FORMATION AND STRUCTURE OF LIGNIFIED PLANT CELL WALL - FACTORS CONTROLLING LIGNIN STRUCTURE DURING ITS FORMATION

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1 69 FORMATION AND STRUCTURE OF LIGNIFIED PLANT CELL WALL - FACTORS CONTROLLING LIGNIN STRUCTURE DURING ITS FORMATION Noritsugu Terashima and Rajai H. Atalla USDA Forest Products Laboratory, One Gifford Pinchot Drive, Madison, WI U.S.A. Abstract. The structure of lignified plant cell wall is dependent on the manner of assembly of polysaccharides and lignin during the formation of the cell walls. Synthetic lignins prepared in the presence of carbohydrates under conditions that approximate cell wall Signification have structures which resemble native lignin more closely than those prepared by conventional methods. From these experiments, following factors were suggested to be effective in controlling lignin structure during its formation: ph of polysaccharide gel in which polymerization of lignols proceeds; association of lignols with polysaccharides; relative concentration of monomers and oligomers. HPLC analysis of of the products in an early stage of polymerization of monolignols indicated that ph and polarity of the medium are important factors that affect the linkage type between monomers. Major dimers formed in solution are ß-O-4, ß-5, and ß-ß dimers, and low ph and low polarity promoted formation of the ß-O-4 dimer which is known to be a prevalent substructure in native lignin. Polysaccharides may play an important role in various ways throughout the formation of polylignols in the cell wall. Keywords. Lignin structure, Monolignols, Dilignols, Oligolignols, Polylignol, Dehydrogenation polymer, DHP, Polysaccharides INTRODUCTION The physical and chemical properties of wood are better understood based on its ultrastructure. Ultrastructure means the three dimensional macromolecular structure of the major components, cellulose, hemicellulose and lignin, and the manner of their assembly in the cell wall. Based on observations of this assembly process during the biogenesis of tree cell walls by various non-destructive methods such as radiotracer method and electron microscopy, a tentative working hypothesis concerning the formation mechanism and ultrastructure of the cell wall has been proposed (1, 2). In this hypothesis, the following proposals were made: 1. At an earliest stage of cell wall differentiation, a cell plate composed mainly of pectic substances (PEC) are formed. On both sides of the cell plate, the primary wall (PW) and the secondary wall (SW) are formed successively. Cellulose microfibrils (CMF) and hemicelluloses (HC) are deposited at the same time so that the CMFs are kept separate by HC gel. CMFs run in a random direction in PW, and in a fixed direction in SW. In the middle layer of the secondary wall, the orientation of CMFs is almost parallel with the main cell axis, while in the outer and inner layers, the CMFs are more inclined to the axis.

