Critical Reviews in Biochemistry and Molecular Biology, 38:305–350, 2003Copyright C© Taylor and Francis Inc.ISSN: 1040-9238DOI: 10.1080/10409230390242443

Lignin: Genetic Engineering and Impacton Pulping

Marie Baucher,1,4 Claire Halpin,2 Michel Petit-Conil,3

and Wout Boerjan4*

1Laboratoire de Biotechnologie Vegetale, Universite Libre de Bruxelles, Brussels,Belgium; 2Plant Research Unit, School of Life Sciences, University of Dundee at theScottish Crop Research Institute, Invergowrie, Dundee, UK; 3Centre Technique duPapier, Grenoble Cedex 9, France; 4Department of Plant Systems Biology, FlandersInteruniversity Institute for Biotechnology (VIB), Ghent University, Gent, Belgium

* Address correspondence to: Wout Boerjan, Department of Plant Systems Biology, Flanders InteruniversityInstitute for Biotechnology (VIB), Ghent University, Technologiepark 927, B-9052 Gent, Belgium; Tel.: 32-9-3313881; Fax: 32-9-3313809; E-mail: wout.boerjan@psb.ugent.be.

ABSTRACT: Lignin is a major component of wood, the most widely used raw material for theproduction of pulp and paper. Although the biochemistry and molecular biology underpinninglignin production are better understood than they are for the other wood components, recent workhas prompted a number of re-evaluations of the lignin biosynthetic pathway. Some of the work onwhich these revisions have been based involved the investigation of transgenic plants with modifiedlignin biosynthesis. In addition to their value in elucidating the lignin biosynthetic pathway, suchtransgenic plants are also being produced with the aim of improving plant raw materials for pulpand paper production. This review describes how genetic engineering has yielded new insightsinto how the lignin biosynthetic pathway operates and demonstrates that lignin can be improvedto facilitate pulping. The current technologies used to produce paper are presented in this review,followed by a discussion of the impact of lignin modification on pulp production. Fine-tunedmodification of lignin content, composition, or both is now achievable and could have importanteconomic and environmental benefits.

KEYWORDS: Chemical pulp, monolignol biosynthetic pathway, pulp and paper, transgenictrees, wood

TABLE OF CONTENTS

I. Introduction 307II. Lignin 307

A. Wood—The Raw Material 307B. Structure of Lignin 309C. Biosynthesis of Lignin 309

III. Mutants and Transgenic Plants with Modified Lignin 313A. Up- and Down-Regulation of Phenylalanine Ammonia-Lyase (PAL) 313B. Up- and Down-Regulation of Cinnamic Acid 4-Hydroxylase (C4H) 317C. Knock-Out Mutation for p-Coumarate 3-Hydroxylase (C3H) 318D. Down-Regulation of Caffeic Acid O-Methyltransferase (COMT) 318

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E. Down-Regulation of Caffeoyl-CoAO-Methyltransferase (CCoAOMT) 319F. Up- and Down-Regulation of Ferulic Acid 5-Hydroxylase (F5H) 320G. Down-Regulation of 4-Coumarate:CoA Ligase (4CL) 321H. Down-Regulation of Cinnamoyl-CoA Reductase (CCR) 321I. Down-Regulation of Cinnamyl Alcohol Dehydrogenase (CAD) 322J. Up- and Down-Regulation of Peroxidases 323K. Down-Regulation of Laccases 324

IV. Papermaking Processes 324A. Mechanical Pulps 325

1. Mechanical processes based on stone grinders 3252. Mechanical processes based on refiners (RMP) 3253. Thermomechanical pulping process (TMP) 3264. Chemimechanical (CMP) and chemithermomechanical (CTMP)

pulping processes 3265. Bleaching of mechanical pulps 327

B. Semi-Chemical Pulps 327C. Chemical Pulps 327

1. Alkaline chemical pulping processes 327a. Sulfate or Kraft process 327b. Other alkaline processes for the production of chemical pulps 328

2. Acidic or bisulfite chemical pulping processes 3283. Bleaching of chemical pulps 328

a. ECF bleaching sequences 328b. TCF bleaching sequences 329

D. Biological Pulps 329V. Effect of Lignin Modifications on Pulping 329

A. Chemical Pulping of Wood from Transgenic Poplars Down-Regulatedfor COMT and CAD 330

B. Chemical Pulping of Wood from Transgenic Poplars Overexpressing F5H 334C. Chemical Pulping of Wood from Transgenic Tobacco Down-Regulated

for CCR and CAD 334D. Chemical Pulping of Wood from a PinecadMutant 334E. Mechanical Pulping of Wood from Transgenic Poplars Down-Regulated

for COMT and CAD 336VI. Conclusions and Perspectives 336Acknowledgments 337Glossary 337References 339

ABBREVIATIONS: CAD: cinnamyl alcohol dehydrogenase;CCoAOMT: caffeoyl-CoA O-methyl-transferase;CCR: cinnamoyl-CoA reductase;C4H: cinnamic acid 4-hydroxylase;C3H: p-coumarate3-hydroxylase;4CL: 4-coumarate:CoA ligase;CMP: chemimechanical pulp;COMT: caffeic acid/5-hydroxyferulic acidO-methyltransferase;CTMP: chemithermomechanical pulp;CWR: cell wall residue;DFRC: derivatization followed by reductive cleavage;DP: degree of polymerization;DRIFT: dif-fuse reflectance infrared Fourier transform;ECF: elemental chlorine free;F5H: ferulate 5-hydroxylase;FTIR: Fourier transform infrared;G: guaiacyl; GC-MS: gas chromatography-mass spectrometry;GM: genetically modified; H: p-hydroxyphenyl;HCT: hydroxycinnamoyl-CoA:shikimate/quinate hy-droxycinnamoyltransferase;HPLC: high-pressure liquid chromatography;LAC: laccase;5OHG: 5-hydroxyguaiacyl;NMR: nuclear magnetic resonance;PAL: phenylalanine ammonia-lyase;PGW: pressuregroundwood;POX: peroxidase;RMP: refiner mechanical pulp;S: syringyl;SAD: sinapyl alcohol dehydro-genase;Sin: sinapaldehyde;Sy: sum of syringaldehyde and syringic acid;Syr: syringaldehyde;TCF: totallychlorine free;TMP: thermomechanical pulp;SGW: stone groundwood;V: sum of vanillin and vanillic acid;Van: vanillin; *: see glossary.

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I. INTRODUCTION

Lignin, along with cellulose, is a major con-stituent of wood and one of the most abundantbiopolymers on Earth. Despite the importance ofwood as an industrial raw material and a renewableenergy resource, investigation into the biochemistryand molecular biology of wood formation is stillin its infancy. Relatively little is known about thedetailed biochemistry underlying the production ofcellulose and hemicellulose wood components, andgenes involved in these processes have only recentlybeen cloned. Lignin biochemistry, on the contrary,has matured over 40 years of research and muchof the molecular biology of lignification has beenelucidated over the past decade. The rapid advancein lignin research over that of other wood compo-nents has been partly due to the relative tractabilityof the problem, but has also significantly benefitedfrom the interest in, and knowledge of, lignin chem-istry gained from the pulp and paper industry. Dur-ing the manufacture of high-quality paper, lignin ischemically separated from the polysaccharide com-ponents of wood during pulping and bleaching reac-tions. Lignin extraction consumes large quantities ofchemicals and energy leading to a poor environmen-tal image for the industry (Higuchi, 1985; Odendahl,1994; Biermann, 1996). The benefits of removingas much lignin as possible in order to avoid residuallignin, which causes discoloration and reduces pa-per brightness (Chianget al., 1988), have to be bal-anced against the loss of pulp quality and strengththat result when cellulose is significantly degraded.For these reasons, new biological approaches topulping are continuously being researched. One ap-proach has been to pre-treat wood chips with en-zymes or fungi that degrade lignin (Messner &Srebotnik, 1994; Viikariet al., 1994; Paiceet al.,1995; Akhtaret al., 1998a, 1998b) and at least onesuch biopulping application is nearing commercial-ization (Kirk & Akhtar, 2000). An alternative, butcomplementary, idea has been to modify lignin con-tent or structure in genetically engineered trees toreduce lignin production or to make lignin easier toextract. Clearly, achieving this goal requires a pro-found understanding of lignin biosynthesis at thebiochemical and molecular levels. Therefore, recentresearch into lignin biosynthesis has been motivatedby the concerns and interests of the pulping industry.This research has equally been invaluable in yield-ing fundamentally new and unexpected insights intothe basic biochemistry and molecular biology of lig-nification. In some cases, the most useful result of

genetic engineering experiments aimed at improv-ing pulping has been the realization that thetra-ditional scheme of the lignin pathway is wrong insome respects, prompting renewed investigation ofthe pathway by classical biochemical and enzymo-logical approaches. In other cases, modification oflignin biosynthetic genes has indeed led to the pro-duction of plants with improved pulping character-istics, but the results have not been wholly intuitive.The ability to predict how specific genetic modifi-cations might influence pulping is still a significantchallenge to our limited understanding of the chem-istry and biochemistry of the plant cell walls thatconstitute wood.

This review presents the current status of ourunderstanding of the reactions and genes involved inlignin biosynthesis and the impact these have in de-termining lignin quality for industrial applications,primarily wood pulping. It provides an introductionto wood and lignin structure and to the lignin biosyn-thetic pathway before focusing on the large body ofinformation that has recently been gained by modi-fying the expression of lignin biosynthetic genesinplanta. Finally, following a brief overview of dif-ferent pulping processes, we evaluate the potentialimpact of changes in wood biochemistry, achievedby genetic engineering of lignin biosynthesis, to cur-rent pulping practices.

II. LIGNIN

A. Wood—The Raw Material

Wood is the major raw material for the pro-duction of pulp and paper (Food and AgricultureOrganization, 2001a). Wood characteristics vary indifferent types of plant; for instance, conifers (gym-nosperms) produce softwood whereas angiospermsproduce hardwoods. Softwoods are mainly com-posed of three cell types, tracheids (which play arole both in rigidity and conduction), and axial andray parenchyma cells. Hardwoods are mainly madeof fibers, vessels, and axial and ray parenchymacells. Vessels transport water and solutes throughthe vascular system whilefibers provide rigidity,and ray cells facilitate centripetal nutrition (Higuchi,1997). Tracheids, vessels, andfibers vary in shapeand size (Table 1). The dimensions and chemicalcomposition of the different cell types of wood de-pend on genetic and developmental factors, but arealso influenced by environmental conditions, suchas the climate and soil type (Vallette & de Choudens,

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TABLE 1Dimensions of the Different Cell Typesof Softwoods and Hardwoods (Fengel &Wegener, 1984)

CellCell Type Character Dimensions

Softwood(Picea abies)

Tracheids Diameter 20–40µmWall thickness 2.1–4.3µmLength 1.7–3.7 mm

Hardwood(Fagussp.)

Fibers Diameter 15–20µmWall thickness 5µmLength 0.6–1.3 mm

Vessels Diameter 5–100µmWall thickness 1µmLength 0.3–0.7 mm

1992). Softwoods and hardwoods differ in theirpulping characteristics. Indeed, the individual celltypes within wood differ in their chemical char-acteristics, reflecting the underlying differences inbiochemistry and molecular biology that are onlybeginning to be appreciated.

The three major components of wood cell wallsare cellulose, hemicellulose, and lignin (Table 2).Long molecules of cellulose provide the skeletonof the walls. Linear cellulose chains are aligned to-gether in structures known as‘elementaryfibrils’or ‘protofibrils’ that, in turn, associate into morecomplex structures called microfibrils (Figure 1).Microfibrils are highly organized and form distinctfibrillar cell wall layers (Delmer & Amor, 1995).

TABLE 2Chemical Components of Softwoods andHardwoods (Aitken et al., 1988)

Components Type Softwoods Hardwoods

Cellulose 42–44% 43–47%Lignin 25–30% 17–26%Hemicelluloses Hexosans±15% 5–8%

Pentosans 10–15% 15–35%Waxes, resins, 1–10% 0.5–2%

fatsMineral <1% <1%

substances

FIGURE 1. Organization of the cellulose skele-tons in the fiber wall (Parham, 1987).

Hemicelluloses and other carbohydrates provide thematrix of the cell wall, whereas lignin, a heteroge-nous hydrophobic phenolic polymer, encrusts theother wall components to waterproof and strengthenthe wall.

In a transverse plane, the parietal structure ofwood cells is made of a primary and a secondarywall, the latter consisting of two or three layers,designated S1, S2, and S3 (Figure 2). Outside theprimary wall, the middle lamella connects adjacentcells. The various cell wall layers differ in chemicalcomposition (Mellerowiczet al., 2001). Studies onthe distribution of lignin by UV microscopy show

FIGURE 2. Structural organization of the cellwalls of fibers (Petit-Conil, 1995).

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that the middle lamella and the primary wall of sec-ondary thickened cells are rich in lignin (70–80%lignin, which is approximately 15–20% of the to-tal lignin in the cell wall). In contrast, the secondarywalls are composed of only 20–25% lignin, but con-tain 80% of the total lignin because they constitutethe largest part of the total cell wall (Ferguset al.,1969).

B. Structure of Lignin

The lignin polymer is produced by the dehydro-genative polymerization of essentially three differ-ent cinnamyl alcohols (p-coumaryl, coniferyl, andsinapyl alcohol) that differ in the degree of methoxy-lation at the C3 and C5 positions of the aromaticring (Figure 3). When incorporated into lignin, thesealcohols are called thep-hydroxyphenyl (H), gua-iacyl (G), and syringyl (S) units of the polymer,respectively. In addition to the three main monolig-nols, lignin contains traces of units from incompletemonolignol biosynthesis and incorporates vari-ous other phenylpropanoid units, such as hydrox-ycinnamyl aldehydes, acetates,p-coumarates,p-hydroxybenzoates, and tyramine ferulate (Sederoffet al., 1999; Boerjanet al., 2003). A variety of chem-ical linkages, including ether and carbon-carbonbonds, connect the units in lignin (Figure 4; Higuchi,1990; Ralphet al., 1998; Boerjanet al., 2003). Thecomplexity and heterogeneity of the polymer de-pend on the relative proportions of the three princi-pal monolignol units as well as the different typesof interunit linkages (Campbell & Sederoff, 1996;Monties, 1998). For example, lignin from gym-nosperms consists mainly of G units and low lev-els of H units, whereas lignin from angiosperms ispredominantly made up of both G and S units alongwith traces of H units. Lignin from grasses incorpo-rates G and S units at comparable levels and moreH units than dicots. Lignin rich in G units, suchas gymnosperm lignin, has relatively more carbon-carbon bonds than lignin rich in S units because thearomatic C5 position of G units is free to make link-ages. As a consequence, wood lignins essentiallymade of G units (softwoods) are less susceptible toKraft delignification than lignins made of G and Sunits (hardwoods) (Chiang & Funaoka, 1990; Onaet al., 1996; Lapierreet al., 1999) because the targetsof the chemical Kraft delignification process are thenon-condensed etherβ-O-4-linkages in lignin (seesection IV.C.1.a), whereas the carbon-carbon bonds(β-β, β-1,β-5, and 5-5) are more resistant to chem-ical degradation.

