WOOD AND CELLULOSIC CHEMISTRYsecond edition, revised and expanded

edited by

David N.-S. HonClemson University Clemson, South Carolina

Nobuo ShiraishiKyoto University Kyoto, Japan

M A R C E L

MARCEL DEKKER, INC.U E K K E R

NEWYORK BASEL

Library of Congress Cataloging-in-Publication Data

Wood cellulosic and chemistry / edited David by N.-S. Hon, Nobuo Shiraishi.-2nd rev. ed., and expanded. p. cm. Includes index. ISBN 0-8247-0024-4 (alk. paper) 1. Cellulose. 2. Wood-Chemistry. I. Hon, David N.-S. 11. Shiraishi, Nobuo. QD323.W662000 572'.56682"dc21 00-060

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Preface

Life and its surroundings are constantly changing within our dynamic world. As we stride into the new millennium, information technology and biotechnology continue to flourish. Rapid economic expansion, social development, and high demands for shelter, clothing, energy, and food for our overpopulated world have resulted in a desperate need for new and yet functional materials to support societys infrastructure. Wood or lignocellulosic-based materials have made a significant contribution to the quality of living for human beings. With new developments in wood chemistry, scientists are confident that wood will continue to play an important role in fulfilling the needs of human beings. Over the past decade, the trend of emphasizingbio-basedtechnologieshasbeen observed worldwide. In February 1998, a long-term development project, PlanVCrop-based Renewable Resources 2020, was implemented among the U.S. Department of Agriculture, U.S. Department of Energy, and many U.S. companies, agricultural associations, and universities. The aim of the project was to obtain novel chemicals from plant- and crop-based renewable resources in order to widen the usage of crops, the yield of which has been significantly increased through bio-technological advancements. The recent movement of producing foods by means of genetically manipulated seeds should enhance the effectiveness of this project. Before the start of this project-which is considered the future of the petrochemicalindustry-majorchemicalcompanies in the UnitedStates,suchasDow Chemical,Dupont, and Monsanto,havebeenchanging their strategies in research and development.Theyhavestrengthened their bio-basedresearch field, trying to yield as many chemicals as possible from biomass. They are developing production technologies for ethanol, sorbitol, lysine, tryptophane, citric acid, lactic acid, poly(lactic acid), erythritol, 1,3-propanediol,etc.,frombiomass.Furthermore, in August of 1998 PresidentClinton issued an executive order, Developing and Promoting Biobased Products and Bioenergy, to further the development of a comprehensive national strategy that includes research. development, and private sector incentives to stimulate the creation and early adoption of technology needed to make bio-based products and bio-energy cost-competitive in national and international markets. Also. there has been research in so-called green chemistry. In this new methodology. biomass is the recommended MW material. Thc importance of wood and cellulose rescarch is thus rccognizcd.iii

iv

Preface

Since the publication of' the first edition of this book, considerablc advancement i n various fields ofwood chemistry has been made, as can be attested by many scientific publications in addition to well-attended international conferences. We contacted the contributors to the first edition, soliciting their opinions on revising and updating the book, and we received tremendous support from them as well as the publisher. Unfortunately, and inevitably, several authorswereunableto participate, buttheyrccomrnended their successors. Although most of the chapters in this new edition carry the same titles as those i n the previous edition, they have all been extensively revised and updated. In addition, this edition includes several new chapters representing important threads in the total fabric of wood chemistry. These new chapters cover the subjects of chemical synthesis of cellulose, preservation of wood, preservation of waterlogged wood, biodegradable polymers from lignocellulosics, recycling of wood and fiber products, and pulping chemistry. As editors, we feel fortunate to have been able to recruit some of the best talent in the field to this endeavor. We thank the contributors for their efforts. Any praise for the content should be addressed to them, and comments and criticisms to us will be welcome.

David N.-S. Hon NoDuo Shiruishi

Contents

1.

Ultrastructure and Formation of Wood Cell WallMinoru Fujittr trrlcl Hirnshi Hcrmdrr

1

2.

ChemicalComposition and DistributionShirr) Strkrr

SI

3.4.

Structure o f Cellulose: Recent Developments in Its CharacterizationFrrrtlittrktr Hot-ii

83

Chemistry of Lignin 109 Akirn Srrkrrkihrrr-rrcrrlrl Yoshillit-o Strrlo175Ttrtltrslli lsllii rrrltl Kuxrt1tr.w Shirr1i:rr

S. Chemistry of Cell Wall Polysaccharides6.

Chemistry of Extractives Toshitrki U r ~ r t w ~ ~ r Chemistry of BarkKokki Sakoi

2 13

7.

243

8. ChemicalCharacterization o f Wood and Its ComponentsJrrirtw Htrexr cult1Jucrrlittr Frret-

275

9.

Color

~tnd Discolorationtrrld

D m i t l N . -S. Horl

3x5 NoDrryrr Mitlcwlrr.cr

IO.

Chemical DegradationKrcrrl-ZotrgLrri

443

1 I. Weathering and Photochemistry o f Wood IltrlGcl N.-S. Hot1

S I3

V

vi

Contents

12. Microbial, Enzymatic, and Biomimetic Degradation of Lignin in Relation to Bioremediation 547 Rrkqfumi Huttnri und Mikio Shimadu 13. Chemical Modification of WoodMisato Nothoto

573

14. Chemical Modification of Cellulose599Akirn Isogai

15.

ChemicalSynthesis of Cellulose627 Furniaki Nukatsubo

16. Wood PlasticizationNohuo Shiruishi

655701

17. Wood-Polymer CompositesHirnshi Mizunztrchi

18.

Adhesion and AdhesivesHiroslli Mizurturc.hi

733

19. Pressure-SensitiveAdhesives and Forest Products765Hiroshi Mizunlcrchi

20. 21.

Wood-InorganicCompositesas Shiro Suku Preservation of WoodD u r r d D . NicAolcrs

Prepared by the Sol-Gel Process

781

795 807 827

22.23.

Preservation of Waterlogged WoodDavid N.-S. Hot1

Biodegradable Plastics from LignocellulosicsMuriko Yr)shioku m c l Nohuo Shirtrishi

74. 25.

Recycling o f Wood and Fiber Products849Tcrkcrrlori Arirrrn

Pulping Chemistry 859Giirn11 Gelle~rstcclt

Contributors

TakanoriArima Department of Biomaterial Sciences, Graduate School and Life Sciences, The University of Tokyo, Tokyo, Japan

of Agricultural

Jaime Baeza Departamento de Quimica, Facultad de Ciencias, Universidad de Concepcicin, Concepcicin, Chile Juanita Freer Departamento de Quimica, Facultad de Ciencias, Universidad deConcepcicin, Concepcicin, Chile Minoru Fujita Division of Forest and BiomaterialsScience,Graduate culture, Kyoto University, Kyoto, Japan Goran Gellerstedt Department of Pulp and PaperChemistry Institute of Technology, Stockholm, SwedenSchool of Agri-

and Technology, Royal

Hiroshi Harada Division of Forest and Biomaterials Science, Graduate School riculture, Kyoto University, Kyoto, Japan TakefumiHattori David N.-S. Hon Carolina FumitakaHoriiWood Research Institute,Kyoto University, Kyoto, Japan

of Ag-

School of Nature Resources, Clernson University, Clemson,South

Institute for Chemical Research,

Kyoto University, Kyoto, Japan

TadashiIshii Division of Bio-Resources Technology, Forestry and Forest Products Research Institute, Ibaraki, Japan Akira Isogai Department of Biomaterial Science, The University of Tokyo, Tokyo, Japan Yuan-Zong Lai Faculty of Paper Science and Engineering, SUNY College of Environmental Science and Forestry, Syracuse, New Yorkvii

viii

Contributors

NobuyaMinemura Hiroshi Mizumachi

Hokkaido Forest Products Research Institute,Hokkaido, Japan Professor Emeritus, The University of Tokyo. Tokyo, Japan

FumiakiNakatsubo Division of Forest and BionlaterialsScience,GraduateSchool Agriculture, Kyoto University. Kyoto, Japan Darrel D. Nicholas State, Mississippi MisatoNorimoto

of

Forest Products Laboratory, Mississippi State University, Mississippi

Wood Research Institute, Kyoto University, Kyoto. Japanof

Shiro Saka Department of Socio-Environmental Energy Science,GraduateSchool Energy Science, Kyoto University, Kyoto, Japan KokkiSakaiFaculty of Agriculture, Kyushu University. Fukuoka, Japan

Akira Sakakibara Laboratory o f Wood Chemistry. Research Group of Bioorganic Chemistry, Division of Applied Bioscience, Hokkaido University. Sapporo. Japan Yoshihiro Sano Laboratory of Wood Chemistry. Research Group of Bioorganic Chemistry, Division of Applied Bioscience, Hokkaido University, Sapporo, Japan Mikio Shimada Wood Research Institute, Kyoto University, Kyoto, Japan Kazumasa Shimizu Division of Wood Chemistry, Forestry and Forest Products Research Institute. Ibaraki, Japan Nobuo Shiraishi Division of Forest and Biomaterials Science. Graduatc riculture. Kyoto University, Kyoto. JapanSchool of Ag-

Toshiaki Umezawa Wood Research Institute. Kyoto University, Kyoto. Japan Mariko Yoshioka Division of Forest and Biolnaterials Science, Graduate School of Agriculture, Kyoto University, Kyoto, Japan

Ultrastructure and Formation of Wood Cell WallMinoru Fujita and Hiroshi HaradaKyoto University, Kyoto, Japan

1.

