advanced wood chemistry 1web.nchu.edu.tw/pweb/users/taiwanfir/lesson/11941.pdf · flattened at ends...

Advanced in Wood Chemistry

Dr. David S. -Y. Wang

Course Outline

1.Formation of Wood Cell Wall

2.Chemical Composition and Distribution of Wood

3.Chemical Characterization of Wood and Its Components

4.Extractives

5.Weathering and Photochemistry of Wood

6.Degradation of Lignin in Relation to Bioremediation

7.Recycling of Wood and Fiber Products

8.Wood Plasticization

The important of trees

• Trees are the longest-lived members of

natural world.

• Trees providing fuel, raw materials, food,

and drugs.

• Moderating the environment and

contribute to climate stability.

Formation of Wood Cell Wall - General structure of wood

• Wood - the secondary xylem formed by cell division in the vascular cambium of both gymnosperms and angiosperms, and especially in Ginkgo.

• Softwood and hardwood

• Sapwood and Heartwood

• Reation wood

Wood cells are produced in the

vascular cambium from two type

of meristematic cells

Fusiform initial

Ray initial

Wood Cell

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The microstructure of wood

showing the various scales. The cell itself is a long “fiber”, about 100 times

its width (which is only 20 to 40 micrometers).

The cell wall is made of microfibrils one of the

most accepted models (we don’t really know)

of which is shown below. The microfibrils are

made of bundles of cellulose chains, mostly

crystalline. These microfibrils are wound in a

spiral or helix within the walls and the closer

this helical angle (microfibril angle) is to the

fibre axis, the stronger and stiffer is the fibre.

Current status of tree cell biology - Hierarchical approach

• Precursor cells - fusiform and ray cambial cells.

• Cell types in the secondary vascular system of the angiosperm tree Aesculus hippocastanum L.

Secondary phloem Vascular cambium Secondary xylem

Axial systemFusiform initial

Axial parenchyma

Fibre

Sieve element

Companion cell

Radial system

Ray parenchyma Ray initial

Axial parenchyma

Fibre

Vessel element

Isolation ray cell

Contact ray cell

Sieve element:篩管分子Aesculus hippocastabum: 七葉樹

Some of the changes that take place as cambial derivatives undergo secondary vascular differentiation in Aeculus hippocastanum

Feature Changed effected Cell type affected

Shape/size Height/width altered All

Flattened at ends Some sieve and vessel elements, axial parenchyma

Widened All

Shortened Axial parenchyma

Cell walls Thickened All

More-ordered All

Multi-layered S1, 2, 3 layers, Xylem cells

Additional components (e.g. liginied) Xylem cells and phloem fibres

± elaborated (e.g. pits, tertiary thickenings, sieve perforation plates)

All

± loss of plasmodesmata (e.q. inter-vessel pits, vessel-ray contact pits)

Contact ray cells and vessel elements

Cytoplasm Nuclei repositioned All

Cytoskeleton All

Total loss of contain Fibres, vessel elements

Selective loss of contents Sieve elementsNo loss of contents Xylem and phloem parenchyma

Current Status of Tree Cell Biology

• Anatomical

• Microscopical techniques

• Transmission electron microscopy (TEM).

• Anatomical/Biochemical

• Immunological procedure is apply to anatomical study.

• Structural biology - the role of the cytoskeleton.

• Biochemistry - cryo-sectioning technique.

• Molecular-genetical.

cryo-sectioning：冷凍切片

Wood Development Xylogenesis - an example of cell differentiation in an exceptionally

complex form, which controlled by a wide variety of factors:

•Exogenous - photoperiod and temperature

•Endogenous - phytohormones

•Interaction between exogenous and endogenous

•It is driven by the coordinated expression of numerous structural genes

involved in cell origination, differentiation, programmed cell death, and

heartwood (HW) formation and by virtually unknown regulatory genes

orchestrating this ordered developmental sequence.

WOOD BIOSYNTHESIS

• Wood (secondary xylem) is manufactured by a succession of five major steps:

• Cell division

• Cell expansion (elongation and radial enlargement)

• Cell wall thickening (involving cellulose, hemicellulose, cell wall proteins, and lignin biosynthesis and deposition)

• Programmed cell death

• HW formation.

22 Physiology of Woody Plants

their relative position within a growth layer or increment.

The boundary between the earlywood and late-wood of the same ring can be very sharp or gradual. The boundary is sharp in hard pines, Douglas-fi r, larch, and juniper. Ladefoged (1952) found an abrupt early-wood–latewood transition in ring-porous angiosperms and a gradual one in diffuse-porous species. Various arbitrary methods of clearly characterizing both early-wood and latewood cells have been advanced. One of the most popular standards for gymnosperms is that of Mork (1928), who considered a latewood tracheid to be one in which the width of the common wall between the two neighboring tracheids multiplied by two was equal to, or greater than, the width of the lumen. When the value was less than the width of the lumen the xylem was considered to be earlywood. All measurements were made in the radial direction. Mork’s defi nition originally was applied to spruce xylem but has been adopted widely for general use with gymnosperm woods. This defi nition is not useful for angiosperm woods because there often are serious problems in distinguishing between earlywood and latewood.

Within an annual xylem increment the width of the earlywood band generally decreases and the width of the latewood band increases toward the base of the tree. In gymnosperms the earlywood tracheids are wider toward the stem base than near the top of the stem within the same xylem increment. The transition between the last earlywood tracheids and fi rst-formed latewood tracheids of the annual increment also is sharper in the lower stem than in the upper stem.

Some tracheids fi t the usual defi nition of latewood because of a decrease in their radial diameter, without appreciable change in wall thickness. Other tracheids, however, become latewood because of an increase in wall thickness without a change in diameter. Both dimensions show continuous change from the top of the stem toward the base until the latewood forms. In upper parts of stems “transition latewood” often forms, which cannot be conveniently classifi ed as either true earlywood or latewood (Fig. 2.18).

Cambial growth of tropical trees is very diverse and appears to be strongly determined by heredity. In many species xylem may be added to the stem during most or all of the year. Hence, many tropical trees, especially those in continually warm and wet tropical climates, lack growth rings or have very indistinct ones. Examples are Agathis macrophylla in Melanesia, many tropical mangroves, and mango in India (Whitmore, 1966; Fahn et al., 1981; Dave and Rao, 1982). Other tropical species produce distinct growth rings, often more than one each year.

The anatomical features that delineate growth rings in tropical woods vary greatly among species. In Acacia catechu, for example, growth rings are outlined by narrow bands of marginal parenchyma, and some-times by thick-walled fi bers in the outer latewood. The growth rings of Bombax malabaricum are identifi ed by radially compressed fi bers and parenchyma cells in the outer latewood. The xylem increments of Shorea robusta have many irregularly shaped parenchyma bands that sometimes are mistaken for annual rings.

Phloem Increments

The annual sheaths of mature phloem are much thinner than the increments of xylem because less phloem than xylem is produced annually. The total thickness of phloem in general also is limited because the old phloem tissues often are crushed, and eventu-ally the external nonfunctional phloem tissues are shed.

In many woody plants the phloem is divided by various structural features into distinguishable growth increments. However, these are not as clearly defi ned as annual xylem increments. Often the structural dif-ferences of early and late phloem are rendered indis-tinguishable by collapse of sieve tubes and growth of parenchyma cells.

In some species the annual increments of phloem can be delineated because early phloem cells expand more than those of the late phloem. In pear, tangential bands of fi ber sclereids and crystal-containing cells are characteristic boundaries of annual growth of phloem (Evert, 1963). Early and late phloem increments some-times can be identifi ed by features of phloem paren-chyma cells. For example, phloem parenchyma cells

FIGURE 2.18. Seasonal variation in formation of earlywood, transition latewood, and latewood at different stem heights of a red pine tree. From Larson (1969).

Ch002-P088765.indd 22Ch002-P088765.indd 22 9/10/2007 3:29:56 PM9/10/2007 3:29:56 PM

Seasonal variation in formation of earlywood, transition latewood, and latewood at different stem heights of a red pine tree.

CAMBIAL GROWTH Vegetative Growth 55

would elongate to form lateral branches (Lanner, 1964, 1966). Lloyd (1914) described such growth as foxtail-ing because the upper part of the abnormally elongat-ing shoot had a conical or “foxtail” appearance (Fig. 3.14). This striking form of exaggerated apical dominance often produces trees with up to 6 m, and occasionally up to 13 m, of branchless stem. Foxtailing is a problem of variable degree wherever pines are grown in the tropics (Kozlowski and Greathouse, 1970).

