Influence of hydrodynamic environment on composition and

Planta (1994) 192:461- 472 Springer-Verlag 1994

Influence of hydrodynamic environment on composition and macromolecular organization of structural polysaccharides in Egregia menziesii cell walls J.M. Hackney1, G.P. Kraemer2*, R.H. Atalla1, D.L. VanderHart3, D.J. Chapman2 1

USDA Forest Service, Forest Products Laboratory, One Gifford Pinchot Drive, Madison, WI 53705-2398, USA Department of Biology, UCLA, Los Angeles, CA 90024, USA 3 National Institute of Standards and Technology, Division 440, Gaithersburg, MD 20899, USA 2

Received: 31 May 1993 / Accepted: 28 July 1993

Abstract. To test whether secondary and tertiary structures of marine-algal structural polysaccharides may be altered during adaptive responses to hydrodynamic stresses, juvenile Egregia menziesii (Turn.) Aresch. sporophytes were cultured under three different regimes: (i) low-energy (LE) specimens were subjected to water motion produced by standard bubbling and circulation of tank water; (ii) high-energy (HE) specimens received additional movement in pumped streams of water; and (iii) stretched (STR) specimens were grown under low-energy conditions but also were subjected to constant, longitudinal tension (0.7 N). After 6-10 weeks growth, cell-wall structural polysaccharides from specimen blades were isolated by solubilizing less-ordered matrix polysaccharides. Neutral-sugar and uronic acid contents of these isolates were measured, and samples were analyzed by x-ray diffraction and by Raman and 13C-nuclear magnetic resonance (NMR) spectroscopy. On average, structural polysaccharides formed about 7.2% of dry-weight biomass. The portion of isolated mass accountable to neutral sugars ranged from an average of 85% for STR sporophytes to 94% for both LE and HE specimens. For all specimens, glucose composed an average of 99% of this fraction. Uronic acids could not be detected in isolates from any treatment group. Cellulose dominance in each isolate was indicated clearly in x-ray diffraction patterns and in Raman and 13C-NMR spectra. These data further demonstrated that both the cellulose I allomorph and the disordered form of the polymer were present in 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. * Present address: Laboratorio di Ecologia del Benthos, Punta S. Pietro, I-80077 Ischia Porto, Napoli, Italia Abbreviations: CP/MAS = cross polarization/magic angle spinning; HE = high energy; LE = low energy; NMR = nuclear magnetic resonance; STR = stretched Correspondence to: J.M. Hackney; FAX: 1 (608) 231 9592; Tel.; 1 (608) 231 9439

each isolate and that the STR isolate contained small quantities of the cellulose II allomorph. In general, the LE and HE samples had very similar crystallinity; lateral order was slightly more developed in LE samples. However, the STR treatment produced cellulose with lowest crystallinity and least lateral order. Results suggest that mechanical stress modified cellulose crystallinity in these kelps by altering levels of disordered cellulose and lateral dimensions of cellulose crystallite and, in one instance, changed the crystallinity qualitatively. Physical disturbances to cell plasma membranes may have instigated these trends. In the STR specimens in particular, such disturbances might have been supplemented by fundamental changes to kelp physiology, affecting both substantial decreases in crystallinity and production of the cellulose II allomorph. Changes in the nature of the cellulose cannot, however, account for changes in the elastic moduli. Key words: Alginate - Cellulose - Cell wall – Crystalline allomorphs – Hydrodynamics – Phaeophyta

Introduction The physical environment is an important determinant of algal morphology and physiology, and variables such as photon-flux density and wavelength (Calvert 1976; Miyachi et al. 1978), salinity (Gessner and Schramm 1971), and inorganic-nitrogen concentration (Neish and Shacklock 1971) elicit an assortment of adaptive responses. Water motion also appears to have an effect, with thallus structural adaptations and changes in productivity reported for marine algae exposed to different flow rates or wave-action levels (e.g. Armstrong 1987, 1989; Leigh et al. 1987; Koehl and Alberte 1988; Wheeler 1988; Kraemer and Chapman 1991a). Our purpose here was to investigate whether adaptive responses by marine brown algae to hydrodynamic variables may be detected within secondary and tertiary

