Biochemistry and Biosynthesis of Wood...

Dr. David S.-Y. Wang

Professor

Department of Forestry, NCHU

Biochemistry and Biosynthesis of

Wood Components

Course Topics

• General introduction of wood

• Chemical composition of wood

• Biosynthesis of cell wall polysaccharides

• Phenylpropane derivatives

• Lipids synthesis

• Isoprenoids synthesis

• Formation and development of wood tissues

• Formation of earlywood, latewood, and heartwood

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Figure 1 Some familiar uses of wood as a material and as a source of other cellulose-derived products: Wood for lumber(a); Wood composites such as waferboard (b); Wood pallets (c); Aerial photograph of pulp and paper mill (Tembec,Temiscaming, Quebec, Canada), with the light-colored wood chip piles in the center of the image used as raw material(d); Paper is wound into large rolls (which can weigh up to !25 tons), with this resulting from processing wood chips to makepulp, compressing the same to remove water and drying pulp (e); Selected pulp and paper products (toiletries/paper towels,paper tissues, packaging (e.g., egg and cardboard boxes, paper tissues) (f); books (i); newsprint (not shown), and so on.Selected examples of wood products in building construction, such as a typical ‘chalet’ in the Alps, France (g); the GoldenTemple in Kyoto, Japan (j); and the main gate of the Kanda Shrine in Tokyo, Japan (n); Pencils (m); toys (o, p); boats (q); utilitypoles (r); and cooking/eating utensils (s); A forested hillside in British Columbia, Canada (h); and in the Lake District of Chile(k); with harvested timber temporarily piled (l); Images from L. B. Davin, Washington State University (a, c, f–s); http://www.plywoodnews.com (b); M. G. Paice, Pulp and Paper Institute of Canada, Pointe Claire, Quebec, Canada (d); andForestWorks, North Melbourne, Victoria, Australia (http://www.forestworks.com.au), with permission (e).

Trees: A Remarkable Biochemical Bounty 1175Some familiar uses of wood as a material and as a source of other cellulose-derived products

(d)(c)(b)(a)

(f)(e)

(i)

(h)(g)

(l)(k)(j)

(m) (n) (p)(o)

Figure 2 Selected miscellaneous uses of woods for artistic and/or functional applications, reflecting various material (woodbiopolymer) properties: Mandolin (a); many musical instruments employ wood from specific species in order to produceparticular sounds/tones. Hokkaido bear carving (b) (provenance: near Lake Toya, Japan); Wood-carved fruit assortment(c); An elephant carving of red cedar (Toona ciliata) wood, with a king and carriage (Visakhapatnam, Andhra Pradesh, India)(d); Decorative carvings on choir stall seating in the Mosteiro dos Jeronimos (Lisbon, Portugal) (e and f); Lion carving in thesame monastery (g); Close-up of an oak choir stall (fifteenth century) by Jorg Syrlin the Elder, in the Ulm Cathedral, Germany(h); Totem pole in the Canadian Museum of Civilization (Gatineau, Quebec, Canada) (i); Wood sculpture in the Chateau deVizille garden (Vizille, France) (j); Artificial flowers made fromwood shavings (k); and a wooden ox-cart drawing logs (l) (Pucon,Chile). Black-faced ibis carvings (m) (Villarıca, Chile); Wooden bananas decoration (n) (Brotas, Brazil); Fine furniture examples,such as a Louis XIV commode (o) and a Louis XVI bureau (p). Images from L. B. Davin, Washington State University (a, c, e–n);H. Moore, Washington State University (b and d); LG Antique Restoration, Los Angeles, CA, USA (http://ifixantiques.com withpermission) (o); and French Accents Antiques (http://www.faccents.com, with permission) (p).

1176 Trees: A Remarkable Biochemical Bounty

Selected miscellaneous uses of woods for artistic and/or functional applications, reflecting various material (wood biopolymer) properties

products (Figure 11)1,2 to mulch to other highly valued spices, for example, cinnamon (Figure 8). Woodyplants also globally serve as a principal source of renewable energy, directly as either fuel, such as wood, or aswood-derived by-products, such as lignins, that are solubilized during wood-pulping operations. In short, muchof our very existence and human expression depends on the woody growth habit.

Our trees and forests contribute extensively to the quality of our diverse environments, from their aesthetic(visual) contributions to our various landscapes (Figure 12), to the quality of the air we breathe, to the manyblossoms and fragrances that we so greatly treasure. Furthermore, woody plants serve as important repositoriesof organic carbon through biological CO2 sequestration and photosynthesis. Indeed, wood-forming plants canbe thought of as nature’s most abundant reservoir of organic carbonaceous substances on land and thus are ofpivotal importance for global climate chemistry. Yet, humanity’s escalating overuse of these resources may leadto long-term environmental crisis. For example, deforestation in the 1990s occurred at a rate of 9.4 millionhectares per year, resulting in up to 10% of all trees being listed as either threatened or endangered, with 1002tree species declared critically endangered (each estimated to be <50 individuals/species) worldwide.3 There isthus now the challenge of sustainably preserving/utilizing these remarkable resources globally and, of course,the corresponding biochemical diversity encapsulated within. Yet history has repeatedly recorded that failureto sustainably husband forest resources can result in collapse of the civilizations in the regions so impacted.4

Indeed, we often take for granted humanity’s critical reliance on the sustainability of the diverse woody habitatsthat extend over the very small thin line(s) of topsoil worldwide, and that of their individual growth habits.