Determining the amount, structure, ormonomeric composition of lignin in a plant isextremely difficult because of the heterogeneity ofthe polymer and the high proportion of covalentbonds linking different monomers. Moreover,during isolation, lignin undergoes secondarymodifications, such as condensation, oxidation,addition, or substitution. Therefore, a combinationof several methods has to be used to obtain reliableinformation on lignin structure. Different histo-chemical, chemical, and spectroscopic methodshave been developed to investigate lignin contentand composition (Monties, 1989; Lapierre, 1993;Dean, 1997; Lu & Ralph, 1997). Each method hasits own limitations (for discussion, see Anterola& Lewis, 2002) and apparent discrepancies inthe literature can often be attributed to the dif-ferent techniques used by different laboratories.Therefore, it is important that the techniques arespecified when, for example, lignin content orstructure evaluations from transgenic plants arecompared. In this review, lignin methods, whetherspecifically referred to or not, will be indicatedby superscript letters and defined in the glossary.Lignin can be quantified either by removing allthe cell wall constituents, with the exception oflignin (for example, Klasone lignin determination)or by extracting the lignin component from thecell wall (e.g., with thioglycolic acidh or acetylbromidea). The main methods used to determinelignin structure and composition are based on theanalysis of the degradation products of lignin [bypyrolysis gas chromatography-mass spectrome-try (pyrolysis GC-MS)g, alkaline nitrobenzeneoxidationb, thioacidolysisi , derivatization followedby reductive cleavage (DFRC)c, or Fourier trans-form infrared (FTIR) spectroscopyd]. The targets ofthese techniques are essentially theβ-O-4 linkagesthat are the most abundant linkages in lignin.Physical methods, such as UV and IR spectroscopy,allow the identification of particular structuresthat absorb light at specific wavelengths. Thanksto recently developed methods, such as NMRspectroscopyf, a more detailed picture of ligninstructure has been obtained, especially clarifyingthe different chemicals bonds in the polymers(Ralphet al., 1998, 1999a).

C. Biosynthesis of Lignin

Lignin monomers, or monolignols, are pro-duced intracellularly, then exported to the cell wall,and subsequently polymerized. The monolignols are

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FIGURE 3. Phenylpropanoid and monolignol biosynthesis pathways. CAD, cinnamyl alcoholdehydrogenase; 4CL, 4-coumarate:CoA ligase; C3H, p-coumarate 3-hydroxylase; C4H, cinna-mate 4-hydroxylase; CCoAOMT, caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA reduc-tase; COMT, caffeic acid O-methyltransferase; HCT, p-hydroxycinnamoyl-CoA:shikimate/quinatep-hydroxycinnamoyltransferase; F5H, ferulate 5-hydroxylase; PAL, phenylalanine ammonia-lyase;SAD, sinapyl alcohol dehydrogenase. The boxed area represents the most likely pathway to theproduction of monolignols. All enzymatic reactions shown have been demonstrated by in vitro as-says and do not necessarily occur in vivo. F5H? and CCR?, substrate not tested; ?, conversiondemonstrated but enzyme unknown; ??, direct conversion not convincingly demonstrated.

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FIGURE 4. Possible intermonomeric linkages in lignin (Ralph et al., 1998; Lapierre et al., 1999;Boerjan et al., 2003). p-hydroxyphenyl (H), R H; guaiacyl (G), R OCH3 at C-3 and R H at C-5;syringyl (S), R OCH3.

products of the phenylpropanoid pathway, startingfrom phenylalanine (Figure 3), and most of the genesinvolved in monolignol production have been clonedor are present in expressed sequence tag/genomicdatabases (for a review, see Christensenet al., 2000;Boerjanet al., 2003; Raeset al., 2003). The ligninbiosynthetic pathway has been the subject of manyrecent reviews (Baucheret al., 1998; Whettenet al.,1998; Grima-Pettenati & Goffner, 1999; Lewis,1999; Boudet, 2000; Dixonet al., 2001; Humphreys& Chapple, 2002; Anterola & Lewis, 2002; Boerjanet al., 2003), but is still being continually revisedand updated to incorporate the results of new re-search. Because of the already extensive coverageof the pathway in the literature, only the most im-portant and most recent revisions to the traditionalpathway will be described here.

The hydroxylation and methylation reactionsthat ultimately determine the monomeric composi-tion of lignin (because the three monolignols differonly in their degree of methoxylation) have longbeen considered to occur at the level of the cin-namic acids (Higuchi, 1985). Several experimentshave demonstrated that the methylation steps canalso take place at the hydroxycinnamoyl-CoA level,mediated by either caffeic acid/5-hydroxyferulicacid O-methyltransferase (COMT) or caffeoyl-CoA O-methyltransferase (CCoAOMT) (Yeet al.,1994; Li et al., 1997; Inoueet al., 1998; Martzet al., 1998; Meng & Campbell, 1998; Maury

et al., 1999). However, recent work based on ra-diotracer andin vitro enzyme assays has shownthat the hydroxylation and methylation reactionsoccur preferentially at the cinnamaldehyde and cin-namyl alcohol level in reactions catalyzed by ferulicacid 5-hydroxylase (F5H; also named conifer-aldehyde 5-hydroxylase or Cald5H) and COMT(alternatively called 5-hydroxyconiferaldehydeO-methyltransferase or AldOMT) (Matsuiet al., 1994,2000; Chenet al., 1999; Humphreyset al., 1999;Osakabeet al., 1999; Mauryet al., 1999; Liet al.,2000; Parvathiet al., 2001; Guoet al., 2002).COMT preferentially methylates caffeyl aldehyde,5-hydroxyconiferaldehyde, and 5-hydroxyconiferylalcohol (Figure 3), although differences exist be-tween the COMTs of different species analyzed(Humphreyset al., 1999; Li et al., 2000; Parvathiet al., 2001; Zubietaet al., 2002). These revisionsto the role and position of COMT within the ligninpathway help to explain the results of previous stud-ies on COMT-suppressed plants where total lignincontents were found to be maintained despite dra-matic reductions in S lignin.

A continuing controversy surrounds the in-volvement of 4-coumarate:CoA ligase (4CL) andsinapic acid in S lignin biosynthesis. Traditionally,sinapic acid was thought to be a lignin precur-sor that was converted to sinapoyl-CoA by 4CL.However, results ofin vitro experiments with 4CLsfrom different plants throw significant doubt on

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this assumption. Whereas 4CL isoforms from someplants have been found to convert sinapic acid tosinapoyl-CoA (Grandet al., 1983; Higuchi, 1997;Lindermayret al., 2002a; Yamauchiet al., 2003),enzymes from other plants apparently are deficientin this activity (Allina et al., 1998; Huet al., 1998;Ehlting et al., 1999; Dixonet al., 2001; Yamauchiet al., 2003). Small differences between 4CL iso-forms can significantly influence its activity. Re-cently, a model based on structural data postulatesthat the substrate specificity of 4CL is determinedby 12 amino acid residues (Schneideret al., 2003).Indeed, deletion of a single Val residue in the soy-bean Gm4CL2 and Gm4CL3 isoforms, which donot normally convert sinapic acid into sinapoyl-CoA, allowed these enzymes to gain this activity(Lindermayret al., 2002b). Similarly, deletion ofthe amino acid Val-355 or Leu-356 in theAra-bidopsisAt4CL2 confers activity toward sinapicacid (Schneideret al., 2003). Interestingly, a novel4CL isoform lacking the Leu residue has recentlybeen identified inArabidopsisby phylogenetic anal-ysis and this isoform efficiently converts sinapic acid(Schneideret al., 2003). These data suggest that theprogression into the monolignol biosynthesis path-way in different plants may depend on the specificityof the particular 4CLs present, and probably also ontheir cell-specific expression patterns.

Perhaps the most significant recent discoveryhas been the realization, based initially on classi-cal enzyme assays, thatp-coumaroyl-shikimate andp-coumaroyl-quinate (and notp-coumaric acid aspreviously thought) are the preferred substrates forp-coumarate 3-hydroxylase (C3H) (Schochet al.,2001). Although recombinant C3H can slowly con-vert p-coumaric acid into caffeic acid in yeast (Nairet al., 2002), this reaction is no longer thought tobe important in the lignin biosynthetic pathway.Instead,p-coumaroyl-CoA is probably convertedinto shikimate and quinate esters by a reversiblehydroxycinnamoyl-CoA:shikimate/quinate hydro-xycinnamoyltransferase (HCT) and these com-pounds subsequently act as preferred substrates forC3H (Schochet al., 2001). Recently, the gene en-coding HCT has been cloned (Hoffmannet al.,2003). After hydroxylation by C3H, the resultingcaffeoyl-CoA esters are converted to caffeoyl-CoAby the same enzyme, HCT, before being methy-lated by CCoAOMT or COMT, as previously en-visaged. Significantly, analysis ofArabidopsismu-tants in theC3H gene provides strong support forthe proposed revised role of this enzyme in thelignin biosynthetic pathway (Frankeet al., 2002a,2002b).

Another potential revision to the lignin path-way concerns the role of cinnamyl alcohol dehydro-genase (CAD), the enzyme thought to catalyze thereduction of the cinnamaldehydes, coniferyl alde-hyde, and sinapyl aldehyde into cinnamyl alcohols.Recently, Liet al. (2001) have identified a sinapylalcohol dehydrogenase (SAD) from poplar that usessinapaldehyde as the preferred substrate and demon-strated that SAD co-localizes with S lignin forma-tion both temporally and spatially. This suggests thatSAD may be the enzyme responsible for the produc-tion of sinapyl alcohol, the S lignin precursor, rele-gating CAD to a role only in the production of the Glignin precursor, coniferyl alcohol (Liet al., 2001).The data provided by Liet al. (2001) are convinc-ing, but circumstantial. Further work, particularlythe production ofsadmutants or SAD-suppressedtransgenic plants, is needed to validate/investigatethe role of SADin vivo in a variety of angiosperms.

The final steps in the biosynthesis of ligninare the oxidation of the cinnamyl alcohols to thecorresponding radicals in the cell wall and theirsubsequent polymerization. The possible mecha-nisms of lignin polymerization have been recentlyreviewed (Lewis, 1999; Christensenet al., 2000;Boerjan et al., 2003). A significant body of re-search over the past decade has proposed that perox-idases, laccases, and other phenol oxidases may beinvolved in the radical formation process (Savidge& Udagama-Randeniya, 1992; Dean & Eriksson,1994; McDougallet al., 1994; Richardsonet al.,1997). Recently,Onnerudet al. (2002) have sug-gested that the monolignols are polymerized by aredox shuttle-mediated oxidation. However, the ex-act role of these different enzymes remains as elu-sive as ever and little real progress has been made.This is in part due to the multiplicity of these en-zymes that exist in plant cells and the probabilitythat there may be some redundancy in function be-tween them. Consequently, determining the role ofindividual enzymes in a process such as lignificationcan be difficult. This lack of progress on the mecha-nisms of lignin polymerization has exacerbated thesurprisingly heated controversy that has developedover whether monolignol coupling is a random or ahighly orchestrated process. With the discovery of adirigent protein inForsythia, capable of catalyzingthe stereoselective coupling of two coniferyl alco-hol radicals into the lignan pinoresinol (Davinet al.,1997), it has been suggested that monolignol radicalcoupling during lignin biosynthesis is also tightlycontrolled in plants (Ganget al., 1999)—a proposi-tion in direct conflict to the well-established andsupported random coupling model (Hatfield &

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Vermerris, 2001). This controversial suggestion,however, still awaits testing by reverse geneticapproaches.

A persistent challenge is to understand howsimilar enzymes in different plants can give riseto the striking natural heterogeneity that exists inlignin. The content and composition of lignin differsnot only among plant taxa, but also between differ-ent cell types of a single tissue and even within asingle cell wall (Joseleau & Ruel, 1997). Lignin canalso be influenced by environmental stress. Currentthinking regards lignin heterogeneity as a productof the spatio-temporal and conditional expressionof the genes involved in the lignin pathway and ofdifferences in the substrate specificity and kineticsof the enzymes they encode (Campbell & Sederoff,1996; Weisshaar & Jenkins, 1998; Chenet al., 2000;Hardinget al., 2002; Zubietaet al., 2002). Theseprocesses may also be regulated by pathway inter-mediates because the concentration of certain in-termediates has been shown to affect enzyme ac-tivity and gene expression (Osakabeet al., 1999;Blount et al., 2000; Liet al., 2000; Anterolaet al.,2002). Despite our increasing knowledge of ligninbiosynthesis, major uncertainties remain (Boudetet al., 1995; Whetten & Sederoff, 1995; Campbell &Sederoff, 1996; Douglas, 1996; Dixonet al., 2001).For instance, little is known about the cell biology ofthe process. Although the monolignols are assumedto be stored as glucosides before being transportedto the cell wall and polymerized into lignin, littlehard evidence supports this assumption. Similarly,the precise subcellular location of most of the ligninbiosynthetic enzymes is still an open question. Fur-thermore, as illustrated above, some of the recentrevisions to the lignin pathway have been madepurely on the basis of classical biochemical experi-ments to determine the substrate specificity and ki-netics of action of particular enzymes. It is crucialto determine whether these experiments, performedin vitro, reliably indicate the role of the respectiveenzymesin vivo. In order to gain a fuller under-standing of how the lignin biosynthetic pathwayoperatesin vivo, many research laboratories havebeen studying mutant and transgenic plants with al-tered expression of lignin biosynthetic genes. Bothmodel (Arabidopsisand tobacco) and economicallyimportant species (alfalfa and poplar) have been ge-netically engineered, and results obtained have gen-erally been in accordance. This research has revo-lutionized our understanding of lignin biosynthesis,having prompted and, in some cases, confirmed therevisions to the pathway made on the basis of bio-

chemical data. It has also opened up new research ar-eas, posed new questions, and indicated how lignincould be modified to improve industrial processes,such as pulping.