CAMBIAL GROWTH

Increase in the diameter of tree stems occurs primar-ily from meristematic activity in the vascular cambium, a cylindrical lateral meristem located between the xylem and phloem of the stem, branches, and woody roots.

Over the years there has been spirited controversy about whether the term cambium should refer exclu-sively to a single layer of cambial initials or whether it should encompass both the cambial initials and their recent derivatives, the xylem mother cells and phloem mother cells. One problem is the diffi culty of identify-ing the single (uniseriate) layer of initials. Although recognizing the existence of such a layer it often is useful to use the term “cambial zone” to refer to the entire zone of dividing cells (the uniseriate layer plus the xylem and phloem mother cells). The cambial zone in dormant trees may vary from 1 to 10 cells in the radial plane of the stem, but in growing trees the width

is extremely variable. Bannan (1962) found the cambial zone to be 6 to 8 cells wide in slow-growing trees and 12 to 40 cells wide in fast-growing trees. A useful ter-minology for the various cell types and tissues involved in cambial activity is given in Fig. 3.15.

Cell Division in the Cambium

Two types of cell division occur in the cambium: additive and multiplicative. Additive division involves periclinal (tangential) division of fusiform cambial ini-tials to produce xylem and phloem mother cells, which in turn divide to produce xylem and phloem cells. Multiplicative division involves anticlinal divisions of fusiform initials, which provide for circumferential expansion of the cambium.

Production of Xylem and Phloem

Following winter dormancy the cambium of tem-perate zone trees is reactivated to produce xylem inwardly and phloem outwardly. New annual incre-ments of xylem and phloem are thus inserted between old layers of these tissues, causing the stem, branches, and major roots to increase in thickness.

Most investigators agree that cambial reactivation occurs in two stages, which involve change in appear-ance of the cambium (change in color, translucence, slight swelling) (Evert, 1960, 1963; Deshpande, 1967), followed by mitotic activity that produces cambial derivatives. As the second phase begins, the fi rst few cell divisions may be scattered and discontinuous at different stem levels in large trees having buds on

FIGURE 3.14. Five-year-old normally branched trees and branchless foxtail forms of Carib pine in Malaysia. From Kozlowski and Greathouse (1970).

FIGURE 3.15. Terminology for describing cell types and tissues associated with cambial growth. From Wilson et al. (1966).


The cambial zone in dormant trees

may vary from 1 to 10 cells in the

radial plane of the stem, but in

growing trees the width is extremely

variable. Bannan (1962) found the

cambial zone to be 6 to 8 cells wide in

slow-growing trees and 12 to 40 cells

wide in fast-growing trees.

Time of Growth Initiation and Amounts of Xylem and Phloem Produced

56 Physiology of Woody Plants

many lateral branches. Nevertheless, once seasonal cambial growth starts, the xylem growth wave is propagated downward beginning at the bases of buds (Wilcox, 1962; Tepper and Hollis, 1967).

Time of Growth Initiation and Amounts of Xylem and Phloem Produced

Undifferentiated overwintering xylem mother cells are rare except in very mild climates or under unusual circumstances (Larson, 1994). Photographs of stem transections taken during the dormant season typi-cally show undifferentiated cambial zone mother cells abutting directly on mature xylem cells. In contrast, immature sieve elements or phloem parenchyma cells very commonly overwinter in partially differentiated states (Evert, 1960, 1963; Davis and Evert, 1968). These cells are the fi rst to expand and mature the following spring (Larson, 1994).

Many studies suggest that cambial reactivation to produce phloem cells precedes xylem production. For example, in black locust phloem production began about a week before xylem production (Derr and Evert, 1967). In many diffuse-porous angiosperms and in gymnosperms phloem production occurs fi rst. In trem-bling aspen, jack pine, red pine, and eastern white pine phloem production preceded xylem production by several weeks (Evert, 1963; Davis and Evert, 1965; Alfi eri and Evert, 1968). In eastern larch, balsam fi r, and black spruce much of the annual phloem incre-ment was produced even before any xylem cells formed (Alfi eri and Evert, 1973). For the fi rst month and a half of cambial activity in pear trees, most of the cambial derivatives were produced on the phloem side (Fig. 3.16). By the middle of May, four to six rows of mature or partially differentiated sieve elements had formed. This amounted to approximately two-thirds of the total produced for the year (Evert, 1963). In horse-chestnut the fi rst cambial divisions to produce new phloem cells began fi ve weeks before any xylem cells were cut off by the cambium (Barnett, 1992).

Patterns of cambial reactivation of tropical species are diverse (Fahn, 1990). In Polyalthia longifolia, phloem mother cells that went through the dormant period differentiated fi rst. Later phloem cells formed by divi-sion of cambial initials. Subsequently phloem produc-tion stopped and xylem production began. Much later production of xylem stopped and phloem production resumed (Ghouse and Hashmi, 1978, 1979). Avicennia resinifera and Bougainvillea spp., which form successive cambia, produce alternating bands of xylem and phloem (Studholme and Philipson, 1966; Esau and Cheadle, 1969). In the evergreen species, Mimusops elangi, the fi rst cambial derivatives formed on the xylem side (Ghouse and Hashmi, 1983).

By the end of the growing season the number of xylem cells cut off by the cambium greatly exceeds the number of phloem cells produced. This is so even in species in which initiation of phloem production pre-cedes initiation of xylem formation. In white fi r the xylem and phloem cells were produced in a ratio of 14 to 1 (Wilson, 1963). In at least some species, xylem production is more sensitive than phloem production to environmental stress. Hence as conditions for growth become unfavorable, the xylem-phloem ratio often declines. In northern white cedar the xylem-phloem ratio fell from 15 to 1 to 2 to 1 as tree vigor decreased (Bannan, 1955). These relations apparently do not hold for certain subtropical species that lack recognizable annual growth rings in the phloem. In Murray red gum, for example, the ratio of xylem to phloem pro-duction changed little under different environmental conditions. A similar xylem-phloem ratio, about 4 to 1, was found for both fast-growing and slow-growing trees (Waisel et al., 1966).

Differentiation of Cambial Derivatives

After xylem and phloem cells are cut off by the cambial mother cells they differentiate in an ordered sequence of events that include cell enlargement, sec-ondary wall formation, lignifi cation, and loss of proto-plasts (Fig. 3.17). These events do not occur stepwise, but rather as overlapping phases. For example, secondary wall formation often begins before growth

FIGURE 3.16. Seasonal changes in cambial activity of pear trees. From Evert (1960). Originally published by the University of California Press; reprinted by permission of the Regents of the University of California.


Seasonal changes in cambial activity of pear trees.

For the first month and a half of cambial activity in pear trees, most of the cambial derivatives were produced on the phloem side. By the middle of May, four to six rows of mature or partially differentiated sieve elements had formed. This amounted to approximately two-thirds of the total produced for the year. In horse-chestnut the fi rst cambial divisions to produce new phloem cells began five weeks before any xylem cells were cut off by the cambium.

Differentiation of Cambial Derivatives Vegetative Growth 57

of the primary wall ends. During cell differentiation most cambial derivatives are altered morphologically and chemically into specialized elements of various tissue systems.

Cambial derivatives that are cut off on the inner side of the cambium to produce xylem may differentiate into one of four types of elements: vessel members, fi bers, tracheids, or parenchyma cells. Vessel members, tracheids, fi ber tracheids, and libriform fi bers develop secondary walls and the end walls of vessels become perforated, but the derivatives of ray initials change little during differentiation. However, the ray tracheids of gymnosperms are greatly altered as they develop secondary walls and lose their protoplasts. Changes in cell size also vary appreciably among different types of cambial derivatives.

The molecular and cellular events that direct xylem element growth and differentiation are complex. For example, Allona et al. (1998) indicated that hundreds of genes are expressed in immature xylem of young loblolly pine trees. Using expressed sequence tag (EST) data derived from this tissue and publicly available sequence database information, the authors identifi ed cDNAs representing several classes of proteins that would be expected in growing regions, including cell wall proteins, lignin biosynthetic and carbohydrate metabolism enzymes and several proteins (e.g., protein kinases and transcription factors) that may regulate cell wall synthesis.

