Changes in Cell Wall Architecture of Differentiating Tracheids of ...

Plant Cell Physiol. 40(5): 532-541 (1999) JSPP © 1999

Changes in Cell Wall Architecture of Differentiating Tracheids of Pinus thunbergii during Lignification Jonas Hafren ', Takeshi Fujino2 and Takao Itoh 2 1 2

Swedish Pulp and Paper Research Institute (STFI), Box 5604, 114 86 Stockholm, Sweden Wood Research Institute, Kyoto University, Uji, Kyoto, 611 Japan

The cell wall architecture, before and after lignification, of differentiating tracheids in Pinus thunbergii has been examined using a rapid-freeze deep-etching technique combined with transmission electron microscopy. Replicas of cells from the cambial zone showed that the unlignified primary cell wall was highly porous with micronbrils extensively interconnected by crosslinks. The unlignified secondary cell wall has unidirectional microfibrils, more or less associated in bundles, forming a wavy pattern around pores of characteristic slit-like shape with narrowing ends. As the lignification progresses, the cell wall structure becomes dense, with no detectable pores. Delignification of wood samples leads to the reappearance of crosslinks, individual microfibrils and pores in the secondary cell wall, although in a somewhat altered shape. In addition, cellulose-synthesizing enzyme complexes (rosettes) have for the first time been detected on the plasma membrane of differentiating xylem cells of softwood.

secondary cell walls as in primary walls. Fengel (1971), Page (1976), Kerr and Goring (1975a), Ruel et al. (1978, 1979) and Scallan (1974) have presented different tentative models of the secondary cell wall. The model presented by Kerr and Goring (1975b), known as "the interrupted lamella model", is the most widely recognized. Agarwal and Atalla (1986, Atalla and Agarwal 1985) suggested that lignin is also orientated in the secondary wall. Recent electron microscopic investigations of the secondary wall have indicated that the cellulose microfibrils are slightly tilted and a "twisted honeycomb model" has been suggested (Terashima et al. 1993), which would be in accordance with a helicodial pattern of deposition of cellulose in the growing cell wall (Neville et al. 1976, Roland and Vian 1979). Electron microscopy has served as an important tool in the development of most models of the secondary cell wall. However, electron microscopy needs elaborate techniques to preserve the true cell-wall structure. In studies of native, water-swollen cell walls, it is important but difficult not to alter the native structure during sample preparation. In 1979, Heuser and Salpeter used the rapid-freezing and deep-etching (RFDE) technique to study neural cells. The method of preparing replicas of RFDE samples has proven useful for structural studies in numerous biological preparations. Very rapid cooling at 20°Cms~ 1 captures most biological specimens in an almost natural state, making dehydrants or chemical fixatives unnecessary. Using the RFDE technique, Goodenough and Heuser (1985) examined the cell-wall structure of Chlamydomonas. McCann et al. (1990) used onion and Itoh and Ogawa (1993) suspension culture cells of poplar, to study primary walls. The first study of the effect of the lignification upon the secondary cell wall (Nakashima et al. 1997) used a suspension of cultured differentiating cells of Zinnia elegans. Recently, native wood fibers of Eucalyptus tereticornis, have also been investigated using the RFDE technique (Fujino and Itoh 1998).

Key words: Cell wall — Delignification — Lignification — Rapid-freezing deep-etching — Pinus thunbergii — Ultrastructure.

Little is known about the three-dimensional structure of the irregular and heterogeneous polymers such as pectin, hemicelluloses and lignin in the plant cell walls. This is in large part due to the insolubilization of the wood components as a result of lignin encrustation of the fiber wall during cell development, hindering the selective disintegration and isolation of wood components, and thereby making polymer-specific analysis difficult. Models of the unlignified primary cell wall have been presented by e.g. Albersheim (1975), Monro et al. (1979) and Lamport (1986). These studies have mostly been performed either on primary walls of intact cells or on suspension cultures of higher plant cells. The size, spatial distribution, and interconnecting covalent bonds of the different cell-wall constituents is not known to the same extent in lignified

