Chemical Characterization of Wood and Its Components

Transcription

1 Chemical Characterization of Wood and Its Components Wood is chemically heterogenous and its components can be divided into two groups: structure components of high molecular weight nonstructural components of low molecular weight To characterize and investigate the distribution of chemical components is very important for understanding the wood properties. In general, hardwoods contain more hemicellulose than softwoods but less lignin. Typical components of softwoods and hardwoods % Cellulose lignin Norway spruce Polysoses Extractives Beech

2 Distribution of Polysaccharides n The distribution of cellulose is probably the easiest to study at the various morphological regions of wood. n A study of the distribution of hemicellulose is different. n In spruce and beech, it was found that xylan concentration is rather high in the S 1 and S 3 for both woods. n Through oxidation of polysaccharides with heavy metal, subsequent electron microscopic observations indicated the highest concentration of hemicellulose in S 1 layer of spruce tracheids.

3 Distribution of Polysaccharides n Experimental approach to study the ultrastructural localization of polysaccharides in wood n Additive approach: cells at different development stage of increase age are analyzed during the deposition of their wall layer. n Microscopic approach: cells are studied by light or electron microcopy before and after fractional extraction of certain polysaccharides or after contrasting them specifically. n Radioautographic approach: certain polysaccharides in the walls are labeled by feeding a radioactive precursor. n Direct approach: fully developed cells are dissected mechanically into single cell wall layers that can then be analyzed separately. Distribution of Polysaccharides n Meier and Wilkie (1959) and Meier (1964) investigated polysaccharide composition in different cell wall layer of pine tracheids, spruce tracheids, and birch fiber. n Young tracheids or fibers in radial sections through the outermost part of the xylem bordering the cambial zone were separated by using a micromanipulator under the polarizing microscope into four layers: M+P, M+P+S 1, M+P +S1+S 2 outer part, M+P+S 1 +S 2 +S 3.

4 Distribution of Polysaccharides n The separated layers were hydrolyzed with sulfuric acid, and from the amounts of the monosaccharides in the hydrolytes, the percentages of the different polysaccharides with known chemical structures were calculated. Metabolism of Sugar Nucleotides l Cellulose and β-1,3-glucan are synthesized on the plasma membrane by mediation of enzymes and are directly deposited onto the cell walls. l Other polysaccharides are synthesized by the Golgi apparatus in the cytoplasm, and are transported in the Golgi vesicles, which fuse with the plasma membrane, depositing the polysaccharides onto the growing cell wall.

5 Metabolism of Sugar Nucleotides n Synthesis of polysaccharides in the Golgi apparatus n Sugar nucleotides are linked to specific carries and penetrate Golgi membranes. n The sugar nucleotides transferred are converted to polysaccharides by sugar transferase associated with the inner surface of the Golgi cisternae. n The synthesis of hemicellulose, pectin, and glycoproteins are completed at the lumen. However, immunoelectron micrographs have suggested that different polysaccharides, such as hemicellulose and pectin are synthesized in different Golgi vesicles.

6 Metabolism of Sugar Nucleotides n Electron micrographs of cell membranes have indicated the occurrence of terminal complexes (TCs) linked to the end of cellulose microfibrils located at the outer layer of the plasma membrane. Rosettes (specially arranged TCs) are located at the inner layers of the plasma membrane. These Rosettes (TCs) are suggested to be cellulose-synthesizing enzyme complexes in higher green plants. n UDP-glucose incorporated as a precursor into the cells is converted to cellulose by the mediation of the TCs and is transported to the cell wall.

7 Polysaccharides Biosynthesis n In the synthesis of other wood polysaccharides both UDP- D-glucose and GDP-D-glucose are involved. n GDP-D-glucose is the principal nucleotide as concerns the formation of mannose-containing hemicellulose, including galacto-glucomannans and glucomannans. n Monomeric sugar components needed are formed from the nucleotides by complex enzymic reactions involving epimerization, dehydrogenation, and decarboxylation. Metabolism of Sugar Nucleotides n Sugar nucleotides are sugar donors involved in biosynthesis of oligo- and polysaccharides. n Glycosyl transferases are involved in polysaccharide synthesis, catalyze transfer of sugar residues of nucleotide sugars to the acceptor using energy released by hydrolysis the high energy phosphate linkage between sugar and phosphate in the nucleotide.