The cell cytoskeleton likely also plays a substantial role in xylem cell growth and differentiation. Chaffey

et al. (1999, 2000) discussed the possible roles of microtubules (MT) and microfi laments in cell wall development of angiosperms. Microtubules in fusi-form cambial initials are arranged randomly. As fi bers begin to differentiate, MT become more organized and assume a dense, single-layered helical arrangement. This pattern persists as the cell passes from primary to secondary wall deposition and orientation of MT coin-cides with that of cellulose microfi brils leading to the widely-held idea that MTs somehow direct the deposi-tion of cellulose, perhaps through positioning of cellulose synthase rosettes (Chapter 7). In vessel ele-ments MT are associated with tertiary helical thicken-ings, pit borders, and edges of perforation plates. Microfi laments (MF) in fusiform intitals have a loose axial orientation that persists after helically-oriented wall deposition begins.

As fi bers undergo axial lengthening during this stage, MF may be involved in delivery of secretory vesicles that play a role in growth in the axial direction. In developing vessel elements, additional MF pres-ences occur in rings that form early in the development of bordered pits, circular arrays at contact pit locations,

FIGURE 3.17. Variations in radial fi les of tracheids of red pine at different times during the growing season. 1: Primary wall zone; 2: Cytoplasm zone; 3: Flattened latewood cells; 4: More latewood cells; 5: Mature earlywood; P: Phloem; L: Latewood of the preceding year. From Whitmore and Zahner (1966). FIGURE 3.18. Epifl uorescent images of the vessel elements in

the late stage of secondary thickening, showing (left) narrow bands of microtubules (darts) and branching microtubules (arrows) and (right) the ring of microtubules associated with development of the perforation (P), rings of microtubules associated with bordered-pit development (darts, left), and strands of microtubules associated with tertiary thickening T. From Planta. A cytoskeletal basis for wood formation in angiosperm trees: The involvement of cortical microtubules. Chaffey, N., Barnett, J., and Barlow, P., 208, 19–30, Figure 5. © 1999 with kind permission from Springer Science and Business Media.


Variations in radial fi les of tracheids of

red pine at different times during the

growing season.

1: Primary wall zone;

2: Cytoplasm zone;

3: Flattened latewood cells;

4: More latewood cells;

5: Mature earlywood; P: Phloem; L:

Latewood of the preceding year.

in a manner so precise that microfibrillar cellulose islargely crystalline. Until recently, not even one singleenzyme involved in cellulose biosynthesis and dep-osition was purified or cloned (Arioli et al., 2000). Afirst breakthrough had been the identification ofgenes encoding the catalytic subunit of the cellulosesynthase (Ces) complex. In Arabidopsis, at least sixgenes encode putative catalytic subunits of Ces. Inaddition, a large gene family of over 20 more dis-tantly related genes, so-called Ces-like (Csl) genes,exists, whose gene products most likely are involvedin the synthesis of other polysaccharides. In higherplants, the substrate for Ces (UDP-d-Glc) is providedby Suc synthase. The complex Ces/Suc synthase isthought to have a cytoplasmic localization and thegrowing cellulose chain may be secreted through themembrane via a pore. Cortical microtubules (mainlycomposed of !- and "-tubulin) may determine thewall pattern by defining the position and orientationof cellulose MFs during the differentiation of con-ducting elements (Chaffey, 2000), probably by guid-ing the movement of the cellulose-synthesizing com-plex in the plasma membrane. However, although inmany cases co-orientation of microtubules and MFswere observed, mathematical models (that remain tobe tested in wood-forming tissue) relying on thegeometry of the cell, have been proposed to challengethis dogma (Mulder and Emons, 2001).

The water-insoluble cellulose MFs are associatedwith mixtures of soluble noncellulosic polysaccha-rides, the hemicelluloses, which account for about25% of the dry weight of wood. They generally occuras heteropolymer such as glucomannan, galactoglu-comannan, arabinogalactan, and glucuronoxylan, oras a homopolymer like galactan, arabinan, and "-1,3-glucan. The biosynthesis of these polysaccharides oc-curs in the Golgi apparatus by a process that can bedivided into two main steps: the synthesis of thebackbone by polysaccharide synthases, and the ad-dition of side chain residues in reactions catalyzed bya variety of glycosyltransferases (Keegstra andRaikhel, 2001).

The third major component of wood (25%–35%) islignin, a phenolic polymer derived from three hy-droxycinnamyl alcohols (monolignols): p-coumarylalcohol, coniferyl alcohol, and sinapyl alcohol, givingrise to H, G, and S units, which differ from each otheronly by their degree of methoxylation. Lignin em-beds the polysaccharide matrix giving rigidity andcohesiveness to the wood tissue as a whole, andproviding the hydrophobic surface needed for thetransport of water. Lignin content and monomericcomposition vary widely among different taxa, indi-viduals, tissues, cell types, and cell wall layers. As anexample, no S units are detected in gymnospermscompared with angiosperms. Lignin biosynthesis has

Figure 1. A, Drawing of a transverse section of the cambial zone of maritime pine (Pinus pinaster) showing the fusiform (F)and ray (R) initial cells in the cambial zone (CZ). X, Centripetal xylem differentiation with radially enlarging, maturing, andmature xylem. P, Centrifugal phloem differentiation with radially enlarging, maturing, and mature phloem. Empty arrowheadindicates a newly deposited radial wall (anticlinal division). Full arrowhead indicates a newly deposited tangential wall(periclinal division). B, Three-dimensional scheme of maritime pine wood showing the relatively homogeneous structure ofconifer xylem. Ninety percent of the wood is made of tracheids, and the remainder is composed of ray parenchyma andlongitudinal parenchyma cells, as well as resin ducts in certain species. Sap water ascends via the xylem and nutritive sapdescends via the phloem. Sap can also be transported radially via the ray cells and tangentially by bordered pits. It isimportant to note the different direction and faces which are needed to describe wood structure: transverse, radial, andtangential sections.

Wood Formation in Trees

Plant Physiol. Vol. 127, 2001 1515

F: fusiform initial cells

R: ray initial cells in the cambial zone

CZ: cambia.

X: Centripetal xylem differentiation with radially

enlarging, maturing, and mature xylem.

P: Centrifugal phloem differentiation with radially

enlarging, maturing, and mature phloem.

Empty arrowhead indicates a newly deposited radial

wall (anticlinal division).

Full arrowhead indicates a newly deposited

tangential wall (periclinal division).

Transverse section of the cambial zone of maritime pine (Pinus pinaster)

in a manner so precise that microfibrillar cellulose islargely crystalline. Until recently, not even one singleenzyme involved in cellulose biosynthesis and dep-osition was purified or cloned (Arioli et al., 2000). Afirst breakthrough had been the identification ofgenes encoding the catalytic subunit of the cellulosesynthase (Ces) complex. In Arabidopsis, at least sixgenes encode putative catalytic subunits of Ces. Inaddition, a large gene family of over 20 more dis-tantly related genes, so-called Ces-like (Csl) genes,exists, whose gene products most likely are involvedin the synthesis of other polysaccharides. In higherplants, the substrate for Ces (UDP-d-Glc) is providedby Suc synthase. The complex Ces/Suc synthase isthought to have a cytoplasmic localization and thegrowing cellulose chain may be secreted through themembrane via a pore. Cortical microtubules (mainlycomposed of !- and "-tubulin) may determine thewall pattern by defining the position and orientationof cellulose MFs during the differentiation of con-ducting elements (Chaffey, 2000), probably by guid-ing the movement of the cellulose-synthesizing com-plex in the plasma membrane. However, although inmany cases co-orientation of microtubules and MFswere observed, mathematical models (that remain tobe tested in wood-forming tissue) relying on thegeometry of the cell, have been proposed to challengethis dogma (Mulder and Emons, 2001).

The water-insoluble cellulose MFs are associatedwith mixtures of soluble noncellulosic polysaccha-rides, the hemicelluloses, which account for about25% of the dry weight of wood. They generally occuras heteropolymer such as glucomannan, galactoglu-comannan, arabinogalactan, and glucuronoxylan, oras a homopolymer like galactan, arabinan, and "-1,3-glucan. The biosynthesis of these polysaccharides oc-curs in the Golgi apparatus by a process that can bedivided into two main steps: the synthesis of thebackbone by polysaccharide synthases, and the ad-dition of side chain residues in reactions catalyzed bya variety of glycosyltransferases (Keegstra andRaikhel, 2001).