In this study, we have observed the gradual changes in the cell wall structure in tracheids during the development from cambium to mature xylem. Numerous rosettes in the fractured face of the plasma membrane of developing cell walls have been observed. The existence of cellulose-synthesizing enzyme complexes, rosettes, in different plants has been reported, and recently also in the differentiating

Correspondence and reprints: Jonas Hafren, Swedish Pulp and Paper Research Institute (STFI), Box 5604, 114 86 Stockholm, Sweden. E-mail: [email protected]. Fax nr: +46 8 4115518. 532

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Cell wall architecture of Pinus thunbergii xylem of a hardwood, Eucalyptus tereticornis (Fujino and Itoh 1998). The rosettes encountered in this work are the first to be reported in differentiating xylem of softwood. The effect of partial chemical delignification on the cell wall structure has here also been studied using the RFDE-technique. Material and Methods Wood samples—Japanese black pines (Pinus thunbergii, three years old) were cultivated in a nursery at the Wood Research Institute, Kyoto University, Uji, Japan. Tangential and radial samples of pine 2 mm3 in size were cut on June 25, 1997, from the cambial zone and differentiating xylem. The bark was stripped off and the samples were then immediately frozen with liquid helium in a Meiwa QF 5000 rapid freezing apparatus (Heuser et al. 1979, Heuser and Salpeter 1979). The frozen samples were then transferred to liquid nitrogen, in which they were stored until they were used for replica preparation. Samples of the same pine tree were fixed in FAA, 50% ethanol: formaldehyde : glacial acetic acid (90 : 5 : 5, v/v/v), dehydrated by solvent exchange in ethanol, and thereafter embedded in LR-White (London Resin Inc.), without removal of the bark. Delignification procedure—The delignification process was performed according to Wise et al. (1946). Small chips ( 2 x 2 x 10 mm) of Japanese black pine (total of 2.5 g) were treated at 75°C in 150 ml acetic acid buffer (0.2 M, pH 3.5) containing 1.5% sodium chlorite for 24 h. The reaction mixture was changed to fresh delignification solution every sixth hour. Afterwards, the samples were thoroughly washed in water, cut and subjected to RFDE preparation. Deep etching and replica techniques—Frozen pine samples were inserted in a Balzers BAF 400 (Liechtenstein) freeze-etching apparatus, fractured therein at — 150°C and then deep etched at - 9 5 ° C for 15 min at 2 x 10" 4 Pa. The sublimed sample surface was coated by rotary shadowing as described by Fujino and Itoh (1994). The 2 nm thick shadowing material coating was obtained by high power (1,900 V and 90 mA) input, for 7 s, to a platinum electrode, mounted at an angle of 25°. Subsequent carbon shadowing at an angle of 85° for 45 s at 2,400 V and 100 mA, reinforced the replica. The wood tissue was dissolved by floating the samples on 50% sulphuric acid containing 5% potassium dichromate overnight. Prior to collecting the replicas on Formvar-coated copper grids, the replicas were washed three times in distilled water. Electron microscopy—A transmission electron microscope (JOEL 2000 EX II), operated at 100 kV was used to observe the replicas obtained. Micrographs were taken on Fuji electron microscopic film (FG, orthocromatic) and reversed to positives before printing. Light and epifluorescence microscopy—Semi-thin sections of the LR-White embedded pine samples were cut using an ultramicrotome (Reichert-Jung, Ultracut E), and observed by light and epifluorescence microscopy (Zeiss, Axioplan). The sections for light microscopy were stained with toluidine blue to enhance the contrast. Lignin and neutral sugar analysis—Klason lignin content and sugar compositions were determined of the mature xylem and delignified xylem samples according to Theander and Westerlund (1986). The wood samples (200 mg) were ground to 60 mesh using a Wiley mill, and then dispersed in 3 ml 72% sulfuric acid (30°C, 1 h). Thereafter, the samples were diluted in destilled water to 2%