GENERAL STRUCTURE OFWOOD AND WOOD CELLS Wood

A.1.

Softwood Hardwood and In introduction it should be understood that the term wood refers to the secondary xylem formed by cell division in the vascular cambium of both gymnosperms (softwoods) and angiosperms(hardwoods). and especially i n Ginkgo. Similarsecondary xylem may be produced by plants of different form and structure, such as vines and shrubs, the xylem of which may be an important resource of pulping material. The structure and formation of the secondary xylem are discussed in this chapter. Both softwoods and hardwoods are widely distributed on earth, from tropical to arctic regions.The xylem of those species present in moderate-temperate to arcticregions is characterized by distinct growth rings, in which some anatomical differences can be noted. In the softwoods consisting mainly of tracheids (approximately 90% of wood volume), the latewood (summer wood) can be distinguished from the earlywood (spring wood) by its smaller radial dimensions and thickercells walls. Theseanatomicaldifferenccsare reflected in the higher density of the latewood compared with the earlywood. In softwoods growing i n tropical or warm areas, growth rings cannot be distinguished due to the indistinct boundary between earlywood and latewood. As with the softwoods, hardwoods are also present i n tropical to arctic regions. In colderregions,hardwoodspeciesaredeciduous, whereas i n tropical regions, they are predominantly evergreen and their growth rings are difficult to recognize. The macroscopic characteristics of hardwoods are reflected in the distribution and number of different ccll typessuch as vessels (pores),parenchyma, and fibers. Although fibers may account for only 25% of wood volume, in some cases, for hardwood, it may be as high as 50-70%. In contrast to the tracheid as the main cell in softwoods,hardwoods have a variety of cells. Some deciduous hardwoods such a s oak or elm have very large vessels concentrated at the beginning of annual rings. Suchwoodsare called ring porous wood,whereas otherdeciduousspecies and almost all evergreen hardwoods in which the vesselsare evenly dispersed over the annual ring are callcd diffuse porous wood. The above dis1

Harada 2

and

Fujita

tinctions represent extremes and there are many intermediate arrangements of the vessels. Variations in arrangements of these vessels with other xylem tissues such as parenchyma are reflected in the figure and grain of the wood itself when it is cut from the tree. The physical properties of wood such as density also result from such arrangements of the cells.

2. Sapwood and Heartwood When a tree stem is cut transversely, a portion of heartwood can be seen frequently as a dark-colored zone near the center of the stem. This portion is surrounded by a lightcolored peripheral zone called sapwood. The sapwood or at least the outer part of the stem conducts water throughthe tissue where the water is transpired, and mineral nutrients are also carried with water from the roots into the wood. In addition, the sapwood has living parenchyma tissue, which often plays some physiological role such as the storage of starch or fat. From this point of view, the sapwood is considered an active xylem tissue. In contrast to sapwood, heartwood is dead xylem. As the tree matures, all parenchyma cells of the sapwood die, and other typesof cells such as tracheids or fibers become occluded with pigment composed of polyphenols and flavanoids supplied mainly from the ray parenchyma. The bordered pits of gymnosperms become aspirated, whereas the vessels are blocked by tyloses or gum in angiosperms. Thus, heartwood does not participate in water conduction. Although the conducting and physiological functions are lost in heartwood. the durability of wood against rot or insect decay is remarkably improved due to an addition of such pigments. Moreover, these pigments confer a variety of beautiful colors on wood.3. Reaction Wood Reaction woods that appear on branches or a leaning stem by any force such as a landslide or snowfall have a peculiar nature. Once reaction wood is formed as a biological response, the living tree tries topreserve the original position of its stemorbranches.For the practical use of woods, the reaction woods have not been appreciated very much because of their different characteristics fromnormalwood in both a physical and a chemical sense. The occurrence and nature of reaction woods contrast quite a bit between softwood and hardwood. In softwood trees, the reaction wood forms at the lower side of a leaning stem or branches, where the compression stress reacts on the xylem. Therefore, this reaction wood is generally called compression wood. compression woodis heavy and appears dark brown on account of its highly lignified tracheid walls (see Section II), which seem to adapt to compression stress. Thus, compression wood is easily distinguished from normal wood by its dark color. The cambial activity at the lower position of a leaning stemorbranchacceleratesveryquicklyanddevelops a widercompressionareathan normal wood on the opposite side. Through the accumulation of compression wood tracheids over many years, a leaning stem will return gradually to the vertical position. The annual rings of such a stem, however, are conspicuously eccentric. On the contrary, reaction wood in many species of hardwoods is formed at the upper side of a leaning stem or branches where the xylem loads the tensile stress. Therefore, such reaction woods are called tension wood. Fibers of tension wood have a slightly lignified cell wall (see Section 11) that is adapted to the tensile stress just like a bowstring. It is not so easy to distinguish this area from a normal one on account of its slightly pale tone, in comparison to the case of compression wood.

Formation Ultrastructure and

Wall

o Cell f

3

In fact, the occurrence of both reaction woods is averytroublesomeproblem in wood utilization. These reaction woods, however, are interesting material for the examination of wood structure and formation, as will be noted often in the following sections.

B. Wood CellsWood cells are produced in the vascular cambium from two types of meristematic cells: the fusiform initial and the ray initial (Fig. l ) . Since cells derived fromthe fusiform initials that are upright in the stem occupy a major part of xylem, woods show remarkable anisotropism. The principal functions of xylem tissue are water conduction from roots to shoots, the mechanical support of a huge tree body, and a physiological role such as the storage of starch. Although these functions are common in both softwoods and hardwoods, the xylem of the latter is more evolved than that of the former, being adapted to each function.

softwood

hardwood

pits

%I

fusiform initials xial parenchyma cells axial parenchyma c e l l s

a

.B@'

ray tracheidFIGURE 1 hardwood.

ray \ ray parenchyma cell initials p

Shapes of major wood cells from fusiform ray the and initials softwood in and

arada 4

and

Fujita

I n softwoodsand Ginkgo, tracheids, beingmajorcells of xylem, are considered relatively underevolvedbecausetheyhavebothconductiveandmechanical properties. Bordered pits, the occurrence of which define a cell as a tracheid, are very important to the regulation of water flow. On the other hand, cell wall thickness is related directly to the strength of tracheids. The earlywood tracheids, therefore, seem to be well adapted to the conducting function whereas the latewood tracheids are loaded with the mechanical property, judging from their peculiar shapes. On the earlywood tracheids, well-developed pit pairs are distributed abundantly between the neighboring tracheids, and the cell walls of latewood tracheids are very thick. Only a small number of fusiform cells are subdivided into strand cells by horizontal partitions and compose an axial parenchyma. These parenchymatous cells survive in the sapwood for many years, being different from the tracheid, in which the protoplast is lost soon after differentiation (see Section III), and are part of some physiological functions. In somegenera of Pinaceae, axial resin canalssurrounded by epitherial cells are constructed. The occurrence and structure of resin canals are often used in the identification of softwoods, although the volume of such resin canals is very slight in wood. Ray cells are derived from the ray initials and elongated radially. A series of these ray cells make a ray parenchyma. Needless to say, these parenchyma cells are alive in the sapwood and are tied to the storage of nutrients such as starch or fat and also the transportation of some metabolites between the phloem and the heartwood. As a result, they must be related to the secretion of heartwood substance into the tracheids. Also, in some genera of Pinaceae, radial resin canals surrounded by epitherial cells are formed in many ray tissues, and more ray tracheids occur in the ray tissues. Hardwood xylem can be characterized by the development of vessel elements and wood fibers specialized for water conduction and the mechanical property, respectively. The vessel elements construct a very long and thick tube, namely, a vessel, being joined vertically with one another by a perforation that has a more developed style compared with the bordered pit pairs between tracheids. The occurrence of perforation distinguishes the vessel elements from the tracheids. Wood fibers elongate remarkably and possess very thick cell walls. The most developed type of cell, having simple pits (see Section II), is called libriform wood fiber. On the other hand, there are some intermediating cells from the tracheids to the vessel elements or wood fibers, i.e., vascular tracheids, vascentric tracheids, andfiber tracheids. The fiber tracheids are often included in the categoryof wood fibers. because there is no need to separate them from the libriform wood fibers in the practical use of wood. Vessel elements, wood fibers, and various types of tracheids in the hardwoods lose their protoplast just after the development of their secondary wall. However. in some hardwood species specialized wood fibers that remain alive for several years and often store starch grains are formed; they are called living wood fibers. Axial parenchyma cells, which are dispersed on the transverse section of softwoods, are clustered at the vessel periphery or form a group that is often linked tangentially. Resin canals that are surrounded by epitherial cells are formed in many genera of Dipterocarpaceae and a few Leguminosae. Ray parenchyma cells sometimes aggregate and develop a so-called broad ray. The broad rays make a peculiar figure on a board. especially on the radial surface, as observed in oak or beech. Cells contained i n the ray also vary in their anatomical features. Some of them are upright or square at the marginal position. These variations are used for the identitication of hardwoods [ l ] . Both axial and ray parenchyma cells are apparently concerned with physiological functions-for instance, the storage of nutrients or heartwood

Formation Ultrastructure and

Wall

of Cell

5

formation. Radial resin canals or latex tubes are formed in the ray tissue of some tropical hardwoods.

II.