In this contribution, we first consider how the woody habit presumably emerged and, by inference, how theirquite distinct phytochemical factories may have evolved. Regardless of how this occurred, these developments haveultimately resulted in humanity’s massive use of tree resources from both material and phytochemical perspectives.Today, the plant sciences are directed mainly to establishing how such diverse processes occur at the molecularlevels, that is, from wood formation to the generation of specialized phytochemicals, including how they ultimatelybecame compartmentalized in different tissues and organs. As the reader will hopefully appreciate from theaccompanying text, there has never been more a need to understand the biochemistries of wood formation than now.

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(c) (d) (e) (f)

(g)

Figure 3 Rubber trees (Hevea brasiliensis) (a) are tapped for latex (b) that is used for rubber production. Selected rubberproducts include tires (c), pencil erasers (d), boots (e), latex gloves (f), and rubber bands (g). Images from D. Nandris;H. Chrestin, Institut de Recherche pour le Development (IRD), Mahidol University, Bangkok, Thailand (a and b), and L. B.Davin, Washington State University (c–g).

Trees: A Remarkable Biochemical Bounty 1177Rubber trees (Hevea brasiliensis)tapped for latex

3.27.2 Evolution of the Woody Growth Habit: Land Colonization and Adaptation

Present-day trees or tree forms are classified under ferns, gymnosperms, and angiosperms, although only thelatter two groups produce wood from a cambium. The total number of extant arborescent species is currentlydifficult to precisely establish because of variations in definitions used.3 Nevertheless, !100 000 are thought tobe in existence globally, this representing up to 25% of all plant species.3,5 Of the 100 000 or so tree species,however, there are less than 1000 that are gymnosperms; the bulk are the angiosperms.6 (Named from theancient Greek, gymnosperm, meaning naked seed, refers to plants with seeds that are borne externally, as on ascale or similar structure, whereas angiosperm refers to those with vessel seed, indicating the carpel in whichthe seed develops.7) The enormous breadth of the topic of tree resources alone limits any substantial discussionof shrubs, bushes, and lianas (woody vines), even though they can have a woody growth habit.

Plants apparently first emerged on land from their forerunner algal ancestors during the mid-Ordovicianperiod, some 460Mya.8,9 Over this lengthy evolutionary period, numerous truly remarkable innovations andadaptations occurred that eventually led to our familiar tree forms. This ultimately afforded the 350 000 or sodistinct plant species in existence today, and thus the fantastic diversity in terms of plant shapes, sizes, habitats, andassociated phytochemical constituents. In achieving this colonization of land, some of the remarkable evolutionarychanges manifested over the passage of time included, among others: generation of specialized cell types, such aslignified (reinforced) secondary cell walls and vascular tissues; formation of protective wood and bark tissues, andother specialized cell types within; the ability to continuously orient/reorient massive photosynthetic canopies;

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Figure 4 Lacquer tree, objects of art and Russian lacquer boxes. The lacquer tree (Rhus vernicifera) (a and b) is the sourceof urushi (c), used to make objects of art such as in d, e, and g. Themost sought after Russian lacquer boxes (f and h) originatefrom four villages – Palekh, Fedoskino, Kholui, and Mstera. Items in (f) are from Fedoskino, whereas that in (h) is from Palekh.They are made of ‘papier mache’ painted with several coats of black lacquer on the outside and red lacquer on the inside.Extremely fine brushes are used to create the fine lines and details in each painting. Finally, when the painting is finished alayer of transparent lacquer is applied. Altogether, this can take several months to complete. Images from S. Ross, UrushiArtist, Japan (a–c, e, and g), and L. B. Davin, Washington State University (d, f, and h).

1178 Trees: A Remarkable Biochemical BountyLacquer tree (Rhus vernicifera)

Urushi

(a)

(c)

(b)

(d)

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Figure 5 Acacia senegal (a, b) is the source of gum arabic (c). Some selected uses of gum Arabic: as an ingredient in softdrinks, candies, weight loss compositions (e.g., Slim Fast), and so on. Gum arabic is also used as a binder (e) for watercolorpaint (f) as it dissolves readily in water. It is also used in shoe polish (g), and as a wettable adhesive, such as in postage stamps(h). Images from D. Lesueur, CIRAD, Tropical Soil Biology and Fertility Institute of CIAT, World Agroforestry Centre, Nairobi,Kenya (a–c), L. B. Davin (d and h), and H. Moore (e–g) Washington State University.

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Figure 6 Maple trees (Acer saccharum) and maple syrup. Trees are tapped annually for their sap in early spring before budsemerge (a), with sap collection (b and c) and large amounts of sap then processed (d) to produce syrup of different grades (e, f).Most trees are tapped once annually (some twice), with each tap yielding!80 l of sapwhich is then concentrated to!1.5 l. Imagesfrom B. Putnam, Putnam Family Farm, Cambridge, Vermont, USA (a–d, f), and L. B. Davin, Washington State University (e).

