The substructure of the cellulose microfibrils from the cell walls of the algae Valonia ventricosa

© 1971 by Academic Press, Inc.

J,, ULTRASTRUCTURERESEARCH36, 725--731 (1971)

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The Substructure of the Cellulose Microfibrils from the Cell Walls of the Algoe Valonia ventricosa K. H. GARDNER AND J. BLACKWELL

Division of Macromoleeular Science, Case Western Reserve Univeristy, Cleveland, Ohio 44106 Received December 18, 1970, and in revised form February 3, 1971 The fine structure of the cellulose microfibrils from the cell wall of the marine algae Valonia ventricosa has been investigated by observation of deformed and undeformed specimens in the electron microscope. On treatment with ultrasonic vibrations, the purified cell wall broke down into microfibrils 210/~ in width. Sections of microfibrils which had been disrupted by the ultrasonic treatment consisted of elementary fibrillar fragments with a regular width of 35 A. Deformation by stretching on Mylar film separated the microfibril into similar fibrils with a width of 35 A or multiples thereof. Striations on the intact microfibrils are interpreted in terms of a substructure consisting of these elementary fibrils. The cellulose from the cell walls of the algae Valonia ventricosa is comprised of long thin ribbonlike microfibrils with a cross sectional width of approximately 200 A (13). It has recently been observed that when these microfibrils are subjected to ultrasonic vibrations, regions along the length are disrupted showing that they are made of smaller fibrils with an approximate cross section of 35 A (4). These smaller fibrils were similar to those first reported by Mtihlethaler (10) for cellulose f r o m the primary cell walls of the root tip Allium cepa and have since been observed in celluloses from a number of different sources (7, 12); they have been termed elementalT fibrils by Frey-Wyssling and Mtihlethaler (3). Observation of elementary fibrils in Valonia cellulose has been thought to conflict with X-ray evidence where the line broadening indicates a much larger crystallite size. For the cellulose from the marine algae Chaetomorpha melagonium, which is very similar to that of Valonia, the crystallite width has been determined as approximately 170 A, which implies that the microfibrils are single crystals (11). Furthermore, no X-ray evidence has ever been observed corresponding to a repeat of the order of 35 A within the structure. It has been suggested that the substructure interpreted as elementary fibrils is an artifact due to splitting of crystalline microfibrils, and that, when 46

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this occurs, v a r i o u s widths are possible d e p e n d i n g only on the n u m b e r of chains in the f r a g m e n t s (11). W e have suspended the cellulose f r o m Valonia ventricosa in water, using ultrasonic vibrations, a n d have e x a m i n e d the suspension in the electron microscope, l o o k i n g for evidence of any regularity in the fragments of the microfibrils. I n a d d i t i o n we have subjected the microfibrils to d e f o r m a t i o n on M y l a r films (6) a n d m e a s u r e d the widths of the subunits so p r o d u c e d .

MATERIALS AND METHODS Samples of the cell walls of the algae Valonia ventricosa were purified by boiling in a large excess of 1% aqueous sodium hydroxide for 6 hours, with a change of alkali solution after 3 hours. They were then rinsed with distilled water and allowed to remain overnight in 0.05 N hydrochloric acid at room temperature, rinsed in water, and allowed to air dry. Sections of the purified cell wall were subjected to ultrasonic vibrations for 4 hours, after which the larger unsuspended fragments were removed. Specimens of the suspension for electron microscopy were prepared on carbon-coated grids and negatively stained with 1% aqueous uranyl acetate solution. Deformation of the microfibrils was achieved following the technique of Geil (6). A drop of dilute suspension was allowed to dry completely on a Mylar film, after which the Mylar was stretched approximately 50%. A few drops of poly (acrylic acid) (PAA) were added and allowed to dry on the stretched sample. The dried P A A film, with the stretched cellulose adhering to it, was stripped from the Mylar, and the side which had been in contact with the Mylar was carbon coated. Finally the film was floated on water, where the P A A fraction dissolved away and the remaining carbon film with the stretched cellulose was left floating on the surface. This was picked up on a grid and negatively stained with 1% uranyl acetate solution. Electron micrographs were obtained using a Hitachi H U 11A and a Zeiss 9A electron microscope. RESULTS A N D DISCUSSION W h e n sections of the cell wall were suspended in water, the cellulose b r o k e d o w n into r i b b o n l i k e microfibrils of i n d e t e r m i n a t e length. M e a s u r e m e n t of the m a x i m u m a n d m i n i m u m widths showed t h a t the r i b b o n s h a d a w i d t h of 210_+ 5 A a n d a thickness of 100-110_+5 A. Some aggregates consisting of a n u m b e r of p a r a l l e l microfibrils were also present, a n d in a few cases, two parallel microfibrils were o b s e r v e d twisted together with a p e r i o d of a p p r o x i m a t e l y 1/~. Similar bundles have been observed for FIG. 1. 210 ~ cellulose microfibril which has been disrupted by ultrasonic vibrations and negatively stained with uranyl acetate. Note that the subunits, particularly the four close to A, have equal widths of 35 +_5 4. All the fibrillar material is believed to originate from the ultrasonic disruption of the single microfibril, the material in region B coming from a layer above or below the layer of the "four" in region A. x 250 000.

