STRUCTURAL POLYSACCHARIDES IN MOLECULAR ARCHITECTURE OF PLANT CELL WALLS- FROM ALGAE TO HARDWOODS

R.H. ATALLA AND J.M. HACKNEY USDA Forest Service, Forest Products Laboratory,1 One Gifford Pinchot Drive, Madison, WI 53705-2398

ABSTRACT The structural polysaccharides are a family of polymers of hexoses and pentoses that occur in all plant cell walls. The distinguishing characteristic of these polymers is a ß-1.4-linked backbone. The most common among these is cellulose. which is the lin­ ear homopolymer of anhydroglucose. These polysaccharides are capable of aggregating into highly ordered structures that are the primary determinants of the mechanical and physical properties of cell walls. An overview of the variations in patterns of structural-polysaccharide aggregation within cell walls is presented here. Among the majority of the algae cellulose is the domi­ nant structural polysaccharide: thus the habit of aggregation is dominated by the patterns of cellulose. Among primitive plants. other structural polysaccharides represent a larger fraction of cell-wall mass and cellulose is less dominant. In woody tissues of higher plants. structural polysaccharides are the major components of the cell wall, and the patterns of aggregation are again dominated by the characteristic habits of cellulose. Within the phylogenetic framework, higher levels of morphological development apparently involve greater complexity in the molecular architecture of the cell walls and a finer level of blending of the components of aggregates at the molecular level.

INTRODUCTION The primary structural components of plant cell walls are a group of polymers with backbones made up of ß-1.4-linked monosaccharides. The dominant polymer is. in most instances, cellulose; it is the homopolymer of anhydroglucose shown here schematically.

The manner of coaggregation of cellulose with other cell-wall constituents varies widely. In material terms, the different forms of coaggregation include composite structures in which the cellulosic component can be viewed as a separate phase embedded in a matrix of other constituents, genuine blends wherein the mixing of the constituents is at the molecular level. and more complex architectures that are intermediate between, or combinations of, these two forms of organization. We recently began a survey of this range of variation and its relationship to the phylogeny of source species from various divisions of photo­ synthetic organisms. We provide here an overview of these findings and discuss levels of

1 The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin. This article was written anti prepared by U.S. Government employees on official time, and it is therefore in the public domain and not subject to copyright. 2 organizational complexity at the molecular level as they relate to the morphological com­ plexity of organisms within the evolutionary scheme of the plant kingdom. To establish an appropriate perspective, we begin by discussing variation in patterns of cellulose aggregation when it occurs either in pure form in the native state or in a form that allows easy isolation with mild procedures that do not perturb its native orga­ nization. In order to provide a framework for assessing the structures in which noncellu­ losic polysaccharides occur in significant quantities. we also consider extensions of the methodologies used to characterize the structures of cellulose. We conclude with an overview of a preliminary survey of cell-wall structural polysaccharides from a wide range of sources representing different levels of morphological development among photosyn­ thetic organisms.