462

structures of cellulose and associated insoluble polysaccharides in cell walls. Such changes develop in terrestrialplant cellulose in response to mechanical stress (Timell 1986). Structural polysaccharides were isolated from juvenile sporophytes of Egregia menziesii grown under different intensities of water motion and under mechanical stress. Isolates were subjected to unit-sugar assays and to diffractometric and spectroscopic analyses to determine macromolecular organization. Cellulose is the main fibrous structural polysaccharide in cell walls of higher plants and most marine algae (Preston 1974). Brown-algal cell walls are organized into multiple layers (lamellae) of microfibrils. These microlibrils are primarily cellulosic and are laid down within the plane of the cell wall. In some species, microfibril orientation is totally random within the planes of lamellae (Cronshaw et al. 1958). In other species, microfibrils are approximately parallel within individual lamellae and shift in angle between subsequent lamellae (Peng and Jaffey 1976). Within each lamella, microfibrils are embedded in a complex matrix consisting of alginic acid and a mixture of sulfated fucans that incorporate units of xylose, galactose, and uronic acids (Kloareg and Quatrano 1988). Many of these polysaccharides are also present, though perhaps in different proportions, in a matrix found between cells (Vreeland 1981; Mariani et al. 1985). Cellulose contribution to brown-algal cell-wall mass often is minimal; a range of 1.5%–20.0% of total dry weight exists in various species in the Fucales or Laminariales (Cronshaw et al. 1958; Quatrano and Stevens 1976; Kloareg 1984; Mabeau and Kloareg 1987). Although cellulose microfibrils probably are the primary load-bearing components of brown-algal cell walls, the matrix polysaccharides that dominate thallus mass also are likely to contribute to the structural function of walls through their gel-forming capacities (Kloareg and Quatrano 1988). Microfibrils in cell walls of brown algae and other cellulose producers are composed primarily of ordered aggregations of cellulose molecules in varying degrees of crystallinity. The isolated cellulose molecule consists of β-1,4-linked anhydroglucose units. This molecular framework allows some degree of variation. particularly in the dihedral angles that define conformations of glycosidic linkages along the chain or in the positioning of the primary alcohol group at C-6. In the solid state, the placement of hydroxyl groups and oxygen atoms on each unit affords numerous opportunities for intermolecular hydrogen bonding as well as some specific patterns of intramolecular hydrogen bonds. These, together with the stiffness of glycosidic linkages between pyranose rings, promote the packing of cellulose chains into three-dimensional, ordered crystalline domains. Crystallinity in cellulose manifests a complex polymorphy (Atalla 1987), e.g. cellulose I (native cellulose), the more recently described celluloses I x and Iβ (Atalla 1989a). and cellulose II (regenerated or mercerized cellulose). Definition of crystalline-lattice forms and the coherence of order in a particular sample characterize cellulose tertiary structure. Shifts in space of the relative positions of individual atoms within the anhydroglucose

J.M. Hackney et al.: Structural polysaccharides of Egregia

units of an isolated chain and shifts in the pattern of prevalent bonds within these units describe cellulose secondary and primary structures, respectively (Atalla 1989a). Conformation of cellulose chains within each crystalline-lattice form may, in principle, vary from one lattice form to the next in response to different patterns of chain packing or hydrogen bonding; moreover, the noncrystalline region contains a range of chain conformations. We anticipate that the techniques of Raman and 13 C-nuclear magnetic resonance (NMR) spectroscopy along with x-ray diffractometry will provide complementary information about differences between the algal cellulose samples under consideration (Atalla 1984, 1989a,b; Atalla and VanderHart 1989). In particular, certain regions of the Raman spectrum are sensitive to changes in skeletal geometry and relatively insensitive to variations in intermolecular packing, whereas 13C-NMR spectral positions are more similar in their sensitivity to these changes. Contrasting signatures for cellulose I, cellulose II, and disordered cellulose appear in all three kinds of measurement, and under conditions of constant humidity, the sharpness of features associated with the various crystalline forms will increase monotonically with coherence of lateral order in crystallite. Thus, we expect to monitor changes both in crystallinity and in crystalline form within these samples. In most current models, a well-defined boundary does not separate less-ordered matrix polysaccharides from the cellulose crystallite that tend to align with the longitudinal axis of a microfibril (Fengel and Wegener 1984). The long-range order of crystalline regions may be disrupted by termini of individual cellulose chains or by intercalations of noncellulosic polysaccharide chains. These periodic disruptions of the crystalline lattice will interfere with a regular packing of cellulose chains, resulting in gradual transitions along the axis between zones of order and disorder. Similarly, a gradual transition occurs from the crystalline regions outward to the paracrystalline cortex of the microfibril, as cellulose chains become less ordered and are dispersed in ever-increasing proportions of noncellulosic polymers. Also, some chemical continuity may exist between microfibrils and surrounding matrix polysaccharides. For brown algae in particular, Kloareg and Quatrano (1988) proposed a model of cell walls in which cellulose chains are bound covalently to xylofucoglycans (Doubet 1983), which in turn are cross-linked via glycoproteins and ascophyllan (a xylofucoglycuronan) to alginates within the matrix. Because of this intimate admixture of cellulose chains and noncellulosic polysaccharides, isolating a pure cellulose fraction from brown-algal cell walls is not possible through nondestructive procedures. Cellulose most commonly is isolated from algal cell walls by dissolving the noncellulosic polysaccharides in boiling alkali. The positions of hydroxyl groups in the crystalline regions of native cellulose protect the glycosidic linkages from attack during such base hydrolyses (Atalla 1979). However, the presence of more than one type of sugar unit, branches of the chain molecule, and various other steric hindrances prevent extensive, ordered packing of most noncellulosic polysaccharicies, leaving them susceptible to degradation