III. MUTANTS AND TRANSGENICPLANTS WITH MODIFIED LIGNIN

A. Up- and Down-Regulation ofPhenylalanine Ammonia-Lyase(PAL)

Phenylalanine ammonia-lyase (PAL) catalyzesthe first step of the phenylpropanoid pathway—the non-oxidative deamination ofL-phenylalanineto cinnamic acid (Figure 3). PAL has been sup-pressed by 85% and>98% in the stems of trans-genic plants with resultant 52% (Sewaltet al.,1997) and 70% (Korthet al., 2001) reductionin Klasone lignin content, respectively (Table 3).Lignin monomeric composition, determined by py-rolysis GC-MSg, was characterized by a lower pro-portion of G units and a 1.7-fold increase in S/G ratio(Sewaltet al., 1997). Similarly, using an indepen-dent methodi, Korth et al. (2001) determined a4-fold increase in the S/G ratio caused by a morepronounced reduction in the level of G units than inS units. Because PAL catalyzes thefirst step of thephenylpropanoid pathway, reduction of its activityresults in a wide range of abnormal phenotypes. Thetransgenic plants were stunted, had curled leaves,and had thinner cell walls in the secondary xylemwith less lignin than those of the control (Elkindet al., 1990; Bateet al., 1994). These plants werealso more susceptible to the fungal pathogenCer-cospora nicotianae(Maheret al., 1994). A slightincrease in Klasone lignin and dry matter contentwas observed in the stem of PAL-overexpressingplants (Howleset al., 1996; Korthet al., 2001).Overexpression of PAL did not lead to changes inlignin composition as determined by pyrolysis GC-MSg (Sewaltet al., 1997), but to a decrease in theamount of S units, yielding a reduction in the S/Gratio when lignin was analyzed by thioacidolysisi

(Korth et al., 2001). The level of chlorogenic acid(3-caffeoylquinic acid) has been correlated withthe PAL enzymatic activity in leaves and stems ofboth PAL-silenced and PAL-overproducing tobacco(Elkind et al., 1990; Bateet al., 1994; Maheret al.,1994; Howleset al., 1996; Blountet al., 2000; Korthet al., 2001).

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TABLE 3Lignin Mutants and Transgenic Plants

Enzyme Activity Lignin LigninPlant Gene In Stems (%) Content Composition References

Arabidopsis thalianaC3H (ref8mutant) — Reduced1,2 Traces of S and G5,6,11, Frankeet al. (2002b)

— principally H5,6,11

COMT(Atomt1mutant) 6.6 Increased1 Traces of S, G Goujonet al. (2003a)increased,5OHG7

COMT(S) 212 No changes1 No Changes7 Goujonet al. (2003a)F5H (fah1mutant) — No changes1 Trace levels of Syr5, Chappleet al. (1992),

Sy5 and S8,11, Meyeret al. (1998),Van5, V5 or G11 Maritaet al. (1999),increased Siboutet al. (2002)

F5H (S) — Reduced1 Sy5 or S8,11 increased, Meyeret al. (1998),V5 or G11 decreased Maritaet al. (1999)

F5H (S) — Reduced1 S increased7, G decreased7 Siboutet al. (2002)4CL (AS) 8 Reduced2 V decreased5, Leeet al. (1997)

Sy/V increased5

CCR(AS) 19 Reduced1 S/G increased or decreased Goujonet al. (2003b)depending on cultureconditions7

CCR(irx4 mutant) — Reduced2,8 — Joneset al. (2001)CAD (Atcad-Cmutant) 38 Reduced1 No changes7 Siboutet al. (2003)CAD (Atcad-Dmutant) 6 Reduced1 S decreased7, G increased7, Siboutet al. (2003)

S/G decreased7, Sin andSyr increased7

Liriodendron tulipiferaLAC (AS) — No changes No changes Deanet al. (1998)

Lycopersicon esculentumPOX(S) — Increased2 — El Mansouriet al.

(1999)Medicago sativa

COMT(AS) 4.5 Reduced1 G decreased7, Guoet al. (2001)S/G decreased7,no S7, no 5OHG7

COMT(S) 15 Reduced1 S7,8,11 and G8,11 decreased, Guoet al. (2001),S/G decreased7,8,11, Maritaet al. (2003)5OHG7,8, Syr/V decreased5

CCoAOMT(AS) 4 Reduced1 S11 and G7,11 decreased, Guoet al. (2001),S/G increased7,8,11, Maritaet al. (2003)Syr/V increased5

COMT(AS)/ 12/5 Reduced1, S decreased7, Guoet al. (2001)CCoAOMT(AS) No changes4 S/G decreased7

COMT(S)/ 11/25 Reduced1, S and G decreased7, Guoet al. (2001)CCoAOMT(S) No changes4 S/G decreased7

CAD (AS) 30 No changes1 More aldehydes, Baucheret al. (1999)S/G decreased7

Nicotiana tabacumPAL (S) 2.3 Reduced1 S and G decreased7, Korth et al. (2001)

S/G increased7

PAL (S) 10 Reduced1,2,13 S/G increased6, Elkind et al. (1990),G decreased6 Bateet al. (1994),

Sewaltet al. (1997)PAL (S) 150 Increased1 S decreased7, Korth et al. (2001)

S/G decreased7

(Continued on next page)

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TABLE 3Lignin Mutants and Transgenic Plants (Continued)

Enzyme Activity Lignin LigninPlant Gene In Stems (%) Content Composition References

PAL (S) 200 Increased1 No changes6 Howleset al. (1996),Sewaltet al. (1997)

C4HI (AS) 20 Reduced1 S/G decreased6 Sewaltet al. (1997)C4HI (S) 200 No changes1 No changes6 Sewaltet al. (1997)C4HII (AS and S) 10 Reduced4 S/G decreased7 Bleeet al. (2001)COMT(AS) 42 Reduced2 No changes5 Ni et al. (1994)COMT(AS) 54 No changes3 Sy/V decreased5, Dwivedi et al. (1994)

Sy decreased5

COMT(S) 2 No changes1 S decreased7, Atanassovaet al. (1995)5OHG7

COMT(S) 322 — No changes7 Atanassovaet al. (1995)CCoAOMT(AS) 25 Reduced1 S/G increased6, Zhonget al. (1998)

S and G decreased7

CCoAOMT(AS) ∼20 Reduced1 No changes7 Pinconet al. (2001a)CCoAOMT(AS)/ 8/14 Reduced1 S/G decreased6, Zhonget al. (1998)

COMT(AS) S and G decreased6

CCoAOMT(AS)/ ∼20/∼30 Reduced1 S decreased7, 50 HG7 Pinconet al. (2001a)COMT(AS)

F5H (S) — Reduced1,2 or S/G increased5, Frankeet al. (2000)lignin more S increased5,11

extractable and G decreased5

4CL (S) <1 Reduced1,4 Syr/Van decreased Kajitaet al.or increased5, (1996, 1997)less Van5 and Syr5,, morep-hydroxybenzaldehyde5,H increased, S and Greduced15, more HCAs8

4CL (AS) ∼20 Reduced4 Syr/Van increased, Kajitaet al. (1996)less aldehydes5

CCR(AS) 30 Reduced1,4 S and G decreased7, Piquemalet al. (1998),S/G increased7, Ralphet al. (1998)tyramine ferulateincreased8

CCR(S) 1 Reduced1 S and G decreased7, O’Connellet al. (2002)S/G increased7

COMT(AS)/ 41/10 Reduced1 S/G increased7 Pinconet al. (2001b)CCR(AS)

CAD2(AS) 7 No changes1 More aldehydes6, Halpinet al. (1994)S/G decreased7

CAD2(AS) 8 No changes1,4 S/G decreased7, Yahiaouiet al. (1998),more cinnamaldehydes8 Ralphet al. (1998)

CAD2(AS) 9 No changes4 More aldehydes10 Stewartet al. (1997)CAD2(AS) 45 No changes4 More aldehydes7 Higuchiet al. (1994),

Hibino et al. (1995)CAD2(AS)/ variable/ No changes1,4 No changes7 Abbottet al. (2002)

COMT(AS) variableCAD2(S)/ 15/36 Reduced1,4 — Abbottet al. (2002)

COMT(S)CAD2(AS)/ 12/32 Reduced1 S/G increased7, Chabanneset al. (2001b)

CCR(AS) S and G decreased7,tyramine ferulateincreased8

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TABLE 3Lignin Mutants and Transgenic Plants (Continued)

Enzyme Activity Lignin LigninPlant Gene In Stems (%) Content Composition References

CAD2(S)/ 4/24/18 Reduced1,4 — Abbottet al. (2002)COMT(S)/CCR(S)

POX(S) 250 Increased2,4 No changes7 Chabbertet al. (1992),Lagrimini (1991)

POX(S) — No changes1 More coniferaldehyde7 Elfstrandet al. (2002)POX(AS) 1 No changes2,4 No changes7 Chabbertet al. (1992),

Lagrimini et al. (1997a)Pinus taeda

CAD (cad-n1mutant) 1 Reduced1 More dihydroconiferyl MacKayet al. (1997),alcohol8, more Ralphet al. (1997)coniferaldehyde8

Populus kitakamiensisPOX(AS) 56 Reduced4,12 Moreβ-O-47 Yahonget al. (2001)

P. tremula x P. albaCOMT(AS) 5 No changes1 S/G decreased7, Van Doorsselaereet al.

G increased7, (1995), Lapierreet al.5OHG7 (1999), Ralphet al.

(2001a, 2001b), Pilateet al. (2002)

COMT(S) <3 Reduced1 S/G decreased7, Jouaninet al. (2000),5OHG7, Ralphet al. (2001b)more coniferaldehyde7

CCoAOMT(S) 10% protein Reduced1 S/G increased7, Meyermanset al.amount S and G decreased7, (2000)

more p-hydroxybenzoicacid8

CCoAOMT(AS) 30 Reduced1,14 S and G decreased6 Zhonget al. (2000)F5H (S) — — S/G increased11, Frankeet al. (2000)

Sy5 or S11 increased,V decreased5

CCR(S, AS) — Reduced1 S/G increased7 J.-C. Leple, C. Lapierreand W. Boerjan

CAD2(AS) 30 Reduced1 More syringaldehyde Baucheret al. (1996),Lapierreet al. (1999),Pilateet al. (2002)

CAD2(S) 30 No changes1 No changes7 Baucheret al. (1996),Lapierreet al. (1999)

POX(S) 800 No changes1 No changes7 Christensenet al.(2001a, 2001b)

LAC (AS) — No changes1,4 No changes7 Ranochaet al.(2000, 2002)

P. tremuloidesCOMT(S) 28 No changes1,4 S/G decreased7, Tsaiet al. (1998)

5OHG7, moreconiferaldehyde7

4CL (AS) 10 Reduced1 No changes7 Hu et al. (1999)4CL (AS) 10 Reduced1 No changes7,8 Li et al. (2003)F5H (S) 280 No changes1 S/G increased7,8 Li et al. (2003)4CL (AS)/ 10/110 Reduced1 S/G increased7,8 Li et al. (2003)

F5H (S)(Continued on next page)

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TABLE 3Lignin Mutants and Transgenic Plants (Continued)

Enzyme Activity Lignin LigninPlant Gene In Stems (%) Content Composition References

Sorghum bicolorCOMT(bmr12and — Reduced1,4 S reduced6,7, 5OHG6,7, Chabbertet al. (1993),bmr18mutants) Syr/V decreased5 Suzukiet al. (1997),

Bout & Vermerris(2003)

COMT(bmr26mutant) — — S reduced6 Bout & Vermerris(2003)

CAD?(bmr6mutant) ∼30 Reduced1,2 S and G decreased6,7, Pillonelet al. (1991),coniferaldehyde Chabbertet al. (1993),increased6,7, Suzukiet al. (1997)Van and Syr decreased5,Syr/V decreased5

Zea maysCOMT(bm3mutant) 10 Reduced1,4 S7 and Syr5 decreased, Kuc et al. (1968),

5OHG7 Grandet al. (1985),Lapierreet al. (1988),Chabbertet al. (1994a,1994b), Vignolset al.(1995), Suzukiet al.(1997).

CAD (bm1mutant) ∼30 Reduced1 S and G decreased7 Halpinet al. (1998)

Numbers in superscript refer to the method used to analyze lignin content or composition;1Klason;2thioglycolic acid;3sum of Klason and acid-soluble lignin;4acetyl bromide;5nitrobenzene oxidation;6pyrolysis GC-MS;7thioacidolysis;8NMR; 9alkaline hydrolysis followed by gas chromatography;10FTIR, 11DFRC; 12permanganate oxidation;13toluidineblue and UVfluorescence;14DRIFT;15Pyrolysis GC; (AS), transgenic plants with an antisense construct; G, guaiacyl; H,p-hydroxyphenyl; HCA, hydroxycinnamic acid; 5OHG, 5-hydroxyguaiacyl; LAC, laccase; POX, peroxidase; S, syringyl;(S), transgenic plants with a sense construct; Sin; sinapaldehyde; Sy, sum of syringaldehyde and syringic acid; Syr,syringaldehyde; V, sum of vanillin and vanillic acid; Van, vanillic acid;—, not determined; ?, gene not identified.

B. Up- and Down-Regulation ofCinnamic Acid 4-Hydroxylase (C4H)

Hydroxylation at the C4 position of cinnamicacid to p-coumaric acid is catalyzed by C4H, acytochrome P450-linked monooxygenase belongingto the CYP73 subfamily (Teutschet al., 1993;Chapple, 1998). In transgenic tobacco plants, C4Hactivity was altered by expressing the alfalfa class IC4H (CYP73A3) (Sewaltet al., 1997; Blountet al.,2000) or the French bean class II C4H (CYP73A15)(Bleeet al., 2001) genes in sense or antisense orien-tation (class I and class II C4H share approximately60% similarity). Overexpression of class I C4H hadno effect on Klasone lignin, nor on the S/G ratiog.In contrast, a 76% reduction in total C4H activ-ity led to a 63% decrease in Klasone lignin and amodification of the lignin monomeric composition.The amount of S unitsg was strongly reduced andS/G decreased by over 90% (Sewaltet al., 1997).Similarly, a reduction by 90% of C4H activity by

down-regulation of the class II C4H resulted in a27% decreased lignin contenta, and one line had adecreased S/G ratioi (Blee et al., 2001). These re-sults are hard to reconcile with an increase in S/Gwhen PAL is suppressed (see previous section) be-cause PAL and C4H are presumed to be sequen-tial enzymes in the lignin pathway. Three possibleexplanations have been proposed by Dixonet al.(2001): (i) the pathway to G lignin may some-how bypass C4H; (ii) C4H may catalyze additionalreactions in the lignin biosynthetic pathway; and(iii) a specific form of C4H might be organized ina complex with other enzymes or in a metabolicchannel committed to S lignin biosynthesis. In sup-port of this last proposition, Rasmussen and Dixon(1999) have shown apparent channeling betweenPAL and C4H, which was reduced by overproduc-tion of PAL. Other evidence potentially support-ing the channeling hypothesis is the appreciationthat regulatory mechanisms probably exist to co-ordinate PAL and C4H expression. For example,

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decreased PAL activity has been shown in transgenictobacco modified to reduce C4H expression (Blountet al., 2000). Regulation may be mediated by path-way intermediates; for example, cinnamic acid mayact as a feedback regulator of the phenylpropanoidpathway. Nevertheless, given the importance of thesuggestion that metabolic channeling may exist onthe lignin pathway, it is surprising that little directproof has yet been provided. The detection of pro-tein:protein complexes, for example, should not bedifficult given the sophistication of modern molec-ular, biochemical, and cell biological methods andwill undoubtedly provide a productive area for in-vestigation in the near future.