STRUCTURE AND STATES OF CELLULOSE AGGREGATION Cellulose generally is regarded as the component responsible for much of the me­ chanical strength of the cell wall. Its unique structural properties result from its ability to retain a semicrystalline state of aggregation even in an aqueous environment; this is unusual for a polysaccharide. Two conceptual frameworks are combined to describe states of cellulose aggregation. The first is one applied frequently to semicrystalline polymers: the other is employed for polymorphic crystalline solids. Within the first of these frameworks. cellulose is typical of the general class of linear homopolymers. usually described as semicrystalline. that can aggregate to form microcrystalline domains. On the other hand. the crystalline domains of cellulose can occur in more than one crystal lattice form: hence its classification as polymorphic. Characterizing the physical structure of cellulose requires identification of the allomorph of the crystalline domains as well as an assessment of the balance between crystalli ne and amorpholis microdomains. Three different levels of structure define a particular state of aggregation. The primary structure refers to the chemical structure that reflects the pattern of covalent bonding; for cellulose it is fairly well established and is usually not in question. The next level, the secondary structure, is that of the conformations of individual molecules. It defines the relative disposition in space of the repeat units of an individual molecule given the constraints imposed by conformational energy considerations and by the packing of the molecules in a particular state of aggregation. This level of structure is important in spectroscopic studies, where the energy levels at which transitions occur are determined by the values of the internal coordinates that define molecular conformations. The final level, that of tertiary structure, reflects the arrangement of the molecules relative to each other in a particular state of aggregation whether this state is amorphous or is associated with one or another of the allomorphs contributing to the polymorphic state of cellulose crystallinity. This is the level of structure probed by diffractometric measurements, which are inherently most sensitive to the three-dimensional organization represented by a particular state of aggregation. Keeping in mind that different levels of structure represent a hierarchy of structures nested within each other is important. The specification of a secondary structure has implicit within it a specification of the primary structure. A precise definition of the tertiary structure, in turn, has implicit within it an equally precise definition of both primary and secondary structures. I n fact, for low -molecular-weight compounds that can form single crystals, the determination of tertiary structure through diffractometric studies can be carried out with sufficient precision to characterize both primary and secondary structures as well. In contrast, the diffractometric, data for polymeric materials are much more lim­ ited in content, and it becomes necessary to complement them with structural infor­ mation derived from studies carried out on monomers or oligomers and information derived from investigative techniques that provide additional independent information about the structure. One consequence of the uncertainties is that different investigators, emphasizing different sources of structural information to complement the diffractometric data, arrive at different views concerning the distinguishing features of the structures of cellulose. The different approaches to structural analysis have been reviewed by Atalla [1,2]. In the following discussion the primary focus will be on the native forms of cellulose.

Native Cellulose Early studies of cellulose structure relied primarily on x-ray diffractometry [3,4]. These necessarily addressed the issue of tertiary structure, and because of the infor­ mation content limitations of the diffractometric data, much uncertainty remained with respect to secondary structure. The difficulty of determining structure was compounded by the finding that different native celluloses possessed different unit cells. Thus, the al­ gal celluloses gave patterns that seemed to require 8-chain unit cells. while the somewhat less crystalline, higher-plant celluloses appeared to possess 2-chain unit cells. The two classes of celluloses also had very different infrared spectra in the O-H stretching region. suggesting different patterns of’ hydrogen bonding [5]. This uncertainty was compensated for in structural calculations by introducing seem­ ingly plausible assumptions that the lattice possessed the symmetry of space group P21 and that the twofold-helix axis was coincident with the chain axis. All disallowed reflec­ tions were assumed negligible in the structural calculations. The validity of these assump­ tions has been questioned by many investigators. More recent diffractometric studies also remain in conflict with respect to the details of the tertiary structure; French et al. have noted that the data do not allow discrimination between parallel up. parallel down. or anti parallel structures [6]. During the past two decades, a number of new structure-sensitive techniques have been developed, and they have been applied to studies of cellulose. These have been particularly valuable because they are capable of probing secondary structure directly. The techniques include Raman spectroscopy and solid-state 13C nuclear magnetic reso­ nance (NMR) in the experimental arena and conformational energy calculations in the theoretical domain. They have been used in more recent analyses of structure-related phenomena in cellulose to complement the information available from the diffractometric measurements.

Raman Spectroscopy Raman spectroscopy is the common alternative to infrared spectroscopy for inves­ tigating molecular vibrational states and vibrational spectra. Its key advantage in the glycosidic linkages in sequence along the chain axes. The left-handed and right-handed linkages were envisioned as representing relatively small departures of the dihedral angles from those prevailing for a twofold helix. This model has been discussed in more detail elsewhere [11,12]. Diffractometric studies are inherently incapable of detecting relatively small departures from the symmetry of the twofold helix because all of the disallowed reflections associated with departures from this symmetry are usually excluded in the analyses of the data.

