulose synthase proteins and several glycosytransferases that add sidechains to the polysaccharide backbones. Recent progress in identifying the proteins needed for polysaccharide biosynthesis should lead to an improved understanding of the molecular details of these complex processes, and eventually to an ability to manipulate them in an effort to generate plants that have improved properties for human uses.     

beta-1,3-Glucan in Developing Cotton Fibers: Structure, Localization, and Relationship of Synthesis to That of Secondary Wall Cellulose

Evidence is presented for the existence of a noncellulosic β-1,3-glucan in cotton fibers. The glucan can be isolated as distinct fractions of varying solubility. When fibers are homogenized rigorously in aqueous buffer, part of the total β-1,3-glucan is found as a soluble polymer in homogenates freed of cell walls. The proportion of total β-1,3-glucan which is found as the soluble polymer varies somewhat as a function of fiber age. The insoluble fraction of the β-1,3-glucan remains associated with the cell wall fraction. Of this cell wall β-1,3-glucan, a variable portion can be solubilized by treatment of walls with hot water, a further portion can be solubilized by alkaline extraction of the walls, and 17 to 29% of the glucan remains associated with cellulose even after alkaline extraction. A portion of this glucan can also be removed from the cell walls of intact cotton fibers by digestion with an endo-β-1,3-glucanase. The glucan fraction which can be isolated as a soluble polymer in homogenates freed of cell walls is not associated with membranous material, and we propose that it represents glucan which is also extracellular but not tightly associated with the cell wall. Enzyme digestion studies indicate that all of the cotton fiber glucan is β-linked, and methylation analyses and enzyme studies both show that the predominant linkage in the glucan is 1 → 3. The possibility of some minor branching at C-6 can also be deduced from the methylation analyses. The timing of deposition of the β-1,3-glucan during fiber development coincides closely with the onset of secondary wall cellulose synthesis. Kinetic studies performed with ovules and fibers cultured in vitro show that incorporation of radioactivity from [¹⁴C]glucose into β-1,3-glucan is linear with respect to time almost from the start of the labeling period; however, a lag is observed before incorporation into cellulose becomes linear with time, suggesting that these two different glucans are not polymerized directly from the same substrate pool. Pulse-chase experiments indicate that neither the β-1,3-glucan nor cellulose exhibits significant turnover after synthesis.     

Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and β-1,3–1,4-Glucans through Distinct Mechanisms

Structural carbohydrates comprise an extraordinary source of energy that remains poorly utilized by the biofuel sector as enzymes have restricted access to their substrates within the intricacy of plant cell walls. Carbohydrate active enzymes (CAZYmes) that target recalcitrant polysaccharides are modular enzymes containing noncatalytic carbohydrate-binding modules (CBMs) that direct enzymes to their cognate substrate, thus potentiating catalysis. In general, CBMs are functionally and structurally autonomous from their associated catalytic domains from which they are separated through flexible linker sequences. Here, we show that a C-terminal CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, does not interact with β-glucans independently but, uniquely, acts cooperatively with the catalytic domain of the enzyme in substrate recognition. The structure of BhCBM46 revealed a β-sandwich fold that abuts onto the region of the substrate binding cleft upstream of the active site. BhCBM46 as a discrete entity is unable to bind to β-glucans. Removal of BhCBM46 from BhCel5B, however, abrogates binding to β-1,3–1,4-glucans while substantially decreasing the affinity for decorated β-1,4-glucan homopolymers such as xyloglucan. The CBM46 was shown to contribute to xyloglucan hydrolysis only in the context of intact plant cell walls, but it potentiates enzymatic activity against purified β-1,3–1,4-glucans in solution or within the cell wall. This report reveals the mechanism by which a CBM can promote enzyme activity through direct interaction with the substrate or by targeting regions of the plant cell wall where the target glucan is abundant.     

The expansin superfamily

The expansin superfamily of plant proteins is made up of four families, designated α-expansin, β-expansin, expansin-like A and expansin-like B. α-Expansin and β-expansin proteins are known to have cell-wall loosening activity and to be involved in cell expansion and other developmental events during which cell-wall modification occurs. Proteins in these two families bind tightly to the cell wall and their activity is typically assayed by their stimulation of cell-wall extension and stress relaxation; no bona fide enzymatic activity has been detected for these proteins. α-Expansin proteins and some, but not all, β-expansin proteins are implicated as catalysts of 'acid growth', the enlargement of plant cells stimulated by low extracellular pH. A divergent group of β-expansin genes are expressed at high levels in the pollen of grasses but not of other plant groups. They probably function to loosen maternal cell walls during growth of the pollen tube towards the ovary. All expansins consist of two domains; domain 1 is homologous to the catalytic domain of proteins in the glycoside hydrolase family 45 (GH45); expansin domain 2 is homologous to group-2 grass pollen allergens, which are of unknown biological function. Experimental evidence suggests that expansins loosen cell walls via a nonenzymatic mechanism that induces slippage of cellulose microfibrils in the plant cell wall.     

Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as “transceptors” with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.