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acidity and hydrolyzed for 1 h at 125°C. The residue was filtered off on a Pyrex No. 2 glass filter while still hot, and gravimetrically quantified (Klason lignin). The amounts of acid soluble lignin derivatives were spectrometrically analyzed. After cooling, an internal standard (myo-Inositol) was added to the sample. Part of the hydrolyzate was transferred to a test tube (1 ml) and made slightly alkaline by adding 12 M ammonia solution (200^1), then 3 M ammonia (0.1 ml) containing 15% (w/v) potassium borohydride was added prior to mixing and left to react (1 h, 40°C). Glacial acetic acid (0.1 ml) was added to the solution to make it acidic. Then two 0.5 ml parts were taken from the sample, which were, respectively, derivatized to alditol acetates when mixed with acetic anhydrid (5 ml) and 1-methylimidazol (0.5 ml) and allowed to react for 1 h at room temperature. When 1 ml absolute ethanol was added, the test tubes were placed in a room temperate water bath. After 10 min, 5 ml distilled water was added. Finally, 7.5 M potassium hydroxide (5 ml) was added; and after 5 min, another 5 ml potassium hydroxide (7.5 M). The tubes were vigorously mixed, and the derivatized sugars dissolved in ethyl acetate-phase were analyzed on a Hewlett-Packard 5890 gas chromatograph equipped with flame ionized detector and J&W DB225-column (25 m x 0.32 mm I.D, film thickness 0.25 fjm). The starting oven temperature was set to 160°C and raised to 220°C at 6°C min" 1 .

Results Anatomic overview—Light microscopy at low magnification gives an overview of the latest annual ring (Fig. la). The latest annual ring contains about 12 cells that have been formed into differentiating xylem and (mature) xylem. The epifluorescence picture indicates the extent, and distribution, of the lignification (Fig. lb). The bright areas show the lignin autofiuorescence (Wardrop 1976). The onset of lignification in the developing xylem zone seems rather abrupt; however, as shown in Fig. la and lb, few cells were formed in the latest annual ring, which subsequently gives a narrow differentiating xylem. When tangential sections of the differentiating xylem were prepared from the wood stem for the RFDE-replicas, the bark was removed. Cells in the radial expansion zone have thin walls and relatively large lumen area, and therefore lower mechanical strength than surrounding tissue. This means that when the bark is separated from the wood, the split occurs in the cell enlargement zone. Thereby active wall-synthesizing cells in the same stage of development are exposed on the surface of the wood sample, whereas the cambium is mainly found on the bark part (Edashige et al. 1995). However, in the radial sections when the bark is maintained, cells in all differentiating stages from the cambium to mature xylem can be observed. Unlignified primary walls—As the cell^wall development proceeds, an early primary wall is deposited as is evident in Fig. 2. Randomly oriented microfibrils of 11 ± 2.3 nm in diameter form a fibril network with a pore size of 8-28 nm. Abundant crosslinks can be noted between the microfibrils. The crosslinks are 2.2±1.3 nm in diameter. Based on RFDE images of sequentially extracted poplar

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Cell wall architecture of Pinus thunbergii

Fig. 1 An overview of the cambial zone, where Fig. la is stained with toluidine blue, and Fig. lb represent an epifiuorencence image in which lignin autofluorencence is shown by the bright areas (bar = 75^m, cambium=c, dx=differentiating xylem, below the arrow = mature xylem). cells in suspension culture, crosslinks with similar features to those observed in this study were shown to consist of pectin and hemicelluloses (Itoh and Ogawa 1993). A more mature primary wall or possibly an early Si wall is shown in Fig. 3. The cell-wall structure displays an increasingly more lateral alignment of microfibrils with considerably greater association of fibrils running almost unidirectional. The fibrils are aggregated into larger bundles of two or more microfibrils with less spacing in between them than in Fig. 2, but the microfibrils and the crosslinks have the same features as in the earlier stage of primary cell-wall formation. Unlignified secondary walls—The appearance of the unlignified secondary wall (S2) can be seen in Fig. 4. The wavy, rather porous, microfibrilar network in the Japanese black pine is very similar to the structure of the early secondary cell wall reported by Fujino and Itoh (1998) in Eucalyptus tereticornis and Nakashima et al. (1997) in