Trees: A Remarkable Biochemical Bounty 1179

Acacia senegal (a, b) is the source

of gum arabic (c). Some selected

uses of gum Arabic: as an

ingredient in soft drinks, candies,

weight loss compositions (e.g.,

Slim Fast), and so on. Gum arabic

is also used as a binder (e) for

watercolor paint (f) as it dissolves

readily in water. It is also used in

shoe polish (g), and as a wettable

adhesive, such as in postage

stamps (h).

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(c)

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(e) (f) (g) (h)

Figure 5 Acacia senegal (a, b) is the source of gum arabic (c). Some selected uses of gum Arabic: as an ingredient in softdrinks, candies, weight loss compositions (e.g., Slim Fast), and so on. Gum arabic is also used as a binder (e) for watercolorpaint (f) as it dissolves readily in water. It is also used in shoe polish (g), and as a wettable adhesive, such as in postage stamps(h). Images from D. Lesueur, CIRAD, Tropical Soil Biology and Fertility Institute of CIAT, World Agroforestry Centre, Nairobi,Kenya (a–c), L. B. Davin (d and h), and H. Moore (e–g) Washington State University.

(d)(a) (b) (e)

(f)(c)

Figure 6 Maple trees (Acer saccharum) and maple syrup. Trees are tapped annually for their sap in early spring before budsemerge (a), with sap collection (b and c) and large amounts of sap then processed (d) to produce syrup of different grades (e, f).Most trees are tapped once annually (some twice), with each tap yielding!80 l of sapwhich is then concentrated to!1.5 l. Imagesfrom B. Putnam, Putnam Family Farm, Cambridge, Vermont, USA (a–d, f), and L. B. Davin, Washington State University (e).


Maple trees (Acer saccharum) and maple syrup. Trees are tapped annually for their

sap in early spring before buds emerge (a), with sap collection (b and c) and large

amounts of sap then processed (d) to produce syrup of different grades (e, f). Most

trees are tapped once annually (some twice), with each tap yielding around 80 L of

sap which is then concentrated to around 1.5 L.

elaboration of a plethora of often species-specific distinct biochemical pathways leading to, for example, chemicaldefense systems; evolution of distinct plant pollination/reproductive strategies and adaptations, and a myriad ofrelated regulatory processes at the genomic/proteomic and metabolic levels.

3.27.2.1 Land Colonization, the Early Phases: Turgor-Based Stem Support Systems

The earliest terrestrial plants had features similar to today’s bryophytes (mosses, liverworts, and hornworts),and were thus nonwoody. They were small in stature, transporting water by hydroid cell types (having primary

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Figure 7 Tree-derived amber (fossilized resin) and jewelry. Amber is found in extensive deposits in different parts of theworld: Fly (family Chironomidae) in amber (a). Amber is used in the making of jewelry, for example, necklace (b) and earrings(c). Amber specimen from Professor R. S. Zack, Washington State University. Images from H. Moore, Washington StateUniversity (a) and Amber Goods – Amber Jewelry (http://www.ambergoods.ie), with permission (b, c).

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Figure 8 Selected examples of common spices from tree species: Cinnamon (Cinnamomum verum; a), cloves (Syzygiumaromaticum; b), nutmeg (Myristica fragrans; c), and bay leaves (Laurus nobilis; d). Cocoa beans (Theobroma cacao; e–f) canbe processed into chocolate (g) and other products. Images from H. Moore, Washington State University (a–d), Consulat deSao Tome & Principe, Marseille, France (e, f) and L. B. Davin, Washington State University (g).

1180 Trees: A Remarkable Biochemical BountyTree-derived amber (fossilized resin) and jewelry




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Cinnamon (Cinnamomum verum)

Cloves (Syzygium aromaticum)

Nutmeg (Myristica fragrans)

Bay leaves (Laurus nobilis)

Cocoa beans (Theobroma cacao)

StrychnosNux-vomica

O

(5) Camphor

(e)

N

HO N

CHH

HMeO

H C

H

Cinchona officinalisbark

(1) Taxol

(3) Quinine

(4) Acetylsalicylic acidSalix alba

Taxusbrevifolia

O N

NO

O

OH

(2) Camptothecin

O

O

O

O

HHO

OHO

O

ONHO

OH

Me

O

Me

OO

O OH

O

O

Prunusdulcis

(9) R-Prunasin

ON

OHO

HO OH

OH O N

OHO

HO OH

O

OHO

HO OH

OH

(8)R-Amygdalin

Camptothecaacuminata

(b)(a)

(d)(c)

(6) Azadirachtin A

O

H

HOO

O

O

OOMe

O H

O

MeO

O

O

O

HO

H

O

O

H

H

Azadirachtaindica

(f)

(7) Strychnine

N

O

H

O

N

H

H

(g) (h)