and solubilization. If chains of a noncellulosic molecule are sequestered within the ordered lattice of a cellulose crystallite or are bound with particular strength to the surface of the crystalline region, they may be isolated as part of the “structural polysaccharide” fraction of cell walls. This fraction is similar to the α-cellulose fraction of wood, which is defined as the fraction of polysaccharide that survives extraction in strong alkali. However, in such a context, the base treatment is applied to wood after the lignin component has been removed by harsh chemical treatments that often involve elevated pressures and temperatures. Despite early reports to the contrary (Reznikov et al. 1978), we have observed that lignin is absent in phaeophytes upon careful analysis (data not shown: Ragan 1984). Although various authors have referred to α-celluloses isolated from algae, we prefer to recognize this fundamental difference in chemical treatments by calling the present fraction of compounds “structural polysaccharides”. Kraemer and Chapman (1991a) extracted and analyzed an alkali-soluble fraction of alginic acid from E. menziesii sporophyte blades to determine whether this polyuronide, the most abundant matrix polysaccharide in brown algae, plays a role in mechanical adaptation to hydrodynamic stress. The authors could not detect a significant relationship between composition of alginic acid and the greater strengths and stiffnesses of sporophyte blades grown under high levels of water flow. They subsequently proposed examining other cell-wall constituents and any links between such constituents for possible influence upon structural adaptations; this prompted us to focus the current study upon structural polysaccharides of the alga. Materials and methods Culture system. Juvenile sporophytes of Egregia menziesii (Turn.) Aresch. (5-10 cm total length) were grown in outdoor tanks in Los Angeles County, California (Kraemer and Chapman 1991a). Holdfasts of these algae were attached to pieces of cinder block with nylon twine and submerged in a large (1220 L) tank, which was bubbled with air and flushed with local seawater at a flow rate of 18 L • min-1 (ca. 21 turnover volumes per day). Specimens were cultured under three different treatments: (i) low-energy (LE) conditions, average water velocity of 0.7 cm • s-1; (ii) high-energy (HE) conditions, average velocity of 120 cm • s-1 (outflow from four submersible pumps continuously directed at sporophytes); (iii) stretched (STR) treatment, tensile force applied to specimen blades (distal ends of blades on some specimens in LE tank were clamped and attached by monofilament to 85-g fishing weights, which exerted constant, longitudinal tensile force of 0.7 N (ea. 40 kN • m-2; cross-sections of STR blades were 3.25-4.35 cm2)). Water velocities in the HE tank were ca. 20% those possible at collection sites. Sporophytes were cultured for 6-10 weeks. Isolation of structural polysaccharides. Structural polysaccharides of specimens in each treatment group were isolated in two stages. To analyze the most accessible fraction of alginic acid, sporophyte blades were first dried, ground, and stirred in 5% Na2CO3 to extract alginates (Kraemer and Chapman 1991a). Remaining biomass was then boiled under reflux and a N2 atmosphere for 2 h in a solution of 1% NaOH, to which ca. 100 mg. L-1 NaBH4 was added to reduce the hemiacetal endgroup and thus minimize degradation via the alkaline peeling reaction (White and Kennedy 1988). The boiling

463 stage, which removed the mujority of unordered polysaccharides, was repeated twice, with distilled-water rinses after each boil. The samples were then soaked for 24 h in the dark in an acidified solution of 0.1 M NaClO2 (1.5 g NaClO2 + 0.5 mL glacial acetic acid + 160 mL H2O) to remove residual chromophores. After a distilledwater rinse, samples were subjected to three 30-min soaks in a solution of diethylenetriaminepentaacetic acid (DTPA) 10 chelate metal ions that might interfere with spectroscopy (1 g DTPA dissolved in 900 mL H2O with sufficient NaOH to achieve dissolution; pH adjusted to 7 with HCl; volume brought to 1 L). Finally, after a distilled-water rinse, the isolated structural polysaccharides were freeze-dried and weighed to calculate percentage of recovery. Analyses of sugars and uronic acids. Subsamples of isolate-s were analyzed in duplicate for neutral sugar monomers, employing a recently modified technique for wood-sugar analysis (Pettersen and Schwandt 1991). Dilute-acid (H2SO4) hydrolyzates of the isolated polysaccharides were injected directly onto an anion-exchange liquid-chromatography column, without prior neutralization or concentration, and were separated using a 0.25-mM NaOH eluent. The post-column stream then was directed through a pulsed-amperometric detector cell, where amounts of each monomer present were measured as increases in the oxidative current passing through a gold electrode. Concentrations were quantified by comparison to standards of individual monomers. Uronic-acid compositions were determined calorimetrically (Scott 1979). Upon heating in concentrated H2SO 4, uronic acids potentially present within each isolate formed 5-formyl-2-furancarboxylic acid. This chromagen then reacted selectively with the calorimetric reagent 3,5-dimethylphenol, producing a chromophore with an absorption at 450 nm. Absorptivity was assumed to be linear with concentration, which was measured by comparison to a D-galacturonic acid standard. X-ray diffraction studies. To study the tertiary structure of compounds in each isolate, powder diffraction patterns were recorded from sample disks exposed to Cu-K x radiation in the reflection mode. Measurements were taken with a Philips1 (Eindhoven, Netherlands) model APD-3720 diffractometer equipped with a graphite monochromator, using a sampling interval of 0.02 2-θ°, sampling time of 1 s per interval, and scanning range of 4-40 2-θ°. X-ray diffraction provided patterns that consisted of various peaks, corresponding to planes of diffraction within crystalline regions, superimposed upon a much broader background generated by disordered components. Individual distraction peaks frequently overlapped one another and on occasion were obscured partially because of low signal-to-noise ratios. To characterize the structures of these samples more accurately, peaks in the reflection patterns were separated with a Savitzky-Golay smoothing function (Savitzky and Golay 1964) to fit parabolas over recorded maxima in a least-squares procedure. Peak positions then were estimated by interpolation between negative inflection points, as were peak intensities and widths at half height after corrections for background counts. Instrumental aberrations and wavelength-dependent contributions to the profile were accounted for mathematically, and these preliminary values were refined for more accurate fits to the original diffraction patterns by a profile-fitting program that employed a Marquardt nonlinear least-squares algorithm (Marquardt 1963; Schreiner and Jenkins 1983). These fittings were confined between 7.5 and 32.5 2-θ° on each diffraction pattern to provide a reasonably linear background and thus increase accuracy of fit. 13