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Zinnia elegans. The microfibrils aggregate in bundles of different sizes, with the individual fibril width of 6.2± 1.3 nm. A distinct feature of the secondary wall in Fig. 4 seems to be a system of "slit-like" pores scattered in the cell wall between the microfibrils. These structures, 8-40 nm in diameter, are of various lengths with narrowing ends. Numerous crosslinks between the microfibrils can be seen in the gaps. The crosslinks are 1.6± 1.4 nm in diameter. Cellulose-synthesizing enzyme complex—When the frozen samples are subject to freeze fracture, the fracture runs through different organelles, or in this case cell wall layers. The fracture often takes place at specific locations making it possible to compare the same areas in different samples. The plasma membrane is a lipid bilayer in which part of the cellulose-synthesizing enzyme complexes, called rosettes, are embedded (Mueller and Brown 1980). The face of the lipid monolayer adjacent to the cytoplasm, is exposed by the fracture of the plasma membrane and is called the PF-face (Branton et al. 1980) The PF-face of the plasma membrane from the differentiating tracheid show a rosette (Fig. 5). Small globular membrane proteins are assembled in a circular structure. Lignifying and lignified cell walls—Fig. 4 and 6 show the secondary wall with different degrees of lignification. In Fig. 4, the microfibrils are unidirectional, but still many pores and crosslinks can be seen. Therefore, the micrograph is likely to show a secondary cell wall under development with only slight lignin encrustation. In the final stage of development when the cell wall deposition have stopped and the mature fiber is fully lignified, as the mature xylem in Fig. 6, the S2 wall is highly compact and the encrustation of lignin seems to have sealed the "pore system" in the cell structure. Hence, any gaps or crosslinks are difficult to distinguish in the xylem. The lignin seems to be deposited around the microfibrils. The microfibrils are on average 9.3±0.5 nm in diameter. Since a diameter of 6.2 nm was measured on the unlignified cell wall, the microfibril structure is about 3.1 nm thicker after the cell wall maturation. The surface of the microfibrils in Fig. 6 features somewhat more irregular and less smooth texture than before the lignification (cf. Fig. 4). Therefore, the remaining deposition of cell wall constituents seems not to be completely evenly distributed on the polysaccharide network. Chemical analyses of native mature xylem (Table 1) indicated 28.2% Klason lignin ( + 0.2% acid soluble), which is normal in Pinus thunbergii (28.3%, Sarkanen and Hergert 1971). Removal of lignin from the tracheid walls—Delignification of wood by sodium chlorite is fairly selective towards lignin, although some polysaccharides are also dissolved. The effect of delignification on the sample from the mature xylem secondary cell wall is shown in Fig. 7. The general appearance seems to be similar to that of the unlignified S2 wall, as shown in Fig. 4. The "slit-like" pores

Cell wall architecture of Pinus thunbergii

535

Fig. 2 Tangential image of a newly synthesized primary wall in the cambium, with a loose multi-directional microfibril structure. The RFDE-micrograph (bar=200 nm) clearly reveals the prominent feature of numerous crosslinks between the microfibrils (crosslinks are indicated by arrowheads).

are again apparent, with explicit crosslinks and gaps between the microfibrils. The microfibril size, 7.7±0.5nm, and porosity pattern are somewhat altered. The delignified microfibrils in Fig. 7 are significantly larger in diameter than the corresponding microfibrils in the unlignified S2 wall (Fig. 4). The crosslinks are again visible after the

removal of lignin and they are of about the same size, 1.9±1.1 nm, as before lignification. The chemical analysis of the delignified wood samples indicated an almost complete delignification. The carbohydrate analyses showed that, arabinose and galactose were degraded to a higher extent than glucose and xylose by the sodium chlorite

Table 1 Carbohydrate and lignin analysis of untreated- and sodium chlorite delignified wood Relative amount of neutral monosaccharides (%) ara xyl man gal glc

Klason and acid soluble lignin (%) Acid soluble Klason

Xylem

3.8

14.7

14.9

5.5

61.1

28.4

0.2

Delignified xylem

2.4

15.0

13.0

3.4

66.2

1.3

0.2

The abbreviations means:ara=arabinose, xyl=xylose, man=mannose, gal=galactose, glc=glucose, Klason=the gravimetrical measured lignin according to the Klason technique, Acid soluble=the spectrometrically determined acid soluble part of lignin from the Klason determination.