Solid- state 13C NMR Spectra The second important spectroscopic method that has been applied in investigating the structure of cellulose is high-resolution 13C NMR of the solid state based on the CP- MAS technique. In this technique, cross-polarization (CP) is used to enhance the 13C signal. Also, high-power proton decoupling is applied to eliminate dipolar couplings with protons and magic-angle spinning (MAS) of the sample around a particular axis relative to the field is conducted to eliminate chemical shift anisotropy. Application of this method results in acquisition of spectra of sufficiently high resolution to distinguish chemically equivalent carbons that occur in magnetically nonequivalent sites. Although the technique has been applied to cellulose by a number of investigators [10,13-16], VanderHart and Atalla developed its application to the study of native celluloses [17-19]. The most significant new information derived from the CP-MAS spectra is that relat­ ing to the ultrastructure of native celluloses. The spectra reveal multiplicities that cannot be interpreted in terms of a unique unit cell, even though they arise from magnetically nonequivalent sites in crystalline domains. The narrow lines observed have relative in­ tensities that are neither constant among the samples of different native celluloses nor in the ratios of small whole numbers as would be expected if they arose from different, sites within a relatively small unit cell. VanderHart and Atalla proposed that native celluloses are composites of two distinct crystalline forms [17,18]. Spectra, of the two forms were resolved through linear combination of the spectra of native celluloses possessing the two forms in different proportions. The two types were designated celluloses The form was found to be dominant in the majority of celluloses from lower plant forms and in bacterial celluloses. while the form was found dominant in celluloses from higher plants. In studies of the Raman spectra of different native celluloses. Atalla [20] concluded that the two forms consist of molecular chains that, have the same molecular conformation. Wiley and Atalla [11] presented evidence suggesting that the hydrogen- bonding patterns differ in the two forms although the molecular conformations are the same. That is, although the secondary structures are the same, there arc subtle differences in the tertiary structures. Additional studies by VanderHart and Atalla, based on observations of spin diffusion and relaxation in the 13C NMR CP-MAS experiments, confirmed the existance of the and forms in native celluloses [19]. More recent studies have shown that the particular tertiary structure occuring in a native cellulose may be correlated with the architecture of the cellulose-synthesizing complexes on the cell membranes [12]. All these studies have helped conclude that the form of tertiary structure dominates in the majority of celluloses from algae or bacteria. and the form dominates among the celluloses from the higher plants.

APPLICATION TO CELLULOSE CHARACTERIZATIONS The studies and considerations briefly reviewed earlier have placed the problem of characterizing the structures of cellulose in a fresh perspective. Until application of the spectroscopic methods was developed, the characterization of states of cellulose aggre­ gation, in both fundamental and applied investigations, was limited to qualitative as­ sessments of the x-ray diffractograms. The much more laborious mesurement of the 5 leveling-off degree of polymerization, used in some instances as a measure of crystallinity, is of limited value. Thus, it, was difficult to develop quantitative measures of the vari­ ation in patterns of aggregation with sample origin and history. With the spectro­ scopic methods, developing more complete and quantitative descriptions of the states of aggregation is possible, thus defining secondary as well as tertiary structures more com­ pletely; correlations with the properties of the celluloses are now possible to a much greater extent. A number of applications of spectroscopic methods to the characterization of pure celluloses have been reported. The first involved quantitative analysis of the degree of intermorphic transformations based on Raman spectroscopy [21]. The technique later was adapted to assess the susceptibility of native celluloses to lattice conversions during different procedures for chemical isolation [22]. Some effects of mechanical action on cellulosic fibers also have been monitored by these applications [23], and effects of heat treatments on amorphous celluloses have been characterized by combining spectroscopic methods with x-ray diffractometry in a complementary manner [24].