The third major component of wood (25%–35%) islignin, a phenolic polymer derived from three hy-droxycinnamyl alcohols (monolignols): p-coumarylalcohol, coniferyl alcohol, and sinapyl alcohol, givingrise to H, G, and S units, which differ from each otheronly by their degree of methoxylation. Lignin em-beds the polysaccharide matrix giving rigidity andcohesiveness to the wood tissue as a whole, andproviding the hydrophobic surface needed for thetransport of water. Lignin content and monomericcomposition vary widely among different taxa, indi-viduals, tissues, cell types, and cell wall layers. As anexample, no S units are detected in gymnospermscompared with angiosperms. Lignin biosynthesis has

Figure 1. A, Drawing of a transverse section of the cambial zone of maritime pine (Pinus pinaster) showing the fusiform (F)and ray (R) initial cells in the cambial zone (CZ). X, Centripetal xylem differentiation with radially enlarging, maturing, andmature xylem. P, Centrifugal phloem differentiation with radially enlarging, maturing, and mature phloem. Empty arrowheadindicates a newly deposited radial wall (anticlinal division). Full arrowhead indicates a newly deposited tangential wall(periclinal division). B, Three-dimensional scheme of maritime pine wood showing the relatively homogeneous structure ofconifer xylem. Ninety percent of the wood is made of tracheids, and the remainder is composed of ray parenchyma andlongitudinal parenchyma cells, as well as resin ducts in certain species. Sap water ascends via the xylem and nutritive sapdescends via the phloem. Sap can also be transported radially via the ray cells and tangentially by bordered pits. It isimportant to note the different direction and faces which are needed to describe wood structure: transverse, radial, andtangential sections.



• Ninety percent of the wood is made of tracheids, and the remainder is composed of ray parenchyma and longitudinal parenchyma cells, as well as resin ducts in certain species.

• Sap water ascends via the xylem and nutritive sapdescends via the phloem. Sap can also be transported radially via the ray cells and tangentially by bordered pits. It is important to note the different direction and faces which are needed to describe wood structure: transverse, radial, and tangential sections.

Three-dimensional scheme of maritime pine wood showing the relatively homogeneous structure of conifer xylem

Cell Walls of Woody Plants - Autoradiography and Ultraviolet Microscopy

• Autoradiography is a useful technique for visualizing physiology and biochemical process within cells and cell wall.

• The incorporation site of radiolabelled precursor is easily identified as black deposits after developing the nuclear emulsion coated on the specimen.

• Light microscopy autoradiographs of transverse sections of differentiation secondary xylem from stem of Cryptomeria japonica fed 3H-phenylalanine.

‣（a）Silver grains are observed

at cell corner regions in the

tracheids of S1 formation stage.

• （b）Numerous silver grains are

distributed on the secondary

walls in the tracheids just after S3

formation.

Cell Walls of Woody Plants - Autoradiography

and Ultraviolet Microscopy

• A UV-microscope is a powerful tool for the detection of

lignin absorbs UV light, whereas other cell wall

components.

• Cellulose, pectins and hemicellulose do not absorbs UV

light.

Transverse section of differentiating

secondary xylem from stem of

Cryptermeria japonica photographed in UV

light at 280 nm

TEM microscopic autoradiograph of differentiating secondary xylem from stem of Cryptomeria japonica. Radiolabelled products are observed on the cell wall and near the Golgi appartus (G).

in the moss Physcomitrella patens where a mannan epitopeoccurs differentially in relation to cell types [30]. Aspectroscopic and biochemical study of cellular differen-tiation in the single-celled alga Acetabularia acetabulum hashighlighted the role of crystalline mannan in this organ-ism and that cell wall polymers vary with cell morphology[31]. The available antibody probes appear to recognizemannnosyl residues but bind to varying degrees to glu-comannans and galactomannans. Members of family 27and 35 CBMs can show better specificity in this regardand bind to cell walls in a more restricted way suggestingthe spatial regulation of mannan structure in xylem cells[9]. The function of mannans in cell walls is not clear, andit is intriguing that the addition of galactoglucomannanoligosaccharides to Zinnia cultures indicates a role forthese polymers in cell differentiation [32].

The polymers of primary cell walls display a widermolecular and functional diversity than those of second-ary cell walls. Differences are beginning to be mapped forall three of the major polymer classes: cellulose, hemi-cellulose and pectins. Cellulose-directed CBMs displaymajor differences in binding to cell types reflectingdifferent cell structural features such as intercellularspaces and collenchyma thickenings [8]. In some cases,mannans are abundant in epidermal cell walls [28,29].The epidermis is an important growth controlling celllayer [33] often with a distinctively thickened outer radialcell wall [34] and the systematic mapping of cell wallmicrostructures of epidermal cell walls in relation togrowth is an important target. Analysis of guard cells ina range of species has indicated that these specializedepidermal cells have a distinctive cell wall composition

310 Physiology and metabolism

Figure 1

Cell type and cell wall diversity in a plant organ. Immunofluorescence analysis of cell walls in transverse sections of young developing stems ofindustrial hemp. Moving in from the stem surface there is an epidermal cell layer, subepidermal cell layers, a collenchyma bundle, a band of developing(primary) phloem sclerenchyma fibres (arrowheads indicate distal edge), a region of parenchyma cells that contains phloem cells and where secondaryphloem fibres develop, a cambium layer and xylem. Interior to the xylem is the pith parenchyma (not shown). Probes used are crystalline cellulose-directed CBM3a [8], xylan monoclonal antibody LM11 [19], pectic HG monoclonal antibody JIM7 [50] and (1 ! 4)-b-galactan monoclonal antibodyLM5 [42]. CBM3a binds to all cell walls although with differing intensities. LM11 binds to cells with secondary cell walls only (phloem fibres and xylemvessel elements) and JIM7 binds to primary cell walls and most abundantly to a subepidermal cell layer and to cortical parenchyma cells. The LM5epitope is notably absent from cell walls of developing phloem fibre cells but abundant in adjacent parenchyma. Scale, 100 mm. Figure developed frommicrographs provided by Blake et al. [21].

Current Opinion in Plant Biology 2008, 11:308–313 www.sciencedirect.com

Cell type and cell wall diversity in a plant organ. Immunofluorescence analysis of

cell walls in transverse sections of young developing stems of industrial hemp

Moving in from the stem surface there is an epidermal cell layer, subepidermal cell layers, a collenchyma bundle, a band of developing (primary) phloem sclerenchyma fibres (arrowheads indicate distal edge), a region of parenchyma cells that contains phloem cells and where secondary phloem fibres develop, a cambium layer and xylem. Interior to the xylem is the pith parenchyma (not shown). Probes used are crystalline cellulosedirected CBM3a, xylan monoclonal antibody LM11, pectic HG monoclonal antibody JIM7 and (1 → 4)-β-galactan monoclonal antibody LM5. CBM3a binds to all cell walls although with differing intensities. LM11 binds to cells with secondary cell walls only (phloem fibres and xylem vessel elements) and JIM7 binds to primary cell walls and most abundantly to a subepidermal cell layer and to cortical parenchyma cells. The LM5 epitope is notably absent from cell walls of developing phloem fibre cells but abundant in adjacent parenchyma. Scale, 100 μm.

Concept illustration of the relationship between the deposition of cell

wall polymers and formation of the heterogeneous structure of lignin

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Structure and synthesis of cell walls

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Liner chains of (1→4)-linked β-D-glucose. Each microfibril may consists of 6 to

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high tensile strength, equivalent to steel. Insoluble, chemically stable. Excellent

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• Multilayers • 45% cellulose, less hemicellulose and pectins • Orientation of the cellulose microfibrils are more neatly aligned • Often impregnated with Lignin: 35%, stronger than cellulose – Phenolic polymer with complex linkage of aromatic alcohol subunits – Made from phenylalanine by peroxidase and laccase – Displaces water from the matrix and form a hydrophobic network – Prevent wall enlargement, increase mechanical strength, reduce susceptibility of walls to attack by pathogens, also reduced the extractibility of cellulose .