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Cell wall architecture of Pinus thunbergii

Fig. 3 RFDE-micrograph of the primary cell wall or S, wall from the cell-enlarging zone (tangential section, bar=200 nm).

treatment (Table 1). Discussion The microfibrils are generally organized in a characteristic way for the particular tissue concerned, but occasionally neighboring microfibril runs together as bundles, at different angles, or in other ways cause ultrastructural variations. The microfibril width has therefore been measured on structures with separated fibrils of approximately constant width. Developing xylem contains both cellulose and hemicelluloses, however, intimately associated, and the dimensions of the fibrils do not therefore refer to the cellulose alone. The measurements of the microfibril widths have been made on electron micrographs, and all data presented represent the measured mean value and standard deviation of 40 measurements, respectively, and the data have been corrected for the 2 nm thick platinum layer added by the shadowing. Previous studies have shown the diameter of the cellulose microfibril to be about 3-4 nm (Ioetovitch 1992, Fengel 1971, Chanzy 1987, 1990)

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in the S2 wall. Cellulose microfibrils in primary walls have been estimated to be smaller, about 2 nm (Chanzy et al. 1979). The absolute values vary depending upon the cellulose source and technique of determination (Fink et al. 1995). Permanganate staining of lignin implying a negative staining of the cellulose/hemicellulose fibril gives average width of about 7 and 10 nm for the hemicellulose/cellulose fibril of softwood and hardwood respectively (Ruel et al. 1978, 1979). In this study of the microfibrils in the developing xylem, the microfibrils are considerably larger than the cellulose microfibrils and the fibril size of the primary wall is also slightly larger than the unlignified secondary wall. The difference in width between primary and secondary microfibrils may be due to the fact that the primary wall contains almost twice as much of hemicellulose as the S2 wall (Meier 1961). Hemicelluloses are highly swollen in the water-saturated state and are closely associated along the cellulose microfibrils, probably obscuring differences in size between the microfibrils of primary and S2 walls. All measured cell structures in the Japanese black pine seem to be smaller than comparable entities in eu-

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537

Fig. 4 RFDE-image of an unlignined S2 wall in the differentiation xylem (radial section). A dense structure with scattered oval pores and crosslinks is shown (crosslinks are indicated by arrowheads, bar=200nm).

calyptus, which also has a higher hemicellulose content (Fujino and Itoh 1998). The crosslinks of the S2-microfibrils, shown in Fig. 4, have a diameter of about 1.6 nm, which is smaller than the diameter of any cellulose microfibril, and also smaller than the estimated diameter of the crosslinks in the primary wall (Fig. 2), although a comparison of the diameters of the crosslinks in primary and secondary cell walls shows no a significant difference (P>0.1). With regard to the origin and chemical composition of the crosslinks, pectin and hemicelluloses are proposed constituents (McCann et al. 1990, Itoh and Ogawa 1993, Donaldson and Singh 1998). Although polysaccharides like pectin and hemicelluloses are heterogene-

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ous and polydisperse, they tend to aggregate and could thereby form crossbridges. Pectin polymers consist mainly of an acid or methylated homogalacturonan backbone, branched at locations containing rhamnose units. Arabinans and galactans are also present, especially in more branched areas (Fry 1988). In the presence of Ca 2+ ions, a series of consecutive acidic galacturonic units can form rigid intra- and inter-molecular structures ("egg-box" gel, Powell et al. 1982). Hemicelluloses have in numerous studies been stated to have properties such as aggregation and crystallization, suitable for forming fibril structures (e.g. Fengel 1967, Yundt 1951). Lignifying cell walls—The lignification front lags be-