ULTRASTRUCTURE OF WOOD CELL WALL

Wood is a natural composite material and a chemical complex of cellulose, lignin, hemicelluloses, and extractives [2]. Cellulose is the framework substance, comprising 40-50% of wood in the form of cellulose microfibrils, whereas hemicelluloses are the matrix substances present between cellulose microfibrils. Lignin, on the other hand, is the encrusting substance solidifying the cell wall associated with the matrix substances. The significance of lignin as the encrusting substance can be demonstrated by examination of the lignin skeleton created by the acid removal of carbohydrates (Fig. 2). The roles of these three chemical substances in the cell wall are compared to those of the constructing materials in the structures made from the reinforced concretein which cellulose, lignin, and hemicelluloses correspond, respectively, to the iron core, cement, and buffering material to improve their bonding.

A.

Cellulose Microfibrils

The crystalline nature of cellulose in wood has been demonstrated by studies with X-ray diffractometry and polarization microscopy. This crystalline nature was also confirmed by the electron diffraction patterns of the secondary walls of wood cells in selected areas [3]. Figure 3a isatransmissionelectronmicrograph of a longitudinalsection of latewood tracheids of Pinus densifloru, showing the intercellular layer (I), and the S, and S, layers. The electron diffraction diagram is of a selected area in S2 (Fig. 3b), which is represented by a small circle. The (101), (loi), and (002) of the equatorial reflections and (040) of

i

FIGURE 2 Electron micrograph of ultrathin transverse section of earlywood tracheids from Pinus densgora, showing thedistribution of lignin inthe cell wall, which was skeletonized using the hydrofluoric acid technique.

6

Fujita and Harada

FIGURE 3 (a) Electron micrograph of ultrathin longitudinal section of tension wood fibers from Pinus densiporu. (b) The corresponding diffraction diagram taken from the encircled area.

ormation Ultrastructure and

Wall

of Cell

7

the meridional reflection can be seen. It should be noted that crystallographic planes are based on the Meyer and Misch (1937) model of the unit cell of cellulose I, i n which the h axis (the fiber axis) is vertical. I t iswell known that in the wood cell wall, celluloseexists in the form ofthin threads with an indefinite length. Such threads are called cellulose microfibrils, and they play an important role in the chemical, physical, and mechanical properties of the wood. The greenalga, Kdorzia. which is oneform of Chlorophyceae,hasbeenstudied intensively by microscopists and crystallographers as an excellent material for the ultrastructural study of cellulose microfibril. Why then is W o t z i a used for the study of the cellulose microfibril of the wood cell wall? Because the cell walls of Valonia are unlignified, their microfibrils are readily isolated. Furthermore, as described later, Vrtlonicc microfibrils are approximately 20 nm in width, which is about five times larger than those of wood, and they are highly crystallized. However, the difference between algal microfibrils such as those of Vhlorli~zand ordinary ones produced by the higher plants also must be stressed. One of the differences is the selectively uniplaner orientation of algal microfibrils, that is, the ( 101) plane facing the cell surface, while cellulose microfibrils of higher plants are randomly oriented, although both microfibrils are laid along the cell surface i n their longitudinal direction [3,4]. The other is the crystallographic heterogeneity in algal microtibrils as detected by NMR [ S ] , and a triclinic system mixed with an ordinary monoclinic system was detected by electron diffraction [6]. The interface between these systems is not yet shown, although the former amounts to about 50%.

1.

Dimensions of the Cellulose Microfibril

As described above, it is clearly demonstrated through electron microscopy that the cellulose molecular chains are organized into strands as cellulose microfibrils. Figure 4 shows transmission electron micrographs of disintegrated cellulose microfibrils negatively stained withuranyl acetate. Figures .la and 4b, respectively, show the microfibrils of klonicr tnc~cmphyscrcell wall and the holocellulose of Pirlus drnsijur-a. A discrepancy in the size of the crystalline region of cellulose, obtained by X-ray diffractometry and electron microscopy, led to differing concepts as to the molecular organization of microfibrils. Frey-Wyssling 171 regarded the microfibril itself as being made up of a number of crystallites, each of which was separated by a paracrystalline region and later termedelementary fibril by Frey-WysslingandMuhlethaler 181. The term elementary fibril is therefore applied to the smallest cellulosic strand. Muhlethaler [ 10,l 11 applied this term to the cellulose fibril with a diameter of approximately 3.5 nm, using the negative-contrastpreparationtechnique for electron microscopy.Preston and Cronshaw [91, on the otherhand,considered the microfibril tohaveasinglecore of cellulose crystallite surrounded by a paracrystalline region. The width of cellulose microfibrils is reported to vary in different cellulose materials [ 121. For instance, as shown i n Fig. 4, Vrrlorzia cellulose microfibrils, being about 20 nm wide, are much larger than those of wood holocellulose. Shown in Table 1 are the crystallite size and microfibril width for several cellulose materials [ 131. The crystallite size was estimated with Scherrers equation at the reflection (002) or (101) of X-ray diffractometry, whereas the microfibril widthsweremeasured directly from the electron micrographs. The width range and mode width are also included in this table. It should be noted that the size of crystallites varies in different sources of cellulose materials, for results from both X-ray diffractometry and electron microscopy. According to Heyn [ 141, the negative stain can penetrate only the regions accessible to water. Thus, the translucent parts seen on the electron micrographs correspond to the

8

Fujita and Harada

FIGURE 4 Electron micrographs of the cellulose microfibrils of Vuloniu mucrophysa (a) and of Pinus densifloru holocellulose (b) (disintegration, negatively stained with uranyl acetate), showing the difference of cellulose microfibril width between wood and Vuloniu.

Formation Ultrastructure and

Wall

of Cell

9

TABLE 1 Crystallite Size and Microfibril Width

Crystallite Microfibril size" width SamplesPinus dens$ora

02 2) 4.1

Untreated 2.76 HolocellulosePopulus euramericana

(2.5)b 2.2 (002) 14.3 11.9 (101)

layer Gelatinous Normal wood Valonia 15-30'Reflection examined. hModewldth. Source: Ref. 12.

(20.0)b

(002)

crystalline regions of cellulose. Therefore, the difference in the microfibril width must be ascribed to that in the size of cellulose crystallites. In addition, the values obtained are not always equal to the 3.5 nm in elementary fibrils proposed by Muhlethaler [ l l ] .

2. Cross-Sectional View of Cellulose Microfibrils Figures 5a and 5b are similar electron micrographs of the ultrathin cross section of cellulose microfibrils from Valonia macrophysa and the gelatinous layer of Populus euramericana tension wood fiber. These were obtained by means of diffraction contrast in the bright-field mode foran epoxy resin-embedded section. This technique reveals a crystalline region as a dark zone dueto electron diffraction. Thus, cellulose microfibrils have a highly

FIGURE5 Electron micrograph of ultrathin transverse section of cellulose microfibrils (diffraction contrast in the bright field mode), showing their cross-sectional views: (a) from Valonia macrophysa; (b) from G layer of Populus eurarnericana.

Harada 10

and

Fujlta

crystalline nature. It is interesting to note in Fig. 5a that a Vuloniu microfibril does not have any subunits corresponding to the elementary fibrils [13,17]. Additionally, cellulose microfibrils appear to be almost square in their cross section in both wood and Vuloniu [15-171.

3. Crystalline Structure of Cellulose Microfibrils Figure6shows Vuloniu macrophysu microfibrilsmechanicallydisintegratedwithacid,taken by diffraction contrast in the bright-field mode. Cellulose microfibrils can be seen as the dark areas, again indicating the highly crystalline structure cellulose. of However, the internal crystalline ultrastructure of cellulose microfibrils is not revealed by electron microscopic techniques such as negative staining and diffraction contrast, because lattice imagesof cellulose microfibrils are not obtained. The most important reason is that cellulose microfibrils are damaged the electron beam and their crystalline by nature is destroyed by irradiation under normal photographing conditions. Recently, the crystalline ultrastructure of cellulose microfibrils in Vuloniu macroby a specially developed technique for taking highphysu cell wall has been revealed resolution lattice images [15,16]. Figure 7 is an example of the lattice fringe substructures from disintegrated cellulose microfibrils. This micrograph shows the lattice image 0.60 of nm, corresponding to thatof the (101) plane. The lattice spacingof 0.60 nm is also shown in the electron and optical diffraction patterns. The lattice lines are observed regularly at about 20 nm width across the cellulose microfibril and are also visible along its length for more than 50 nm without any disruption. Figure 8 shows images of the cross section of cellulose microfibrils obtained using ultrathin sections. Lattice lines at0.60, 0.54, and 0.39 nm are visible in this figure. Therefore, a single microfibril is indicated as the individual crystal. Accordingly, it is suggested that the crystal line subunits as 3.5 nm elementary fibril and periodicity in its length does not exist inside the cellulosemicrofibril. Unfortunately, lattice images of cellulose microfibrils have not yet been taken in wood cellulose, since wood cellulose has low crystallinity and the size of the cellulose

FIGURE6 Electron micrograph of cellulose microfibrils fromVuloniu mucrophysu (disintegration, diffraction contrast in the bright-field mode), showing the crystalline nature of cellulose microfibrils.

Formation Ultrastructure and

Wall

of Cell

11

FIGURE 7 Lattice image of a disintegrated cellulose microfibril of Valonia mcrophysa, showing the lattice spacing of 0.60 nm.

FIGURE 8 Lattice images of the cross-sectional face of cellulose microfibrils from Valonia macrophysa, showing the lattice spacings of 0.60, 0.54, and 0.39 nm, respectively.