C. Knock-Out Mutation forp-Coumarate 3-Hydroxylase (C3H)

The gene encodingp-coumarate 3-hydroxylase(C3H) has only recently been cloned by two in-dependent research groups. Using a functional ge-nomics approach, Schochet al. (2001) identi-fied CYP98A3 as a possible candidate for C3H.CYP98A3 was highly expressed in developingxylem as determined by immunolocalization andhad high activity when using 5-O-(4-coumaroyl)D-quinate and 5-O-(4-coumaroyl) shikimate as sub-strates and low activity when usingp-coumaroyl-CoA (Figure 3). In parallel, by screeningAra-bidopsis mutants under UV light, Frankeet al.(2002a) isolated the reduced epidermalfluores-cence 8 (ref8) mutant. By positional cloning, theREF8gene was identified as the cytochrome P450-dependent monooxygenaseCYP98A3. Theref8mu-tant had collapsed xylem vessels, a higher cell walldegradability, and a higher susceptibility to fungalcolonization (Frankeet al., 2002b), associated withthe accumulation ofp-coumarate esters instead ofsinapoylmalate and with a reduction in lignin con-tent of 60-80%e,h. A range of analyses showed thatlignin composition was dramatically altered, be-ing almost entirely made ofp-coumaryl alcoholunitsb,c,g (Frankeet al., 2002b). This work con-firmed that CYP98A3 is C3H and added furthersupport to the idea that the quinate and shikimateesters ofp-coumaric and caffeic acid are probablyimportant intermediates in lignin biosynthesis.

D. Down-Regulation of Caffeic AcidO-Methyltransferase (COMT)

Down-regulation of COMT activity has beenachieved using either antisense or sense transgenes

in tobacco (Dwivediet al., 1994; Niet al., 1994;Atanassovaet al., 1995), poplar (Van Doorsselaereet al., 1995; Tsaiet al., 1998; Jouaninet al.,2000), and alfalfa (Guoet al., 2001). In all threespecies, drastic reductions in the lignin S/G ratioc,i

were apparent and an unusual phenolic compound(5-hydroxyguaiacyli , 5OHG; Figure 3) was presentin the polymer (Atanassovaet al., 1995; VanDoorsselaereet al., 1995; Tsaiet al., 1998; Lapierreet al., 1999; Jouaninet al., 2000; Guoet al., 2001;Maritaet al., 2003). 5OHG has been described in thelignin of maizebm3(Chabbertet al., 1994a, 1994b;Suzuki et al., 1997), sorghumbmr6 and bmr18(Suzuki et al., 1997), andArabidopsis Atcomt1i

(Goujon et al., 2003a) mutants. In the lignin ofthe transgenic poplars described by Jouaninet al.(2000), the level of 5OHG units even exceeded thatof S units, whereas lignin of theArabidopsismu-tant totally lacked S units and had increased lev-els of 5OHG and G unitsi . A novel dimer com-posed of a G unit linked to a 5OHG unit by anα-β diether bond has previously been identifiedas a normal, but minor, component of the ligninpolymer by thioacidolysisi (Figure 5) (Hwang andSakakibara, 1981). These data, together with NMRf

experiments showing the presence of benzodiox-ane units in lignin, demonstrate that 5OHG is in-corporated into lignin as a monolignol (Figure 5)(Jouaninet al., 2000; Ralphet al., 2001a, 2001b;

FIGURE 5. Molecules found in alteredamounts in the lignin of transgenic plants, asdetermined by NMR. 1Ralph et al. (2001b);2Ralph et al. (1997); 3Ralph et al. (1999b);4Ralph et al. (1998).

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Marita et al., 2003). In COMT-suppressed trans-genic plants, the yield of thioacidolysisi products(S+G+5OHG) was lower than in wild-type plants,indicating a reduced proportion of the targets of thisdegradative procedure, theβ-O-4 linkages, and acorrespondingly increased proportion of C-C link-ages. Indeed, an enrichment in biphenyl (5-5) andphenylcoumaran (β-5) linkages has been detectedi

(Lapierreet al., 1999; Jouaninet al., 2000; Guoet al., 2001). The increased frequency of thesedimeric structures corresponds with an increase inthe degree of lignin condensation, which makes thelignin more similar to softwood lignin. In COMT-suppressed alfalfa,β-β, β-1, andβ-5 linkages in-volving S units, were absenti (Guo et al., 2001),whereas in COMT-down-regulated poplar, free phe-nolic groups inβ-O-4-linked G units (Figure 4)were less abundant (Lapierreet al., 1999). In poplar,when the bark was removed, the wood of the COMT-suppressed lines had a rose (Van Doorsselaereet al.,1995) or red-brown color (Tsaiet al., 1998; Jouaninet al., 2000) compared to the whitish wild-typewood—this coloration has been ascribed to an in-creased amount of coniferaldehyde (Tsaiet al.,1998).

In the maizebm3and the sorghumbmr6 andbmr18 mutants, which have little COMT activ-ity (Kuc et al., 1968) because of mutations inthe COMT gene (Vignolset al., 1995; Bout &Vermerris, 2003), lignin content is reduced. InCOMT-suppressed alfalfa, Klasone determinationsshowing that lignin content is reduced are at oddswith data from acetyl bromidea determinations inwhich no change in lignin amount is found (Guoet al., 2001; Maritaet al., 2003). Similarly, inCOMT-suppressed poplar, reduced lignin contente

has been reported (Jouaninet al., 2000) as wellas no change in lignin amount in poplare (VanDoorsselaereet al., 1995) or aspena,e (Tsai et al.,1998). Reports describing COMT-suppression intobacco also differ on whether lignin content ish

(Ni et al., 1994) or is note (Dwivedi et al., 1994;Atanassovaet al., 1995) reduced. In theArabidop-sis Atcomt1mutant, even a slight increase in lignincontente was detected (Goujonet al., 2003a). Theexplanation for these discrepancies is not knownand may be related to the actual level of COMTsuppression achieved in each case. However, thesedata also highlight the caution that has to be exer-cised when comparing lignin data from plants grownfor different lengths of time under varying envi-ronmental conditions and analyzed using differenttechniques.

Despite these small discrepancies, the data fromall of the COMT-suppressed tobacco and poplarplants indicate that COMT plays a predominantrole in determining the incorporation of S unitsinto the lignin polymer in these species. These re-sults are consistent with those of Liet al. (2000),who showed, for several angiosperm species, that5-hydroxyconiferaldehyde is the preferred substratefor COMT in vitro. In alfalfa, a reduction inCOMT activity affected both the content of G andS units (Guoet al., 2001; Maritaet al., 2003),in accordance with the results of Parvathiet al.(2001), who found that in alfalfa, COMT is alsoinvolved in the methylation of caffeyl aldehyde(Figure 3).

E. Down-Regulation ofCaffeoyl-CoA O-Methyltransferase(CCoAOMT)

Until recently, the methylation reactions at theC3 and C5 hydroxyl functions of the lignin pre-cursors were thought to occur mainly at the cin-namic acid level by bi-functional COMT. However,the association of CCoAOMT expression with lig-nification (Pakuschet al., 1991; Yeet al., 1994;Ye & Varner, 1995; Ye, 1997; Martzet al.,1998; Chenet al., 2000) and the observation thatdown-regulation of COMT preferentially affectedthe amount of S units (see section III.D) sug-gested the existence of an alternative pathway forthe methylation of the lignin precursors at thehydroxycinnamoyl-CoA level (Figure 3). Down-regulation of CCoAOMT affected the Klasone lignincontent by 12–50% in transgenic tobacco (Zhonget al., 1998; Pincon et al., 2001a), alfalfa (Guoet al., 2001; Marita et al., 2003), and poplar(Meyermanset al., 2000; Zhonget al., 2000). Intobacco and poplar, the decreased lignin contentwas due to reduction of both G and S units asdetermined by pyrolysis GC-MSg (Zhong et al.,1998, 2000) or thioacidolysisi (Meyermanset al.,2000). Because the decrease in G units was morepronounced, the S/G ratio increased (Zhonget al.,1998; Meyermanset al., 2000). In contrast, theS unit amount was not reduced in transgenic al-falfa (Guoet al., 2001) nor in other, independentlyproduced, tobacco (Pinconet al., 2001a), as deter-mined by thioacidolysisi . In addition, Zhonget al.(2000) have shown by diffuse reflectance infraredFourier transform (DRIFT)d spectroscopy that the

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lignin extracted from wood of the transgenic poplarswas less cross-linked than that of the control. Incontrast to the transgenic poplars, which were notaffected in growth or morphology, the transgenictobacco plants down-regulated for CCoAOMT hadcollapsed vessel walls (probably because of thereduced lignin content) and altered growth andflower development (Pincon et al., 2001a). Otherconsequences of CCoAOMT down-regulation intransgenic poplar were an enhancedfluorescenceof the vessel cell walls and the accumulation ofp-hydroxybenzoic acid esterified to lignin. In ad-dition, increased amounts of methanol-extractablephenolics, namelyO3-β-D-glucopyranosyl-caffeicacid, O4-β-D-glucopyranosyl-sinapic acid, andO4-β-D-glucopyranosyl-vanillic acid, were de-tected in the wood of the transgenic poplars(Meyermanset al., 2000). Similarly, soluble caf-feoyl glucoside accumulated in stem extracts oftransgenic alfalfa (Guoet al., 2001). The ac-cumulation of glucosides of caffeic and sinapicacid results most probably from a detoxifica-tion of free caffeic and sinapic acid, as indicatedby feeding experiments with these two hydrox-ycinnamic acids (Meyermanset al., 2000). Theobservation that lignin content was reduced inpoplars down-regulated for CCoAOMT, whereasO4-β-D-glucopyranosyl-sinapic acid accumulated,is in agreement with the hypothesis that sinapicacid is not the main precursor for S unitsinvivo. The incorporation in the cell wall ofp-hydroxybenzoic acid may be responsible for theincreasedfluorescence (Meyermanset al., 2000).A red (Meyermanset al., 2000) or a light orange(Zhonget al., 2000) color has been noticed in thexylem of the transgenic poplars, but its origin isunknown.

Simultaneous down-regulation of both COMTand CCoAOMT in tobacco (Zhonget al., 1998;Pinconet al., 2001a) and alfalfa (Guoet al., 2001)resulted in combinatorial and/or additive effects.In comparison with the respective single transfor-mants, a greater reduction in Klasone lignin contentwas measured in tobacco (Pinconet al., 2001a) butnot in alfalfa (Guoet al., 2001). In both species, thelignin S/G ratio was reduced although in tobaccothis was due to decreases in both G and S unitsg

(Zhonget al., 1998), whereas only S units decreasedin alfalfai (Guoet al., 2001). To explain the relativepreservation of G units in alfalfa, Guoet al. (2001)suggest that additional enzymes may be involved inthe methylation of the monolignol precursors of Gunits.

F. Up- and Down-Regulationof Ferulic Acid 5-Hydroxylase (F5H)

For many years, it was generally acceptedthat the 5-hydroxylation of the monomethoxylatedlignin precursor, catalyzed by the enzyme ferulicacid 5-hydroxylase (F5H), would take place atthe hydroxycinnamic acid level (Figure 3). How-ever, Osakabeet al. (1999) and Humphreyset al.(1999) demonstrated that the 5-hydroxylation ofthe monomethoxylated precursor occurs prefer-entially at the cinnamaldehyde level, leading tothe renaming of the enzyme to coniferaldehyde5-hydroxylase or Cald5H. A number of reports haveindicated that the hydroxylation step at C5 mayalso occur at the cinnamyl alcohol level (Matsuiet al., 1994, 2000; Daubresseet al., 1995; Chenet al., 1999; Humphreyset al., 1999; Parvathiet al.,2001).

An Arabidopsismutant deficient in F5H (fah1)was described more than 10 years ago. The mu-tant produced a lignin deficient in S units (Chappleet al., 1992) with a consequently increased fre-quency of phenylcoumaran (β-5) and biphenyl (5-5) linkages (Maritaet al., 1999) (Figure 4). Onthe other hand, whenArabidopsisF5H was over-expressed from theC4H promoter in the mutant, alignin that was almost entirely composed of S unitslinked by β-O-4 linkages was produced (Meyeret al., 1998; Maritaet al., 1999). The proportion ofS units in the lignin of these plants was the highestever reported for any plant (Ralph, 1996). Similarly,lignin of tobacco and poplar transformed with thesame chimeric gene was enriched in S units (Frankeet al., 2000). Recently, Liet al. (2003) have overex-pressed a sweetgum F5H (Cald5H) under the controlof a xylem-specific promoter (Pt4CL1P) in trans-genic aspen and reported a 2.5-fold increase in theS/G ratioi and no changes in lignin contente. Inter-estingly, an accelerated maturation/lignification ofstem secondary xylem cells was noted in these F5H-overexpressing plants, a phenomenon attributed tothe involvement of oligomeric S lignin moieties insignaling mechanisms that promote secondary wallthickening (Liet al., 2003). More research is neededto explore and potentially confirm this intriguingpossibility.

In F5H-overexpressingArabidopsis, benzodi-oxane structures were detected in lignin, prob-ably as a consequence of the increasedflux to5-hydroxyconiferaldehyde and 5-hydroxyconiferylalcohol (Ralphet al., 2001b). A 25–35% reduc-tion in Klasone lignin content was observed in

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F5H-overexpressingArabidopsis (Marita et al.,1999) and tobacco (Frankeet al., 2000). However,the apparent reduction in Klasone lignin may bean artefact because S units have fewer radical cou-pling sites, reducing the possibilities for couplingand branching and thereby making S-rich ligninsmore easily degraded and extracted (Maritaet al.,1999; Frankeet al., 2000). These studies demon-strate that F5H plays a pivotal role in determininglignin monomer composition. Therefore, modifyingF5H expression and altering the relative amount of Sunits may offer opportunities for engineering ligninquality in hardwoods and softwoods.