8 Metabolism of Sugar Nucleotides l In plants, a number of kinases exist that phosphorylate free monosaccharides to from the monosaccharide-1-phosphate. l Glucose incorporated into cells is phosphorylated to glucose-6-phosphate, which is converted to glucose-αphosphate by phosphoglucomutase. l Glc-1-P reacts with nucleoside-3-phophate to give nucleotide glucose which is converted to several sugar nucleotides by mediation of dehydrogenase, carboxylase and epimerase. Hypothetical Model of the Mechanism of Cellulose Synthesis in Plants sucrose synthesis UDP glucose pyrophosphorylase phosphoglucomutase hexokinase

9 Formation of uridine diphosphate glucose (UDP-Dglucose) Metabolism of Sugar Nucleotides Sugar donors in higher plant: Uridine diphosphate (UDP) Guanosine diphosphate (GDP) Adenosine diphosphate (ADP) (ADP-glucose is not involved in biosynthesis of wood polysaccharides but in starch biosynthesis) Sugar derivatives of nucleoside-1-phosphate, nucleoside diphosphate oligosaccharides, and sugar derivatives of nucleosides other then U, G, and A are found in plant cells, but their physiological roles are not known at present.

10 Metabolism of Sugar Nucleotides Biosynthesis of the monosaccharide units in polysaccharides occur mostly by isomerization of sugar residues of UDP-sugars, and is regular in a complex fashion. Properties of the Enzymes Associated with the Major UDP-sugars n UDP-Glucose Pyrophosphonylase UTP + Glc-1-P UDP-Glc + Pyrophosphate n Glucose-1-phosphate uridyltransferse n Metal ion (e.g. Mg 2+ ) are required to activate the enzyme. n UDP-glucose occupies a central position in sugar nucleotide metabolism. It serve as a precursor in sucrose synthesis and of sugar nucleotides required for the synthesis of cellulose and hemicellulose.

11 Disaccharides Cellobiose Maltose Sucrose Properties of the Enzymes Associated with the Major UDP-sugars n Sucrose synthase UDP-Glucose + Fructose Sucrose + UDP n The enzyme catalyze sucrose synthesis by transfer of a glucose residue of UDP-Glc to fructose. It is a tetramer of identical subunits of about 90 KDa. n In most higher plant, sucrose is the major source of carbon translocated to nonphotosynthetic tissues. The enzyme is frequently found in sink tissues where elevated concentrations of sucrose are available. It seems that the enzyme is involved in the cleavage of sucrose to afford UDP-Glc for glucan synthesis in tissues.

12 Properties of the Enzymes Associated with the Major UDP-sugars n UDP-Glucose Dehydrogenase UDP-Glc + 2 NAD + + H 2 O UDP-Glucuronic acid + 2 NADPH + 2 H + n UDP-glucuronic acid formed by this reaction is a precursor of UDPgalacturonic acid, UDP-xylose, and UDP-arabinose, which are used in the synthesis of pectin and hemicellulose. Properties of the Enzymes Associated with the Major UDP-sugars n UDP-Glucuronate UDP-Glucuronate + NAD + UDP-xylose + CO 2 + NADPH + H + n This enzyme catalyzes the decarboxylation of UDP-glucuronate to give UDP-xylose.

13 Properties of the Enzymes Associated with the Major UDP-sugars n UDP-Glucose 4-epimerase UDP-glucose UDP-galactose n The enzyme catalyzes epimerization of the hydroxyl group of the asymmetric carbon 4 of the glucose residue of UDP-glucose to give UDP-galactose. glucose galactose Properties of the Enzymes Associated with the Major UDP-sugars n UDP-Glucuronate 4-epimerase UDP-Glucuronate UDP-Galacturonate n Catalyzing epimerization of the hydroxyl group at C4 of a glucuronic acid residue of UDP-Glucuronate to give UDP-galacturonate.