12

Fujita and Harada

microfibril is rather smaller compared with that of Valonia. In the near future, beam damage at room temperature against wood cellulose microfibrils would be reduced at least 10 times with cryo-electron microscopy. The cellulose microfibrils of the gelatinous layer of poplar (Populus eurntnictrna) tension wood in disintegrated samples are found to have many kinks, suggesting that the cellulose microfibril is highly crystalline [13]. However, the cellulose microfibrils of the gelatinous layer, about 100 nm in length prepared by ultramicrotome, become shorter than their original length upon hydrolysis [ 131. As a result, the crystalline regions in the cellulose microfibril of wood cell wall are thought to havesomecrystallinedislocations caused by chain ends [ 181. The cellulose microfibrils consist of a core crystalline region of cellulose surrounded by paracrystalline cellulose and short-chain hemicellulose. Lignin encases them and binds them into a rigid structure of wood cell wall.

B. Cell Wall Layers and LamellaeAt the first step of differentiation of a woody cell, the living protoplasm produces a primary wall (P) that can be extensively increased in its surface as the cell develops. The substance between the primary walls of adjacent cells is called the intercellular layer (I) or the middle lamella. Since it is difficult to distinguish the region between the I layer and the P wall in the mature cell wall, the termcompoundmiddlelamella (CM) is generally used to designate the combined I layer and the two adjacent P walls. After the enlargement of the cell ceases. the cell wall layers are formed by the apposition of wall substances onto the inside of the primary wall. These wall layers are called the secondary wall (S) Although the primary wall andsecondarywallare classified by the ontogenetic process of plant cells, actual layered structures have been examined by the orientation of cellulose microfibrils. As a result the concept of lamellae, which are composed ofvery thin layers of only one or two cellulose microfibril width, is introduced. Cell walls were thickened by the appositional supply of these lamellae from the protoplast, so cellulosic interlamellae bridges are not accepted in the concept. The lamnella structure on the secondary wall is interesting in both physical and chemical properties of wood. Kerrer and Goring proposed a composite model with hemicelluloses and lignin [IS]. Although it is very intelligent, actual microfibril orientation on a lamella may fluctuate more [2O,2 I ] .

Tracheids and Fibers Figures 9 and I O are polarized photomicrographs at crosscd polars of transverse sections of tracheids and fibers, respectively. Both reveal the three-layered structure of the cell wall due to the differences in the orientation of cellulose microfibrils. According to the concept of Kerr and Bailey [22], normal wood cell wall consists of P and S walls, and the S wall is composed of a relatively narrowor thin outer layer (S,), an inner layer (S3), and a relatively thick middle layer (S?). However, the P wall cannot be distinguished in the figure due to the strong birefringence of the S , layer adjacent to the P wall. The S , and S, layers appear bright in the photographs, whereas the S, layer is at total extinction. That the birefringence of the S, layer occurs to a lesser degree than that of S , in the fibers of F q u s crewtcl indicates the poordevelopment of the S,. Despitesubsequentextensive studies with electron microscopy, the concept and terminology described above are still commonly accepted. Figure 1 1 is an electron micrograph ofan ultrathin transverse section from Cty7tomer-iajapotzicn, stained with silver protenate. It shows the intercellular layer (I), different1.

Ultrastructureand Formationof Cell Wall

13

I

FIGURE 9 Polarized-lightphotomicrograph of transverse section from earlywood tracheids of Pinus c/ensiforu, showing thethree-layeredstructure of the cell wall due to the birefringence of cellulose microfibrils.

layers of the secondary wall (S), and the warty layer (W) in an earlywood tracheid. The same layering structure from an earlywood tracheid of Pinus densiflot-a is shown more clearly in a longitudinal section that was skeletonized by the hydrofluoric acid technique (Fig. 12). Figure 16, (pg. 18). shows the texture of the P wall diagrammatically. The microfibril orientation in the primary wall was interpreted by the multinet growth hypothesis proposed by Roelofsen [23] and supported for the differentiating conifer tracheids by Wardrop[24].

FIGURE 10 Polarized-lightphotomicrograph of transverse section from wood fibers of Fugus crenatcr, showing the same structures as in Fig. 9.

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FIGURE 11 Electronmicrograph of ultrathintransverse section of an earlywood tracheidfrom Cryptomeria japonica, showing I, S,, S*, S1, and W (warty layer) at the final differentiating stage of the cell wall.

FIGURE 12 Electronmicrograph of ultrathinlongitudinal section of earlywood tracheidsfrom Pinus densifom (skeletonized cell wall with the hydrofluoric acid method), showing the I, P, S,, Sl, and S3 of the cell wall.

Formation Ultrastructure and

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15

In the multinet hypothesis, the microfibrils are first deposited transversely to the cell axis and passively shifted longitudinally during cell extension. From an opposite viewpoint, an orderedfibrilhypothesiswasproposed by Roland et al [25] in order to interpret the . crossed polylamellated structure in the primary wall of parenchyma cells. According to this hypothesis, whether the orientation of microfibrils becomes transverse, oblique, or longitudinal is determined at the time of deposition of cell wall and may not be changed thereafter. Recently, Fujii et al. [26] proposed a modified multinet hypothesis of microfibrils orientation in the primary wall. The difference between this conceptand Roelofsens theoryisthattheshift of microfibrilorientation during cell extension is made in the individual lamella and each lamella becomes thin on the outer surface of the P wall due to extension. The three layers of the secondary wall, designated S,,S2, and S3,are organized in a plywood type of construction. The S , or S3, with a large microfibril angle to the cell axis, is designated as a flat helix, and the S2,with a small angle, as a steep helix (see Fig. 16). It is also shown that the layers themselves are of lamellae of microfibrils with varying amounts of shift in orientation, visible in the transmission electron micrograph. The S , is composed of several lamellae with alternating S and Z helices of microfibril orientation [28,29], and this structure in the S , is termed crossed fibrilar texture [28]. Figure 13 is

FIGURE 13 Electronmicrograph of theradialinnersurfacein a differentiatingtracheidfrom Pirzus densgoru (direct carbon replica), showing the microfibrillar orientation newly deposited of the microlamella crossing that of the underlying microlamella in S,.

Harada16

and

Fujita

a transmission electron micrograph of a replica of the inner surface of the differentiating early wood tracheid of Pinus densgora forming the S,, showing the criss-crossed texture of the microfibril orientation in the two different lamellae. The middle layer of the secondary wall (S,) is the thickest within the layers of the secondary wall. Therefore, the S, contributes most to the bulk of the cell wall material and is a compact region in which a high degree of parallelism of microfibrils exists. The S, isathinlayer of flathelices of microfibrilorientationasseenin S,. As opposed to the highly oriented S,, the S, is loosely textured. The S,, birefringent to a somewhat lesser degree than the in wood fiber, shows that this layer poorly developed. S, is Althoughthe S2 exhibitsamicrofibrillarorientationwithsteephelices,there are transition lamellae on its inner and outer surfaces. Several lamellae in these regions show agradualshift of microfibrilanglesbetween S, and S, andbetween S, and SJ [30]. However, the gradual shift of microfibril angles is more abrupt between S, and S, than between S, and S,. The transition lamellae in the secondary wall are not detected in TEM micrographs of ultrathin sections, since this lamella is relatively thin compared with the S, and S,. The method for evaluating microfibril angles in the secondary wall of wood cells was proposed by Yamanaka [31]. Figure 14 is a "EM micrograph of a transversely oblique section of an earlywood tracheid from Pinus dens.ijZora (stained with KMnO,). The curve through black dots shows the changes of the microfibrillar angle with respect

FFIGURE 14 Electron micrograph of ultrathin oblique section of an earlywood tracheid in Pinus densijfflora and microfibril angles in the secondary wall: top, I. bottom, lumen side.

Formation Ultrastructure and

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17

to the tracheid axis from the top S, to the bottom S,. The horizontal line in the upper part of the figure shows the angles of microfibrillar orientation, the symbols (-) and (+), respectively, referring to Z and S helices. The gradual changes of the microfibrillar angles from S, to S, and from S, to S, are shown there. The helical cellulose microfibril orientationin the S, is typically demonstrated in Xray diagrams of wood [32]. The arcs at 0.39, 0.54, and 0.60 nm in the X-ray diagram of wood show that the cellulose crystallites (microfibrils) lie in a helix around each wood fiber or tracheid. The microfibril orientations are Z helices in the S, and S helices in the S,, although S, is the crossed arrangement of S and Z helices. Preston [32] suggests that the structure with various microfibril angles in the secondary wall passes through only one cycle, but this may be the brief duration of wall thickening in higher-plant cell walls compared with that in algae. Roland and Mosiniak [33] presented a diagram regarding the changesof cellulose microfibril angles in the case of a secondary wall of tracheids and wood fibers (Fig. 15). Figure 15 illustrates the case between the S, and S, layer. The change of microfibril angle is regular and continuous between the S , and S, layers,butitstopsduringthe deposition of the S , layer. Afterwards the change of microfibril angle reopens toward S, layer deposition. The texture of cellulose microfibrils in the P and S walls of softwood tracheids and hardwood fibers is shown as a schematic diagram in Fig. 16. The thin primary wall (P) consists of a loose aggregation of microfibrils oriented more or less axially to the cell axis on the outer surface. The S, layer is a flat helix but with crossed structure, whereas the S, layer is a steep helix and the S, layer is a flat helix. There are intermediate layers: the S,,, present between the S , and S, layers; and the S,,, between the S, and S, layers. The spiral thickening is the ridge of microfibrils that exist on the inner surface of the S, layer. The spiral thickening is considered partthe S, layer becauseof its continuity of with the S, layer and parallel arrangement to the S, layer microfibrils.