Figure 9 Phytochemical treasures: T. brevifolia (a) andC. acuminata (b) accumulate the cancer chemotherapeutics, taxol (1)and camptothecin (2), respectively. Cinchona spp. (e.g., Cinchona calisaya) (c) and S. alba (d) are sources of the medicinalsquinine (3) and acetylsalicylic acid (4). The medicinal compound, camphor (5), used as a cough suppressant, is derived fromCinnamomumcamphora (e). The neem tree (A. indica; f) harbors the insecticide, azadirachtin A (6). Poisons, such as strychnine(7) and cyanogenic compounds, (R)-amygdalin (8), (R)-prunasin (9), are fromS.Nux-vomica (f) and the almond treeP. dulcis (g),respectively. Images from R. E. B. Ketchum, Washington State University (a), J. Manhart, Department of Biology, Texas A&MUniversity, College Station, TX (b), The Garden of Medicinal Plants, Kyoto Pharmaceutical University, Kyoto, Japan (c), A. M.Patten, Washington State University (d), L. B. Davin, Washington State University (e), Cal Lemke, University of Oklahoma,Norman, OK (f), Kohler’s Medizinal Pflanzen, 1887 (g), R. Sanchez-Perez; F. Dicenta, CSIC-CEBAS, Murcia, Spain (h).


Phytochemical treasures: T. brevifolia (a)

and C. acuminata (b) accumulate the

cancer chemotherapeutics, taxol (1) and

camptothecin (2), respectively. Cinchona

spp. (e.g., Cinchona calisaya) (c) and S.

alba (d) are sources of the medicinals

quinine (3) and acetylsalicylic acid (4). The

medicinal compound, camphor (5), used

as a cough suppressant, is derived from

Cinnamomum camphora (e). The neem

tree (A. indica; f) harbors the insecticide,

azadirachtin A (6). Poisons, such as

strychnine (7) and cyanogenic compounds,

(R)-amygdalin (8), (R)-prunasin (9), are

from S. Nuxvomica (f) and the almond tree

P. dulcis (g), respectively.

REVIEWS

Genomics of cellulosic biofuelsEdward M. Rubin1,2

The development of alternatives to fossil fuels as an energy source is an urgent global priority. Cellulosic biomass has thepotential to contribute tomeeting the demand for liquid fuel, but land-use requirements and process inefficiencies representhurdles for large-scale deployment of biomass-to-biofuel technologies. Genomic information gathered from across thebiosphere, including potential energy crops and microorganisms able to break down biomass, will be vital for improving theprospects of significant cellulosic biofuel production.

The capture of solar energy through photosynthesis is a pro-cess that enables the storage of energy in the form of cell wallpolymers (that is, cellulose, hemicellulose and lignin). Theenergy stored in these polymers can be accessed in a variety

of ways, ranging from simple burning to complex bioconversionprocesses. The high energy content and portability of biologicallyderived fuels, and their significant compatibility with existing pet-roleum-based transportation infrastructure, helps to explain theirattractiveness as a fuel source. Despite the increasing use of biofuelssuch as biodiesel and sugar- or starch-based ethanol, evidence sug-gests that transportation fuels based on lignocellulosic biomass rep-resent the most scalable alternative fuel source1. Lignocellulosicbiomass in the form of plant materials (for example, grasses, woodand crop residues) offers the possibility of a renewable, geograph-ically distributed and relatively greenhouse-gas-favourable source ofsugars that can be converted to ethanol and other liquid fuels.Calculations of the productivity of lignocellulosic feedstocks, in partbased on their ability to grow onmarginal agricultural land, indicatesthat they can probably have a large impact on transportation needswithout significantly compromising the land needed for food cropproduction2.

Lignocellulosic biofuel production involves collection of biomass,deconstruction of cell wall polymers into component sugars (pre-treatment and saccharification), and conversion of the sugars tobiofuels (fermentation) (Fig. 1). Partially because of the historicallylow demand for biologically based transportation fuels, each step inthis process is in the early stages of optimization for efficiency andthroughput. The crops from which biomass is currently derived havenot been domesticated for this particular purpose and the presentmethods for saccharification and fermentation are inefficient and

expensive. However, the recent and pressing desire to develop alter-natives to fossil fuels has made the rapid improvement of biofuelproduction a high priority, in which biologically derived energy(‘bioenergy’)-relevant genomic insights and resources will have animportant role (Table 1).

BiomassFrom the perspective of transportation fuels, plants can be viewed assolar energy collectors and thermochemical energy storage systems. Itis the storage of energy in a form that can later be accessed viathermochemical or enzymatic conversion that distinguishes biomassfrom other renewable energy sources. Cellulosic biomass, sometimesreferred to as lignocellulosic biomass, is an abundant renewableresource that can be used for the production of alternative trans-portation fuels3. The three main components of lignocellulose arecellulose, hemicellulose and lignin (Fig. 2), with the relative propor-tions of the three dependent on the material source4. Cellulose, themain structural component of plant cell walls, is a long chain ofglucose molecules, linked to one another primarily by glycosidicbonds5. Hemicellulose, the second most abundant constituent oflignocellulosic biomass, is not a chemically well defined compoundbut rather a family of polysaccharides, composed of different 5- and6-carbon monosaccharide units, that links cellulose fibres intomicrofibrils and cross-links with lignin, creating a complex networkof bonds that provide structural strength5. Finally lignin, a three-dimensional polymer of phenylpropanoid units, can be consideredas the cellular glue providing the plant tissue and the individual fibreswith compressive strength and the cell wall with stiffness6, in additionto providing resistance to insects and pathogens.