XYLOGENESIS

317

Figure 2 Structural changes in organelles during autolysis of differentiating Zinnia TEs. [Courtesy of Y Watanabe.]

Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 1996.47:299-325. Downloaded from arjournals.annualreviews.orgby National Taiwan University on 09/15/09. For personal use only.

Structural changes in organelles during autolysis of differentiating Zinnia TEs

been the most studied pathway, resulting in the clon-ing of several structural genes (Whetten et al., 1998;Christensen et al., 2000). However, it is somewhatsurprising that recent attempts at engineering ligninbiosynthesis have demonstrated that our currentmodels of the pathway are incomplete (Grima-Pettenati and Goffner, 1999).

Cell wall proteins and pectins are among the otherminor compounds of the cell wall. Although differentproteins are present in the cell wall at different timesduring development, the amount of protein remain-ing in the wood is small. Nevertheless, such proteinscould play important roles determining the compo-sition and morphology of xylem cell walls (Cassab,1998). Abundant cell wall associated proteins havebeen found in many plants and have traditionallybeen classified into four main groups: Gly-rich pro-teins, Pro-rich proteins, arabinogalactan proteins,and Hyp-rich glycoproteins (or extensins). These pro-teins are cross-linked into the cell wall and probablyhave structural functions. Pectins are thought to playa fundamental role in the regulation of cell wallextensibility. They are also thought to be exportedfrom the Golgi apparatus as highly esterified galac-turonan and then de-esterified in muro by cell wall-bound pectin methylesterases, thus allowing the for-mation of intermolecular bonds through calcium ions(Guglielmino et al., 1997; Higuchi, 1997).

When lignification is completed, conducting xylemelements undergo programmed cell death, involvingcell-autonomous, active, and ordered suicide, inwhich specific hydrolases (Cys and Ser proteases,nucleases, and RNase) are recruited (Roberts andMcCann, 2000). Several factors (auxins, cytokinins,and Suc) prepare the cell to die by determining theprofile of hydrolases synthesized by the cell. Thesehydrolases are inactive in the vacuole. By a signalthat remains unknown, a calcium flux provokes thevacuoles to collapse with the release of hydrolases(Jones, 2001) that degrade all of the cellular contentbut not the secondary cell wall.

Regulation of Wood Biosynthesis

The role of phytohormones in procambium initia-tion, cambial cell division, primary cell wall expan-sion, and secondary wall formation has been re-viewed by Sundberg et al. (2000) and Mellerowicz etal. (2001). Recent findings have demonstrated theexistence of an auxin (indole-3-acetic acid [IAA]) gra-dient across the developing vascular tissues of pineand poplar. This IAA concentration gradient seemsto have a function in positional signaling, i.e. cambialderivatives develop according to their position alongthe gradient, and neighboring cell files receive thesame dose to develop in a synchronized manner. ASuc gradient has been observed as well and mayprovide additional positional information for xylemand phloem differentiation. Other hormones than

auxins have been shown to be involved in xylogen-esis by interacting with IAA in a synergetic (gibberel-lins, cytokinins, and ethylene) or inhibitory (abscisicacid) manner.

Knowledge of the molecular events that determinethe differentiation pathway of a cambial derivative isembryonic. Even if the intervening signal transduc-tion steps remain mysterious, we can assume thatthese signaling inputs result in altered patterns ofgene transcription, which in turn requires the activityof specific transcription factors. In particular, consid-erable progress has been made in understanding theroles of transcription factors in controlling lignifica-tion. The analysis of lignification genes have alsoshown the presence in the promoter of conservedmotifs that have been demonstrated to be importantin xylem localized gene expression (Lacombe et al.,2000). Proteins that can bind this motif and activatethe transcription, belong to the MYB family. TwoMYB genes preferentially expressed in Pinus taedaxylem were proposed to be involved in regulatingtranscription during xylogenesis (Newman andCampbell, 2000).

Wood Cell Walls Are Highly Structured

The cell wall is composed of several layers that arefabricated at different periods during cell differenti-ation (Fig. 2). The first layer to be developed after celldivision is called the middle lamella, which is foundbetween the wood cells, and ensures the adhesion ofa cell with its neighbors. The middle lamella is only0.5 to 1.5 !m thick and is made up of pectic sub-stances to which lignin may be added during thedifferentiation period. At the beginning of cell differ-entiation, the primary cell wall forms. This new,highly elastic layer is attached to the middle lamellaand is approximately 0.1 !m thick. The primary cellwall is made up of several layers of MFs, which are

Figure 2. Three-dimensional structure of the secondary cell wall of atracheid (xylem cell). The cell wall is divided into different layers,each layer having its own particular arrangement of cellulose MFs,which determine the mechanical and physical properties of the woodin that cell. These MFs may be aligned irregularly (as in the primarycell wall), or at a particular angle to the cell axis (as in layer S1, S2,and S3). The middle lamella ensures the adhesion between cells.

Plomion et al.

1516 Plant Physiol. Vol. 127, 2001

Wood Cell Walls Are Highly Structured

Ultra-structural arrangement

• It is well known that the prime factor determining mechanical properties of

wood fibres is the arrangement of the cellulose microfibrils at different

angles with respect to the longitudinal fibre axis with variations in different

layers, as well as arrangement into microfibrillar aggregates.

• Cellulose aggregates from that of a single microfibril of 3.5 nm to aggregates

of a side length of 30 nm have been measured within the structure of the S2.

These findings are in good agreement with those obtained by small-angle X-

ray scattering. The aggregates, averaging in size around 16–20 nm, have also

been shown to be able to take up moisture and thus change laterally their

dimensions quite substantially.

122 L. Salmen and I. BurgertArticle in press - uncorrected proofArticle in press - uncorrected proof

Figure 1 Schematic picture of the cell wall of a softwood fibre (tracheid) indicating the cellulose microfibril angle (MFA) of thesecondary wall (S2), the concentric lamellar arrangement of cellulose aggregates interspaced by matrix lamella, the lenticular undu-lating cellulose aggregate structure and the variability of cellulose aggregate sizes, as well as the arrangement of matrix componentsfrom glucomannan (non-substituted xylan in hardwoods) closest to the cellulose microfibrils that outwards associates to a condensedtype of lignin followed by the xylan (more highly substituted xylan in hardwoods) associated to a more non-condensed type of lignin.Modified following an original drawing by Akerholm and Salmen (2003).

obtained by small-angle X-ray scattering (Jungnikl et al.2007). The aggregates, averaging in size around16–20 nm, have also been shown to be able to take upmoisture and thus change laterally their dimensions quitesubstantially (Salmen and Fahlen 2006). Based on this, itcan be supposed that amorphous structures are inte-grated in the aggregates, being either paracrystalline cel-lulosic surfaces or consisting of hemicelluloses, primarilythose of glucomannan in softwood. A close connectionbetween cellulose and glucomannan in softwood hasalso been indicated by dynamic Fourier transform infra-red spectroscopy (FT-IR), where the molecular co-per-formance during mechanical load is demonstrated to bestrong between these components (Akerholm and Sal-men 2001). In hardwoods, occurrence of two types ofhemicelluloses, one low and one more substituted xylan,have been found. The low substituted one is first depos-ited and associated to the cellulose microfibrils andfavours their aggregation (Joseleau 2007).

Along the fibre, the structural arrangement is also var-iable as indicated from transversely fractured rapid freez-ing deep etched (RFDE) samples combined with TEM. Itwas demonstrated by examining surface replicas of part-ly delignified softwood fibres by RFDE-TEM that theypossess an undulating aggregate structure of cellulosefibrils (Bardage et al. 2004). The spacing between theaggregates were characterised as lens shaped with atangential diameter between 3 and 14 nm (Bardage et al.2004). This finding agrees well with early views of Boyd(1982) concerning a lenticular microfibril arrangement, asillustrated in Figure 1. Such a structure with alternatingclose proximities or true aggregations of cellulose fibrilsmay well explain the fact that heat treatment under moistconditions (Fahlen and Salmen 2003) or removal of some

of the matrix components under such conditions maycause an increased cellulose aggregation (Duchesne andDaniel 2000; Hult et al. 2001; Fahlen and Salmen 2003).This may be explained as an increased area of contactbetween fibrils in the aggregates in their longitudinaldirection (Salmen 2006).