FIGURE 15 Schematic diagram of the change of microfibrilorientationfrom three-layered structure of the cell wall. (From Ref. 32.)

S, and Sz in the

Harada 18

and

FuJlta

FIGURE 16 Schematic diagram of the microfibril orientation in the primary wall and different layers of the secondary wall from tracheids and fibers: Po,PI; outer and inner parts of the primary wall; SlzrS23, intermediate layers between S , and S , and between S2 and S,, respectively.

The warty layer is one of the major structural features of wood cells found by in in electron microscopy [34]. It was first found softwood tracheids and later the tracheids, vessels, and wood fibers of hardwoods (see Fig. 11). The major chemical constituents of warts arereported to be lignin and hemicelluloses according to examination by component removal treatment of ultrathin wood sections [35]. The warts are believed to arise from the extra wall materials and remainscytoplasm that are deposited on the layer through of S, the plasma membrane [36,37]. The warty layer is not found in all softwoods and hardwoods [30,38]. Parham and Baird [39] have pointed out that the appearance of warts in wood has a phylogenetic trend. Softwood tracheids and primitive hardwood cells nearly always have warts, but as the cell types become more advanced or specialized, they become wart-free.

2. Vessels The texture of cellulose microfibrils in the walls of specialized cells such as vessel elements and parenchyma cells cannot be readily described as in softwood tracheids and hardwood fibers. A concept of standardized cell wall organization in vessel elements was, however, represented by Kishi et al. [40,41]. The microfibrils in the primary wall extend straight and are arranged parallel to one another within one lamella, and the wall consists of three parts, P-outer, P-middle, and P-inner, each showing a different microfibril orientation. The microfibrils are oriented transversely with respect to the vessel axis in the Pouter and are oriented at random in the P-middle. The P-inner consists of a crossed polylamellatedstructure. It isalsoreportedfromthe examination of vessel elements from nearly 30 Japanese hardwoods with polarizing and electron microscopy that the layered structure of the secondary wall can be classified into three categories: the typical threelayered structure, an unlayered structure, and a multilayered structure. The typical threelayered structure consists of S,, S2,and S3 similar to those of softwood tracheids and hardwood fibers, although the S, and S3 layers are thicker than those of tracheids and

Formation Ultrastructure and

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19

wood fibers. The unlayered structure has only microfibrils, with the orientation of a flat helix. The multilayered structure has more than four layers, in which microfibril angles to the vessel axis change. This type of structure contains in some cases the so-called bowshaped pattern. Figure 17 isa TEM micrograph of the transverse sectionof Cinnamomum camphora and shows the microfibril angle and helix in the part of the bow-shaped pattern appearing on the vessel wall of the multilayered type of structure. As shown in Fig. 17, the pattern results from the progressive changes of microfibrillar orientation in the wall from 90" to 0" and from 0" to 90".

3. Parenchyma CellsIn spite of the fact that parenchyma cells had been generally considered to have only primary wall, thoseof wood are reported sometimes to develop secondary wall, in addition to complicated primary wall. It is evident from recent studies that ray and axial parenchyma cells in both softwoods and hardwoods have variations or complexities in their wall structure that are not observed in the cell walls of tracheids and wood fibers. In softwoods, the cell wall structure of the ray parenchyma cells was divided into five categories by Fujikawa and Ishida [42]. However, as shown in Fig. 18, it is fundamentally classified into two types; the first type consists of the primary walland protective

b

90FIGURE 17 Electron micrograph of ultrathin oblique sectionof a vessel wall stained with KMnO., from Cinnamomum camphora, showing a bow-shaped pattern (a) and the microfibril angles and helices (b).

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Fujita and Harada

L-

I l

I I

i

P

S 1

i

S2

1

I

FIGURE 18 Schematicdiagram of themicrofibrilorientation in the cell wall of softwood ray parenchyma cell: (a) the first type; (b) the second type. (From Ref. 41.)

layer (Fig. 18a), and the second type consists of the primary wall, secondary wall, and protective layer (Fig. 18b) [42]. However, the protective layer and a random arrangement of microfibrils is omitted in this figure. The P, appears with microfibrils of almost parallel orientation to the ray cell axis, the P, with the network appearance of microfibrils, and the P3 with several crossed polylamellate at microfibrillar angles of 30-60". It is interesting to note that the ray parenchymacell wall in thediploxylem of Pinus develops in two stages: that is, the primary wall and inner protective layer are fornled in the sapwood, and just before the heartwood is developed, the secondary wall and protective layer are deposited. In the axial parenchyma cells of softwood, the cell wall texture is very similar to that of ray parenchyma cells, except that the microfibrils are arranged in a flat helix with respect to the cell axis in the P , . In hardwoods, the primary wall ofray parenchyma cells has the so-called polylamellated structure proposed by Chafe and Chauret 1431. It was pointed out by Chafe and Chauret [43] that an isotropic layer and protective layer characterize the layered structure of the secondary wall of xylem parenchyma cells in hardwoods. According to examinations of thechemicalcomponents of these two layers using aseries of treatments on serial ultrathin sections, both a protective layer and an isotropic layer are rich in hemicelluloses and contain some pectic substances and cellulose microfibrils, but they have little lignin at the first stage of their developing process and become lignin-rich after the deposition of the inner secondary walls on them [44]. Consequently, both layers are considered the

Ultrastructureand Formationof Cell Wall

21

same in their origin and are called amorphous layer by Fujii et al. [M].Figure 19 shows electron micrographs of transverse sections by ray parenchyma cell from Tiliu juponicu; Fig. 19a shows cell walls skeletonized with hydrofluoric acid, while Fig. 19b shows sodiumchloride-treatedcellwalls.Blackzonesshow an amorphous layer indicating the presence of much lignin (Fig. 19a), but these disappear through delignification as seen in Fig.19b. It has been reportedbyFujii et al. [45] fromtheexamination of ray and axial parenchyma cell walls from about 50 species of Japanese hardwoods that the secondary wall is composed of a lignified cellulosic layer (CL) and an amorphous layer (AL) and that the cell wall structure can be classified into three types according to the presence and organization of these two kinds of layers. Figure 20 is a schematic diagram of the cell wall organization of hardwood ray parenchyma cells: (1) 3CL-type, (2) 3CL+AL-type, (3) 3CL+AL+IL-type. CL refers to the lignified cellulosic layer that is similar to the ordinary wood cell wall, whereas ICL refers to the lignified cellulosic layer inside the amorphous layer (AL). The 3CL-type wall structuremay be considered thestandard structure of parenchyma cells of hardwoods, whereas the 3CL+AL-type wall structure occurs in cells that have extensive pit contact with vessels.

FIGURE 19 Electron micrographs of ultrathin cross section of the ray parenchyma cell from Tilia japonica, showing the amorphous layer (AL) of the secondary wall: (a) delignified cell wall; (b) cell wall skeletonized using hydrofluoric acid treatment.

Harada22 3CL

and

AL

1-qICLx :

Fujita

.: ...........,..,..........:....(C)

(a)

FIGURE 20 Schematic diagram of the cell wall organization of hardwood ray parenchyma cell, showing three types of wall structure: (a) 3CL; (b) 3CL AL,(c) 3CL + AL ICL.

+

+

4.

Reaction Wood Cell Wall

As described above (see Section I), softwood reaction wood is called compression wood and hardwood reaction wood is called tension wood. Figure 21 is a polarizing micrograph of compression wood tracheids from Pinus densijlora, and it demonstrates that the S, layerpresent in normal wood tracheids is lacking. This is clearly shown in an electron micrograph of a cross section of a compression wood tracheid from Pinus densijioru (Fig. 22), and the presence of deep spiral checks in the S layer is also revealed. The microfibrillar orientation of the S layer is nearly 45", , ,

Harada 24

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Fujita

cellulose of the G layer is highly crystalline. Its microfibrils are oriented parallel to the longitudinal axis of the fiber and the G layer is easily separated from the remainder the of fiber wall. Another structural feature of the tension wood fiber wall is that the G layer deposits on any one of the normal three secondary wall layers, S,, S2, and S,. The secondary wall of the tension wood fiber consists of three types, that is, S, G , S, S, G , and S, Sz + S, + G, depending on the wood species or part within a stem. Consequently, the G layer is called the S, layer when we refer to the S,, Sz,and S, layers.

+

+

+

+

C.

Sculpturing of the Wood Cell Wall

Cellulosic fibers such as cotton, ramie, jute are relatively simple, smooth-walled comand posites of lamellae, but in wood the cell walls are almost invariably interrupted by gaps (pits) and sculpturing features.

1 Pit Structure . Pits are gaps in the secondary wall of wood cells. There are two types of pits: bordered pits and simple pits. Generally, pits are present as pairs between two adjacent cells: bordered pit pairs, simple, and half-bordered pit pairs. In softwood, the pit border region of the cell wall is composed of border thickening (BT), S,, S2, and S, from the outer part of the cell wall as shown in Fig. 24. The presence of BT and thicker S, are features of the pit border wall. The microfibrils circle at theBT and sweep around thepit at the individual layers S,, Sz,and S,. In softwood bordered pit pairs, many species show a thickening at the center of the pit membrane. The torus suspended from fine cellulosic strands to form is a margin around the torus as shown in Fig. 25. The margin consists of an open net of

FIGURE 24 Schematic diagram of pit border organization in bordered pits of softwood tracheids. BT, initial pit border.