G. Down-Regulationof 4-Coumarate:CoA Ligase (4CL)

Transgenic plants with reduced 4CL activityhave been produced in tobacco (Kajitaet al., 1996,1997),Arabidopsis(Leeet al., 1997), and aspen (Huet al., 1999; Liet al., 2003). In tobacco, reduction of4CL by over 90% resulted in 25% less lignina,i . Inpoplar andArabidopsiswith a>90% reduced 4CLactivity, lignin content was reduced by 45–50%i.

In tobacco, the low 4CL activity was associ-ated with browning of the xylem tissue (Kajitaet al.,1996). The monomeric composition of ligninb wasaltered and characterized by a 3-fold increase in theamount ofp-hydroxybenzaldehyde and an 80% anda 67% decrease in the amount of syringaldehyde(Syr) and vanillin (Van), respectively, resulting ina 40% reduction in the Syr/Van ratio (Kajitaet al.,1997). The amount of the ester- and ether-linkedp-coumaric, ferulic, and sinapic acids increased dra-matically in the brown xylem tissue (as determinedby alkaline hydrolysis of the cell walls followedby gas chromatography and NMRf of milled woodlignin). In contrast, in transgenicArabidopsis,theSy/V (Sy is the sum of syringaldehyde and syringicacid and V is the sum of vanillin and vanillic acid)ratiob was increased because of a 40% reduction inthe amount of V units, suggesting that 4CL is re-quired for the synthesis of G, but not S units, as pos-tulated by Leeet al. (1997) and Huet al. (1998).In transgenic aspen down-regulated for 4CL, Huet al. (1999) also detected an increase in non-lignin alkali-extractable wall-bound phenolics (p-coumaric acid, caffeic acid, and sinapic acid), andshowed by NMRf that these acids were not incorpo-rated into the lignin polymer. However, they did notdetect any difference in lignin S/G composition us-ing thioacidolysisi , in contrast to the data obtained

for Arabidopsisand tobacco. Another discrepancybetween the results published by Kajitaet al. (1997)and Huet al. (1999) is that the transgenic tobaccolines with the most severe reduction in lignin con-tent (25%) were characterized by a collapse of vesselcell walls and reduced growth (Kajitaet al., 1997),whereas the transgenic poplars with a 45% reduc-tion in lignin content had a normal cell morphologyand a higher growth rate than the control (Huet al.,1999). However, the increased growth was probablydue to pleiotropic effects caused by the constitutivedown-regulation of 4CL governed by the CaMV35Spromoter, because it was not observed in the trans-genic aspen reported by Liet al. (2003), in which theantisensePt4CLwas under the control of an aspenxylem-specific promoter,Pt4CL1P. The increasedlevel of hydroxycinnamic acids as non-lignin cellwall constituents has been suggested to contribute tothe cell wall strength in transgenic poplar (Huet al.,1999). Because several 4CL isozymes exist with dif-ferent cell-specific expression, down-regulation ofseveral or all isozymes simultaneously may perturbmetabolite levels other than those involved in lignin,with a secondary effect on growth as a consequence.

Interestingly, antisense inhibition of 4CL in as-pen trees led to a 15% increase in cellulose content.These results suggest that lignin and cellulose depo-sition are regulated in a compensatory fashion andthat a reduced carbonflow toward phenylpropanoidbiosynthesis increases the availability of carbon forcellulose biosynthesis (Huet al., 1999; Li et al.,2003).

A combinatorial down-regulation of 4CL alongwith an overexpression of F5H in xylem has beenachieved by co-transformation of twoAgrobac-teriumstrains in aspen (Liet al., 2003). Additive ef-fects of independent transformation were observed,in particular a 52% reduction in lignin content asso-ciated with a proportional increase in cellulose anda higher S/G ratio. The results show that stackingtransgenes allows several beneficial traits to be im-proved in a single transformation step (Halpin &Boerjan, 2003).

H. Down-Regulationof Cinnamoyl-CoA Reductase(CCR)

CCR catalyzes the reduction ofhydroxycinnamoyl-CoA thioesters to the cor-responding aldehydes, a reaction considered tobe a potential control point that regulates the

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overall carbonflux toward lignin (Lacombeet al.,1997). Transgenic tobacco (Piquemalet al., 1998;Ralph et al., 1998; O’Connell et al., 2002) andArabidopsis(Goujonet al., 2003b) down-regulatedfor CCR, are characterized by an approximate 50%decrease in Klasone lignin. The lignin S/G ratioi

was increased (mainly because of a decrease in theG unit amount) in transgenic tobacco and variable,depending on the growth conditions, in transgenicArabidopsis. In tobacco, an orange-brown colorwas observed in the xylem. The presence ofunusual phenolics (such as ferulic acid and sinapicacid) in the cell wall may account for this color,because semi-in vivo incorporation of these twohydroxycinnamic acids into stem sections resultedin a comparable phenotype (Piquemalet al., 1998).A change in the lignin structure was also indicatedby the higher amount of alkali-labile material thatcould be released from the extractive-free ligninpolymer of the transgenic lines (O’Connellet al.,2002). The transgenic plants with the lowest CCRactivity and 50% reduced lignin had abnormalphenotypes, such as collapsed vessels, stuntedgrowth, and abnormal leaf development. Importantalterations in thefiber cell walls were observed,such as a loosening in the arrangement of thecellulose microfibrils, that resulted in reduced cellwall cohesion (Pinconet al., 2001b; Goujonet al.,2003b). Chabanneset al. (2001a) have shownthat the reduction of lignin deposition in tobaccowas not uniform in the cell wall, but that the S2and S3 layers of thefibers and the vessels weremainly influenced. Similarly, lignin depositionwas mainly affected in the inner S2 layer in CCRdown-regulatedArabidopsisplants (Goujonet al.,2003b). A decrease in thioacidolysisi yield wasmeasured in the lignin of the CCR down-regulatedtobacco plants, indicating that the remaininglignin was more condensed (Piquemalet al.,1998; O’Connell et al., 2002). Also an increasedamount of tyramine ferulate (Figure 5), an unsualcomponent of tobacco cell walls that is probablya sink for feruloyl-CoA, was incorporated into thelignin of the CCR-down-regulated tobacco plants(Ralph et al., 1998). Down-regulation of CCR intransgenic poplar led to an orange coloration of thexylem, a 20% decrease in Klasone lignin content,and an increase in the S/G ratioi (J.-C. Leple,C. Lapierre, & W. Boerjan, unpublished results).A ccr mutant, designated irregular xylem (irx4),has been identified in Arabidopsis(Joneset al.,2001). Like the CCR-down-regulated tobaccoand Arabidopsisdescribed above, this mutant ischaracterized by a 50% reduced lignin contentf,h, a

collapse of the vessels, and an altered growth andmorphology. Taken together, the results obtainedby altering the expression of CCR indicate thatthis gene controls the quantity of lignin produced,although the adverse phenotypes associated withCCR suppression will limit its use as a target formodifying the lignin content in plants.

By crossing transgenic tobacco down-regulatedfor COMT (Atanassovaet al., 1995) with tobaccodown-regulated for CCR (Piquemalet al., 1998), asimultaneous reduction in COMT and CCR expres-sion was achieved (Pincon et al., 2001b). Progenywere inhibited in both CCR and COMT activitiesand had intermediate phenotypes. The line withmost down-regulated expression of both genes hadlignin characteristics similar to those of the CCR-down-regulated parent (low lignin content and highS/G ratio) (Table 3), but characteristics typical ofCOMT down-regulation, such as a low S/G andthe incorporation of 5OHG, were not found (Pinconet al., 2001b). These observations could result frominsufficient COMT down-regulation to trigger thephenotype. Alternatively, the down-regulation ofCCR, which is upstream in the pathway, may pre-vent the accumulation of 5OHG derivatives.

I. Down-Regulation of CinnamylAlcohol Dehydrogenase (CAD)

CAD catalyzes the last step in the biosynthe-sis of the monolignols, which is the reduction ofcinnamaldehydes to cinnamyl alcohols. Transgenicplants with reduced CAD activity have been pro-duced in tobacco (Halpinet al., 1994; Hibinoet al.,1995; Stewartet al., 1997; Yahiaouiet al., 1998),poplar (Baucheret al., 1996), and alfalfa (Baucheret al., 1999), whereascad mutants exist in pine(MacKayet al., 1997), maize (Halpinet al., 1998),andArabidopsis(Siboutet al., 2003). CAD suppres-sion has been associated with a red or red-browncolor of the stem xylem. An unusual monomer, di-hydroconiferyl alcohol (Figure 5), was shown to beincorporated into the ligninf of the pinecadmutantand accounted for 30% of the lignin compared toonly 3% in wild-type lignin (Ralphet al., 1997).Accordingly, higher amounts of arylpropane-1,3-diol structures (Figure 5), arising from dihydro-coniferyl alcohol, have been found in the lignin ofthe pinecadmutant by NMRf analysis (Ralphet al.,1999b, 2001a). In contrast, no dihydroconiferyl al-cohol has been found in the lignin of transgenicangiosperms down-regulated for CAD, such as to-bacco and poplar (Ralphet al., 1998).

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A higher amount of cinnamaldehydes has beendetected in the lignin of CAD-down-regulated to-bacco by pyrolysis GC-MSg (Halpin et al., 1994)and NMRf (Ralphet al., 1999a, 2001a), in CAD-down-regulated poplars by thioacidolysisi (Kimet al., 2002), in the pinecadmutant by NMRf (Ralphet al., 1997), and in theArabidopsis Atcad-Dmu-tant by thioacidolysisi (Sibout et al., 2003). Us-ing the CAD-down-regulated tobacco, Kimet al.(2000) demonstrated by NMRf that sinapaldehydecan makeβ-O-4 linkages with both G and S units,whereas coniferaldehyde cross-couples only withS units in the lignin. The cross-coupling of cin-namaldehydes into the lignin polymer results prob-ably in a more extended conjugated system in thepolymer, which might cause the red color of thexylem (Higuchiet al., 1994). The lignin of plantswith low CAD activity was more extractable inalkali (Halpin et al., 1994; Baucheret al., 1996;Bernard-Vailhe et al., 1996; Yahiaouiet al., 1998;MacKayet al., 1999). When fed to sheep, transgenictobacco and alfalfa with suppressed CAD activityhad slightly improvedin situ cell wall degradabil-ity, indicating that CAD may be a suitable targetfor modifying forage digestibility (Bernard-Vailheet al., 1995; Baucheret al., 1999).

In contrast to what might be expected, onlya slighty lower Klasone lignin content was mea-sured in the wood of transgenic poplar lines down-regulated for CAD (Lapierreet al., 1999; Pilateet al., 2002), in the pinecad mutant (MacKayet al., 1997), and in theArabidopsis Atcad-Dmutant(Siboutet al., 2003). These results may be explainedby the incorporation into the lignin polymer of otherphenolics, such as aldehydes and dihydroconiferylalcohol, as described above. In transgenic poplar, theS and G unit composition and the percentage ofβ-O-4 linkages did not differ from those of the control,but the lignin was enriched in free phenolic groups inboth S and G units and in diarylpropane (β-1) struc-tures (Lapierreet al., 1999) (Figure 4). Similarly,the proportion of G units with free phenolic groupswas higher in theAtcad-Dmutant than in the control(Siboutet al., 2003). The relative proportion of thefree phenolic groups may be an important parame-ter in determining the solubility of lignin (Lapierreet al., 1999). In contrast, the S/G ratio of the ligninof transgenic tobacco (Ralphet al., 1998) and trans-genic alfalfa (Baucheret al., 1999) was reduced,suggesting that in these plants the uncondensed Sstructures are more affected than their G analogs.These data are in apparent conflict with the recentproposal that SAD, and not CAD, is involved in Slignin biosynthesis in angiosperms (Liet al., 2001).

A simultaneous down-regulation of CAD andCCR has been achieved by crossing homozygoustransgenic lines in which either CAD (Halpinet al.,1994) or CCR (Piquemalet al., 1998) was down-regulated (Chabanneset al., 2001b). The lignincontente was decreased by approximately 50% intobacco with 32% of wild-type CCR activity and12% of wild-type CAD activity, a reduction that iscomparable to that in the homozygous, but not in thehemizygous, parental line down-regulated for CCR.This observation suggests that down-regulation ofboth genes has synergistic effects on the reductionof lignin quantity (Chabanneset al., 2001b). Thelignin S/G composition of the double transformedlines had increased to a level similar to that ofthe down-regulated CCR parent, when determinedby thioacidolysisi . Surprisingly, the NMRf spectrashowed that the lignin structure of the double trans-formants was closer to that of wild-type plants thanto the CCR- or the CAD-down-regulated parent.The phenotype of the double transformants was nor-mal with only slight alterations in the vessel shape,showing that, similarly to the results of Zhonget al.(1998) and Huet al. (1999), plants can tolerate im-portant reductions in lignin content.

A simultaneous suppression of COMT (to 24%of wild-type level), CCR (to 18% of wild-type level),and CAD (to 4% of wild-type level) was achieved intobacco by a single chimeric construct, consisting ofpartial sense sequences for the three different genes.The transgenic lines were stunted and had charac-teristics of COMT, CCR, and CAD suppression inlignin; for example, the xylem was red (indicativeof CAD suppression), contained collapsed vessels(indicative of CCR suppression), and had reducedstaining for S lignin (indicative of COMT suppres-sion) (Abbottet al., 2002). This approach to reduc-ing expression of several genes simultaneously by asingle construct was found to be more efficient thancrossing different transgenic lines altered in the ex-pression of single genes.

J. Up- and Down-Regulationof Peroxidases

Although peroxidases are believed to catalyzethe final condensation of cinnamyl alcohols in theformation of lignin, no definitive proof has been pre-sented yet for the involvement of any specific per-oxidase isozymein vivo, mainly because of the highnumber of genes that encode peroxidases (Tognolliet al., 2002) and the typically low substrate speci-ficities of these enzymes.