2 70 2. Deposition of lignin occurs in three distinct stages. First, deposition occurs in a short period mainly in the compound middle lamella (CML) and cell corner (CC) regions preceded by deposition of PEC and arabinose-galactose-rich HCs. The second stage is a very slow deposition while CMFs and HC are deposited in SW. Finally, intensive signification occurs after deposition of most of the polysaccharides in the SW completed. 3. Monolignols, p- coumaryl alcohol (HA), coniferyl alcohol (CA) and sinapyl alcohol (SA), are supplied to the differentiating cell wall as their stable and water soluble derivative, p- glucocoumaryl alcohol, coniferin, and syringin. Hydrolysis of these glucosides by ß-glucosidase liberates monolignols and glucose, and oxidation of the liberated glucose by glucose oxidase and oxygen may produce hydrogen peroxide. Polymerization of monolignols is effected by the hydrogen peroxide and peroxidase, or by laccase and oxygen. The kind of monolignol supplied to the cell wall changes with the age of the cell in the order of HA, CA and SA. This causes structural heterogeneity of protolignin in the cell wall, so that there is a higher content of p- hydroxyphenyl- and guaiacyl lignin in the CML and CC, and a higher content of guaiacyl and syringyl lignin in SW regions. The CML and CC lignins contain more condensed structures than SW lignin does. 4. Polymerization of monolignols occurs in a swollen hydrophilic gel of PEC or HC. The deposition of oligolignols in the HC gel changes the system from hydrophilic to hydrophobic, and water is ejected from the system toward the interior of the differentiating cell wall. As a result, the swollen gel shrinks, and the distance between oligomers becomes short enough to form new linkages between oligomers. Calcium ions and enzymes bound to HC are replaced by oligolignols and ejected toward the interior of the cell wall with the ejected water. Based on the above hypothesis, guaiacyl-type synthetic lignins (dehydrogenative polymer of monolignol, DHP) were prepared from coniferin by the action of ß-glucosidase and peroxidase, with hydrogen peroxide generated in situ through the action of oxygen and glucose oxidase on the glucose liberated from the coniferin. Polylignols were also prepared from CA under the condition of continuous removal of water from the polymerization system. The structure of these novel polylignols approximated that of native lignin more closely than did the structure of polylignols prepared by the conventional method from CA. The significant differences were higher contents of ß-O-4, 5-5,4-O-5 and ß-1 substructures, and lower content of ß-ß, ß-5 substructures (Fig. 1) in the novel DHPs which approximate native lignin more closely than does conventional DHP (3). From the observation of signification processes in plant cell wall, and from the results of in vitro preparation of DHPs, the ph of the polysaccharide gel, and the behavior of the oligomers in the polysaccharides were suggested as possible factors controlling lignin structure during its formation. The purpose of the present work is to examine the effect of these factors on the in vitro polymerization of monolignols under simplified reaction conditions.

3 71 Fig. 1 Dimers of coniferyl alcohol EXPERIMENTAL Materials and methods Monolignols CA, SA and HA were prepared by the method of Quideau and Ralph (4). Polymerization of CA CA (1.8 mg, 0.01 mmol) was dissolved in a mixture of diglyme (bis[2- methoxyethyl] ether) and water (8/2-2/8, v/v, 1 ml) at various ph adjusted with H 3 PO 4 and Na2HPO 4, then peroxidase (0.1 u/ 0.1 ml, Type 1, Sigma Chem. Co.) and H 2 O 2 (0.5 mg, mmol/0.1 ml) were added at 40 C. After predetermined reaction time, an aliquot of the reaction mixture was injected directly to HPLC column to analyze the products. Effect of kind of solvent was examined by changing diglyme to dioxane, glycerol, and McIlvaine buffer. HPLC analysis of polymerization products Hewlett Packard 1090 Series II/L Liquid Chromatography equipped with diode-array UV detector and a reversed phase column (Inertsil phenyl, 5µ, 250 X 4.6 mm, MetaChem Technologies Inc., USA) was employed at 40 C using acetonitrile-water (37/63, v/v) containing 0.1 % phosphoric acid as a solvent (flow rate: l ml/min). Determination of dimers Identification of compounds corresponding to major peaks on the elution diagram of CA dimers was carried out by comparison of the UV spectra of the eluted compounds and those of authentic ß-O-4, ß-5, and ß-ß dimers that were prepared by the following method. The quantity of the dimers were determined from the integrated peak area at 275 nm making correction of absorbance of each dimer at the same wave length.