CHARACTERIZATION OF STRUCTURAL POLYSACCHARIDES In the work discussed so far, the focus has been on relatively pure celluloses from different biological sources: many of these sources are commercially important. In the study of cell-wall architecture. the analysis of structure is complicated further by the presence of noncellulosic constituents that can interact with cellulose to varying degrees. The other polysaccharides can be part of the matrix within which cellulose fibrils are imbedded and may be separated easily from the cellulose by mild extractive treatments, or they can become so fully integrated into the structure of the fibrils that they must be viewed as modifying the state of aggregation. When the architecture of cell-wall polysaccharides is examined, this range of interactions is seen to form the basis for a hierarchy of complexity at the level of intermolecular integration. In order to explore this hierarchy, the conceptual framework and instrumental met hods developed for the characterization of cellulose have been adapted for assessing the states of aggregation of structural polysaccharides isolated from cell walls. For the purposes of this study. the structural polysaccharides are defined in terms of well-etstablished procedures for isolating nonsoluble components from cell walls. These procedures reflect varying degrees of complexity of the aggregations. In these procedures, biomass is treated first with an organic solvent to remove extractives and then with aqueous alkali to solubilize nonfibrillar matrix polysaccharides. For materials such as seed fibers or the cell walls of algae and certain lower land plants, the structural polysaccharides are not linked extensively to other components. Therefore, this solubilization step can be conducted directly. For the lignified woody tissues of higher land plants. however, a harsh chemical pretreatment, usually an oxidative technique, is required in order to break down the lignin and provide access to the polysaccharides of the cell wall [25-28]. Isolates prepared by procedures such as these may then be characterized by various chemical and physical analyses. The first step in the characterization consists of an analysis of the monosaccharides obtained through acid hydrolysis. This allows a determination of the degree to which polysaccharides other than cellulose are incorporated in the insoluble component of the cell wall. The state of aggregation then is characterized by x-ray diffractometry. Raman spectroscopy, and solid-state 13C NMR. The diffractograms and spectra are assessed by comparing them to corresponding analyses of pure native celluloses. These comparisons allow judgments concerning the degree to which the lattice order of a particular sample departs from that observed in the most crystalline of pure celluloses.

6 SURVEY The primary objective of our survey was to expand the fund of information available on cellulosic cell walls to include the much broader category of organisms and tissues in which. although cellulose is a major or dominant component. significant quantities of other polysaccharides also occur. The basis for the approach we adopted was the finding. in the course of earlier studies. that in most instances diffractometric and spectral patterns clearly were related to those of pure native celluloses. We became interested in establishing how broadly the patterns characteristic of cellulose dominate those of the total structural­ polysaccharide content of cell walls. In order to systematize the survey, we related it to a scheme of phylogenetic relationships linking the plant kingdom to the algae. The phylogenetic relationships between the organisms examined in the survey (Figs. 1 and 2) provide a framework for tracing the development of cell-wall complexity among photosynthetic organisms [29,30]. Although a large number of algae exist as microscopic unicells, many brown. red. and green algae are organized into multicellular bodies with forms that range from simple filaments to more massive, three-dimensional structures reaching lengths of over 50 m. Certain green algae in the Class Charophyceae, commonly believed to share ancestors with early forms of land plants, display particularly differenti­ ated morphologies. However, no alga shows evidence of a true vascular system. Although the most primitive land plants. the bryophytes. occasionally possess cells with structures that favor the transport of food and water, representatives of this group lack the special­ ized vascular tissues found in higher plants. As a result, the diminutive bryophytes are confined to moist environs where adequate water is available for direct absorption over the entire surface of the plant body. Well-developed conducting tissues make their first ap­ pearance among the pteridophytes; true leaves. stems. and roots have developed entirely from the maturation of apical meristems in the tips of roots and shoots. (A meristem is a form of undifferentiated plant tissue from which all new cells arise.) Finally. among the gymnosperms and certain angiosperms in the Class Dicotyledons. significant portions of conducting tissues arise from the maturation of lateral meristems. which significantly thicken the stems. branches. and roots of these plants. Wood, or secondary xylem. is prominent among the tissues arising from such meristems. Lignin typically constitutes 20 to 30 percent of the secondary cell walls of woody tissues; it is a complex heteropolymer of variously substituted phenylpropane units that appears to be integrated into the tertiary structure of the structural polysaccharides. When the structural polysaccharides from representatives of the different phyloge­ netic groupings were examined, a number of different analyses suggested that variation in the structure of polysaccharide aggregates correlated with the complexity of cell-wall architecture in the organisms. After appropriate isolation procedures were conducted, each sample of structural polysaccharides was characterized by sugar analyses. by x-ray diffractometry. and by Raman and solid-state 13C NMR spectroscopy. Based upon these analyses, cellulose was confirmed as the primary component of structural polysaccharides in each isolate: the green algae of the Class Ulvophyceae with noncellulosic cell walls were not included within this survey [31]. However, the level of cellulose within these isolates appeared to vary generally with phylogenetic placement. One such indication was provided indirectly by the percentages of monosaccharides con­ tributing to sample mass. Glucose. the only monosaccharide component of cellulose. constituted approximately 70 to 90 percent of isolates derived from the brown and red algae and from green algae in the Classes Chlorophyceae and Ulvophyceae. This percent­ age dropped among certain members of the Class Charophyceae. showing a consistently lower contribution of glucose to samples from those divisions constituting the pterido­ phytes: glucose typically provided 40 to 65 percent of total mass. Among the samples derived from woody tissues of gymnosperms and dicotyledonous angiosperms. the values once again ranged above 70 percent. These data were supported by indications of crystallinities gained from x-ray diffrac­ tion patterns (the “CrI” index of Ahtee et al. [32]). While the diffraction patterns were typical of native celluloses, the reflections were greatly broadened by lattice defects