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Both anionic and cationic peroxidases havebeen implicated in lignification based on their affin-ity for coniferyl alcohol, their location in the cellwall, and their expression in lignified tissue (Mader& Fussl, 1982; Lagriminiet al., 1987; El Mansouriet al., 1999). Nevertheless, no change in lignincontent was obvious in transgenic tobacco plantsthat were deficient in the major anionic peroxidase(Lagrimini et al., 1997a). However, transgenicpoplar with a 44% reduction in the activity of astem-specific anionic peroxidase (PRXA3a) had a21% reduced lignin content and a higher contentin β-O-4 linked (uncondensed) structures in lignini

(Yahonget al., 2001).The overexpression of peroxidase genes in

transgenic poplar (PXP 3-4; Christensenet al.,2001a, 2001b) and in tobacco (spi 2; Elfstrandet al.,2002) resulted in 800-fold and 5-fold increased to-tal peroxidase activity, respectively. No effects ofthe genetic modification on the overall phenotype,the Klasone lignin content, or the recovery yieldof G and S units after thioacidolysisi were identi-fied. However, in the transgenic tobacco producedby Elfstrandet al. (2002), a higher frequency ofconiferaldehyde was detectedi and reduced stemflexibility was noted, suggesting modifications ofthe cell wall properties. In addition, the tobaccoplants with higher peroxidase activity were moresusceptible toPhytophthora parasitica, but allowedless growth ofErwinia carotovora.In contrast, in-dependently produced transgenic tobacco lines witha 10-fold higher peroxidase activity were character-ized by increased lignin contenta,h in leaves, stems,and roots (Lagrimini, 1991; Chabbertet al., 1992).No changes in lignin monomeric compositioni weredetected, but the amount of monomers involved inβ-O-4 linkages was lower in the transgenic lines,suggesting that the lignin was more condensed(Chabbertet al., 1992). These plants had reducedroot mass and fewer root branches, probably be-cause of altered auxin metabolism (Lagriminiet al.,1997b) and were characterized by wilting of theleaves and a rapid browning of wounded tissues(Lagriminiet al., 1990; Lagrimini, 1991). Similarly,overproduction of an anionic peroxidase in tomatoled to an increase in lignin content in leaves and fruit(Lagrimini et al., 1993; El Mansouriet al., 1999).

K. Down-Regulation of Laccases

The precise role played by laccases in ligni-fication is not yet understood, but there is correl-ative evidence that laccase and oxygen participate

in the polymerization of monolignols. For instance,when peroxidase is inhibited either in the absenceof H2O2 or in the presence of H2O2 scavengers(catalase and superoxide dismutase), coniferyl al-cohol is still oxidized and O2 consumed in tobaccoxylem (McDougallet al., 1994). Because laccasesoperate in the absence of toxic H2O2, these en-zymes could be involved in the early stages of lig-nification (Sterjiadeset al., 1993). Five divergentlaccase genes have been cloned and characterizedfrom poplar (Ranochaet al., 1999, 2000). Trans-genicLiriodendron(Deanet al., 1998) and poplar(Ranochaet al., 2000, 2002) down-regulated in lac-case had no altered phenotype nor any change inlignin amounta,e or S/G compositioni. However, intransgenic poplar down-regulated for one of the lac-case genes (lac3), the walls of xylem cells were ir-regular in contour when compared with the controland had adhesion defects either at the primary cellwall of adjacent cells or within the secondary cellwall of a given cell (Ranochaet al., 2002).

As described above, many different trans-genic plants and a few mutants are now availablewith altered lignin content, altered lignin composi-tion/structure, or both. Whether or not these changesin cell wall biochemistry could have advantages inindustrial operations, such as pulping, can only bedetermined experimentally. A few such experimentshave been performed. To put the results into context,we will first describe briefly the different methodsand parameters important in the production of pulpand paper before reviewing the impact that specificgenetic engineering can have on pulping properties.

IV. PAPERMAKING PROCESSES

Two major categories of processes exist for theproduction of paper pulp: chemical and mechani-cal. The former process uses chemicals to removelignin from fiber cell walls to obtain long andflexi-ble fibers that consist of polysaccharides only. Thelatter pulping process focuses on the mechanicalseparation offibers without the removal of lignin.Mechanical pulping gives the highest pulp yield,but the pulp has limited bleachability and can re-vert in brightness upon exposure to light (the paperbecomes yellow as it ages because of the presenceof lignin). In contrast, chemical pulping yields in-dividual intactfibers that can, due to the fact thatthe hydrophobic lignin has been removed, inter-act with otherfibers via hydrogen bonds, makinga very strong paper. Nowadays, the Kraft process isthe most widely used chemical procedure for the

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TABLE 4Main Wood Pulping Processes

Chemical Mechanical Cooking Pulp YieldPulping Process Treatment Treatment T (◦C) pH Wood∗ Time (h) (%)

Mechanical ProcessesStone groundwood (SGW) — Grinder 130–180 S — 93–95Pressure groundwood (PGW) Steam Grinder 105–125 S — 93–95Refiner mechanical (RMP) — Disc refiner S — 93–95Thermomechanical (TMP) Steam Disc refiner 110–130 S — 80–90Chemithermomechanical Steam+ Na2SO3, Disc refiner 120–130 S/H — 80–90

(CTMP) NaOHChemimechanical (CMP) Na2SO3, NaOH Disc refiner 150–170 S/H — 80–90

Semimechanical ProcessesNeutral sulfite (NSSC) Na2SO3 and Na2CO3 Disc refiner 160–185 7–10 H 0.5–3 70–85

or NaHCO3

Green liquor (GLSC) Na2SO3 and Na2CO3 Disc refiner H 70–85Non-sulfur NaOH and Na2CO3 Disc refiner H 70–85

Chemical ProcessesKraft NaOH and Na2S — 170–175 13–14 S/H 2–5 45–55Soda NaOH — 170–175 H 40–50Soda-anthraquinone NaOH and anthraquinone — H 45–55Soda-oxygen NaOH and oxygen — H 45–55Sulfite or bisulfite Ca(HSO3)2, NaHSO3, — 120–150 1.5–5 H/S 6–8 45–55

NH4HSO3 or Mg(HSO3)2

and H2SO4

H, hardwood; S, softwood;—, not appropriate;∗wood mainly used.

production of paper. However, this process isgradually being replaced by thermomechanical(TMP) and chemithermomechanical (CTMP) pulp-ing methods that give higher pulp yields and con-sume less water. In 2000, the world wood pulp pro-duction has been estimated at 135,852 thousandsof metric tons, from which 6.3% are of mechanicalpulp, 15.6% TMP pulp, 3.6% semimechanical pulp,and 74.5% chemical pulp (Food and Agriculture Or-ganization, 2001a).

Pulping methods can be classified according totheir yield (Table 4) and will be briefly explainedbelow. Examples of properties of pulp obtained bydifferent pulping methods with several wood sam-ples are given in Table 5. Explanations for the ter-minology used to describe pulping processes or pa-rameters are given in the glossary and are indicatedby an asterix* in the text. For a detailed overviewon pulp and paper production, we refer the reader toBiermann (1996).

A. Mechanical Pulps

1. Mechanical Processes Based onStone Grinders

In the stone groundwood (SGW) process, de-barked wood logs are submitted to the action of a

rotating stone covered with abrasive grains of siliconcarbide, aluminum oxide, or quartz in ceramics. Me-chanical energy is transformed into heat and steam,inducing a local increase in the contact temperatureat the stone/wood interface that reaches the glasstransition point of lignin (approximately 140˚C) andresults infiber separation. When thefibers are lib-erated and separated, the pulp is removed from thestone surface by sprinklers and collected in a pit un-der the grinder. Subsequently, the pulp is screened*to remove the shives (fiber bundles) andfines (fiberfragments or ray cells) that have a negative effect onpulp quality.

In the pressure groundwood (PGW) process,the grinder is pressurized with steam and the woodis heated and softened prior to grinding. The highercontact temperature between the wood log and thestone enhances thefiber separation and reduces theamount offines generated. The resulting pulp isstronger than that derived from the SGW process, asmeasured by the tensile*, burst*, and tear indexes*(Table 5).

2. Mechanical Processes Based onRefiners (RMP)

The refiner mechanical pulp (RMP) procedureis based on the use of rotating metal discs with

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TABLE 5Properties of Different Wood Pulps (Data from Holder and Murray, 1970; Leask et al ., 1987;Vallette & de Choudens, 1992; Petit-Conil, 1995)

Pulping Drainage Tensile Burst Tear Breaking ScatteringProperties and Index Index Index Index Length Coefficient BrigthnessWood Type (ml CSF) (mN/g) (kPam2/g) (mNm2/g) (km) (m2/kg) (%ISO)

SpruceSGW 100 27 1.10 3.6 — 68 —PGW 100 32 1.60 4.8 — 68 —TMP 68 — 2.35 8.6 4.20 43.5 53.0CTMP 68 — 2.90 8.2 5.30 38.0 57.0

AspenTMP 68 23 0.90 3.1 — 68.0 58.0CTMP 68 51 2.60 6.2 — 41.0 49.5

BalsamfirSGW 68 — 1.90 6.2 3.69 — —TMP 72 — 3.00 8.2 4.97 — —

Black spruceSGW 65 — 1.20 5.6 2.90 — —TMP 68 — 3.00 9.0 5.40 — —

Pulps from softwoodsKraft 40 — 5.8 12.0 8.9 — 28

Pulps from hardwoodsKraft 40 — 4.5 8.0 7.5 — 29

Pulps from maritime pineSulfate 40 — 5.8 12.0 8.9 — 28Bisulfite 40 — 4.0 7.5 6.5 — 60

CTMP; chemithermomechanical pulp; PGW, pressure groundwood; SGW, stone groundwood; TMP, thermomechanicalpulp; —, not determined.

specific bars and grooves that enable separation ofthe fibers from the wood matrix. The passage ofwood particles over the disc bars induces repeatedcycles of compression and expansion. When carriedout in the presence of water, these cycles generatesteam that softens the chips and mechanically rup-tures thefibers at the middle lamella. As a result,fewer fines and shives are formed and, therefore,RMP pulps are stronger than SGW pulps.

3. Thermomechanical PulpingProcess (TMP)

The TMP process involves a presteaming ofthe chips prior to two refining stages. The main ef-fect of the preheating is softening of lignin, allowingthe recovery of longerfibers with fewerfines andshives than in the RMP process. Thefirst refining

stage, called defibering, is carried out at an elevatedtemperature (generally between 110 and 130˚C) un-der pressure to promotefiber liberation. The secondrefining stage is carried out at ambient temperature,either at atmospheric or at elevated pressure.

4. Chemimechanical (CMP) andChemithermomechanical (CTMP)Pulping Processes

To improve pulp quality for high-quality pa-per grades, mechanical, thermal, and chemical treat-ments can be combined. A chemical pretreatmentof the wood chips prior to a mechanical treatmentenhances defibering and renders the lignin more hy-drophilic. After the mechanical treatment, thefibersare longer and moreflexible and the properties ofthe produced paper are better than without chemical

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pretreatment (Table 5). Furthermore, these benefi-cial effects are associated with a higher pulp yieldand a decrease in the pulp opacity*.

As with the TMP process, the electrical energyis mainly transformed into steam in the refiner discs.The steam is recovered, cleaned, and used in the dry-ing part of the paper machine. It is worth noting thatthe different stages of all these mechanical processes(SGW, TMP, CTMP, and CMP) are integrated intoa single paper machine.

5. Bleaching of Mechanical Pulps

Bleaching involves treatment of the pulp withchemicals to increase its brightness*, which can beimproved in two basic manners. Whereas chemicalpulp is bleached by lignin removal, bleaching of me-chanical pulp preserves the lignin, but removes thechromophores present in lignin. Therefore, bleach-ing of mechanical pulps is usually referred to asbrightening. Brightness is not permanent becauseUV light and oxygen create more chromophores andcause yellowing (brightness reversion).

The brown color of mechanical pulp is mainlycaused by the presence of lignin. In addition, thehigh temperature, atmospheric oxidation, and me-chanical action during the pulping also favor theformation of the brown color. The chemicals thatare generally used to bleach mechanical pulp areoxidizing compounds, such as hydrogen peroxide(H2O2), and/or reducing agents, such as dithion-ite (sodium hydrosulfite [Na2S2O4]). Usually, theperoxide bleaching is carried out in a single stepafter potential pretreatments, such as chelation ofmetallic cations (e.g. Fe2+, Mn2+, and Cu2+), whichdecrease the stability of peroxide. However, forthe production of printing-writing paper (such asnewsprint), high levels of brightness are needed andbleaching sequences of two or three steps that com-bine both oxidizing and reducing chemicals have tobe used.

B. Semi-Chemical Pulps

Semi-chemical pulps are essentially mechan-ical pulps that have been mildly pretreated withsodium sulfite (Na2SO3) and sodium carbonate(Na2CO3) to partially remove lignin and hemicel-luloses and reduce the energy requirement dur-ing the processing. The most common methodsin this pulping category are the neutral sulfitesemi-chemical (NSSC) and the Kraft semi-chemical

methods. Semi-chemical pulps are mostly bleachedwith H2O2.

C. Chemical Pulps

Chemical pulps represent the largest part of theworld pulp market and are used to produce nearlyall paper and board grades (Food and AgricultureOrganization, 2001a). The aim of chemical pulp-ing is to dissolve and remove the lignin from thefiber wall and to separate thefibers at the middlelamella without mechanical damage. Because of theinsoluble and cross-linked nature of lignin, deligni-fication requires harsh pulping conditions, includ-ing high temperatures, a combination of chemicals,and a long chemical treatment time. The differ-ent chemical reactions that occur in the productionof chemical pulps have been described by Gierer(1985).

1. Alkaline Chemical PulpingProcesses

a. Sulfate or Kraft Process

The most widely used pulping process for manylignocellulosic raw materials is the Kraft process.It is based on the use of NaOH and sodium sul-fide (Na2S), which act as delignifying agents. Dur-ing cooking, Na2S is hydrolyzed into NaOH, NaHS,and H2S. The different sulfur compounds react withlignin, forming thiolignins that are more soluble.The liquor applied to the chips is called whiteliquor and the liquor extracted from the reactor andcontaining the removed compounds is called blackliquor, a complex mixture of inorganic and organiccompounds.

For the white liquor, an active alkali* value be-tween 18 and 22% (on oven-dry weight of wood)and a sulfidity* value between 20 and 35%, areused. This chemical treatment cleaves theβ-O-4linkages and the methoxy groups, the latter being re-leased as mercaptans. A high sulfidity increases therate of delignification and protects the pulp againstcarbohydrate degradation in alkaline medium bybuffering the cooking solution, although decreasedcooking times also result in less carbohydrate degra-dation. When the sulfidity of the cooking solution istoo low, the pulp may have a higher lignin contentand significant carbohydrate degradation. When thesulfidity is excessive, the emission of reduced sulfurcompounds may be too high.