The arrangement in space of the cellulose aggregateshas been debated to some extent in the last decade,where the hypothesis of a radial arrangement asopposed to that of the concentric lamellar type was pro-posed (Sell and Zimmermann 1993; Zimmermann et al.1994). However, based on AFM analysis of the fracture,in which radial structures are visible, it is more probablethat these structures are a consequence of energyrelease during fracture (Fahlen and Salmen 2002). Thus,the traditional concepts of a concentric lamellar structureof cellulose aggregates seem to hold. The lamellas havea large variability concerning the cellulose aggregate siz-es. Nevertheless, the average size is the same across theS2 from the lumen side to that of the primary wall (Fahlenand Salmen 2005; Salmen and Fahlen 2006), i.e., thesupramolecular structure is uniform across the S2 wall.

In characterising the structural arrangement of thepolymer matrix, immunolabelling has revealed newinsights with regard to the arrangement of hemicellulosesand lignin in between the cellulose aggregates (Ruel andJoseleau 2005). More non-condensed guaiacyl/syringyland enriched condensed guaiacyl, respectively, wasfound by means of specific polyclonal antibodies againstlignin to be deposited at different stages of cell wall for-mation. TEM on immunogold labelled specimens of pop-lar permitted to follow the deposition of different types ofxylans during the developing phase of the secondary wallby means of polyclonal antibodies against xylan (Ruel

Schematic picture of the cell wall of a softwood fibre (tracheid) indicating the cellulose microfibril angle (MFA) of the

secondary wall (S2), the concentric lamellar arrangement of cellulose aggregates interspaced by matrix lamella, the

lenticular undulating cellulose aggregate structure and the variability of cellulose aggregate sizes, as well as the

arrangement of matrix components from glucomannan (non-substituted xylan in hardwoods) closest to the cellulose

microfibrils that outwards associates to a condensed type of lignin followed by the xylan (more highly substituted

xylan in hardwoods) associated to a more non-condensed type of lignin.

Newly Developed Wood Is Stretched Longitudinally and Compressed Tangentially

arranged randomly within this wall. Pectic sub-stances, lignin, and hemicelluloses can be found be-tween these MFs. As the developing cell reaches itsdefinitive size, a new layer is formed inside the pri-mary cell wall, which is the most important layer forthe cell, in terms of mechanical strength. This newsecondary cell wall is divided into three differentlayers, S1, S2, and S3 (Timell, 1986). Each of theselayers is composed of cellulose MFs, aligned in anordered, parallel arrangement, which differs from Slayer to S layer. Hemicelluloses and lignin are alsopresent in each of these layers. These three S layerscan be modified during cell maturation, which lastsfor several days after the birth of the wood cell, e.g.the amount of lignin and cellulose laid down in thesecondary cell wall may be influenced by abioticfactors such as mechanical stress, i.e. wind and stemlean.

The S1 layer is the thinnest of the S layers, beingonly 0.1 to 0.35 !m thick, and representing just 5% to10% of the total thickness of the cell wall. This layeris considered as an intermediate between the primarycell wall and the S2 and S3 layers. The MF angle withregard to the cell axis is 60° to 80°. The S2 layer is thethickest layer in the secondary cell wall, and is themost important, with regard to mechanical support.The thickness of the S2 layer varies between 1 and 10!m, and accounts for 75% to 85% of the total thick-ness of the cell wall. The MF angle in this layer is 5°to 30° to the cell axis, and can be even higher, de-pending on external mechanical stress (see later sec-tion on reaction wood). The angle of the celluloseMFs in the S2 layer can influence greatly the physicaland mechanical properties of the cell and even stemwood as a whole. As the MF angle increases, withregard to the cell axis, wood becomes less rigid, andthe longitudinal modulus of elasticity decreases, as inthe case of juvenile and compression wood (CW).The innermost layer of the secondary cell wall, the S3layer, is relatively thin, being only 0.5 to 1.10 !mthick. The MFs are ordered in a parallel arrangement,but less strictly than in the S2 layer, and the MF angleis 60° to 90° with regard to the cell axis.

Newly Developed Wood Is StretchedLongitudinally and Compressed Tangentially

Immediately after cell birth, the newly developedcells undergo a several day long “maturation” pe-riod, in which two mechanisms take place in the cellwall: (a) as lignin is deposited, the amorphic cellulosematrix swells transversely, and (b) when cellulosecrystallization occurs, MFs shrink longitudinally. Thecombined effect of these two mechanisms results in alongitudinal shrinking of the cell (Fig. 3). However,as the maturing wood cells are attached to older,already lignified cells, they cannot shrink completely.Hence, these maturing cells are held in a state oftensile stress, and it is only on cutting the wood, that

these “maturation stresses” can be released in theform of residual strains along the longitudinal axis(Archer, 1986). The wood cells at the surface of a treeare therefore stretched longitudinally and com-pressed tangentially and can be said to be held intension. However, as more and more wood is addedto the tree surface, the wood cells inside the trunk areslowly compressed, until they are completely held incompression, toward the center of the trunk. Thisgradient of mechanical stress in a trunk, whereby theoutside is held in tension, and the center in compres-sion, is called growth stress, and can be highly det-rimental to wood quality, resulting in warping andtwisting of boards and planks.

HW: The Final Transformation of Xylem Cells

On cutting a mature tree, several different coloredzones may be observed, a lighter colored externalzone: the sapwood (SW), and often a darker coloredwood: the HW situated at the center of the tree (Fig.4). A third zone, often intermediate in color—thetransition zone, TZ—may exist between the two. SWis known as the functional part of the tree and issometimes termed the “living” part of the tree. Al-though SW contains living cells, most of its mass iscomprised of terminally differentiated non-living tra-cheids or libriform fibers. Soon after their birth in thecambium, wood cells die, except for the longitudinaland radial parenchyma cells, which remain alive andfunctional until several years later, when they toodie. It is when these parenchyma cells become dys-functional, that HW forms. A specific role for HWhas not yet been determined, although it has beensuggested that it forms to provide long-term resis-tance to pathogens or even provides a mechanicalrole in tree support. Recent research suggests thatHW forms as a response to a hydraulic stimulus andthat HW may even develop irregularly, both radially

Figure 3. As the newly developed wood cell (i) begins to differentiate(ii), the deposition of lignin and cellulose in the secondary cell walltends to stretch the cell laterally and cause it to shrink longitudinally(black arrows). However, as the differentiating wood cell is attachedto older wood (iii), it cannot deform completely, thereby setting up amechanical stress in the cell wall (empty arrows). In normal wood(NW), this translates into a tensile stress, therefore, the wood in theouter surface of a tree (iv) is usually held in tension (! and emptyarrows). However, as the new wood accumulates year after year,each new layer exerts a tangential force on the wood already presentin the tree, thereby gradually placing the wood inside the tree undercompression (" and full arrows).



As the newly developed wood cell (i) begins to differentiate (ii), the deposition of lignin and cellulose in the secondary cell wall tends to stretch the cell laterally and cause it to shrink longitudinally (black arrows). However, as the differentiating wood cell is attached to older wood (iii), it cannot deform completely, thereby setting up a mechanical stress in the cell wall (empty arrows). In normal wood (NW), this translates into a tensile stress, therefore, the wood in the outer surface of a tree (iv) is usually held in tension (+ and empty arrows). However, as the new wood accumulates year after year, each new layer exerts a tangential force on the wood already present in the tree, thereby gradually placing the wood inside the tree under compression (- and full arrows).

Heart Wood: The Final Transformation of Xylem Cells

and longitudinally in the trunk, to maintain a con-stant and optimal proportion of SW in the tree stem(Berthier et al., 2001). As yet, there is no clear expla-nation as to why or how HW forms, although atheory has been put forward by Higuchi (1997),which is supported by recent research. Higuchi(1997) proposed that endogenous factors trigger ex-pression of genes involved in phenolics biosynthesisin parenchyma cells bordering the TZ. As the paren-chyma cells die, these phenolics are released anddiffused into the neighboring cell walls and lumens.