4 72 Preparation of CA dimers. CA (900 mg) was dissolved in dioxane-water (2/3, v/v, 250 ml, ph 4.5 adjusted with dil. H 3 PO 4 ), and peroxidase (120 u/10 ml) and 5 % hydrogen peroxide was added in portions monitoring the disappearance of CA. After most of the CA reacted, water (100 ml) was added and extracted with ethylacetate (100 ml X 5). Ethyl acetate solution was dried with anhydrous sulfate and concentrated under reduced pressure. The residual mixture was separated by silica gel column chromatography using methylene chloride-methanol (97/3-90/10, v/v gradient) as a developer to elute dimers in the order, ß-ß, ß-5, ß-O-4 dimers. RESULTS Monitoring AND DISCUSSION polymerization by HPLC Fig. 2 HPLC diagrams of reaction products from CA in a) diglyme-water (2/3, v/v) and b) glycerol-water (2/3, v/v) Fig.2a shows a diagram of HPLC analysis of the reaction mixture of CA (0.9 mg) in diglymewater (1/1, v/v, 1 ml, ph 5.0) containing peroxidase (1u) and H 2 O 2 (0.5 mg/0.1 ml) at 40 C for 10 min. Because the all reaction products were in solution and the whole mixture was injected directly to the analytical column, experimental errors inherent in the process for separation of products were minimized. At the point when most of the monomer is consumed, the major products are three dimers, ß-O-4, ß-5, and ß-ß, and the content of other types of dimer or higher oligomers is small. The identification of the compounds corresponding to those peaks was carried out by comparison of their UV spectra with those of authentic compounds prepared by the method described in the experimental section. This type of monitoring by HPLC gives us useful information on the behavior of monolignols and dilignols in the early stages of their polymerization under various conditions. Dimerization of CA Fig.3 shows the composition of dimers of coniferyl alcohol at different reaction time. It was confirmed that no significant change of ph occurred during the reaction. Due to the low peroxidase activity, the reaction became slow after 2 hours, and addition of extra amount of peroxidase and H 2 O 2 almost completed the dimerization. It is noted that the major products are ß-O-4, ß-5, and ß-ß dimers even after an excess of H 2 O 2 was added, and that the relative

dilute solution. These facts may be understood from various viewpoints.

5 ratios of the three dimers are almost constant regardless of reaction time except in the early stages of the reaction. This indicates that the reactivity of monomers are greater than that of dimers, and that formation of new bonds between monomer and dimers, or between two dimers do not occur as long as they are in dilute solution. These facts may be understood from various viewpoints. One is from steric hindrance which may be greater in formation of 5-5,4-0-5 and ß-1 bonds and linkages between oligomers than in formation of ß-O-4, ß-5, and ß-ß dimers. Another possible explanation is the difference in substrate specificity of peroxidase between monomer and dimer, though it is not yet confined whether the formation of oligomer radical is essential for the growth of lignin macromolecule. Fig. 4 Composition of CA dimers formed in diglyme-water mixtures. Fig.4 shows the composition of CA dimers and oligomers produced in diglyme-water of different ratios. In the solvent of lower diglyme content, the reaction was fast, and the reaction mixture became cloudy due to deposition of less soluble dimers. A part of the separated dimers reacted further with monomer or with dimers to increase the amount of higher oligomers. On the other hand, in a solvent of higher diglyme content, the reaction was slow, but the dimers remained in solution without participating in further reactions. Thus the amount of oligomers is low. It is noted that with increase of diglyme % (decrease of polarity), the ß-ß dimer decreased. When diglyme was replaced with dioxane, almost the same effect of solvent polarity on the dimer composition was observed though the enzyme

6 74 activity was inhibited more than it was in diglyme. Tanahashi and Higuchi examined the effect of polarity of the solvent on dehydrogenative polymerization of 3,5-disubstituted p- coumaryl alcohol with ferric chloride (5), and found that the polarity of solvent decreased, the amount of ß-ß dimer decreased and ß-O-4 dimer increased. Effect of ph on the dimerization of CA Fig. 5 shows the effect of ph on the dimerization of CA in diglyme-water. It is clear that the ratio of the three dimers is greatly affected by the ph. At low ph, the content of ß-O-4 dimer is high and the content of of ß-ß dimer is low. Among the contributing structures to the resonance hybrid of the CA radical shown below, the contribution of the phenoxy radical structure Ra is greater under acidic than under neutral conditions. Thus the participation of Ra in coupling reaction will be greater at low ph to give more ß-O-4 dimer.