1DOE Joint Genome Institute, 2800Mitchell Drive,Walnut Creek, California 94598, USA. 2Lawrence BerkeleyNational Laboratory, 1 Cyclotron Road, Berkeley, California 94720, USA.

Solar energy

Feedstock

Sugars

Biofuels

Fuel-producingmicroorganisms

Physical pre-treatment,chemicals and enzymes

Figure 1 | Biology of bioconversionof solar energy into biofuels. Solarenergy is collected by plants viaphotosynthesis and stored aslignocellulose. Decomposition ofthe cellulosicmaterial into simple 5-and 6-carbon sugars is achieved byphysical and chemical pre-treatment, followed by exposure toenzymes from biomass-degradingorganisms. The simple sugars canbe subsequently converted intofuels by microorganisms.

Vol 454j14 August 2008jdoi:10.1038/nature07190

841 ©2008 Macmillan Publishers Limited. All rights reserved

Biology of Bioconversion of Solar Energy into Biofuels

Solar energy is collected by plants via photosynthesis and stored as lignocellulose. Decomposition of the cellulosic material into simple 5- and 6-carbon sugars is achieved by physical and chemical pretreatment, followed by exposure to enzymes from biomass-degrading organisms. The simple sugars can be subsequently converted into fuels by microorganisms.

compared to that of teosinte. Some of the most rapid increases haveoccurred in the past 40 years, both from advances in agronomicpractices and, importantly, from the application of modern genetics.The optimization of bioenergy crops as feedstocks for transportationfuels is in its infancy, but already genomic information and resourcesare being developed that will be essential for accelerating theirdomestication. Many of the traits targeted for optimization in poten-tial cellulosic energy crops are those that would improve growth onpoor agricultural lands, to minimize competition with food cropsover land use.

Populus trichocarpa (poplar), the first tree and potential bioenergycrop to have its genome sequenced (Table 1)9, illustrates some of theissues and potential of applying genomics to the challenge of optim-izing energy crops. The traits for which the genetic underpinningswill be sought in the genomes of bioenergy-relevant plants, such aspoplar, include those affecting growth rates, response to competitionfor light, branching habit, stem thickness and cell wall chemistry.Significant effort will go into maximizing biomass yield per unit landarea, because this more than any other factor will minimize theimpact on overall land use. One can imagine trees optimized to haveshort stature to increase light access and enable dense growth, largestem diameter, and reduced branch count to maximize energy den-sity for transport and processing. Trees have evolvedwith highly rigidand stable cell walls due to heavy selective pressure for long life and anupright habit. Plants domesticated for energy production, with a

crop cycle time of only a few years, would have less need for a rigidcell wall than wild plants with lifetimes of a hundred years or more.Alterations in the ratios and structures of the various macromole-cules forming the cell wall are a major target in energy crop domest-ication to facilitate post-harvest deconstruction at the cost of a lessrigid plant.

Already, by comparing several of the presently available plant gen-omes (poplar9, rice10,11,Arabidopsis12; see Table 1) coupled with large-scale plant gene function and expression studies, a number of can-didate genes for domestication traits have been identified13,14. Theseinclude many genes involved in cellulose and hemicellulose synthesisas well as those believed to influence various morphological growthcharacteristics such as height, branch number and stem thickness15.In addition to homology-based strategies, other genome-enabledstrategies for identifying domestication candidate genes are beingused. These include quantitative trait analysis of natural variationand genome-wide mutagenesis coupled with phenotypic screensfor traits such as recalcitrance to sugar release, acid digestibilityand general cell wall composition. The availability of high-through-put transgenesis in several plant systems16 will facilitate functionalstudies to determine the in vivo activities of the large number ofdomestication candidate genes. Using these strategies, genes affectingfeatures such as plant height, stem elongation and trunk radialgrowth, drought tolerance, and cell wall stability are but a few ofthe features that are likely to be identified as targets for domestication

OH

Macrofibril

Plant cell

Plant

Cellwall

Lignin

Lignin

Hemicellulose

Pentose

Crystallinecellulose

HydrogenbondCellodextrin

n-3

n-3

n-3

n-3

n-3

Glucose

Hexose

10–20 nm

Macrofibril

OH

OH

OH

OH

p-Coumaryl alcohol Coniferyl alcohol Sinapyl alcohol

OH

O

H G S

OO

Figure 2 | Structure of lignocellulose. The main component oflignocellulose is cellulose, a b(1–4)-linked chain of glucose molecules.Hydrogen bonds between different layers of the polysaccharides contributeto the resistance of crystalline cellulose to degradation. Hemicellulose, thesecond most abundant component of lignocellulose, is composed of various5- and 6-carbon sugars such as arabinose, galactose, glucose, mannose andxylose. Lignin is composed of three major phenolic components, namely

p-coumaryl alcohol (H), coniferyl alcohol (G) and sinapyl alcohol (S). Ligninis synthesized by polymerization of these components and their ratio withinthe polymer varies between different plants, wood tissues and cell wall layers.Cellulose, hemicellulose and lignin form structures called microfibrils,which are organized into macrofibrils that mediate structural stability in theplant cell wall.