C-Nuclear magnetic resonance spectroscopy. To discern tertiary structures, solid-state 13C-NM R spectra were collected on a custom-built NM R spectrometer operating at a magnetic field of 2.35 T, which corresponds to frequencies of 25.193 MHz for 13C and 1

The use of trade or firm names in this publication is for reader information and does not imply endorsement by the U.S. Department of Agriculture of any product or service

464

100.179 MHz for protons. Rotating reference-frame field strengths for both protons and carbons were in the range of 60-65 KHz for each nucleus. Samples were equilibrated to a standard humidity prior to collection of spectra. The 13C spectra were recorded using the various strategies included in the cross-polarization magic-angle-spinning (CP/MAS) technique (Andrew et al. 1958; Hartmann and Hahn 1962; Pines et al. 1973; Schaefer et al. 1975). Cross-polarization (1.5 ms CP time) was employed to enhance 13C-signal strengths, high-power proton decoupling was used 10 eliminate the otherwise-dominant broadening resulting from dipolar interactions between 13C nuclei and neighboring protons, and samples were spun at the “magic” angle (54.74°) to the static field to improve resolution by collapsing what would othertwise be overlapping, chemical-shift anisotropy patterns. Spinning speeds were in the range of 3 KHz. Raman spectroscopy. Raman spectra were recorded as a particularly sensitive means of assessing secondary structure. In this technique, sample disks are excited by a focused laser beam and resultant scattered light is analyzed with a multiple monochromator system. A small fraction of the scattered light is shifted in frequency; magnitudes of such shifts (Raman shifts) are expressed as wavenumber (cm-1) and represent differences between the absolute frequencies of the shifted bands and that of the incident radiation. Because Raman shifts are related directly to vibrational frequencies of a sample, Raman and infrared spectra provide similar information. However, dominant peaks in Raman spectra correspond to vibrations of bonds that are mostly covalent, whereas vibrations of highly polar bonds generate comparatively weak peaks. The opposite relationship is observed in infrared spectra. Therefore, Raman spectroscopy is sensitive primarily to skeletal vibrations of the cellulose chain and is influenced much less by vibrations of hydroxyls, the groups most directly involved in intermolecular hydrogen bonding. Thus, Raman spectroscopy is the most sensitive to the mode of packing into crystalline lattices, and for this reason, it is suited particularly well to studies of molecular conformations. Raman spectra were collected using a Jobin Yvon Ramanor HG2S system (Instrument S. A., Inc., Metuchen, N.J., USA). The excitation source was the 514.5-nm radiation of an argon ion laser. Spectra were recorded by rotating the grating in a step-wise fashion, allowing discrete segments of the spectrum, one wavenumber at a time, to be directed to a photomultiplier detector for measurement. Spectra were scanned in this manner between Raman shift wavenumbers 250-3700 cm 1, a spectral region that encompasses most fundamental frequency shifts. To maximize signal-to-noise ratios, 12-16 scans were recorded using a Tracer Northern (Middleton, Wis., USA) TN1500 data acquisition system. Because isolates had residues that fluoresced in the region scanned, each spectrum was superimposed on a broad background curve with a maximum located between 1600-2000 cm -1. The fluorescence background was removed by applying personally written curve-flattening software. A five-point Savitsky-Golay smoothing function (Bevington 1969) also was applied to each set of data three to five times. After these treatments, spectra were analyzed visually for location and intensity of each frequency peak, which identified and described the structural polysaccharides composing each isolate.


Results

Isolations and chemical analyses. Dry weights of isolated structural polysaccharides averaged 0.7% of sporophyte fresh weights (standard deviation (SD) = ± 0.1%, n = 3 isolations). The sporophytes were not dried before processing, preventing accurate weight determinations. As a result, these data were not analyzed statistically for differences between treatments. However, based upon moisture contents for E. menziesii sporophytes determined elsewhere (data not shown), the average weight of structural polysaccharides is equivalent to ca. 7.2% of total sporophyte dry-weight biomass. An insoluble residue persisted in each isolate following the acid-hydrolysis stage of sugar analyses and was most pronounced for the STR isolate. X-ray dispersive analysis and infrared absorption spectroscopy (Skoog 1985) showed the residue consisted primarily of silicon and carbohydrate, with trace amounts of bromine, potassium, sodium, and sulfur. Preliminary results of 13C-NMR spectroscopy and scanning electron microscopy suggest this residue consists of ascophyllan with portions of diatom frustules and other mineral components (data not shown). Structural polysaccharides of all isolates primarily consisted of glucose (Table 1). On average, xylose was the most abundant nonglucose monosaccharide detected, followed in decreasing order by fucose, mannose, galactose, and arabinose. Uronic acid accounted for less than 0.1% of dry mass for each sample, the lower limit of detection for this analysis. X-ray diffraction studies. Visual analyses of diffraction patterns suggested that cellulose was the primary structural polysaccharide in each isolate. This was based on identifications of peaks at ca. 14.5, 16, and 22.5 2-θ°, which correspond to the 110, 110, and 020 diffraction planes of cellulose I, respectively. Visual analysis of the STR pattern suggested additional peaks at ca. 12,20, and 22 2-θ°, positions that may be assigned to the corresponding peaks of cellulose II. Each pattern also showed a much broader peak, centered at ca. 21 2-θ°, which typically is generated by disordered cellulose (Alexander 1969). The mathematical refinement of preliminary peak characteristics during profile-fitting resulted in the calculation of individual curves describing multiple component peaks in each pattern (Fig. I). When patterns of cellulose I and II standards (untreated and mercerized Whatman CF1, respectively) were analyzed in this manner, plots of resultant curves displayed close correlations