Formation Ultrastructure and

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25

FIGURE 25 Electronmicrograph of the surface of pitmembranefrom Cryptomeria japonica (direct carbon replica), showing the pit membrane structure. T, torus; M, margo.

radially oriented microfibrils superimposed on an unoriented primary wall network, and it extends from the torus to pit border. The torus is generally convex lens-shaped in cross the section. On the other hand, the torus is seldom thickened in other cases. The former is true in species of the Pinaceae and Sciadopityaceae families, and the latter case involves species of Ginkgoaceae, Taxaceae, Chephalotaxaceae, Cupressaceae, Podocarpaceae, and Araucariaceae. The pit membrane of a half-bordered pit pair between tracheids and ray or axial parenchyma cells is quite thick. There is no torus in the center of the pit membrane, and no openings can be seen even at high magnification with an electron microscope. The central feature of the membrane structure of simple pit pairs in the interparenchymatous pits is the presence of plasmodesmatal pores. In hardwoods, the cell wall the pit border of consists of BT, P, S,, S*, and S3in tracheids and fiber tracheids of hardwood, like softwood tracheids. However, the pit border of vessels lacks not only BT but also S, in some parts of the pit border region [47]. The pit membrane of the bordered,half-bordered, and simple pit pairs in hardwoods is equal in thickness, exhibiting the primarywall texture, and there is usually no evidence of a torus. However, the presence of a torus in the intervessel pit membrane is reported in several species of hardwoods [48]. The pit membrane of simple pit pairs has plasmodesmatal pores as seen in softwoods.

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2. Vesture Pits In hardwoods, the pit chamber and pit apertures that are decorated by outgrowths of wall material are known as vestured pits. The outer growths of vestured pits are constructed chemically of lignin, hemicelluloses, and a little pectin [35].The shape and size of the outgrowths of vestured pits are variable. The development of vestured pit outgrowths is regarded as similar to that of warts.111.A. GENERAL DEVELOPMENT OF WOOD AND WOOD CELLS Vascular Cambium and Cambial Activity

One of the characteristic features ofa tree is the formationof the vascularcambium cylindrically surroundingastem,branches,and roots. The vascularcambiumproduces xylem inward and phloem outward. This sequence allows a tree to make itself a huge body. The cylindrical vascular cambium occurs through a series of developing meristem, namely, the apical meristem, the occurrence of procambium in the ground meristem, the growth of the vascular bundle, and the connection of intrafasicular cambium by the development of interfasicular cambium. The vascular cambium is composed of two types of meristematic cells. One is the fusiform initial occupying the major part of meristematic cells, and the other is the ray initial. Through their active cell division, parts of xylem and phloem are produced. However, since their activity in cell division is to a great extent affected by the season and weather, the result is the formation of annual rings in temperate regions. These initials must also multiply themselves on the tangential plane according to the increment of stem diameter. These two types of cell divisions can be distinguished by the direction of the division. The former division is defined as periclinal division, and the latter is called anticlinal division (see Figs. 26 and 27). Periclinal division is the mostimportant in view of woodformationandthus is discussed in detail. Cell division of the initial is extremelyrapid in spring. Moreover, several derivative cells (xylem mother cells) just inside the initial also have the ability to multiply through periclinal division. It is practically impossible to determine the true initial cell among these dividing cells. Therefore, just for convenience, a group of these cells is consideredcambialcellsand their area is called the cambialzone. In softwoods, the fusiform cells derived from the cambial zone differentiate directly into the tracheids except in onlyafewcasesinvolving the formation of the parenchymastrand,whereasthey differentiate into vessel elements, wood fibers, and several types of tracheids and parenchyma cells in hardwoods. Carnbial activity and the derivative differentiation are very important sequences in the growth of trees, environmental preservation of forests, and production of wood as a biomaterial. That is, they are the major sink of organic substances which are synthesized on leaves by CO, fixation, and then the major source of other life activities such as insects and also human beings. A detailed review of the vascular cambium has been published by Larson [491.

B.

Differentiation of Wood Cells

The tern1 differentiation has several meanings in the fieldof biology. I n this chapter, the term will be applied to the restricted case of the process of cell devclopment from the just-forming state i n the meristematic tissue to the mature state at which it is accomplished.

P

R.

..

l!

IfFIGURE 26 (a) Light micrograph around the cambial zone ( C ) ,phloem (Ph), and enlarging xylem (E) from a transverse section of Robinia pseudoacacia. Most fusiform cambial cells are undergoing periclinal division, except for a trace experiencing anticlinal division (arrow). Electron micro(b) graph of fusiform and ray cambial cells. (c) Cytoplasmic feature of enlarging cells.27

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Fujlta and Harada

FIGURE 26 Continued

For instance, the differentiation of tracheids implies their maturing process from birth at the cambial zone to death after the secondary wall formation, by which both water-conducting functions and mechanical properties are given to the tracheid. The method of differentiation of parenchyma cells is quite different from that of tracheids, because they have only the primary wall or an underdeveloped secondary wall on the primary wall. They may be already functioning at the cambial zoneand have the ability to redifferentiate. Therefore, their differentiation is not addressed here. First of all, the differentiationof softwood tracheids, which is the most basic process of wood cell formation, will be discussed in detail. When a specimen block around the cambial zone is taken from the stem of a living tree and then a transverse section is observed under a light microscope, it is noticed that cells are piling up on a radial row from the mature phloem to the mature xylem through the cambial zone (Fig. 27a). The differentiating zoneof tracheids is located between the cambial zone the mature xylem and area. If the whole life of a particular tracheid from birth to death could be traced in situ in a tree stem, the tracheid differentiation would be clearly elucidated. However, it is really impossible to do so because the cells must be fixed with some reagent to preserve their cytoplasmic structure. Regrettably, their dynamic cell actions evolve into static phase by fixation. Therefore, the differentiating process of a tracheid must be deduced from the static cell structure of a series of differentiating tracheids. From this point of view, the differentiating zone of earlywood tracheids is favored for the precise examination their of differentiation. In the spring, the production of tracheids from the cambial zone is very

30

D

Q7

n3

0

s3

FIGURE 27 (a) Light micrograph of the cambial zone (C) and the derivative tracheids in five differentiating stages (RE, S,, S,, S3, and F) between phloem (Ph) and mature xylem (MX)from Cryprorneriujuponicu. (b) Enlarged view of S, depositing cells.

W

h)

Harada 30

and

Fujlta

FIGURE 27 Continued

constant and, as a result, a series of differentiating tracheids is lined up in an orderly fashion along a radialrow from the just-formed stage to the mature stage. This series can be considered a good substitute for the life story of a tracheid, and since it is possible to trace the series using many microscopic techniques, the differentiating process a tracheid of can be grasped dynamically by tracing these differentiating tracheids along radial rows (Fig. 27a). Thedifferentiation of tracheids will be separatedintoseveral developing stages. Tracheids are pushed out in an inward direction from the cambial zone so as to begin enlargement. In the case of tracheids, the enlargement proceeds mainly in the radial direction, whereas enlargement in the tangential and longitudinal directions is very slight. Therefore, it may be appropriate to call this stage the radial enlarging (RE) stage. This fact results in the thinner radial walls of tracheids and the reorientation of cellulose microfibrils that may occur during the extension of the wall. The tracheid in this stage is composed of primary wall similar to the wallof cambial stage (C) cells. The thinned wall is recovered by the supplement of new wall materials on the inner surface. The extended wall is so fragile that it is often damaged and tom off during sampling of a specimen block from a living stem. After the enlargement of cell size, tracheids thicken secondary wall layers with the formation of the S,, S?, and S, layers. These stages are performed by the active deposition of cellulose microfibrils. However, the outermost region of the cell wall, including the intercellular layer, the cell comers, and the primary wall, is lignified during the S, stage. This lignification, which will be called intercellular layer W i g n i fication, may play an important role in stabilizing the cell size and conjugating the differentiating cells with one another. This I-lignification is accomplished in the middle phase of the S2 stage. Hemicelluloses are also supplied just after the deposition of cellulose

Formation Ultrastructure and

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31

microfibrils (see Section IV). The secondary wall, which is still porous and flexible after the deposition of hemicelluloses, is encrusted with lignin and becomes very rigid. The lignification of the secondary wall, which willbe called S-lignification in contrast to I-lignification, is the most active after the S, stage,namely, in thefinal (F) stage of differentiation, although its initiation can be detected already during the S, stage. In this F stage some decorative elements such as warts or helical thickenings are added on the inner surface of the wall. After the wall layers develop, tracheids lose their cytoplasm by autolysis. Amorphous substances that have embedded the pit membrane also dissolve enzymatically sometime in the F stage. Tracheid differentiation is completed as this point and water conduction is achieved in the mature xylem (MX). The differentiation of vessel elements is characterized by enormousexpansion in both the radial and tangential directions. Although the developing stages of tracheids cannotbe applied directly to those of vessel elementsdue to a different secondary wall structure, the relationship of enlargement to secondary wall thickening and lignification is consideredsimilar to the sequence of tracheid differentiation. Needless to say, the formation of perforation pores is completed by the disappearance of the membrane itself, apart from the removal of only an embedding substance in the bordered pit pairs. On the contrary, the differentiation of wood fibers is characterized by the remarkable elongation in cell length that occurs at cell tips [50], and the other properties of differentiation are quite similar to those of tracheids. In hardwood, although the differentiation of both vessel elements and wood fibers proceeds simultaneously, vessel elements differentiate faster than wood fibers. How long a wood cell needs for its differentiation is also an important question. The time requirement for differentiation has been deduced by several methods, but the results are conflicting. A detailed timerequirementwascalculated for young trees of several softwoods by means of periodic inclinations for the internal date marking on the xylem. By these markings and the cell numbers contained in each differentiating stage, a time requirementofabout three weeks for passingthrough the five developingstages of a tracheid (RE, S , , S?, S,, and F) was calculated [51].