NATUREjVol 454j14 August 2008 REVIEWS

843 ©2008 Macmillan Publishers Limited. All rights reserved

Structure of lignocellulose

nHow do trees (plants) synthesis

”Natural Products”?

nWhy do plants synthesis them?

Our Understanding of How Wood Develops Is Not Complete

n With a few exceptions, very little is known about the cellular,

molecular, and developmental processes that underlie wood

formation.

n Xylogenesis represents an example of cell differentiation in an

exceptionally complex form.

Xylogenesisn The formation of wood.

n Controlled by a wide variety of factors both exogenous (photoperiod and

temperature) and endogenous (phytohormones) and by interaction between them. 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.

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Seasonal changes in cambial activity of pear trees

Major groups of endogenous plant hormones

Indole-3-acetic acid (IAA) Gibberellin

Cytokinins

trans-Zeatin Benzylaminopurine

Abscisic acid (ABA)

CH2=CH2

Xylogenesisn 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.

Arabidopsis undergoes secondary growth in roots,hypocotyls and stems. Routine removal of inflorescencestems induces secondary xylem at the root–hypocotyl junc-tion [18,19], which is used for generating ESTs [20]. Ara-bidopsis plants grown under a combination of short- andlong-day conditions can also produce extensive secondaryxylem in hypocotyls and inflorescence stems [21–23]. Thesecondary xylem tissues induced artificially in Arabidopsishypocotyls and stems are remarkably similar to those ofthe poplar tree [18,19,21,23] (Figure 1b,c). In addition,Arabidopsis inflorescence stems develop interfascicularfibre cells with thick secondary walls when internodes ofthe stems cease to elongate. Recently, a new in vitroxylogenic system was established in which Arabidopsissuspension cells were induced to differentiate intotracheary elements by culturing in the presence of brassi-nolide [24,25] (Figure 1d).

The cDNA clones that were sequenced for EST analysiswere used for comprehensive transcriptional profiling bycDNA microarrays (or macroarrays) in loblolly pine [3,26],black locust [9,27], Eucalyptus [10,28], poplar [29–31] andzinnia [15,17]. In addition to the cDNAmicroarray analysis,other methods such as serial analysis of gene expression[32], cDNA-amplified fragment length polymorphism[16,33,34] and differential display [35], have been success-fully adopted for transcriptional profiling during wood for-mation.Genome-wide expression profiling usingAffymetrixGeneChip array ATH1, which represents !23 750 Arabi-dopsis genes [36], was carried out with wood-forming Ara-bidopsis tissues and organs [22,23,37,38], as well as withdifferentiating tracheary elements in the in vitro xylogenic

culture [24]. Moreover, laser microdissection [39,40] andfluorescence-activated cell sorting analysis have proven,in combination with microarray analysis, to be useful toolsfor global gene expression profiling in specific cell types [41].

These analyses have uncovered several genes whoseexpressionwaschangedsignificantlyduringwood formation(Table 1), including genes encoding cell wall structuralproteins and various enzymes associated with the biosyn-thesis of secondary cell wall polysaccharides (e.g. cellulose),the degradation and modification of primary cell walls, thebiosynthesis of lignin precursors, the polymerization of lig-nin in secondary walls, and programmed cell death [14,42].Because the expression of these genes is highly coordinated,it is expected that specific transcription factors mightregulate their expression in a coordinated fashion. Indeed,transcriptionalprofiling indicatesthatmanygenesencodingtranscription factors are expressed preferentially duringwood formation in various plant species (Table 1).

Transcription factors regulating wood formationThe characterization of Arabidopsis mutants with defectsin vascular development, and reverse genetic analysis ofvascular tissue-related genes revealed by transcriptionalprofiling has furthered our knowledge of transcriptionalregulation during wood formation [14,43–45]. As a result,several classes of transcription factors involved in woodformation have come under the spotlight.

AUX/IAAs and auxin response factorsMutation of the MONOPTEROS/AUXIN RESPONSEFACTOR 5 (MP/ARF5) gene, which encodes a transcription

Figure 1. Wood formation from procambium and vascular cambium. (a) Schematic model of xylem (wood) formation. Procambial cells and daughter cells produced bycambial initials differentiate into phloem cells and xylem (wood) cells. Xylem (wood) cells include tracheary elements and fibres. Tracheids and vessels are constituents oftracheary elements. Two types of vessels are observed in angiosperms: protoxylem vessels that commonly have annular and spiral secondary wall thickenings andmetaxylem vessels that usually have reticulate and pitted thickenings. (b) Cross-section of a poplar stem. (c) Cross-section of an Arabidopsis hypocotyl. (d) Trachearyelements induced in the Arabidopsis xylogenic culture. (e) Vessel elements transdifferentiated from the cortex cells of Arabidopsis roots overexpressing the VND7 protein.