465

between component peaks of the two allomorphs and those of the three samples. Diffraction peeks generated by cellulose I and disordered cellulose were observed in each sample pattern, whereas those of cellulose II were also present in the STR pattern. Peak position and width-at-half-height values for fitted component peaks also demonstrated correlations between patterns of isolates and standards (Table 2). Peak positions for isolates generally deviated from those for standards by less than 1.0 2- θ°, although disordered-cellulose peak positions in the two standards themselves differed by 1.34°. The disordered-cellulose peaks were by far the widest of the component peaks and may be difficult to describe accurately by a single curve; Isogai and Usuda (1990) identified two overlapping peaks in disordered cellulose that were described by separate Cauchy distribution functions. In the present study, profile-fitting analyses showed that the integrated areas of disorderedceh,dose peaks contributed 37.2%, 41.7%, and 44.9% of the total area beneath all component peaks in the HE, LE, and STR patterns, respectively. The crystallinity index of Ahtee et al. (1980), which is calculated by comparing the 020-peak height to that of the lowest point on the pattern between the 020 peak and the 110/ 110 composite peak (essentially the height of the disordered-cellulose peak), showed values of 0.76, 0.74, and 0.68 for the HE, LE, and STR samples, respectively. To estimate the integral breadth of cellulose crystalltes, peak position and width-at-half-height values for the 020 peak were entered into the Scherrer equation (Alexander 1969). When these terms were measured directly from diffraction patterns, values of 1.97, 2.83, and 2.84 nm were calculated for the STR, LE, and HE isolates, respectively. When terms associated with the deconvoluted peaks were utilized, somewhat larger values of 2.77, 4.07, and 3.82 nm were calculated for the STR, LE, and HE isolates, respectively. 13

C-Nuclear magnetic resonance spectroscopy. The 13 CNMR spectra also indicated the presence of cellulose I in each isolate as well as a small amount of cellulose H and a marked increase in disordered cellulose in the STR sample. These results were determined by analyzing various features of peaks in each spectrum (Fig. 2a-c), here the 50-120 ppm regions encompassed resonances assigned to C-6 (60-70 ppm), C-2,3,5 (70-81 ppm), C-4 (8193 ppm), and C-1 (102–108 ppm) of the anhydroglucose repeat unit (Atalla et al. 1980; Dudley et al. 1983; Fyfe et al. 1983). In CP/MAS spectra of cellulose I, the broad resonances (wings) that occur just upfield of the crystalline C-4 and C-6 peaks at 88–92 and at 65-67 ppm are at-

Fig. la-e. Distraction patterns for cellulose I standard (a), LE sample (b), HE sample (c), STR sample (d), and cellulose II standard (e). Each pattern overlies component peaks generated by the application of a profile-fitting routine; peaks associated with both cellulose I and cellulose II allomorphs were detected in the STR sample. Sharp spikes were generated by salt contaminants and were not included in profile-fitting analyses

466

Fig. 2. The 13C-NM R spectra of LE (a), HE (b), and STR (c) samples: resonances assigned to C-1, C-4, C-2,3,5, and C-6 of cellulose anhydroglucose molecule. Difference spectra for HE minus LE (d) and HE minus STR (e) were calculated after each spectrum was normalized to remove contributions by disordered cellulose. When compared to the HE spectrum, somewhat larger lateral dimensions of LE cellulose crystallites are indicated by greater levels of resolution in spectrum a and the larger area enscribed below the horizontal line in spectrum d, whereas pronounced C-4 and C-6 upfield wings in spectrum c indicate largest levels of disordered cellulose in STR sample. Features associated with cellulose II allomorph are present at 108, 77.5, and 63-64 ppm in both spectra c and e


tributed to the corresponding C-4 and C-6 carbons in disordered-cellulose domains. These wings are used in particular to judge relative contributions made by the crystalline and disordered phases of the polymer, because they overlap less strongly with their respective crystalline peaks than do those of the other four carbons (VanderHart and Atalla 1987). The wings are of roughly equal size in both the LE and HE spectra (Fig. 2, spectra a and b), though this comparison is hindered by the somewhat greater noise within the LE spectrum. However, certain other details of the LE spectrum, such as the general width of peaks or the greater resolution evident in the C-4 peak and in the upfield side of the C-2,3,5 doublet, suggest that cellulose crystallite of this isolate have slightly greater lateral dimensions than do those of the HE sample. Moreover, by normalizing the LE and HE spectra to a common noncrystalline (disordered) intensity, one can subtract the LE spectrum from the HE spectrum to obtain a difference spectrum (Fig, 2, spectrum d), which indicates that the LE sample has higher crystallinity (more area is enscribed below the baseline than above it). The STR spectrum (Fig. 2, spectrum c) indicates that the amount of disordered cellulose increased markedly in this sample, displaying the most pronounced noncrystalline upfield wings for C-4 and C-6 and also showing greater widths and lower resolutions in its crystalline resonances, particularly in the C-6 peak near 66 ppm. For relatively rigid molecules like cellulose, 13C resonances associated with disordered regions are broader and crystalline resonances are narrower (VanderHart and Atalla 1984). The presence of cellulose II in the STR sample maybe recognized directly from the most distinct resonances assigned to the crystalline form of this allomorph relative to crystalline cellulose I (Dudley et al. 1983). Such features occurred at 108 ppm (one-half of a doublet that replaced the single peak for C-1 in cellulose I), at 77.5 ppm (a distinct minor peak in the C-2,3,5 cluster), and at 6364 ppm (an emerging doublet associated with C-6). When the STR and HE spectra were first normalized to a common disordered-cellulose contribution, subtraction of the STR spectrum from the HE spectrum produced a difference spectrum with no contribution from noncrystalline cellulose (Fig. 2. spectrum e). Negative peaks in these resonance positions verify the existence of cellulose II, which is estimated to constitute ca. 10%- 15% of the total STR isolate.