C. Cytology of Wood CellsCambial cells, the differentiating cells of the tracheid, vessel element, and wood fiber, and also living parenchyma cells possess protoplast in their cell lumens. The most peculiar cytoplasmic structure of the fusiformcambialcells is the existence of ahuge central vacuole (CV) (Fig. 26b). This vacuole is maintained during the differentiation of tracheids (Fig. 27b), vessel elements,and wood fibers, whereas the cytoplasmicregion(Cy) is restricted to the very narrow area between the plasma membrane (Pm) and the vacuole membrane tonoplast (T) (Fig. 26c). On the contrary, the ray cambial cells and their derivative parenchyma cells are full of cytoplasm in their cell lumen, although several smaller vacuoles sometimes occur (Fig. 26b). In the axial parenchyma cells formed by the redivision of a young fusiform derivative, the central vacuole becomes small, and the cytoplasmic area expands in the reverse way. In spite of the cambial zone and differentiating xylem existing under the circumstance of very high pressure between the bark and mature xylem, the cellscontained in this areahaveonlya thin wall. Althoughvacuolation is generally considered a symptom of cell decay, the conspicuous vacuolation of these cells is supposedtoplay a very important role in the sustainment of their cell shapeunder presure. The enlargement of cell volume also depends on the turgor pressure of the vac-

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uole. In fact, it can be pointed out by arealneasurements that the vacuole is the best developed of the cells at the RE stage, when cells are just expanding (Fig. 28a). A nucleous is located around the central position of a fusiform cell in the longitudinal direction [SO], but on the transverse plane it is still pushed to one side of the cell lumen by vacuolation (Fig. 26c). In the cytoplasm, ordinary cell organelles such as Golgi bodies (Go),rough and smooth endoplasmic reticula (r-ER and S-ER). mitochondria (M), plastids (P), small vesicles (v), ribosomes, microtubules, and so on, are contained in a very narrow cytoplasmic region, although the occurrence of these cell organelles except microtubules between plasma membrane (Pm) and tonoplast (T) is not so abundant during the diflerentiation of tracheids, wood tibers, or vessel elements. On the contrary, the cytoplasm of differentiating ray and axial parenchyma cells is crowded with many cell organelles. Especially, starch grains in the plastids and lipid droplets are very abundant, and r-ER are also well developed, whereas microtubules are very scarce. Ray cambial cells and mature ray cells arc almost identical to the differentiating parenchyma cells in their cytoplasmic features(Fig.26b). However, the number and size of starchgrains and lipid droplets contained in mature parenchyma cells change during a year 152-541. Thecytoplasmicfeatures of tracheidschange 21 little both i n quality and quantity according to their differentiation. The increase and decrease of the cytoplasmic area and its constituents of cell organelles were revealed by the combined use of light microscopy (Fig. 28a) and electron microscopy (Fig. 2%) on the differentiating zones of normal and compression woods of Cqptmtwricr juponicu. Areas of cell outline (A,,,,,,,;,,),cytoplasmicsurface (A,,,,,,,,,),and central vacuole (A,,,,,,,,,) were measured on an enlarged light micrograph of the transversesections o f differentiating tracheids using a digitized system connected to a computer (Fig. 28a). The nxasuretnent was performedalongthedifferentiatingtracheids, which were numbered from the initiation of the S , stage, and about 30 radial rows were surveyed. These radial rows of tracheids were sectioned at random in their longitudinal direction, so that the average value of tracheids of the same cell number reflects the makeup of volume i n each region. Areas of cell wall (A,v,J and cytoplasm (A,,.,,,,,,,,,,,) be calculated by finding the can remainder between those of the cell outline, the cytoplasmic surface. and the central vacuole, respectively. These values are diagramed in Figs. 29 and 30. I t should be noted that the cytoplasmic volume of both the normal and compression woods has two peaks during tracheid differentiation. The earlier peak i n both cases is at the intermediating phase from the S , stage to the S, stage, whereas the later one is located just prior to the initiation of the S , or F stage. On the contrary, during the S2 thickening, the cytoplasm is rather poor. Following this, proportions i n RE, S , , early S?, middle S,, late S,, and S, stages (Fig. 29) show the relative constituents of major cell organelles surveyed by electron microscopy (Fig. 2%). The general change in these cell organelles can be grasped by inultiplying the relative value by the total area of cytoplasm diagramed in Fig. 29. In addition to the changes in these cell organelles, the plasma membrane, important to the transportation of materials in and out of the cytoplasm, is always observed during tracheid differentiation and disappears after the development of the cell wall. The cytoplasmic features of differentiating wood fibers and vessel elements are also similar to those of tracheid differentiation, although the vacuolation of vessel elements is Inore extreme. In some species, such as acer or black locust, the living wood tibers are formed during the later period of a growing season. Their protoplast remains after the development of a cell wall and stores many starch grains in the cytoplasm for several years. Therefore, the mature xylem i n the sapwood is composed of ray and axial parenchymacells and sometimes the living wood fibers as the cells have aprotoplast.The

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8 5

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FIGURE 28 Light (a) and electron (b) micrographs of transverse sections of S? depositing tracheids from Cryptomeria japonica. Areas of some cell organelles, for instance, Golgi bodies (Go), were measured with electron micrographs such as those shown in (b).

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B

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0

c e l l number

C

RE

SI

s?r

Sam

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FIGURE 29 Changes of cytoplasmic volume during the differentiation of normal wood tracheids and proportions of some cell organelles at the stages of RE, S , , early S2, middle S,, late S?, S,, and F in Cryptonzeriu jqoniccr (see Figs. 28a and 28b).

FIGURE 30 A change of cytoplasmic volume during the differentiation of compression wood (see Fig. 28a).

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cytoplasmic features of these living cells are affected by the season, and also some of them seem to be specialized in their cell shape and cytoplasm. That is, the cells surrounding a vessel, particularly those directly contacted, become envelope-shaped and are very rich in Golgi bodies, r- and S-ERs, ribosomes, and mitochondria common to cells of active phase. On the other hand, their storage function seems to decay. These vessel-associated parenchyma cells are shown to concern the transportation of materials with vessel lumens [52,53] and also the formation of tyloses or gum that plugs the vessel lumen [55-571.

IV.

FORMATION OF WOOD CELL WALL

There is no doubt that cell walls are formed by the actions of cell organelles contained in each cell, even though some precursors of wall materials such as sugars may be supplied by the intercellular transport system.Therefore, cell wallformation is realized by the careful observation of cytoplasm that is undergoing cell wall development. It is also very important to select proper plant materials for precise examination of cell wall formation, becausegeneral plant cellsbearmanyphysiologicalfunctions in addition to cell wall formation. Moreover, the cell wall is composed of several types of chemical materials that are supposed to be metabolized by different cell actions, and their deposition on the wall may overlap. These complicated factors the major reason that the formation mechanism are of plant cell walls has not yet been explained clearly, in spite of many investigations. Differentiating wood cells such as tracheids are very useful materials from this point of view. That is, they construct a very thick secondary wall, of which the ultrastructure and chemical components have been examined in detail, and the general sequence of cell wall formation can be traced through the series of differentiating cells along a radial row. Besides, the cell organellespossessed by thesecells are concernedonlywith cell wall formation, except vacuolation for the turgor pressure. In addition, if the depositing phase of individual wall materials such as cellulose, lignin, or hemicelluloses can be detected separately in differentiation, the relationship of cell organelleswith the metabolism of those materials would be grasped more clearly.

A.

CelluloseMicrofibrilDeposition

Cellulose is the mostbasic cell wall material in thewhole plant and it constructsthe framework structure of cell walls in the form of crystalline microfibrils as mentioned in Section 11. The formation has been studied using various plant cells from lower plants such as fungi or algae, to higher plants. Plasma membranes located just inside developing cell walls seem to be the most important cell organelles in relation to cellulose microfibril deposition. Although cross-sectional structures composed of unit membraneshadbeen observed by ordinary electron microscopic methods such as chemical fixation and ultrathin sectioning, faceviewsalongthemembranebecamepossiblewith the development of freeze-fracture or etching methods coupled with replication. Small particles on the outer surface of plasma membranes had been reported in various plant cells. The epoch-making discovery, however, was the characteristic assembly of granules located in the interior of the plasma membrane and revealed on the fractured surface in green algae such as Oocystis, Myclusteriu, or Vcloniu [58-601. Interesting structures have been reported using mainly single or naked cells such as algae [62], actobacteria [63,64], and cotton fiber [61], which can be frozen rapidly.