Review TRENDS in Plant Science Vol.12 No.2 65

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Wood formation from procambium and vascular cambium

potential of the genomics approach to forestry research tobe realized. It was also necessary to obtain a betterunderstanding of the expression patterns of differentgenes, as EST frequency gives rather rudimentary dataon gene expression. Furthermore, it is often far moreinteresting to obtain temporal and spatial descriptions ofthe expression patterns, and this type of information isespecially important for biotechnological purposes. Forinstance, it may be desirable to modify a metabolic path-way in a small subset of cells. Thus, a platform for globaltranscript profiling was established in poplar using thefirst set of EST sequences. The first microarray slidecontained about 2500 features and was used to investigatethe molecular basis of xylem development [5!!]. In addi-tion, a new amplification technique was developed, allow-ing RNA to be isolated from submilligram amounts oftissues to generate probes for microarray analysis [6!].Since then, a further generation of microarray slides hasbeen produced with over 13 500 features representing33 000 ESTs; a further generation is planned to accom-modate over 20 000 features derived from 100 000sequenced ESTs.

Genomic sequencingAlthough EST sequencing is a cheap and quick way toidentify expressed genes, we also need to know thecomplete genomic sequence of one or more tree species.The genomic sequence is necessary for several reasons.Firstly, it is highly unlikely that all the genes of any treewill be identified by EST sequencing alone. Secondly,even if there are several hundred genes unique to trees, itwould be extremely useful to identify their individual

contributions to the observed architectural and otherdifferences between simple annual weeds like Arabidopsisand trees. Thirdly, acquisition of a full tree genomesequence would be very valuable for quantitative traitlocus (QTL) analysis, marker-assisted breeding and,importantly, the genome sequence from one tree couldbe used as a platform for identifying synteny between treespecies, as has been done for Arabidopsis and Brassica andother species. Therefore, the news that the United StatesDepartment of Energy (DOE) has decided to sequencethe genome of poplar is welcome to all tree biologists(www.ornl.gov/ipgc). The DOE’s Joint Genome Institute(www.jgi.doe.gov) is expected to produce six times cover-age of the entire genome sequence during 2003. How-ever, without further advances in sequencing andbioinformatics, it seems unlikely that we will obtaingenomic information from any gymnosperm in the nearfuture. This is because gymnosperms have a massivegenome with haploid DNA contents of, on average,15 500 Mbp [7], as compared with 125 Mbp for Arabidop-sis [8] and 550 Mbp for poplar [9].

Genetic mappingGenetic maps of varying quality have been generated forseveral forest tree species, using a variety of approaches.QTLs have been identified for a range of traits, such aswood density, fibre length and resistance [10–13,14!!,15].However, the gains from QTL mapping for breedingpurposes have been severely restricted by the greatdifficulties in identifying the gene (or genes) located atthe QTL. There are two reasons for these difficulties.Firstly, the long time to flowering hampers repeated

Figure 1

Current Opinion in Biotechnology

Conifers (left-hand side) exhibit many aspects of unique biology, but unfortunately are not practical as models in functional genomics. By contrast,Arabidopsis (right-hand side) and poplar (centre) are two model systems that, in combination, provide an excellent platform for functionalgenomics studies of ‘tree’-related biology. These systems can also be used for research to follow up observations made in conifer systems, as bothpoplar and Arabidopsis have secondary growth as exemplified by the cross-sections.

Forest biotechnology enters the genomic era Bhalerao, Nilsson and Sandberg 207

www.current-opinion.com Current Opinion in Biotechnology 2003, 14:206–213

Conifers (left-hand side) exhibit many aspects of unique biology, but unfortunately are not practical as models in functional genomics. By contrast, Arabidopsis (right-hand side) and poplar (centre) are two model systems that, in combination, provide an excellent platform for functional genomics studies of ʻtreeʼ-related biology. These systems can also be used for research to follow up observations made in conifer systems, as both poplar and Arabidopsis have secondary growth as exemplified by the cross-sections.

WOOD BIOSYNTHESIS

n Cell division

n Cell expansion (elongation and radial enlargement)

n Cell wall thickening (involving cellulose, hemicellulose, cell

wall proteins, and lignin biosynthesis and deposition)

n Programmed cell death

n HW formation.

Wood Cells Originate from Vascular Cambium Activity

n The vascular cambium is a secondary meristem derived from the procambium,

which in turn develops from the apical meristem.

n The cambium plays a major role in the diametral growth of gymnosperm and

angiosperm shoots and roots and is of great significance, particularly in respect to

the wood that is produced.

n Cambial activity ensures the perennial life of trees through the regular renewing of

functional xylem and phloem.

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,

and mature xylem. P, Centrifugal phloem

differentiation with radially enlarging, maturing,

and mature phloem. Empty arrowhead

indicates a newly deposited radial wall. Full

arrowhead indicates a newly deposited

tangential wall.

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, andtangential sections.

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 sap descends via the phloem.