14 Properties of the Enzymes Associated with the Major UDP-sugars n UDP-Arabinose 4-epimerase UDP-Arabinose UDP-xylose n The enzyme catalyzes epimerization of the hydroxyl group at the asymmetric C4 of an arabinose residue of UDP-arabinose to give UDPxylose. Simplified Representation of the formation of Hemicellulose Precursors from UDP-D-glucose

15 Model Structure of Cellulose Microfibrils

16 Intramolecular linkage (H-bond): Hydrogen bonds between OH-groups of adjacent glucose units in the same cellulose molecule. These linkages give a certain stuffiness to the single chain. O(3)H to ring oxygen (or O(3)H to O5 ; O(6) to O(2) Intermolecular linkage (H-bond): Hydrogen bonds between OH-groups of adjacent cellulose molecules. These linkages are responsible to the formation of supramolecular structures O(6)H to O(3)

17 Structure of Cellulose. β-d-glucopyranose chain units are in chair conformation (4C1) and the substituents HO-2, HO-3, and CH 2 OH are oriented equatorially.

18 Crystalline Structure of Cellulose Crystalline structure of cellulose has been characterized by X-ray diffraction analysis and by methods based on the absorption of polarized infrared radiation CP-MAS spectrum of the cellulose from Chaetomopha sp.

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20 Cellulose Synthesis Study in the Early Decades ( ) n Greathouse (1959) found that cellulose- 14 C was produced from D-glucose-1-14 C and ATP, mediation of a cell-free system isolated from Acetobacter xylinum. n Hestrin s group (1962) in Israel pioneered study with the gram-negative bacterium Acetobacter xylinum. n Hestrin s group established conditions for growth of A. xylinum and optimization of cellulose production, and also described the basic pathways of carbon metabolism. n In the early 1950s, Leloir et al made the seminal discovery of UDP-glucose, and showed that this high energy molecule could serve as a donor for a number of glycosyltransferases.

21 Cellulose Synthesis Study in the Early Decades ( ) n Glaser (1958) demonstrated that membrane preparations derived from A. xylinum could synthesize limited amounts of β-1,4-glucan. n In A. xylinum, it proved difficult to enhance the very low rates observed by Glaser. In higher plants, little or no cellulose synthase activity was detectable above, a very high background of callose (β-1,3-glucan) synthase activity. This latter enzyme, located in the plasma membrane of plants, uses UDP-glc as substrate to synthesize callose in a reaction that is dependent upon the presence of Ca 2+ and a β- glucoside. n Furthermore, callose, like cellulose, can be highly insoluble in alkali, and many early reports claimed synthesis of cellulose ( alkali-insoluble glucan ) when in fact the product was callose. Cellulose Synthesis Study in the Early Decades ( ) n Advances in microscopy allowed visualization of the cellulose microfibrils in the walls of algae and plants, and, hence, the realization that the patterns of cellulose deposition can often be highly ordered. n In expanding plant cells, microfibrils are usually deposited in arrays transverse to the direction of cell expansion, leading to the concept that whatever controls the pattern of microfibril deposition plays a key role in determining directions of cell expansion. n In the thick cell walls of many cellulosic algae and secondary walls of higher plant cells, microfibril deposition often occurs in layers that alternate in direction, thus creating walls of great strength.

22 Freeze fracture A technique used to look at membranes that reveal the pattern of integral membrane proteins. General outline of technique: Cells are quickly frozen in liquid nitrogen (196 C), which immobilizes cell components instantly. Block of frozen cells is fractured. This fracture is irregular and occures along lines of weakness like the plasma membrane or surfaces of organelles. Surface ice is removed by a vacuum (freeze etching)

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24 Cellulose Synthesis Study in the Early Decades ( ) n Discovery of microtubules by Ledbetter and Porter (1963) were numerous papers showing that the orientation of the cortical microtubule network often paralleled that of the most recently deposited cellulose microfibrils. n Preston (1969) proposed the ordered granule hypothesis that envisioned cellulose synthases as multisubunit complexes in which an ordered array of catalytic subunits would function together to synthesize glucan chains that would then self-assemble to form a microfibril.