Possible candidates that may trigger the molecularsignal include an increase in ethylene, which isneeded for polyphenol production, or a rapid changein water content near the TZ resulting in bordered pitaspiration, and even external mechanical stress, i.e.wind loading or stem lean, as trees which have beensubjected to such a stress, show greater HW forma-tion near the zone of mechanical loading (Berthier etal., 2001). However, HW formation is also underspecies-specific genetic control as some species existwhich do not even produce HW, even at a veryadvanced age (Hillis, 1987). Once genes in the TZhave been activated by a molecular signal, a cascadeof events occurs resulting in eventual cell death. Verylittle work has been carried out on the identificationof the genes involved in this process. However, re-cent research has shown that in black walnut (Juglansnigra), flavanol biosynthesis was up-regulated in the

TZ. Flavonoids are highly soluble polyphenols and inblack walnut, the flavanol production was found tobe highly correlated with the transcript levels ofgenes encoding the enzymes chalcone synthase(CHS), flavanone 3-hydroxylase (F3H), and dihy-droflavonol 4-reductase (DFR), all of which are im-plicated in the flavonoid pathway (Beritognolo et al.,2001). At the same time that these genes are beingexpressed in the TZ, carbohydrate content decreasesdrastically in this zone. Magel et al. (1994) found thatin black locust (Robinia pseudoacacia) non-structuralstorage carbohydrates (Glc, Fru, Suc, and starch) de-crease from the outer to the inner SW, with only traceamounts present in the TZ, thereby suggesting thatthese sugars and starch are necessary for polyphenolsynthesis.

To elucidate the mechanism by which HW forms,future research should concentrate on the genes ex-pressed in the TZ of different species, as well as thefactors, endogenous or exogenous, inducing molecu-lar activity in this zone.

Wood Is a Highly Variable Raw Material

Wood differs among trees. Use of the terms “soft-wood” for gymnosperms and “hardwood” for dicot-yledon angiosperms is a crude acknowledgment ofthis difference. This variability is due to the hetero-geneity of the cell types that make up the differentwoods and the structure of the individual cells. An-atomical, chemical, and physical differences in woodcharacteristics within a single tree are also a commonfeature. These include: (a) variation within the an-nual ring in temperate zones, i.e. early versus latewood, (b) variation due to juvenile wood (JW) withextremely variable properties, ranging from the pithto the bark, particularly in the early years of cam-bium activity, and (c) variation between normal andreaction wood. Wood can also be modified by dam-age from pathogens and by wounding.

Wood Formation Varies during the Growing Season

In temperate regions, the annual course of cambialactivity (dormancy and activation) is induced bytemperature and/or photoperiod (Uggla et al., 2001and refs. therein). In temperate zones, EW is formedearly in the growing season when temperature andphotoperiod are favorable for active growth. EW hasshorter cells and a lower density resulting from thin-walled tracheids or fibers of large radial diameter(Fig. 4B). LW is formed in the late summer or autumnwhen cambial cell division and expansion declines.LW has high density resulting from the small trac-heid/fiber radial diameter and large tangential wallthickness. Having narrower lumens in the woodcells, the LW is much less vulnerable to water-stress-induced xylem embolism, and so increases the safetyof water conductance. The passage of one type of

Figure 4. A, Photograph of a wood disc (Pinus nigra var. Laricio)showing the different types of wood which can be present within atree (photograph courtesy of P. Rozenberg). B, A higher power viewof the wood cells shows the transition between early wood (EW) andlate wood (LW). LW cells have smaller lumens and thicker cell walls.

Plomion et al.

1518 Plant Physiol. Vol. 127, 2001

A: Photograph of a wood disc

(Pinus nigra var. Laricio) showing

the different types of wood which

can be present within a tree

( p h o t o g r a p h c o u r t e s y o f P.

Rozenberg).

B: A higher power view of the

wood cells shows the transition

between early wood (EW) and late

wood (LW). LW cells have smaller

lumens and thicker cell walls.

Identification of wall-related carbohydrate active enzymes in Populus

• Carbohydrate active enzymes (CAZYmes): CAZYmes are involved in the formation and modification of the carbohydrate matrix of wood cell walls.

• Populus has approximately 1600 genes encoding CAZYmes, compared to approximately 900 in Arabidopsis.

• Some gene families have also been characterized in more detail in Populus and other deciduous trees pecies, including genes encoding

• Cellulose synthases (CESAs)

• Cellulose synthase-likesynthases(CSLs)

• Pectin methyl esterases (PMEs)

• XTH genes encoding xyloglucan (XG) endo-transglucosylases and hydrolases (XETs and XEHs)

• Expansins

Modifications of primary cell walls, and implications for wood cell morphology

• Primary-walled developing wood cells of Populus are rich in pectins, cellulose, and XG. During the primary-walled stage, wood cells grow to their final shape by a unique combination of symplastic growth (when the neighboring cells grow together) and intrusive growth (when they move one past another, e.g. fiber elongation).

• Wall plasticity and variations in cell adhesion are key features of the mechanisms involved in wood cell growth.

• Vascular cambium (VC) and adjacent radial expansion (RE) zone are the sites of highest expression of genes encoding wall-modifying enzymes, including expansins, XETs, cellulases, PMEs, polygalac-turonases (PGs), and pectin/pectate lyases (PL1s).

• PMEs modify the methylesterification pattern of homo-galacturonan (HG) in the pectin matrix, thus affecting both wall plasticity and cell–cell adhesion, hence cell growth. Spectra of PME isoforms vary across the developmental gradients of wood-forming tissues, suggesting that the sequential expression of different PMEs may be important in the developmental dynamics of cell expansion.

Secondary wall biosynthesis and modification

• Secondary wall deposition requires a total reprogramming of wall

biosynthesis.

• Recent research has identified fiber-specific and vessel element-specific

master switches that activate transcription factors responsible for inducing

secondary wall programs involving coordinated expression of cell-wall-

related genes.

• CML (compound middle lamella) is cellulose-poor but rich in pectin and XG

while S-layers are rich in xylan and cellulose. CML has much higher lignin

concentrations than the S-layers, for example in Populus, lignin comprises

68% of the CML compared to 6–8% and 25% of the S-layers of fibers and

vessel elements, respectively.

• Most wood biomass is in S2-layers of fibers, which thus should be the main

focus of attempts to modify wood properties for practical uses.

Cellulose biosynthesis and microfibril angle

• Cellulose in plants is biosynthesized by rosettes in the plasma membrane, each

consisting of six globules, each of which is supposed to contain six CESA proteins.

• The Arabidopsis genome contains 10 CESA genes, and 18 CESA genes were identified

in Populus genome.

• Mutant and/or expression analyses have shown that three CESA proteins, different

from those involved in primary wall biosynthesis, play major roles during secondary

wall biosynthesis in xylem tissues in Arabidopsis (AtCESA4, 7, and 8; denoted

secondary wall CESAs). Expression analysis indicates that AtCESA4, 7, and 8

homologs are also the most abundant CESAs during wood formation of Populus and

Eucalyptus.

• An RT-qPCR analysis of all 18 CESAs in various tissues of Populus trichocarpa has

confirmed a high expression of secondary wall CESAs (including gene pair members)

in developing xylem tissues in relation to their expression in other tissues.

Cellulose biosynthesis and microfibril angle

• The cellulose MFs in Populus and Eucalyptus have been estimated to be 3.1 and 3.2 nm in diameter, respectively, by X-ray diffraction. The MFs aggregate in the cell wall into macrofibrils, which seem to be organized in tangential, or sometimes random, sectors.

• The macrofibril size was 15–18 nm in the S2-layer in Populus fibers, and seems to depend on the lignin and hemicellulose contents in the wall. The MFA of the S2-layer typically varies longitudinally and radially within the tree stem, and is important for mechanical properties of the wood fiber.

• Observations of alignments between MFs and cortical microtubules (MTs) in primary and secondary walls, in conjunction with findings that disruption of MT by drugs is followed by MF disorganization, led to the hypothesis that MTs guide the rosettes, thereby affecting the MFA.

• Several microtubule-associated proteins (MAPs) putatively involved in this scaffolding are currently being identified and investigated. Evidence for the involvement of two MAPs in determining the secondary wall MFA of fibers has emerged from FRA mutants in Arabidopsis. FRA1 encodes a kinesin-like protein, and the fra1 mutation results in altered MFA in interfascicular fibers’ inner walls, whereas FRA1 overexpression results in aberrant secondary wall formation. FRA2 encodes a katanin MT-severing protein, and the fra2 mutant has disorganized MTs, thinner cell walls and distorted MF patterns in inner walls of its interfascicular fibers.