467

Fig. 3. Raman spectra of LE (a), HE (b), and STR (c) samples, plotting intensity of signal against shift in exciting frequency (Raman shift), which is expressed in units of wavenumber (1/cm). The STR peak doublet at 300-400 cm-1 and triplet at 400-500 cm-1 are associated with cellulose II

Raman spectroscopy. Based upon distributions of peaks that correspond to skeletal stretching and bending vibrations for cellulose (Atalla 1976), the three Raman spectra (Fig. 3) provided further evidence that this polymer dominates structural polysaccharides in each isolate. Of the three isolates, the HE material was least fluorescent and consequently had the spectrum with the highest signalto-noise ratio (Fig. 3, spectrum b). The 3150-3650 cm-1 band, associated with O-H stretching, was notably largest in the LE spectrum (Fig. 3, spectrum a), suggesting a greater degree of hydration in this sample; this interpretation must be qualified by the uncertainty introduced during correction for underlying fluorescence. The STR spectrum (Fig. 3, spectrum c) displayed a clear signal at 1500-1750 cm-1, a region frequently associated with vibrations of carboxyl groups (Bellamy 1975) and unusual in Raman spectra of celluloses. As evidenced by spectral features closely associated with the two crystalline allomorphs (Atalla 1981, 1983, 1984, 1989b; Atalla and VanderHart 1989), the LE and HE isolates shared characteristics of cellulose I, whereas the STR spectrum displayed features that suggest appreciable quantities of cellulose II and an appreciable increase in the disordered component. Much evidence was drawn from the low-frequency region (250-700 cm-1) of the vibrational spectra, where shifts in frequencies of certain peaks are most readily apparent. Specifically, comparing the LE and HE spectra to that of the STR isolate shows that composition of the 300-400 cm-1 region changes from a triplet of peaks, each increasing sequentially in intensity, to a doublet having roughly equal intensities. This doublet in the STR spectrum is interpreted as a composite feature that reflects contributions from spectra of both cellulose II and disordered cellulose, wherein the three cellulose I peaks collapse into a single peak located just beyond 350 cm-1. Conversely, the 400500 cm-1 region of the LE and HE spectra consists of a doublet of unequal intensities, whereas that of the STR spectrum shows three separate peaks emerging at

wavenumbers characteristic of cellulose II spectra. The broad background resonances in this region of the STR spectrum also occur in the 400-500 cm-1 bands of disordered-cellulose spectra (Atalla 1983). Additional features associated with the spectrum of a highly crystalline cellulose II (Atalla 1984) were evident at higher frequencies in the STR spectrum. The doublet peaks centered at 1350 cm-l displayed more similar intensities here than in the other two spectra. Though high noise levels hindered a firm identification, the broad peak of the O-H stretching region appeared slightly skewed towards higher wavenumbers, as is usually the case in cellulose II spectra. As with NM R spectroscopy, resolution of bands from crystalline domains in Raman spectra increases monotonically with enhancement of lateral order in crystalline domains. Based upon visual analyses, the HE and LE spectra appear to have comparable peak resolutions and, hence, probably have comparable crystallinity. Evaluating line widths of sharper features in the STR spectrum is more difficult because of a large contribution by broad background bands that arose from a greater amount of disordered cellulose. Discussion Our objective was to examine secondary and tertiary structures of cellulose from cell walls in these kelps for changes that may form part of an adaptive response to hydrodynamic stresses. Results clearly indicate that changes occur, though the adaptive potential of these changes remains undetermined. Although cellulose formed a relatively small part of original algal biomasses, cellulose dominated each structural-polysaccharide isolate. The potential identification of a carboxyl-group signal in the STR Raman spectrum prompted us to consider whether residual uronic acids in this isolate may have been derived from breakdown of