25 Cellulose Synthesis Study in the Early Decades ( ) n Preston s suggestions was realized as being most likely true with the discovery in the alga Oocystis by Brown and Montezinos (1976) of so-called linear terminal complexes (TCs) multisubunit arrays at the ends of microfibrils that were seen using the new technique of freeze-fracture of the plasma membrane. n This was followed by the finding in plants of the first smaller, hexagonal structure called a rosette, now believed to represent the type of synthase found in higher plants and some algae. n Until 1982, no breakthroughs were achieved in in vitro synthesis of cellulose; thus, no synthase was purified, nor genes identified that encode the synthase or any other important proteins involved in the process. Visualization of cellulose-synthesizing sites n Electron microscopy studies have demonstrated that cellulose exists in the form of submicroscopic rod-like structures known as microfibrils. n Brown and Montezinos (1976) applied the freeze-fracture technique to visualize the organized site of cellulose synthesis. In these studies, an organized macromolecular complex - the terminal complex (TC) - was observed at the tip of cellulose microfibrils in the greenalga, Oocystis apiculata: the aggregate of subunits was a linear arrangement of particles in three rows. Linear TC (1) from the green alga, Oocystis apiculata, associated with the growing tip (2) of a microfibril. Bar represents 0.5 µm.

26 The rosette terminal complex (TC) from Zea mays root: note the hexagonal arrangement of subunits within the encircled TCs. Bar represents 0.5 µm.

27 Rosette TC n In vascular plants, the cellulose synthase complex is known by its ultrastructural morphology as a rosette TC which was first described in vascular plants by Mueller and Brown in 1980 in Zea mays. n Rosette TCs has a six-fold symmetrical arrangement of transmembrane particle subunits from which the crystalline cellulose I emerges. The TC moves in the plane of the membrane as the crystalline cellulose is generated. n Rosette TCs seem to be highly conserved in their morphology in all vascular plants so far examined.

28 Acetobacter xylinum as a Model Organism to Study Cellulose Synthesis n Benziman et al (1982) obtained rates of in vitro synthesis of cellulose that approached those observed in vivo in A. xylinum. n To obtain these high rates, membranes had to be prepared in the presence of polyethylene glycol (PEG) and supplied with GTP. n Benziman s group showed that PEG was precipitating a soluble enzyme, a diguanylate cyclase, that converted GTP to a unique activator of the cellulose synthase cyclicdiguanylic acid (c-di-gmp).

29 cellulose synthase cyclicdiguanylic acid (cdi-gmp). Cellulose Synthesis and Its Regulation by Acetobacter xylinum Ross et al., 1987

30 Acetobacter xylinum as a Model Organism to Study Cellulose Synthesis n The discovery of c-di-gmp paved the way for subsequent purification of the synthase by a technique called product entrapment. n Kang et al (1984) had found that when detergent-solubilized chitin synthase from yeast was incubated with substrate, the enzyme remained tightly associated with the chitin product and could be effectively purified by centrifugation of the enzyme with the product. n This technique worked well for the A. xylinum synthase, and two groups (Lin et al., 1990; Mayer et al., 1990) obtained a synthase preparation of relatively high purity. A prominent polypeptide of 83- kd was shown to bind the substrate UDP-glc (Lin et al., 1990) and has been accepted to be the catalytic subunit. Using sequence information derived from this polypeptide. Acetobacter xylinum as a Model Organism to Study Cellulose Synthesis n Brown s laboratory (Saxena et al., 1990) isolated a gene from A. xylinum, AcsA, that encodes the catalytic subunit. n In 1990, Wong et al (1990) used the genetic approach of complementation of a cellulose-deficient mutant of A. xylinum to isolate an operon of four genes called BcsA D. Analyses of mutant strains demonstrated that genes A- C are essential for cellulose synthesis. n The BcsA gene isolated by Wong et al shows high homology to the AcsA gene isolated by Brown s group and therefore also encodes the catalytic subunit.