Expression profiles of CAZYme genes in Populus involved in XG, pectin, and xylan

biosynthesis and modification during successive stages of wood development

Modifications of primary cell walls, andimplications for wood cell morphologyPrimary-walled developing wood cells of Populus are richin pectins, cellulose, and XG [1]. During the primary-walled stage, wood cells grow to their final shape by aunique combination of symplastic growth (when theneighboring cells grow together) and intrusive growth(when they move one past another, e.g. fiber elongation)[16!]. Thus, the wall plasticity and variations in celladhesion are key features of the mechanisms involvedin wood cell growth. Consequently, the VC and adjacentradial expansion (RE) zone are the sites of highestexpression of genes encoding wall-modifying enzymes,including expansins, XETs, cellulases, PMEs, polygalac-turonases (PGs), and pectin/pectate lyases (PL1s)

(Figure 1 [16!,17–19]). Genes encoding several of theseenzymes have been upregulated and downregulated intransgenic Populus, shedding some light on wood cellgrowth.

The function of cellulase in wood cell growth is not wellunderstood. A secreted Populus leaf cellulase (PopCel1)has been demonstrated to stimulate cell expansion instem internodes and leaves [20] by loosening the XGnetwork [21]. However, overexpression of a secretedcellulase (AtCel1) in Populus stimulated height growth,but caused no change in wood cell anatomy [22]. Bycontrast, transgenic Populus overexpressing EXPA1showed increased fiber width (9–16%), but neither fiberlength nor vessel radial growth was significantly affected


Figure 1

Expression profiles of CAZYme genes in Populus involved in XG, pectin, and xylan biosynthesis and modification during successive stages ofwood development (A–E). Wood developmental zones are shown on micrograph. A = vascular cambium (VC); B = radial expansion (RE);C = transition between RE and maturation; D = secondary wall formation; E = late maturation. Expression is in log2 ratios and is normalized against apooled sample from all tissues (A–E). Poplar gene names (according to 7, 8, 11, and 15) — Arabidopsis closest homologs. Data from 5. Eachgene is represented by one PU number. XTH: xyloglucan transglucosylase/hydrolase; PL1: pectin/pectate lyase; PG: polygalacturonase.


network co-exists with a network consisting of pecticpolysaccharides. A covalent linkage between xyloglucanand pectin, interconnecting the two networks, appears tobe widespread among Angiosperm taxa [17].

Although relatively few detailed structural analyses havebeen performed on cell walls from non-Angiosperm taxa,it appears that seed plants have cell walls with similar,though not identical, compositions. The quantitatively

Cell wall diversity Popper 287

Figure 1

Key transitions in cell wall components mapped onto a land plant phylogeny (adapted from references [44,45]). Changes in composition aresymbolised as: (+) appearance or substantial increase in occurrence; (#) either a reduction or loss; ( ) (1 ! 3), (1 ! 4)-b-D-glucan [18]; ( ) xyloglucan[13]; ( ) mannose; ( ) 3-O-Methylgalactose [13]; ( ) 3-O-Methylrhamnose [13]; ( ) galacturonic acid; ( ) glucuronic acid; ( ) tannins [12]; ( )branched 4-linked xylan [6]; ( ), rhamnogalacturonan II [11]. Representatives of taxonomic groups shown are (a) Poales, wheat (Triticum aestivum L.);(b) Angiosperms (excluding Poales), Ox-eye Daisy (Leucanthemum vulgare Lam.); (c) Gymnosperms, Larch (Larix decidua L.); (d) Pteridophytes, Treefern; (e) Lycopodiophytes, Selaginella (Selaginella martensii Spring.); (f) Bryopsida, mosses (Polytrichum sp.); (g) Marchantiopsida, Liverworts(Lunularia cruciata (L.) Lindb.); (h) Anthocerotopsida, hornworts (Phaeoceros carolinanus (Michx.) Prosk.); (i) Charophytes, (Nitella sp.)

www.sciencedirect.com Current Opinion in Plant Biology 2008, 11:286–292

Key transitions in cell wall components mapped onto a land plant phylogeny

predominant monosaccharides present in the walls are D-glucose (Glc), D-galactose (Gal), D-mannose (Man), D-xylose (Xyl), L-arabinose (Ara), L-fucose (Fuc), L-rham-nose (Rha) and D-galacturonic acid (GalA). Poales wallscontain more Xyl and less GalA, Gal and Fuc than otherAngiosperms and Gymnosperms contain more Man resi-dues. Differences in cell wall composition are correlatedwith diversification of specific plant taxa. The lycopodio-phytes form a distinctive, basal, monophyletic cladewithin extant vascular plants whose cell walls uniquelycontain the unusual monosaccharide residue 3-O-methyl-galatose [12] that is likely to be a component of manylycopodiophyte primary cell wall polysaccharides in-cluding xyloglucan (MA O’Neill, personal communi-cation).

Additional variation of cell wall composition exists at thepolysaccharide level, (1! 3), (1 ! 4)-b-D-linked glucansare restricted to the Poales [18] and xyloglucan is presentin the cell walls of all land plants but appears to be absentfrom the charophytes, their closest extant ancestors [13].Structural diversity is also seen within specific polysac-charides. Bryophyte and charophyte cell walls are rich inuronic acids [13]. In xyloglucans the presence of the a-L-Fucp-(1-2)-b-D-Galp-(1-2)-a-D-Xylp side-chain appearsto be conserved, but novel side-chains have been charac-terised including uronic acid-containing side-chains inbryophytes, equisetophytes and lycopodiophytes (MJPena et al., abstract in Physiol Plant 2007, 130:18).

Rhamnogalacturonan II (RGII) is required for growth anddevelopment in Angiosperms, and comparable amountsoccur in members of the most primitive extant plantslycopodiophytes, equisetophytes, psilotophytes and pter-idophytes. By contrast, gametophytes of bryophytes con-tain only !1% of the amounts present in vascular plants.In addition the glycosyl sequence of RGII appears to beconserved with the exception that the non-reducing Rharesidue present on the aceric acid containing side-chain ofRGII is replaced by 3-O-methylrhamnose (3-O-MeRha)in some lycopodiophytes and pteridophytes [11]. RGII islinked with the ability to form upright stem and formationof lignified cell walls, which correlates well with itsincreased concentration in vascular plants.

Secondary cell wall polysaccharidesVariation in secondary cell wall composition is perhapsthe most pertinent in the context of biomass production.Secondary cell walls are composed of cellulose, xylan andlignin and, in Gymnosperms, glucomannan.

Cellulose is the most abundant biopolymer on the planet,and cellulose microfibril structure is largely determinedby cellulase synthase catalytic subunits encoded by CesAgenes. Despite conserved regions of CesA genes in plantsand bacteria, mosses lack vascular tissue appear to lacksecondary cell wall specific CesA orthologues [19]. Truesecondary cell walls are perhaps, therefore, restricted tovascular plants.


Table 1

Occurrence of cell wall components shown to vary, with phylogenetic significance, between land plant taxa

Plant group Monosaccharides Polysaccharides Proteins

3-O-MeRha

3-O-MeGal

Uronicacids

Xylan Mannan Xylo-glucana

RGII Pectin (1 ! 3),(1 ! 4)-b-D-glucan

Expansins Ces/Csl

Charophytes + " + " + " # ++ "Hornwort + " + + + ++ # +++ "Liverworts and basal mosses + " + " + ++ # ++ "Advanced mosses + " # + + ++ # ++ " EXPA (conserved

function) andEXPB

CesA, CslA,CslC, CslDb

Homosporous lycopodiophytes + + # + + ++ + + "Heterosporous lycopodiophytes + + # + + ++ + + "Eusporangiate ferns " # # + + ++ + + "Leptosporangiate ferns " # # + # ++ + + "Gymnosperms " # # + # ++ + + "Dicotyledonous Angiosperms " # # + # ++ + + " EXLA, EXLB,

EXPA, EXPBCesA, CslA,CslB, CslC,CslD, CslE,CslG

Poales members ofmonocotyledonousAngiosperms

" # # + # + + + + CesA, CslA,CslC, CslD,CslE, CslF,CslH

", not detectable; #, trace; +, present at low concentration; ++, present at moderate concentration; +++, present at high concentration.a Xyloglucan shows diversity in glycosyl composition.b CslD is highly represented among P. patens ESTs that may reflect their involvement in tip growth of moss protonemata [16].


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