538

Cell wall architecture of Pinus thunbergii

'33£l Fig. 5 Inside the marked box, a rosette is shown on the plasma membrane. The RFDE-micrograph shows the PF-face of a tangential section of an unlignified enlarging xylem cell in the differentiating xylem (bar=50 nm). hind the hemicellulose and cellulose deposition until the completion of the fiber wall, which lead to a phase-separated deposition pattern of the cell components and to a " dynamic cell-wall environment (Wardrop 1957, Fergus and Goring 1970, Saka and Goring 1985). The polysaccharide matrix in the cell wall is hydrophilic, although, some results indicate that there are also weak hydrophobic regions in polysaccharides (Shigematsu et al. 1994). The newly formed hydrophilic cell wall, rich in water (Inomata et al. 1992) gradually turns hydrophobic as the water molecules are displaced by dehydrogenation products of monolignols making the cell wall less porous. These changes in the media surrounding the polysaccharides may affect the microfibril network and its spatial structure, as can be seen in Fig. 4 and 6. The pores and crosslinks cannot be seen in the lignified secondary wall (Fig. 6) but they are again visible in the delignified secondary wall (Fig. 7), although in a somewhat altered shape. This suggests that crosslinks are a native structure of importance for the three-dimensional fibril network of the secondary wall, and that they exist in the mature S2 wall too, although their existence is obscured by the lignin encrustation. A similar kind of pore distribution was also noticed in the study of the secondary cell wall of Eucalyptus tereticornis (Fujino and Itoh 1998) and Zinnia elegans (Nakashima et al. 1997). The cellulose-synthesizing enzyme complex has previously been shown in various plants. The rosettes encountered in the differentiating xylem of Pinus thunbergii, Fig. 5, have the same features as those of the reported rosettes on the PF-face in enlarging xylem cells of eu-

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calyptus (Fujino and Itoh 1998). Formations of rosettes have also been accounted for in e.g. maize, mung bean, and pine root tips (Mueller and Brown 1980) and cress (Scheider and Herth 1986). Effect of lignin removal—The fact that the microfibrils have a larger diameter in the delignified samples (Fig. 7) than in the lignified S2 walls in Fig. 4 may be the result of swelling of hemicellulose due to the release of lignin and some polysaccharides from the microfibrils. A comparison between the Fig. 4, 6 and 7 reveals the reappearance of the crosslinks between the microfibrils. This is a strong indication that they are native structures in the secondary wall with a chemical composition at least partly undegraded by the delignification conditions. Donaldson and Singh (1998) have recently shown crosslinks in the S2wall of pulp fibers. Kraft pulp fibers of Radiata pine were embedded, sectioned and stained with lead citrate. The staining patterns were analyzed by TEM. Digital image processing of the micrographs indicated crosslinks of 4.8±0.4nm in diameter between microfibrils of 5.7±0.5 nm. Using the same technique on native Radiata pine, thinner cellulose microfibrils were found (3.8±0.5 nm). As in this study no crosslinks could be distinguished in the native lignified cell wall due to lignin encrustation of the structures. They found a lesser difference in size between the microfibrils and crosslinks than in this study, which probably is on account of the methods used. However, the results corroborate the idea of interfibrillar bridges, or crosslinks, between the cellulose microfibrils as being a real structure, present in both in primary and secondary cell walls. The microfibrils are more clearly separated in the delignified (Fig. 7) than in the unlignified cell wall (Fig. 4). After removal of lignin, the wavy appearance of the microfibrils is less pronounced than in the unlignified S2 wall as seen in Fig. 4, suggesting a straightening effect on the microfibrils surrounding the slit-like pores, either by the lignification per se or by the delignification process. The sodium chlorite treatment did not result in any increased lateral association of microfibrils into large bundles. It has been shown that the removal and degradation of lignin (Stone and Scallan 1967, Kerr and Goring 1975a) increase the pore size gradually, creating larger pores, which is an effect similar to that seen in Fig. 7. Alkali extraction of the primary walls of onion cells has also shown to initially increase the cell wall porosity. But when enough hemicelluloses and pectin have been removed, the microfibrils become more laterally associated and the pore size decreases (McCann et al. 1990). Implication of the RFDE results on the ideas of cell wall ultrastructure—No fully accepted model of the cell wall exists, but lignin and hemicellulose are often considered to constitute a supporting matrix to the cellulose microfibrils. Hemicellulose and lignin together make up a larger part of the cell wall than cellulose, but in the

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539

Fig. 6 The closed structure of a fully lignified S2 wall in the mature xylem. Neither pores or crosslinks are visible. The RFDE-micrograph shows a radial view of the wood sample (bar = 200 nm). Fig. 7 Delignified S2 wall from the mature xylem as shown by an RFDE-micrograph (bar=200 nm). Between the microfibrils crosslinks are visible, and pores formed like gaps (crosslinks are indicated by arrowheads).