7 75 Effect of ph on the dimerization of SA and HA Dimerization of SA gave only two syringyl (S) dimers (ß-O-4 and ß ß ) under present reaction conditions (in solution state). As shown in Fig. 6, SA dimers found at different ph demonstrated the same ph effect as that found in the dimerization of CA. HA also gave similar results, though the effect of ph on the relative ratio of dimers was not as large as found in the dimerization of CA. Effect of presence of carbohydrates In the presence of pectin or glucomannan in McIlvaine buffer, major products from CA were also ß-O-4, ß-5 and ß-ß dimers. Depending on the ph, their ratio was changed. However the number and amount of oligomers increased. When the reaction was carried out in glycerolwater (4/6, v/v), the volubility of dimers is low, and the reaction mixture became cloudy. The number and amount of higher oligomer also increased (Fig.2b), and a compound presumed to be α glycerol ether of ß-O-4 dimer (peak e) was formed. Preparation of a DHP close in structure to native lignin has been achieved under the condition in which water was removed continuously so that dimers and oligomers are more concentrated and the steric hindrance is overcome to form 5-5, 4-O-5 and ß-1 linkages (3). The polysaccharides may play an important role in various ways in the Dignifying cell wall. a). Phenolic acid esters of polysaccharides may provide a lignin anchor onto which initial polymerization of monolignols may occur (6). b). Addition of polysaccharides to quinone methides can form α benzylic ether or ester which provides another anchor for deposition of lignin. c). Polysaccharides provide acidic and less polar conditions under which polylignols containing frequent ß-O-4 linkages are formed. From the stereochemical studies on the formation of the diastereomers of ß-O-4 dimers, Brunow et al. proposed that the ph of the medium in which lignin biosynthesis occurs is lower than has been assumed (7). d). Polysaccharides adsorb monolignols and dilignols (8) and can thus accelerate reactions by making better contact with each other and with the enzyme bound to polysaccharides. e). CMFs adsorb lignols and hold their aromatic ring in particular orientation (8). This may be one of the causes for preferential orientation of aromatic rings of lignin relative to the cell wall surface (9). f). Polysaccharide gels shrink with deposition of oligolignols on them, and bring the oligomers closer to form variety of linkages including 5-5, 4-O-5 and ß-l linkages which are not formed in dilute solution because of steric hindrance. Thus a three dimensional network between oligomers is formed. g). The assembly of CMFs and PEC or HC in the cell wall matrix plays a role as the template for lignin deposition. Thus they determine the size and shape of the lignin macromolecule.

76 Differences in the kind and property of the gels between ML (PEC) and SW (HC) may be an important factor causing the differences in structure between CML lignin and SW lignin.

microenvironments provided by the polysaccharide constituents.

8 76 Differences in the kind and property of the gels between ML (PEC) and SW (HC) may be an important factor causing the differences in structure between CML lignin and SW lignin. CONCLUSIONS Since lignin is formed by polymerization of monolignols within a polysaccharide matrix, it is anticipated that the structure of the polylignol will depend on reaction conditions with the microenvironments provided by the polysaccharide constituents. Whithin this context, the present study clearly points to the likely effects of ph and matrix polarity on the distribution of inter unit linkages between the monolignols. It is quite likely also that association of the oligomers with polysaccharide matrix constituents will have a significant effect. Acknowledgements. This work was supported by grant No. DE-AI-02-89ER A001 from the Division of Energy Biosciences, Office of Basic Energy Sciences, United States Department of Energy, and by Forest Service, United States Department of Agriculture. Both are gratefully acknowledged. REFERENCES

New Preparations of Lignin Polymer Models under Conditions that Approximate Cell Wall Lignification

N. Terashima et al.: New Preparations of Lignin Polymer Models 521 Holzforschung 49 (1995) 521-527 New Preparations of Lignin Polymer Models under Conditions that Approximate Cell Wall Lignification I.