7 Figure 1 -A scheme illustrating the phylogenetic relationships linking ancestral single-celled organisms, algae, and the land plants. In particular, the development of ancestral green phototrophs into the three recognized classes of green algae is illustrated in the upper half of the scheme; similar developments occurred with the red and brown phototrophs. Based upon details of mitosis and the construction of flagellated cells, the phylogeny of land plants may be linked most directly to those green algae within the Class Charophyceae. associated with the other polysaccharide constituents coaggregated with cellulose. The diffractograms collected on samples from certain charophycean algae and from represen­ tatives of the pteridophytes displayed broader peaks than did those of the remaining algae and the woody land plants. signifying a more extensive defect structure within the crystalline lattice. The width and resolution of bands within the Raman and NMR spectra also distin­ guished the states of aggregation by reflecting the level of perfection within the crystalline domains of each isolate. These spectra again suggested a division among the various rep­ resentatives tested in the survey, with the noncharophycean algae and woody land plants having the more highly ordered lattices. When the ratios of the forms were determined among these two groups of photosynthesizers, a previously perceived trend [12] was supported in which the I, form predominated in bacterial and algal celluloses, whereas the form was most, common in celluloses isolated from higher plants. However,

8 Figure 2-Proposedphylogenetic associations linking the ancestral green algae of the Class Charophyceae to the bryophytes and higher vascular plants within the Kingdom Plantae. The Division Bryophyta includes the nearest terrestrial relatives of the algae; the seedless pteri­ dophytes show the first true vascular tissues, which developed in some unknown evolutionary sequence. the larger contribution by defect structures among isolates from the pteridophytes and some of the charophycean algae resulted in the broadening of the distinguishing features in the NMR spectra. thereby preventing the determination of such ratios. The results of the various chemical. diffractometric, and spectroscopic analyses conducted in this pre­ liminary survey are summarized in Table I. The results of this survey indicate variation in the extent to which noncellulosic poly­ mers and cellulose were aggregated within isolated fractions of structural polysaccharides. In isolates from the red, brown, and noncharophycean green algae. such aggregates con­ sistently appeared to be dominated to the greatest degree by the habit of cellulose. In isolates from representatives of the pteridophyte divisions. noncellulosic polysaccharides made a relatively larger contribution. At higher levels of morphological complexity. the structural aggregates isolated from the woody tissues of gymnosperms and dicotyledonous angiosperms appeared to be dominated once again by cellulose. The structural polysaccharides from the charophycean algae examined in this survey seemed to be divided between the two patterns of aggregation. That is, while some clearly reflected the dominance of cellulose, others were more like the pteridophytes in