468

alginates. However, appreciable levels of uronic acids were not detected in any sample during calorimetric analyses. The signal may have arisen from some byproduct of NaClO2 bleaching; Uhlin (1990) reported an inconsistent occurrence of the peak in spectra of similarly prepared bacterial cellulose. X-ray diffraction and spectroscopic analyses indicated that the cellulose I allomorph predominated in each isolate and identified low levels of cellulose II in the STR sample. However, these procedures were less consistent in ranking cellulose crystallinities. Though all analyses identified the STR isolate as having the lowest crystallinity, profile-fitting results and crystallinity-index values generated from the diffraction patterns showed more disordered cellulose in the LE sample than in the HE sample, opposite the ranking provided by NM R spectra. Because quantitation from x-ray diffraction patterns is less precise for mixed systems (Kakudo and Kasai 1972), we place greater confidence in NM R measurements of ordered and disordered domains in these isolates. The upfield wing of the C-4 resonance may be removed from each NM R spectrum by using the noncrystalline resonance line shape of ball-milled cellulose to null this region of the spectrum. Then, contribution by crystalline domains is quantified by calculating the ratio of remaining C-4 intensities to total original C-4 intensities in each spectrum. In this manner, crystallinity values of 0.46, 0.44, and 0.27 were obtained for the LE, HE, and STR isolates, respectively. Based upon these figures, we conclude that mechanical stress caused the crystallinity of cellulose I to decrease slightly in the HE isolate. Mechanical stress on STR specimens caused more pronounced decreases in cellulose I crystallinity and resulted in production of cellulose II crystallite. Given the value of 27°6 total crystallinity and the estimate of 10%–15% cellulose II content, the STR cellulose I content, by difference, would range from 12% to 17%. Thus, cellulose II formed a substantial fraction of STR crystalline material, albeit the crystalline fraction itself was small. The STR sample fraction (15%) unaccounted for in the sugar analysis (Table 1) cannot explain the substantial drop to 27% crystallinity, even if one assumes that all this fraction consists of some insoluble, saccharide-like substance. To interpret these trends fairly, we first must recognize that this study was based on analysis of isolates. In principle, therefore, we cannot be certain that the forms of cellulose observed reflect their native state within intact blades. Yet, the mild and consistent isolation procedure argues substantially that differences between samples were derived from variations in secondary and tertiary structures of cellulose within intact tissues. We assume here that crystalline cellulose I forms close to cellular sites of microfibril synthesis and that the isolation procedure does little to generate additional crystals of this allomorph, However, the formation of cellulose II is a more open question. Although we proceed by assuming that cellulose II was present within intact blades of stretched kelps. individual cellulose chains, previously scattered throughout blade tissues, could have aggregated into this allomorph during exposure to the mildly alkaline conditions of isolation.


Although cellulose II production has been attributed to various organisms (summarized by Roberts et al. 1989a), certain investigations may have been compromised by contamination or by inappropriate methods of cellulose isolation. After re-examining several such reports, Roberts et al. (1989b) could verify cellulose II production only by the green alga Halicystis and by certain mutants of the bacterium Acetobacter xylinum. To our knowledge, the present study is the first to suggest that mechanical disturbance of an organism may trigger cellulose II production. Mechanical stress frequently is linked to changes in

cellulose crystallinity during studies of woody tissues. Gymnosperm compression wood results from compressive axial stress to stem and branch undersides, and both gymnosperm opposite wood and angiosperm tension wood may develop directly above such tree parts, where longitudinal tensile stress is imposed at least intermittently. Relative to normal woods, compression woods repeatedy display crystallinity decreases, whereas crystallinity increases are detected in both opposite and tension woods (Lee 1961; Parham 1971; Marton et al. 1972; Tanaka et al. 1981; Timmell 1986). Tension woods and dried opposite woods also have higher tensile strengths (Cockrell and Knudson 1973), suggesting that increased cellulose crystallinity in these tissues provides an adaptive response to tensile stresses. When intact blades of the E. menziesii sporophytes were stretched along their longitudinal axes in tests that immediately preceded the chemical treatments of the present study (Kraemer and Chapman 1991a), moduli of elasticity (stiffnesses) were significantly greater for HE and STR sporophytes (0.013 and 0.012 GPa, respectively) than for LE sporophytes (0.007 GPa). However our current findings suggest a trend of lowered cellulose cystallinity in response to mechanical stress, which ordinarily would predict a decreased modulus (Page 1983; Billmeyer 1984). We speculate on two possible origins for this contradiction. First, the modulus of any polymeric system may be enhanced by biasing chain orientations along the direction of an applied stress. Therefore, increased moduli may have resulted from a biasing of cellulose-chain orientations along blade axes of HE and STR kelps during experimental treatments. Secondly, increased moduli may have been associated with some cellwall constituent that was removed in large part during the isolation procedure. The fact that intact blades of HE and STR kelps displayed decreased extensibility in addition to increased moduli (Kraemer and Chapman 1991a) would be consistent with the first explanation. Before removal during chemical treatment, alginic acid composed an average of 14%–17% of blade dry weights (Kraemer 1989), approximately twice the structuralpolysaccharide component estimated here. This, coupled with the measurement of blade moduli that are several orders of magnitude smaller than the modulus calculated for the cellulose unit cell (134 GPa; Waterhouse 1987). would support the second explanation, possibly indicating that nonfibrillar alginates, not cellulose, dominated tensile behavior of these tissues. The finding that crystallinity and crystallite size de-