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In the Kraft process, most of the cooking chem-icals from the black liquor are recovered. Additionof sodium sulfate (Na2SO4) to the black liquor al-lows Na2S and CO2 to be recovered. A reaction be-tween CO2 and NaOH produces sodium carbonate(Na2CO3). Subsequently, these compounds are dis-solved in water to produce the green liquor. WhenCa(OH)2, formed from lime (CaO), and Na2CO3 aresupplemented to the green liquor, NaOH and CaCO3

are produced. Finally, the white liquor is regener-ated by sedimentation of CaCO3 and filtering foradditional clarification. In addition to the regenera-tion of the white liquor, the combustion of organicmaterials generates steam that is used to produceelectrical energy. Therefore, a Kraft pulp mill has aperfect electrical autonomy.

The low yield of Kraft pulp (45–55% of theinitial biomass) is due to the removal of lignin andsome of the hemicelluloses. The kappa number*(determined by oxidation of the pulp by a solutionof potassium permanganate) is directly related tothe residual lignin content and, before bleaching, istypically between 28 and 32 for softwoods and 18to 22 for hardwoods.

The pulp quality of chemical pulps is clearlyhigher than that of mechanical pulps, mainly withrespect to paper strength (Table 5). However, be-cause of the perfectfiber separation, thefines areless numerous and, hence, the chemical pulps havea lower opacity.

b. Other Alkaline Processes for theProduction of Chemical Pulps

In soda pulping, NaOH is used as a delig-nifying agent in the pulping liquor. In the soda-anthraquinone and Kraft-anthraquinone processes,catalysts are used, such as quinonic compounds (forinstance, anthraquinone), that increase delignifica-tion and decrease carbohydrate degradation. There-fore, the cooking time can be reduced and the pulpyield increased (by 1–3%). Another process to pro-duce pulps with a lower kappa number and reducethe charge of bleaching agents consists of prolong-ing the alkaline cooking step.

2. Acidic or Bisul fite ChemicalPulping Processes

The acidic or bisulfite process is mainly usedfor softwoods and is based on the reaction of thewood chips with calcium [Ca(HSO3)2], sodium[NaHSO3], ammonium [NH4HSO3], or magnesium

[Mg(HSO3)2] hydrogenosulfite, or bisulfite, in com-bination with free SO2.

The chemical reactions taking place during thecooking induce the dissolution of lignin and hemi-celluloses. Cellulose is also hydrolyzed when theSO2 charge* is higher. The lignin is removed by sul-fonation and hydrolysis, resulting in lignosulfonicacids. The pulp quality is higher than that of mechan-ical pulps but always lower than that obtained byKraft pulping (Table 5). However, the high bright-ness of the pulp, which is due to the fact that thesepulps can be bleached more easily than Kraft pulps,allows its utilization for some paper grades.

3. Bleaching of Chemical Pulps

The unbleached chemical pulps, containingresidual lignin, can be used for some paper and boardgrades, such as wrapping papers and boxes; how-ever, for the production of high-quality papers, suchas printing-writing papers, the chemical pulps needto be bleached. Bleaching of chemical pulp aimsat the total removal of the residual lignin withoutextensive polysaccharide degradation. Generally, ableaching sequence consists of alternative steps oflignin oxidation and alkaline extraction steps. Eachbleaching step is followed by a washing step toremove the dissolved compounds that would oth-erwise consume bleaching chemicals in the laterstages.

Until the 1980’s, the bleaching sequences wereas follows: C-E-D-E-D [with C, chlorine (Cl2); E,alkaline extraction; and D, chlorine dioxide (ClO2)].ClO2 was used at the end of the bleaching sequencefor its bleaching effect associated with its specificdelignifying action, although ClO2 is 10- to 15-foldmore expensive than Cl2. At the end of the 1980’s,ClO2was used instead of Cl2 for environmental rea-sons: the use of high Cl2 charges (3 to 6% on pulpweight) can induce the formation of organic com-pounds containing as much asfive molecules ofchlorine, such as dioxin or other chlorinated organicchemicals that generate toxicity problems in efflu-ents. At the beginning of the 1990’s, new bleach-ing sequences were developed, such as elementalchlorine-free (ECF) sequences that avoid the use ofCl2, and totally chlorine-free (TCF) sequences thatdo not use chlorinated chemicals at all.

a. ECF Bleaching Sequences

Two ECF bleaching sequence categoriesexist: (i) the sequences based on ClO2, such as

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TABLE 6Conditions Used in the Different Potential Stages of ECF Bleaching Sequences (Petit-Conil,1995)

Conditions D O E P Z Q

Temperature (◦C) 50–80 90–110 60–80 70–110 30–60 50–80Duration 45 min-3 h 30–60 min 45–60 min 1–4 h Few min 30–60 minpH Acidic Alkaline Alkaline Alkaline Acidic NeutralPulp consistency (%) 3–12 10–12 10–12 10–20 10–35 3–12Chemicals ClO2 O2 NaOH H2O2 O3 EDTA% on o.d. pulp 0.5–4% 3–5 bar 1–3% 0.5–5% 0.1–0.7% <0.5%

NaOH NaOH1–3% 1–3%

D, O, E, P, Z, and Q, see text for explanation; o.d., oven dry.

D-E-D-E-D or D-E-D-D, and (ii) the sequencescombining ClO2 and oxygenated chemicals, such asoxygen (O2) (O stage), hydrogen peroxide (H2O2)(P stage), or ozone (O3) (Z stage). The efficiency ofbleaching with O2, H2O2, or O3 can be negativelyaffected by the presence of metal ions (Fe, Cu, orMn) in the pulp. Therefore, it is necessary to applya chelation stage (Q) to complex the ions and to re-move them from the pulp. The bleaching conditionsof ECF sequences are given in Table 6. Frequentlyused sequences are: O-D-E-D-D, O-Q-P-D-E-D, orO-Z-E-D-D.

b. TCF Bleaching Sequences

In the TCF bleaching sequences, O2, O3, H2O2,and peracids (Pa), such as peracetic acid (CH3CO3Hand H2O2) or permonosulfuric acid (H2SO5), areused as bleaching chemicals. Frequently used se-quences are O-Q-P-P, O-Q-P-Z-Q-P, or O-Q-P-Pa-P.

D. Biological Pulps

Biopulping is defined as the treatment of woodchips with lignin-degrading fungi prior to pulping.Such a pretreatment reduces energy requirementsand improves the paper strength. In addition to theeconomical benefits, the biopulping process is notharmful to the environment, because only benignmaterials are used and no additional waste streamsare generated (Reid, 1991; Akhtaret al., 1998a;Highley & Dashek, 1998). Although the brightnessof the pulp is significantly reduced after fungal pre-treatment, pulps can easily be bleached with eitheralkaline hydrogen peroxide or sodium hydrosulfite.Biomechanical pulping is on the way toward be-

ing commercialized (Akhtaret al., 1998b; Breen &Singleton, 1999).

A fungal pretreatment in chemical pulping hasalso been shown to be beneficial for subsequentpulping processes because part of the lignin is re-moved or modified (Akhtaret al., 1998a). Althoughthe results are too preliminary for the process to becarried out on an industrial scale, biochemical pulp-ing could lead to pulps with lower kappa number,which are more easily bleachable, consume fewerchemicals, and need less waste effluent loading.

Biobleaching has been shown to be an effec-tive treatment in which hemicellulases (such as xy-lanases and mannanases) and lignin-degrading fungior their enzymes (such as laccases and peroxidases)are utilized to delignify and brighten the pulp, thusreducing the use of chemicals (reviewed by Onysko,1993; Reid & Paice, 1994; Viikariet al., 1994).Several procedures, such as preatreatment with xy-lanase prior to bleaching, have been scaled up to fullindustrial scale (Viikariet al., 1994, 1998).

V. EFFECT OF LIGNINMODIFICATIONS ON PULPING

Despite the fact that much of the research on ge-netic modification of lignin biosynthesis was moti-vated by a desire to improve wood for more efficientand environmentally benign papermaking, very fewlignin-modified plants (either transgenic or mutant)have yet been tested in pulping and bleaching pro-cesses. The reasons for this regrettable situation areunclear, but many factors are likely to be involved.Most molecular biology laboratories where trans-genic plants have been produced are unlikely to haveeasy access to expertise in pulping technology. It is

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no accident that most of the instances where pulpingcharacteristics of transgenics have been tested comefrom EU-funded consortia projects where the kindof interdisciplinary contacts that are necessary forsuch work are actively fostered. Obviously, pulp-ing data that best approximate real industrial situa-tions will come from reasonably large amounts ofplant material grown in thefield, which again posesproblems for small laboratories that may not haveaccess tofield trial facilities. Similarly, navigatingthe regulatory process governing the controlled re-lease of genetically modified (GM) plants is some-thing many researchers wouldfind daunting. De-signing trials to the rigorous standards necessary togain regulatory approval requires significant effortboth in terms of the paperwork involved and in theimplementation, maintenance, and eventual clear-ance of the trials themselves. The strongly negativeattention such trials can attract from anti-GM ac-tivists is another factor that inhibits efforts in thisarea. The real prospect that such trials might be at-tacked and vandalized (as in the case of a poplartrial in the UK in 1999 and many recent trials in theUS) or that laboratories perceived to be involved insuch work might befirebombed (as happened at theUniversity of Washington in 2001; Luce, 2002), notsurprisingly puts many people off investing their re-search grants and efforts in this area. The end resultis that some elements of the anti-GM lobby deliber-ately prevent the collection of the very data on theenvironmental impact of GM plants that more ra-tional commentators agree is needed as part of thewider exploration of potential GM solutions to cur-rently insoluble environmental problems. It is theresponsibility of society as a whole, not scientiststhemselves, to assess this type of criminal activityaimed atfield trial and laboratory destruction, whichwastes significant amounts of public money and in-hibits scientific advance. Unless a serious effort ismade to tackle this problem, progress is bound to beslow.

The above comments notwithstanding, severalpulping studies have been performed on lignin-modified GM plants including one involving treesgrown for 4 years in thefield (Pilateet al., 2002).The results of thisfield experiment will be discussedin some detail because they are currently the onlydata that can be drawn upon to predict the poten-tial performance of GM lignin-modified trees in thefield, in terms of both stability of transgene expres-sion and persistence of lignin changes and pulpingbenefits. Pulping analyses have also been performedon the same poplar transgenics when grown in thegreenhouse (Baucheret al., 1996; Lapierreet al.,

1999, 2000; Petit-Conilet al., 1999, 2000; Jouaninet al., 2000), on the pinecad mutant (MacKayet al., 1999), on greenhouse-grown transgenic to-bacco down-regulated for CAD or CCR (O’Connellet al., 2002), and on transgenic poplars overexpress-ing F5H (Huntleyet al., 2003). The poplar and to-bacco plants were analyzed by simulated Kraft pulp-ing and/or mechanical pulping and the pine mutantboth by Kraft and soda pulping.

A. Chemical Pulping of Woodfrom Transgenic PoplarsDown-Regulated for COMTand CAD

Laboratory-scale Kraft pulping has been per-formed on greenhouse-grown transgenic poplars fortwo lines down-regulated for COMT (ASB2B andASB10B) and two lines down-regulated for CAD(ASCAD21 and ASCAD52) (Baucheret al., 1996;Lapierreet al., 1999, 2000). Subsequently,field tri-als of the same tree lines were established at twosites, one in France and one in the United Kingdom(Petit-Conilet al., 2000; Pilateet al., 2002). Woodfrom the French trial was harvested after 2 and 4years (i.e. after 2 years regrowth of the previouslyharvested trunks), while trees of the UK trial wereharvested after 4 years of growth only. This har-vesting strategy allowed an assessment of whetherthe Kraft pulping characteristics varied with ageand enabled evaluation of the stability of trans-gene expression and lignin modification over sev-eral years of growth. Table 7 (COMT lines) andTable 8 (CAD lines) pull the data from publicationson greenhouse- andfield-grown trees together forease of comparison.

For COMT-suppressed lines, enzyme activitywas higher infield-grown trees than in youngergreenhouse-grown plants (Table 7), whereas CAD-suppressed lines showed similar or lower enzymeactivity in the field compared to the greenhouse(Table 8). Only ASCAD21 had altered lignincontente (a slight reduction) in greenhouse andfield.Antisense COMT lines had a lower frequency oflignin units only involved inβ-O-4 bondsi, greatlyreduced S/Gi, and significant amounts of 5OHGi

units in lignin (Table 7). The lignin S/G ratio was un-modified in ASCAD lines, but altered lignin struc-ture was indicated by the increased frequency offree phenolic groups inβ-O-4 linked S or G unitsi

(Table 8). These data demonstrate that trees mod-ified in either COMT or CAD showed similar

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TAB

LE7

Lign

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R)

51c

——

——

50c

——

——

51c

——

——

Bul

k∗(c

m3/g

)1.

10c

——

——

1.07

c—

——

—1.

07c

——

——

Tens

ilein

dex

(Nm

/g)

82.3c

——

——

92.6

c—

——

—82

.6c

——

——

Bur

stin

dex

(kP

am2/g

)5.

4c—

—6.

1e6.

3e5.

8c—

——

—5.

5c—

—6.

0e5.

8e

Tear

inde

x(m

Nm2

/g)

4.5c

——

6.4d

4.3d

4.7c

——

——

5.1c

——

6.2d

6.4d

Fib

erle

ngth

(mm

)—

—0.

93b

——

——

0.91

b—

——

—0.

90b

——

Bre

akin

gle

ngth

(km

)—

——

8.6d,

e9.

2d,e

——

—9.

1e8.

9e—

——

9.0d,

e9.

3d,e

12m

,24

m,4

8m

,mon

ths

ofag

e;F,

grow

nin

the

field

;Fr,

Fra

nce;

G,g

row

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the

gree

nhou

se;U

K,U

nite

dK

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om;

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es.

a,b,

c,d

and

e :da

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ree

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l.(1

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l.(2

000)

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ilet

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(199

9),

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l.(2

002)

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ilet

al.

(200

0),

resp

ectiv

ely.

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ftpu

lps

wer

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oduc

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smal

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ssur

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aro

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goi

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ated

bath

.The

cond

ition

sus

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ere

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a,b,

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%su

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,liq

uor

tow

ood

ratio

=4,

tem

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rais

edto

170◦ C

over

90m

inan

dm

aint

aine

dfo

r90

min

.The

pulp

sw

ere

then

was

hed

and

scre

ened

ona

150-

µm

siev

eto

reta

inun

cook

edpa

rtic

les.