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Fro. 2. Twisted cellulose microfibril which shows the ribbonlike nature of the fibril. Negative staining with uranyl acetate reveals parallel striations on the microfibril (arrows). The striations divide the fibril into four approximately equal sections, each of which is ~ 35/~ wide. x 130 000.

bacterial cellulose microfibrils (2). Almost all the aggregates separated into the individual microfibrils on deformation involving a 50 % stretch of the Mylar. Also present in a typical micrograph were a few fibrils of shorter length with cross sections less than 200 A which were thought to be fragments of disrupted microfibrils (see below). Figure 1 shows the end of a microfibril which reveals the presence of substructure. All the material in this region of the micrograph is believed to originate from the ultrasonic disruption of a single microfibril, which has apparently broken down into smaller units. These subunits, particularly the four close to A in Fig. 1, can be seen to have equal widths of 35_+ 5 A. Fibrils in groups of four have been found to be quite common in micrographs of this type and the origin of these groups is somewhat puzzling. It appears that the four fibrils in region A are from the same layer of the original

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Fro. 3. 210 A cellulose microfibrils after negatively staining with uranyl acetate. Single 35 A elementary fibrils can be seen peeling away without any change in overall width of the parent microfibril. x 190 000.

microfibril and that those in region B come from above or below this layer. When measured, all of the fibrils in Fig. 1 and similar micrographs have been found to have widths of 35 A or multiples thereof. Examination of some of the intact microfibrils revealed striations parallel to their lengths. An example of these striated microfibrils is shown in Fig. 2. The markings divide the microfibril into four approximately equal sections, each of which is ~ 35 A wide. The widths of the striated microfibrils are 210_+ 5 A. Observations of this type were relatively rare, and even then, striations were observed only when the microfibrils were flat so that the measured width was maximal. When twisting occurred, corresponding to a decrease in the measured width, the striations were not observed. Four 35 elementary fibrils would have a width of 140/k, which is larger than the microfibril thickness of 100 A, and thus the "fours" cannot correspond to the substructure in an orientation perpendicular to the width of the microfibril. The measured microfibril width of 210 A would correspond to six 35 N elementary fibrils. It is possible that the corner elementary fibrils are absent from the six-unit surface layer of the microfibril, either by accident or design, and the remaining four would be spread over the full 210 A. In Fig. 3, 210 A microfibrils can be seen from which single 35 A fibrils are

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FIG. 4. Cellulose microfibril which has been deformed by stretching on Mylar. The microfibril has broken into at least 12 subunits. Note that in an untwisted region (arrow) the regular 35/~ subunit fibrils lay six across, parallel to the fiber-axis, x 220 000.

peeling away without a change in the overall widths of the parent microfibrils. Further work is being done to elucidate this matter. An electron micrograph of a microfibril which has been deformed by stretching on Mylar is shown in Fig. 4. The microfibril appears to have broken into at least 12 subunits. The straight regions of most of these fibrils have a width of 35 ± 5 A. A few of the fibrils (one or two in each region of deformation) are wider than 35 A, being approximately 70 A and 100 ~ in width; this suggests that they are aggregates of two or three of the 35 A subunits. The smaller fibrils seen along with the intact microfibrils above also had widths corresponding to multiples of 35 A, and are presumably aggregates of elementary fibrils. For a few of these deformed samples, fibrils have been seen with widths of 320 ~ divided by striations into six approximately equal sections, where the width of the unstained sections was ~ 35 A. It seems possible that these are "complete" 210 A microfibrils which have been swelled to the present width by the deformation and the penetration of the stain. The absence of such striations from many of the fibrils suggests that, in the natural microfibril, the subunits are closely bound together and stain cannot penetrate between them.

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We conclude, therefore, that the microfibrils of VaIonia ventricosa are made of up a number of elementary fibrils with a regular cross section of 35 A. No evidence has been seen to support the theory that the microfibrils can be broken down into smaller units of variable size and cross section depending only on the number of chains in the fragment. Evidence will be presented elsewhere (5) which shows that the chitin of the spines of certain diatoms (1) also consists of microfibrils which can be broken down by deformation into regular subunits. The regular substructure for the microfibrils of Valonia can be compatible with the measured crystallite size only if the elementary fibrils are bound together in a regular crystalline manner. It has been suggested that the basic structural fibril for cellulose is synthesized as one unit, all the chains being built up simultaneously (13). If this basic fibril is the elementary fibril, then a number of these must amalgamate to form the microfibril. If the coagulation is imperfect in parts, then this imperfection would serve as a "built-in m e m o r y " for the elementary fibrils. On the other hand, there is no reason to suggest that the hydrogen-bonding network between elementary fibrils, formed on the coagulation into microfibrils, would be the same as that within the elementary fibrils themselves. In this case the unit cell would contain a single elementary fibril and the cells proposed by Honjo and Watanabe (8) and Meyer and Misch (9) would be pseudo cells. It is not unreasonable to suggest that the binding between elementary fibrils would be weaker than within the individual elementary fibrils themselves. Thus distortion would lead to the appearance of regular subunits. We thank Dr P. H. Geil for his comments and advice during this work and Dr V. Tripp of Southern Regional Research Laboratory U.S.D.A., New Orleans, Louisiana, for the supply of specimens of purifed cell walls of Valonia ventricosa. This research was supported by NSF Grant No. GK 5662. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

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