RFDE-micrographs the fibril texture derived from the cellulose fibrils is typical of the cell structure. This indicates that hemicelluloses are associated around the cellulose fibrils and that lignification finally occurs mainly on the surface of the polysaccharide network and possibly partly within in the hemicellulose gel, i.e. a lignification that merely seals a pre-existing polysaccharide-based structure

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by hydrophobation. The structure of lignified cell walls is evidently tight (Fig. 6). It is not clear whether the distance between the microfibrils is real or whether it is affected by the knife removing overlaying fibrils in the freeze fracturing (Fujino and Itoh 1998). The overall cell-wall architecture differences, of random oriented microfibrils in the primary walls, and the unidirectional orientated micro-

540

Cell wall architecture of Pinus thunbergii

fibrils of the secondary walls, can be seen on the micrographs (Fig. 2-6). Abe et al. (1995a) have also shown that the microfibrils are deposited in different orientations during the cell development. After the cell expansion the microfibrils displayed a more orderly deposition pattern, as found in the secondary cell wall. The major reorientation of the microfibril angle during the tracheid development throughout S r S2-S 3 (semi-helicoids, Roland et al. 1979) have shown co-alignment with the microtubules (Abe et al. 1995b). Detailed analysis of the secondary wall reveals a small regular deviation of the microfibril angle in the zhelix. Dunning (1969) showed a spread fan appearance of the S2 wall, which he found most likely to be caused by a transition of the microfibril lamellae orientation. In most models of cell wall structure the cellulose microfibrils are represented by stiff rods. However, in native wood the single cellulose microfibril seems partly to have some flexibility. Albeit the overall microfibril direction is uniform in Fig. 4, the individual microfibrils seems to occasionally run in a slightly sinuous mode. Kataoka et al. (1992) also found deviations in microfibril angles within the lamella forming a "woven" texture, and suggested that superimposed stripes of microfibrils, the lamellae, in the S2 wall becomes compressed by a continuos cellulose deposition, and formed into a somewhat bent, pressed pile of microfibrils. Therefore, the over all secondary cell-wall structure is probably given by both the transition of the microfibril lamellae orientation, and that the microfibrils in an individual lamella are occasionally bent. A hydrated gel of hemicelluloses has been localized adjacent to the outside of the plasma membrane of actively cell-wall synthesizing cells (Inomata et al. 1992). Thereby, the new cellulose microfibrils, growing from the terminal complexes on the plasma membrane, are exposed to, and regularly spaced by the interaction with hemicelluloses, which is an prerequisite to form the individually separated cellulose microfibrils as in the pressed pile model, or the twisted honeycomb model (Terashima et al. 1993). This is in agreement with the cell wall structures found in this study. The way in which the lignification proceeds and alters the cell-wall structure lead to some interesting aspects of the structure and topochemistry. The mathematical estimation of the relative volumes of the cell-wall constituents, based on the density and percentage distribution of the wood components, is a delicate task. The lignified microfibrils in S2 have about 50% larger diameter than the unlignified microfibrils in S2 (cf. Fig. 4, 6). The lignification adds about 20% lignin to secondary wall, which indicates that lignin is deposited in low density. However, it is an arduous task to elucidate the native ultrastructural conditions. Kellog et al. (1975) concluded that there must be a difference in density between isolated and native wood polymers.

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Financial support from the graduate school Wood and Wood Fibre (sponsored by the Swedish Council for Forestry and Agricultural Research and the Swedish University of Agricultural Sciences) and Swedish Pulp and Paper Research Institute is gratefully acknowledged (for J.H). This work was also supported by a Grant-in-Aid for "Research for the future" program (nos. JSPS-RFTF-96L00605) from the ministry of education, science and culture of Japan (for T.F). Valuable discussions with Professor Ulla Westermark and Dr. Noritsugu Terashima have been greatly appreciated.

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(Received August 28, 1998; Accepted March 10, 1999)

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