J.M. Hackney et a1.: Structural polysaccharides of Egregia

crease with mechanical stress prompts us to consider whether such changes reflect disturbance of the cell plasma membrane. Cellulose assembly occurs at the plasma membrane of most eukaryotes, where it is associated specifically with particulate complexes. These complexes are believed to be sites of the nearly simultaneous processes of cellulose polymerization and crystalliziition (Brown 1985; Delmer 1987; Hotchkiss 1989; Emons 1991). A single row of such particles is assumed to form the microfibril-assembly complex in Pelvetia, the one brown alga thus far examined with such intent (Peng and Jaffe 1976). The geometrical placement of these complexes within the membrane and the organization provided by individual complexes likely regulates the size, shape, and crystalline structure of nascent microfibrils (Haigler 1985, 1991; Kuga and Brown 1991). We extend such proposals by suggesting that disturbance of the STR plasma membranes promoted disordered-cellulose production and the aggregation of some glucan chains into the cellulose II allomorph. Extensive disruption of fluid-mosaic membranes cannot be a strict requirement for disordered-cellulose production. Although disordered cellulose was most prevalent in the STR isolate, it contributed substantially to each sample of structural polysaccharides and clearly is associated with cellulose I under normal conditions of growth. Nor is disordered cellulose totally without organization when recovered from native sources; it is not homogeneously disordered and possesses some degree of structure, though to a lesser extent than either of the two crystalline allomorphs discussed here (Agarwal and Atalla 1986). Crystallization of A. xylinum cellulose may be prevented by direct dyes (Ross et al. 1991), and Haigler (1991) suggested that, because cellulose I forms once such dye is washed away, the disordered state preserves the chain orientation and the conformation of anhydroglucose units associated with this allomorph. These observations suggest conditions of the plasma membrane that give rise to disordered-cellulose production are not delimited clearly from those supporting formation of cellulose I. Transitions between the production of cellulose in these two states of organization are most probably gradual, with various factors acting perhaps in concert to determine the balance between the forms. The possibility that disrupted organization of STR microfibril-assembly complexes also promoted the aggregation of glucan chains into cellulose II is supported by numerous associations of this allomorph with systems of decreased order. The recrystallization of either macromolecular cellulose (Kolpak and Blackwell 1978) or cello-oligodextrins (Henrissat et al. 1987) from solution almost always produces cellulose II or other allomorphs within the cellulose II family (sensu Hayashi et al. 1975), although cellulose I may be recovered from phosphoric acid under certain highly specific conditions of regeneration (Atalla and Nagel 1974). Should A. xylinum cellulose synthetases be isolated by rupturing and separation of cytoplasmic membranes, only cellulose II is produced in vitro (Bureau and Brown 1987), and mutants of this bacterium that produce cellulose II appear to lack organized sites of cellulose extrusion on their outer membranes

469

(Roberts et al. 1989a). As Hotchkiss (1989) observed, such evidence suggests that production of cellulose I requires particular spatial arrangements of cellular components and, as such, may be disturbed easily. Although we propose here that disturbances to STR microfibril-assembly complexes affected cellulose tertiary structure, studies suggest that primary structure also may be susceptible to mechanical perturbations. Severe disruptions of plantcell plasma membranes have been linked to imprecise transglucosylation reactions during glucan polymerization, which results in establishment of β - 1,3-linkages between anhydroglucose units and formation of callose rather than cellulose. Within this model, cellulose and callose-synthase enzymes share common catalytic subunits that may be activated differentially, enabling an effective response to cell-wall damage by rapid conversion to callose formation (Delmer 1987, 1989; Delmer and Stone 1988; Northcote 1991). In our view, physical disturbances of plasma membranes would not have affected microfibril assembly in STR specimens unilaterally but may have been supplemented substantially by changes in kelp physiology. Not only did STR blades develop markedly corrugated surfaces during these studies (Kraemer and Chapman 1991a), but sporophytes previously exposed only to control conditions significantly increased carbon incorporation when subjected to the STR treatment during 4-h incubations, fixing an average of 170% more carbon into both alginates and isolated structural polysaccharides (Kraemer 1989; Kraemer and Chapman 1991b). (Such observations probably reflect physiological responses to an essentially unnatural (i.e. continuous) mechanical disturbance. Although the tensile force provided by attached weights corresponds to commonly encountered water velocities of ca. 4-8 m • s-1, this stress was imposed without the enhancement of dissolved nutrients and inorganic carbon that accompanies discontinuous current regimes in the natural marine environment). The polymerization of certain matrix polysaccharides occurs concurrently with that of cellulose in higher-plant cell walls (Mullis et al. 1976; Dalessandro et al. 1988; Northcote 1989), and increased production of noncellulosic polysaccharides in stretched kelps may have been timed to affect crystallization of microfibrils. Noncellulosic polymers with β-1,4-linkages are probably most effective in disrupting the cellulose crystalline lattice. Hayashi et al. (1987), and Hayashi (1989), consider it likely that xyloglucan is dispersed between glucan chains of disordered cellulose in primary cell walls of pea stems, and have demonstrated that this polymer readily binds to the surface of A. xylinum microfibrils in liquid culture to disrupt the normal assembly of cellulosic ribbons (Haigler and Benziman 1982; Haigler 1985). X-ray diffraction shows that various other β-1,4-linked hemicelluloses are occluded within A. xylinum cellulose crystalline lattices (Atalla et al. 1993), causing decreased crystallinity and changes to various physical and chemical properties. We recognize that mechanical stress influences the crystalline structure of cellulose in these kelps. However, the role that cellulose structure plays in determining mechanical properties (e.g. modulus) in E. menziesii sporo-

470

phytes remains uncertain, primarily because of incomplete knowledge about how cellulose chain orientation was affected by stress. Thus far, studies of cellulose and alginic acid from this alga have been based upon separate chemical isolations of these components, which together account for just 20–25% of total thallus biomass. It seems doubtful that further studies would demonstrate control of structural adaptations by any single component within or between cell walls. Rather, mechanical properties of this alga likely are derived from complex interactions between both structural and matrix polysaccharides or from the occurrence of orientational changes in these polymers. More extensive study is required before we understand the molecular origins of changes in material properties that accompany adaptations to stress. However, as our study showed, increasing modulus does not correspond with increasing cellulose crystallinity. References



471

472


Printed on recycled paper