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Small granule assemblies at the tips of microfibrils are called the terminal complex. These granules are considered to be the enzyme for the polymerization of cellulose molecules and their alignment i n the conlplex in relation to crystallization [64]. In underevolvcd algae such as vrtlonicr, the assemblies are large and linear, corresponding to their thick microfibrils [62]. On the other hand, evolved plants have small groups called rosettes [6 11. Thus, the form of the complex is considered to be related to the shape of the cellulose microfibrils and also to the evolution of plants. The polymerization and crystallization of cellulose microfibrils have been surveyed in detail using AcetoDactor- . x - y l i t z ~ ( ~which pro~/, duces a thin cellulosic thread [6S] and has various mutants (661. The sequence of cellulose synthesis described above has not been traced in differentiating wood cells yet, because the freeze-fracture method is difficult to apply to them. However, cellulosic frameworks of wood cell walls are supposed to be constructed by a similar way. perhaps by a rosette. Although the freeze-fracture method isvery effective for visualizing characteristic structures such as the terminal complex on the membrane, the overall structure of differentiating wood cells depositing cellulose microfibrils must be examined by ordinary sectioning methods. Especially in wood cells depositing secondary wall layers, a control lnechanism for microfibrillar orientation is a very interesting viewpoint. Also, as the cellulose deposition is accompanied by the synthesis and accumulation of hernicclluloses and lignin, actions of various cell organelles must be traced in detail. Hence, the deposition phase of cellulose microfibrils i n differentiating tracheids can be traced in both normal and reaction woods. The phase can be detected by the increment of cell wall thickness (671, by means of autoradiography [68-70] (Fig. 3 l ) , and by chemical analysis of selectively collected rnaterials in some developing stagesof tracheids [ 7 I 731 and wood fibers [74](Fig. 32). The results obtained by these methodsshow that cellulose microtibrils are supplied to the wall mainly in the early and middle phases of the S, and S2 deposition stages. In addition to these deposition stages of cellulose microfibrils, most noticeable were the deposition of the G layer in the tension wood fibers and the S, thickening stage in the compression wood tracheids. This stage is composed of the deposition of cellulose microfibrils and is followed by the depositing stages of hemicelluloses and lignin. Compared with other differentiating stages of tracheids and wood fibers, the cytoplasm of cells forming the G layer isvery poor in its activity due to the fact that the region between the plasma membrane and the tonoplast is very narrow and cell organclles are rare there (Fig. 3%) 1751. The exceptionally abundant cell organelle in the cytoplasm is microtubules (MT). They are regularly distributed just inside the plasmamembrane (Pm), keeping a constant space of approximately 8 nm to the inner membrane and also between themselves (Figs. 33a and 33b). They are exactly oriented parallel to the depositing cellulose microfibrils in the stages of the G layer as well as the S , and S2 layers (Fig. 33b). The diameter of the microtubules is approximately 23 nm, and their numbers increase up to 20 per I p m of the plasma membrane, as calculated by their transverse direction. This abundant distribution results in the covering of about 40% of the cytoplasmic surface (Fig. 33b). A feasible link between the microtubules and the inner layer of plasma membrane is also discernible (Fig. 3321).These characteristics strongly suggest that microtubules and plasma membrane comprise the outermost complex of cytoplasm. On the other hand, there are only traces of Golgi bodies, S- and r-ERs, and the vesicles derived from them in the cytoplasm, in spite of the very active synthesis of cellulose microfibrils in this phase of the cell. On the contrary, in the beginning of the S, thickening stage of compression wood tracheids, in which cellulose microfibrils are supplied to the wall at 45" to the cell axis,

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FIGURE 31 Serial light microscopic autoradiographs of before section treatment (a) and after sectiontreatments(b)withsodiumchloriteandhot 1.3% H,SO, from the differentiating compression wood tracheids in Cryptomeria japonica administered with 3H-glucose. Silver grains in (b) show the specific incorporation of radioactivity only on the inner surface of S , and S2 thickening tracheids, which reflects the deposition of cellulose by way of apposition. Removed activity can be detected in the intercellular layer of cells in the, stage (arrows) and in the preexisting secondary S wall of cells in the late S2stage (cells marked by an asterisk)by a comparison between (a) and (b) that implies lignin and hemicelluloses are supplied to the wall by wall of intussusception.

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FIGURE32 Electron microscopic autoradiographs showing the incorporation of H-phenylalanine in the transitional cell from S , to Sz,namely, in the stage of I-lignification (a), and from S , to S, in the stage of S-lignification in Cryptomeria japonica. Radioactivity can be observed around the intercellular layer and also within the Golgi bodies and vesicles in (a). In (b), vesicular inclusion is supplied to the wall by exocytosis (arrows) and radioactivity is often detected in such vesicles and the secondary wall.

cytoplasm isvery dense andwide. A similar complex between the microtubules and plasma membrane, however, is still observed [76]; microtubules are very abundant beneath the plasma membrane, having a link to it (Fig. 34a). The direction of microtubules is also in this case parallel with depositing cellulose microfibrils. Various cell organelles in the cytoplasm, such as Golgi bodies or ER, were shown to be involved in the synthesis lignin of precursors for the succeeding lignin deposition into the S, layer (see the next section). The characteristic appearance of microtubules is always applied to the cells depositing other wall layers such as S , , S2,and S3 without exception. It is interesting to note that the reorientation of microtubules precedes that of depositing cellulose microfibrils in the transition from the completion of a wall layer to the initiation of the next layer (Fig. 35). Moreover, the treatment of colchicine in which microtubule construction is obstructed by the formation of conjugation with microtubule protein tubulin resulted in a remarkable disturbance of depositing cellulose microfibrils (Fig. 36) [77,78]. These results clearly show that although microtubules present inside the plasma membrane cannot readily synthesize long and rigid microfibrils, these are involved in a great extent in the control of depositing cellulose microfibril orientation.

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FIGURE 32 Continued

B. Lignin DepositionLignin is a very important cell wall component, particularly in wood cells, for the enhancement of the physical properties of cell walls and also for sealing the wall from prevention of waterleaks.Fortunately,lignincan be detected under an ordinary light microscope with the use of several stains such as a Wiesner reagent and Maule color reaction. Ultraviolet microscopy is especially useful for the quantitative analysis of lignin distribution in the cell wall [79] and more useful for studying the types of lignins present in the cell wall [80,81]. Needless to say, transmission electron microscopy coupled with potassium permanganate staining [82] or hydrofluoric acid treatment [83], electron probe microanalysis [84], and autoradiography [85-901 are also very useful for the observation of lignin from various points of view. The lignification of tracheid walls is generally known to last for a long period, from the S, stage to theF stage [79]. During this period, the lignification starts at the cell comer, spreads into the intercellular layer, and extends centripetally to the secondarywall. Lignin deposition, however, should be examined more closely in relation to the deposition of cellulose and hemicelluloses on each wall layer. This was attempted in the differentiating tracheids of compression wood, which were convenient for separating the lignification of the I region and S region because of their conspicuously highly lignified secondary wall, especiallyattheouterregion of the S, layer [67]. It has been shownthatthelignin deposition can be separated into two lignification stages, namely, I- and S-lignification. The former is active only during the early stage of secondary wall thickening, mainly at

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FIGURE 33 Electron micrographs of the cytoplasm-cell wall region of a transverse section from a tension wood fiber of Populus euramericana depositmg G layer (a) and of an obliquely sliced section from a S,-depositing fiber (b).

the S , stage, and is soon finished. The shape of I-lignification seems to stop the enlargement of cell size and adheres firmly between neighboring cells. On the other hand, the latter proceeds mainly after the development of a secondary wall framework, even though it begins at the middle phase of S , thickening. At any rate, lignin precursors permeate deeply into the cellulose microfibril framework of both primary and secondary walls and accumulate by way of intussusception. These two types of lignification were also applied in the differentiationof normal wood tracheids[86,91,92]. Moreover, when the speed

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.

..... .,v*

(b) FIGURE 33 Continued

of lignin accumulation and the distribution of peroxidase were compared between the two regions [86], I-lignin seemed to be richer in the condensed-type lignin caused by the bulk polymerization than the S-lignin. This would be so because lignification proceeds with the higher content of lignin monomers and peroxidasein a rather large space without microfibrils. This assumption was confirmed selectively labeled precursors coupled with by light microscopic autoradiography [89,90]. When the lignifying cells are observed from the viewpoint of cytology, the cytoplasm is wider and denser than that of cells depositing cellulose microfibrils. Especially in the compression wood tracheids, an enormous amountof lignin precursors must be synthesized

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FIGURE 34 Electron micrographs of 45"-inclined sections from compression wood tracheids in Cryptomeria japonica. (a) Shows the cytoplasmic feature in the cell just beginning S , deposition. At the cytoplasm-cell wall region (enlarged view), the distribution of microtubules (MT) is similar to that in Fig. 33a, although the cytoplasm is full of cell organelles, especially Golgi bodies. (b)

Shows the huge ridges and cavities in the of Golgi vesicles.

S , layer and poor cytoplasm after the active exmytocis

in their cytoplasm and thentransported from the cytoplasm to the wall. The area of cytoplasm becomes wider at the S , stage and also at the transition from Szto F (Fig. 30), where the cytoplasm becomes rich in Golgi bodies (Go) and ER (Fig. 34a). Although small vesicles (v) are produced mainly from Golgi bodies, they do not move to the cytoplasmic surfaceyet. These small vesicles increase number and grow larger, occupying in

Ultrastructure and Formation Cell Wall of

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I

FIGURE 34 Continued

the largest part of the cytoplasm during the following late phase of the S, stage. S-lignification at the F stage is characterized by the active fusion of the well-developed vesicles to the plasma membrane and by the release of the vesicle inclusion to the wall area, namely, exocytocis. The cytoplasmic area results the formationof an empty region after in lignification (Fig. 3