31 Proposed nomenclature for genes involved in cellulose synthesis Acetobacter xylinum as a Model Organism to Study Cellulose Synthesis n The B gene (AxCeSB), the second gene in the operon, is now presumed to encode a regulatory subunit that binds c- di-gmp, although the evidence for this is not completely firm. n In addition to the 83-kDa polypeptide, another polypeptide of 90 kda co-purifies with the synthase in A. xylinum (Mayer, 1991). n Although sequencing of this polypeptide supports the notion that it is encoded by AxCeSB, the gene product does not bind c-di-gmp. n However, a 67-kDa polypeptide, also found in the purified synthase preparation, did bind c-di-gmp, and this was presumed to be derived from the 90-kDa polypeptide since it cross-reacted to antibodies prepared against the larger polypeptide. n It is unclear why the 90-kDa polypeptide does not bind c-di-gmp, and the relationship between the two is based only upon immunological cross-reactivity.

32 Structure of the Operon of Cellulose Synthesis (bcs) from A. xylinum The operon is 9217 bp in length, and is comprised of the genes bcsa, bcsb, bcs C, and bcs D, which encode the proteins of molecular masses 84.4, 85.3, 141.0, and 17.3 kda, respectively. The gene products by bcsa and bcs B have been characterized as the UDPG-binding and activator (c-di-gmp)-binding subunits of the cellulose synthase complex. bcsc and bcsd genes provide functions essential for microfibril crystalization or extrusion. ATG initiation condon for translation; TGA and TAA termination condons for translation. Volman et al., 1995 acs operon of cellulose synthase from A. xylimmun ATCC showing organization of the acsab, acsc, and acsd genes and the predicted size of polypeptides encodes by these genes. Mutant analysis showed that while the acsab and acsc genes were essential for cellulose production in vivo, the acsd mutant produced amounts of two cellulose allomorphs, suggesting that acsd gene is involved in cellulose crystallization.

33 Formation of Microfibrils n It has been suggested that UDP-glucose, a precursor of cellulose, is transported from the cytoplasm to the plasma membrane, where cellulose is synthesized by enzyme complex (TCs) located on the membrane. n TCs can only be observed by freeze-fracture method, it can be classified into two kinds, rosetes and linear types, based on their arrangement and shape. Structure of the Operon of Cellulose Synthesis (bcs) from A. xylinum The operon is 9217 bp in length, and is comprised of the genes bcsa, bcsb, bcs C, and bcs D, which encode the proteins of molecular masses 84.4, 85.3, 141.0, and 17.3 kda, respectively. The gene products by bcsa and bcs B have been characterized as the UDPG-binding and activator (c-di-gmp)-binding subunits of the cellulose synthase complex. bcsc and bcsd genes provide functions essential for microfibril crystalization or extrusion. ATG initiation condon for translation; TGA and TAA termination condons for translation. Volman et al., 1995

34 acs operon of cellulose synthase from A. xylimmun (ATCC 53582) showing organization of the acsab, acsc, and acsd genes and the predicted size of polypeptides encodes by these genes. Mutant analysis showed that while the acsab and acsc genes were essential for cellulose production in vivo, the acsd mutant produced amounts of two cellulose allomorphs, suggesting that acsd gene is involved in cellulose crystallization. General model showing the molecular organization of the cellulose synthase molecules from the molecular level of organization to the rosette TC

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36 Generalized concept for polymerization reactions leading to glycosylation forming α-1,4-glucan chains in cellulose biosynthesis Four different models to demonstrate possible diversity in glycosylation reactions in cellulose biosynthesis The most acceptable model in which the catalytic subunit is on the cytoplasmic side and the growing polymer chain must some how be directed through the plasma membrane interface. While not shown, six of the cellulose synthases could form a close packing structure which delimits a tunnel-like for export of the glucan chain sheets, in this instance, to the cell surface

37 This model depicts the catalytic subunit on the cell surface. There is no problem with extrusion in this model; however, an independent transport protein for UDP-Glc has to be invented. This model assumes the formation of glycosidic bonds only through lipid intermediates, and the extrusion mechanism still needs to be evoked.

38 This model is a combination of models 2 and 3 above in which lipid intermediates through flippases could possibly translocate glucose or cellobiose to the surface where the catalytic subunit of cellulose synthase could receive these precursors for further processing into long glucan chains. This model does not take into account the ordering of the polymer chains which must occur if the metastable crystalline form of cellulose I is to be generated.