The Differentiation of Xylem Cells Involves Four Major Steps

n Cell expansion

n Deposition of a thick multilayered secondary cell wall

n Lignification

n Cell death

n Derivative cells expand longitudinally and radically to reach their final size

during the formation of the primary wall. Xyloglucan endotransglycosylases,

endoglucanases, expansins, pectin methyl esterases, and pectinases are among

the primary determinants of this process.

n Once expansion is completed, the formation of the secondary cell wall begins,

driven by the coordinated expression of numerous genes specifically involved

in the biosynthesis and assembly of four major compounds: polysaccharides

(cellulose, hemicelluloses), lignins, cell wall proteins and other minor soluble

(stilbenes, flavonoids, tannins, and terpenoids), and insoluble (pectins and cell

wall proteins) compounds in a neutral solvent.

n Between 40% and 50% of wood consists of cellulose. The fundamental

structure units are the microfibrils (MFs), which are the result of a strong

association of inter- and intrachain hydrogen bonds between the different

chains of β-linked Glc residuesin a manner so precise that microfibrillar

cellulose is largely crystalline.

n A first breakthrough had been the identification of genes encoding the

catalytic subunit of the cellulose synthase (Ces) complex. In Arabidopsis, at

least six genes encode putative catalytic subunits of Ces. In addition, a large

gene family of over 20 more distantly related genes, so-called Ces-like (Csl)

genes, exists, whose gene products most likely are involved in the synthesis of

other polysaccharides.

n In higher plants, the substrate for Ces (UDP-d-Glc) is provided by Suc

synthase. The complex Ces/Suc synthase is thought to have a cytoplasmic

localization and the growing cellulose chain may be secreted through the

membrane via a pore.

n Cortical microtubules (mainly composed of α- and β - tubulin) may determine

the wall pattern by defining the position and orientation of cellulose MFs

during the differentiation of conducting elements, probably by guiding the

movement of the cellulose-synthesizing complex in the plasma membrane.

n However, although in many cases co-orientation of microtubules and MFs

were observed, mathematical models relying on the geometry of the cell, have

been proposed to challenge this dogma.

n The water-insoluble cellulose MFs are associated with mixtures of

soluble noncellulosic polysaccharides, the hemicelluloses, which account

for about 25% of the dry weight of wood.

n They generally occur as heteropolymer such as glucomannan,

galactoglucomannan, arabinogalactan, and glucuronoxylan, or as a

homopolymer like galactan, arabinan, and β-1,3- glucan.

n The biosynthesis of these polysaccharides occurs in the Golgi apparatus

by a process that can be divided into two main steps: the synthesis of the

backbone by polysaccharide synthases, and the addition of side chain

residues in reactions catalyzed by a variety of glycosyltransferases.

n The third major component of wood (25%–35%) is lignin, a phenolic

polymer derived from three hydroxycinnamyl alcohols (monolignols): p-

coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, giving rise to H,

G, and S units, which differ from each other only by their degree of

methoxylation.

n Lignin embeds the polysaccharide matrix giving rigidity and cohesiveness

to the wood tissue as a whole, and providing the hydrophobic surface

needed for the transport of water. Lignin content and monomeric

composition vary widely among different taxa, individuals, tissues, cell

types, and cell wall layers.

n Lignin biosynthesis has been the most studied pathway,

resulting in the cloning of several structural genes.

n However, it is somewhat surprising that recent attempts at

engineering lignin biosynthesis have demonstrated that

our current models of the pathway are incomplete.

n Cell wall proteins and pectins are among the other minor compounds of

the cell wall. Although different proteins are present in the cell wall at

different times during development, the amount of protein remaining in the

wood is small.

n Nevertheless, such proteins could play important roles determining the

composition and morphology of xylem cell walls. Abundant cell wall

associated proteins have been found in many plants and have

traditionally been classified into four main groups: Gly-rich proteins, Pro-

rich proteins, arabinogalactan proteins, and Hyp-rich glycoproteins (or

extensins). These proteins are cross-linked into the cell wall and probably

have structural functions.

n Pectins are thought to play a fundamental role in the

regulation of cell wall extensibility. They are also thought

to be exported from the Golgi apparatus as highly

esterified galacturonan and then de-esterified by cell wall

bound pectin methylesterases, thus allowing the formation

of intermolecular bonds through calcium ions.

n Pectin

¨ A mixture of polymers from sugar acids, such as D-galacturonic acid,

which are connected by (α1,4) glycosidic links.

¨ Some of the carboxyl groups are esterified by methyl groups.

¨ The free carbonyl groups of adjacent chains are linked by Ca and Mg

ion.

¨ Preparing jellies and jams.

n When lignification is completed, conducting xylem elements undergo

programmed cell death, involving cell-autonomous, active, and

ordered suicide, in which specific hydrolases (Cys and Ser proteases,

nucleases, and RNase) are recruited.

n Several factors (auxins, cytokinins, and Suc) prepare the cell to die by

determining the profile of hydrolases synthesized by the cell. These

hydrolases are inactive in the vacuole. By a signal that remains

unknown, a calcium flux provokes the vacuoles to collapse with the

release of hydrolases that degrade all of the cellular content but not

the secondary cell wall.

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