9 Table I -Characteristics of structural polysaccharides isolated from assorted algae and land plants

Portion of isolate accounted for by Cellulose Dorminant glucose monomer crystallinity- cellulose I Phylogenetic groupingsa (percent) index value allomorph Red algae Brown algae 72.4-91.5 0.7869- 0.9864 Noncharophycean green algae

Certain charophycean green algae Mosses 29.2-67.1 0.6032-0.7829 -b Pteridophytes

Certain charophycean green algae Gymnosperm woody tissues 71.9-88.4 0.7923-0.8542 Angiosperm woody tissues

a Groupings recognized from data collected during the survey conducted in this study. b Cellulose crystallinities were too low to determine the predominating allomorph. appearing to have a very high level of defect structure. Such an inconsistency might emerge in other algal taxonomic groupings during a more extensive examination. These results may correlate with the position the Class Charophyceae occupies in the overall scheme of plant phylogeny. In addition to cytological evidence that links these algae to the development of more advanced photosynthetic organisms: some facts indicate that the rosette/globule form of cellulose-synthesizing complex. a feature associated with land plants. first appears among the charophytes [33]. When compared to the linear form of complex found in noncharophycean algae. the rosette/globule form appears to produce cellulose crystallites that are interrupted more frequently by disordered or paracrystalline regions [34]. The aggregation patterns noted in this survey corresponded to certain previously observed phylogenetic variations in the primary structures of cell-wall polysaccharides. The more highly branched structures common to many algal glycans cause these polysac­ charides to form slimes or gels. which are particularly well suited to prevent desiccation during low tide or to enable the organisms to withstand the shock of violent wave action. This type of structure also would interfere with the ability of these polysaccharides to aggregate with cellulose molecules. The morphological complexity that, developed among plants exploiting the terrestrial environment was accompanied by a reduction in the de­ gree of branching and in the variety of sugar residues and linkages occurring within the main chains of the glycans. This simplification of primary structure allowed the devel­ opment of more-ordered secondary structures, frequently directed towards the structural support required for effective competition in the terrestrial environment [35,36]. However. the more-ordered secondary structures in noncellulosic polysaccharides also increase the frequent). with which these polymers can enter into aggregations with cellulose chains. by either an intercalation of large segments of chain backbone within the lattice or the insertion of individual chain termini into the crystalline lattice. Within this model. we would consider that the data for the celluloses from gymnosperms and dicotylodenous an­ giosperms indicate that cellulose constitutes a greater proportion of cell walls in these woody tissues. Regardless of the complexity of the structures of these noncellulosic polymers, their relative contribution to the mass of structural polysaccharides isolated from woody materials appears to decrease when compared to those from lower land plants.

10 The appearance of such an aggregation pattern in wood is associated with an increase in the lateral dimensions of cellulosic crystalline domains. and such a development would be complemented by the extensive lignification that is observed in these tissues. By establishing an extensive network of covalent linkages with polysaccharides, the lignin macromolecule provides an effective three-dimensional framework of support within the cell wall [37].

ACKNOWLEDGMENTS

For their support. we gratefully acknowledge the Weyerhaeuser Company, the DOE Division of Energy Biosciences. the DOE University Research Instrumentation Program, the USDA Forest Service. Forest Products Laboratory. and the Institute of Paper Science and Technology.

REFERENCES

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12 Structural Polysaccharides in Molecular Architecture of Plant Cell Walls- From Algae to Hardwoods

R.H. Atalla, Supervisory Chemical Engineer J.M. Hackney, General Physical Scientist

USDA Forest Service Forest Products Laboratory One Gifford Pinchot Drive Madison, WI 53705-2398 USA

Published in Materials Research Society Symposium Proceedings, Vol. 255: 387-397, 1992

Keywords: Structural polysaccharides, cellulose, cellulose I allomorphs, algae, land plants, x-ray diffraction of cellulose

The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin. This article was written and prepared by U.S. Government employees on official time. and it is therefore in the public domain and not subject to copyright.