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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 53705 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.
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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 phydroxyphenyl- 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.
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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[2methoxyethyl] ether) and water (8/2-2/8, v/v, 1 ml) at various pH adjusted with H3PO4 and
Na2HPO4, then peroxidase (0.1 u/ 0.1 ml, Type 1, Sigma Chem. Co.) and H2O2 (0.5 mg,
0.015 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.
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Preparation of CA dimers. CA (900 mg) was dissolved in dioxane-water (2/3, v/v, 250 ml, pH
4.5 adjusted with dil. H3PO4), 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 (97/390/10, v/v gradient) as a developer to elute dimers in the order, ß-ß’, ß-5’, ß-O-4’ dimers.
RESULTS AND DISCUSSION
Monitoring polymerization by HPLC
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 H2O2 (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
Fig. 2 HPLC diagrams of reaction products
monitoring by HPLC gives us useful
from CA in
information on the behavior of monolignols and
a) diglyme-water (2/3, v/v) and
b) glycerol-water (2/3, v/v)
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 H2O2 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 H2O2 was added, and that the relative
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 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 %
Fig. 4 Composition of CA dimers
formed in diglyme-water mixtures.
(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
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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.
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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.
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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 14068 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