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panu

mbe

rand

cellu

lose

degr

eeof

poly

mer

izat

ion

(DP

)w

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cord

ing

toin

tern

atio

nals

tand

ards

.The

pulp

sw

ere

subs

eque

ntly

blea

ched

with

anE

CF

sequ

ence

,O-D

-E-D

at10

%pu

lpco

nsis

tenc

y.

331

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TAB

LE8

Lign

inC

hara

cter

istic

san

dP

rope

rtie

sof

EC

F-B

leac

hed

Kra

ftP

ulps

for

CA

D-D

own-

Reg

ulat

edP

opla

r

Con

trol

AS

CA

D21

AS

CA

D52

3m

24m

24m

48m

48m

3m

24m

24m

48m

48m

3m

24m

24m

48m

48m

Cha

ract

eris

tics

GG

F,F

rF,

Fr

F,U

KG

GF

F,F

rF,

UK

GG

FF,

Fr

F,U

K

CA

Den

zym

atic

acti

vity

(%)

100a

——

100e

—30

a—

—15

e—

30a

——

47e

—L

igni

nch

arac

teri

stic

sK

laso

nlig

nin

cont

ent

100a

100b

100b

——

100a

96b

96b

——

106a

100b

99b

——

(%of

cont

rol)

Yie

ldin

S+

G(µ

mol

/glig

nin

414a

∗—

2580

b—

—31

1a∗

—22

35b

——

323a

∗—

2360

b—

—or

µm

ol/g

CW

R∗)

S/G

1.71

a—

1.78

b—

—1.

32a

—1.

86b

——

1.63

a—

1.94

b—

—R

elat

ive

prop

ortio

n37

/63/

tra—

36/6

4/trb

——

43/5

7/tra

—35

/65/

trb—

—38

/62/

tra—

34/6

6/trb

——

ofG

/S/5

OH

GF

ree

OH

inβ

-O-

——

26.4

b26

.4e

——

—33

.6b

29.2

e—

——

30.5

b—

—4-

linke

dG

units

Fre

eO

Hin

β-O

-—

—3.

0b3.

0e—

——

4.5b

4.5e

——

—3.

5b—

—4-

linke

dS

units

Pul

ppr

oper

ties

Kap

panu

mbe

r20

.8a118

.7c2

17.6

d220

.4e3

19.7

e319

.2a1

15.6

c216

.3d2

18.6

e313

.9e3

18.8

a115

.7c2

——

—C

ellu

lose

DP

1010a1

1900

c214

40d2

2090

e321

90e3

1140

a119

10c2

1670

d221

40e3

2270

e399

0a118

00c2

——

—P

ulp

yiel

d(%

wt)

47.9a1

45.9

c251

.3d2

51.9

e354

.8e3

47.5

a147

.4c2

50.9

d254

.4e3

56.9

e343

.4a1

——

——

Pul

ppr

oper

ties

afte

rE

CF

blea

chin

gB

right

ness

(%IS

O)

—90

.6c

87.9

cd89

.8e

89.3

e—

91.0

c89

.6c,

d90

.1e

90.7

e—

91.0

c89

.2c

——

Cel

lulo

seD

P—

1510

c—

——

—15

00c

——

——

1470

c—

——

Dra

inag

ein

dex

(◦ SR

)—

54c

——

——

53c

——

——

56c

——

—B

ulk

(cm

3/g

)—

1.12

c—

——

—1.

14c

——

——

1.10

c—

——

Tens

ilein

dex

(Nm

/g)

—91

.8c

——

——

84.6

c—

——

—96

.7c

——

—B

urst

inde

x(k

Pam

2/g

)—

5.8c

—6.

1f6.

3f—

5.5c

—6.

1f6.

1f—

5.6c

—5.

9f5.

9f

Tear

inde

x(m

Nm2

/g)

—5.

7c—

6.4e,

f4.

3e,f

—5.

4c—

5.9e,

f5.

1e,f

—5.

6c—

6.3f

5.9f

Kap

paaf

ter

O—

12.5

c—

——

—8.

1c—

——

—8.

6c—

——

Cel

lulo

seD

Paf

ter

O—

1600

c—

——

—15

60c

——

——

1530

c—

——

Fib

erle

ngth

(mm

)0.

42a—

0.93

d—

—0.

43a

—0.

92d

——

——

——

—B

reak

ing

leng

th(k

m)

——

—8.

6e,f

9.2e,

f—

——

9.0e,

f9.

9e,f

——

—9.

2f9.

3f

3m

,24

m,4

8m

,mon

ths

ofag

e;a ,b,c ,

d,e

and

f :da

tafr

omB

auch

ereta

l.(1

996)

,Lap

ierr

eeta

l.(1

999)

,Pet

it-C

onile

tal.

(199

9),L

apie

rree

tal.

(200

0),P

ilate

eta

l.(2

002)

,and

Pet

it-C

onile

tal.

(200

0),r

espe

ctiv

ely.

Con

ditio

nsof

Kra

ftpu

lpin

gan

dbl

each

ing

wer

eas

desc

ribed

inTa

ble

7.1,2

2%ac

tive

alka

li;2,2

0%ac

tive

alka

li;3,1

8%ac

tive

alka

li;—,

notd

eter

min

ed;t

r,tr

aces

.CW

R,c

ellw

allr

esid

ue;F

,gro

wn

inth

efie

ld;F

r,F

ranc

e;G

,gro

wn

inth

egr

eenh

ouse

;UK

,Uni

ted

Kin

gdom

.

332

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FIGURE 6. Evolution of kappa number with ac-tive alkali charge for Kraft pulping of 4-year-oldpoplar trees from the French field trial.

changes to lignin whether grown in the greenhouseor in two differentfield environments.

Chemical pulping of the COMT antisensepoplars demonstrated that their wood was more re-sistant to Kraft delignification than wood from con-trol trees, as indicated by the higher kappa num-ber of pulp from the transgenic plants (Table 7).Pulp brightness, after bleaching with an ECF se-quence, was lower than that of the control (Table 7),although paper strength was similar according toits degree of depolymerization* (DP) of celluloseand its mechanical properties. Pulp from CAD anti-sense plants, however, was more easily delignifiedand had a lower kappa number than control pulp,a difference that was maintained after the oxygendelignification stage (O), thefirst step in the bleach-ing sequence (Table 8). Cellulose DP, pulp bright-ness, and paper strength were similar in control andCAD-suppressed lines (Table 8).

In parallel to the pulping experiments describedabove, wood from the French trial was Kraft pulpedunder a range of active alkali charges from 16 to26% (Figure 6). Increasing the active alkali removesmore lignin and yields lower kappa numbers. Com-

FIGURE 7. Evolution of cellulose DP with ac-tive alkali charge for Kraft pulping of 4-year-oldpoplar trees from the French field trial.

FIGURE 8. Evolution of screened pulp yieldwith active alkali charge for Kraft pulping of4-year-old poplar trees from the French fieldtrial.

pared with the control, less chemical was needed toreach a given kappa number for the pulp made fromCAD antisense lines and more chemical was neededfor COMT antisense poplars. The extent of cellu-lose degradation, for a given alkali charge, was thesame for all lines (Figure 7), suggesting that down-regulation of COMT or CAD does not affect cellu-lose biosynthesis. Another important characteristicof the Kraft process is the evolution of the screenedpulp yield with active alkali charge. Whereas a de-crease in the screened pulp yield was observed atlower alkali charges for the control and COMTdown-regulated poplars, the pulp yield remainedhigh for CAD down-regulated poplars (Figure 8).The selectivity curve (Figure 9) shows the bene-ficial effect of CAD down-regulated lines on Kraftpulping in terms of cellulose preservation and delig-nification: for a given kappa number, the cellulosewas less degraded for the CAD lines, particularlyfor ASCAD21. In contrast, when wood of COMT-down-regulated poplars was similarly pulped, thecellulose DP was lower than that of control wood.

The conclusions from these experiments arethat suppression of CAD activity leads to the

FIGURE 9. Selectivity curve: kappa numberversus cellulose DP for Kraft pulping of 4-year-old poplar trees from the French field trial.

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formation of wood with better Kraft pulping prop-erties, probably because of the slightly decreasedlignin content and modified lignin composition. In-terestingly, ASCAD21, the line with the best pulp-ing characteristics, had an increased proportion offree phenolic groups in both G and S lignin units,possibly influencing the solubility of the polymerduring Kraft pulping. Savings in chemical use andincrease in pulp yield for this line have been esti-mated at 6% and 2–3%, respectively, which is com-mercially valuable (Pilateet al., 2002). Most impor-tantly, this line performed as well as wild-type treesin thefield and showed no adverse effects of the ge-netic modification. In contrast, down-regulation ofCOMT has a negative effect on Kraft pulp prop-erties, supporting the hypothesis that lignin richin G units is difficult to extract by the Kraft pro-cess because of the more condensed lignin. Thelower proportion of free phenolic groups inβ-O-4-linked G units in COMT-suppressed poplars mayalso contribute to the higher kappa numbers (seesection III.D). Although incorporation of 5OHG(with two hydroxyl functions) into lignin in COMT-suppressed plants might be expected to be beneficialfor pulping because of an increase in solubility, thisapparently did not counterbalance the detrimentaleffects of a more condensed lignin in these lines.The results obtained from bothfield trials demon-strated that, contrary to original expectations, de-pleting COMT activity reduces wood quality forKraft pulping.

B. Chemical Pulping of Wood fromTransgenic Poplar OverexpressingF5H

Lignin of transgenic poplars overexpressingF5H is significantly enriched in S units (Frankeet al., 2000). Kraft pulping experiments on woodof 2-year-old greenhouse-grown poplars resulted ina pulp with lower kappa number and an increase inbrightness (Huntleyet al., 2003). This genetic im-provement may increase pulp throughput by 60%,concomitantly with a decreased consumption ofpulping chemicals (Huntleyet al., 2003).

C. Chemical Pulping of Wood fromTransgenic TobaccoDown-Regulated for CCR and CAD

Although tobacco is not normally used for pulp-ing, its value as a model for species that are pulped

has been investigated in laboratory-scale simulatedKraft pulping experiments. Stems of four transgenictobacco lines, two down-regulated in CCR and twodown-regulated in CAD were pulped. For each gene,enzyme activity was suppressed moderately in oneline and severely in the other. Thus, lines CCR34and CCR86 had 48% and 1% residual CCR activity,whereas lines CADJ40 and CADJ50 had 20% and7% residual CAD activity, respectively. As shown inTable 9, the pulps of both types of transgenic plantshad a lower kappa number than those of the con-trol plants (O’Connellet al., 2002). The greatest re-duction in kappa number was obtained for the lineswith the lower CCR or CAD activity. The degreeof cellulose DP was similar for the pulp of all linestested. Micrographs offibers from the unbleachedpulps showed a higher level of cell separation forthe transgenic than for the control lines. Whereasthe CADJ40 pulp was bleached to a higher bright-ness than the control, the pulp from CCR86 was lessbright than the control. This lower brightness wasapparently due to a higher content of unextractedchlorophyll in CCR-suppressed stems (O’Connellet al., 2002). Because the pulping data obtainedwith CAD-down-regulated tobacco are comparableto those from CAD-suppressed poplar (both specieshad reduced kappa number and better bleachingwith no modification of other pulp properties), thesedata indicate that tobacco may be an appropriatemodel for rapid evaluation of the impact of specificgenetic modifications on pulping. In this respect, itwill be interesting to see whether or not the results ofpulping experiments on CCR-down-regulated trees,when these become available, are comparable withthose already obtained in CCR-suppressed tobacco.

D. Chemical Pulping of Wood froma Pine cad Mutant

Different pulping experiments have been per-formed with wood from a 12-year-old pinecadmu-tant (MacKayet al., 1999). As shown in Table 10,soda pulping (26–28% active alkali) resulted in pulpwith a lower kappa number than that of the wild type.These results are in agreement with the increasedlignin reactivity observed with mild alkali treat-ment (MacKayet al., 1999). In contrast to the re-sults obtained with the transgenic CAD-suppressedpoplars, no improved delignification was obtainedusing either the Kraft-Pressure bomb* (20–24% ac-tive alkali) or the Kraft-micro autoclave* (17.5–25%active alkali), possibly because of the abundant pres-ence of dihydroconiferyl alcohol in the lignin of

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TABLE 9Lignin Characteristics and Properties of Bleached Kraft Pulps for CCR- and CAD-Down-Regulated Tobacco

Characteristics Control CCR34 Control CCR86 Control CADJ40 Control CADJ50

Enzymatic Activity (%) 100a 48a 100a 1a 100∗b 20∗b 100∗b 7∗b

Lignin CharacteristicsKlason lignin content 100 100 100 58 100 100 100 100

(% of control)Yield in S+ G (µmol/g 2106c 1695c 1295c (1711d) 766e (726d) — — — —

Klason lignin)S/G 0.72 0.78 0.72 1.61 — — — —Free OH in linked — — 20.2 20.9 — — — —

β-O-4 G unitsPulp Properties

Kappa number 21.6 20.6 20.3 17.7 20.0 18.0 27.0 24.0Cellulose DP 1430 1430 1330 1220 1575 1600— —Cellulose yield (% wt) 35.9 36.6 34.0 37.0 34.3 36.6 33.0 32.4

Pulp Properties After BleachingBrightness (% ISO) — — 82.8e 77.5e 88.9f 88.9f — —Breaking length (m) — — 5960e 8210e 9730f 9270f — —Tear index (mNm2/g) — — 4.9e 3.4e 5.3f 6.2f — —Fiber length (mm) — — 0.93e 0.98e 0.77f 0.72f — —

All data are from O’Connellet al. (2002) except∗ which are from Halpinet al. (1994). All data were obtained by usingmature stems as plant material excepta,b, andc; adetermined on 34- to 45-day-old plantlets regeneratedin vitro; bdeterminedon 60- to 70-day-old greenhouse-grown plants;cdetermined on 7-week-old greenhouse-grown plants;ddetermined on21-week-old greenhouse-grown plants. Conditions of Kraft pulping were 28% active alkali, 25% sulfidity. eBleachingwas performed using an O-D-E/O-D-E-C-F sequence;f Bleaching performed using an C-E-D-E-D sequence;—, notdetermined. CCR34 and CCR86 are suppressed CCR plants whereas CADJ40 and CADJ50 are suppressed CAD plants.

the pine mutant, which is involved in C-C linkages(MacKay et al., 1999). The latter compound hasnot been detected in the transgenic poplars, possiblyreflecting the differences in the lignin biosyntheticpathway between gymnosperms and angiosperms.Interestingly, the reduced kappa number in the mu-tant after soda pulping opens up the possibility oflowering consumption of sulfide during pulping,

thereby reducing the production of volatile organicsulfur compounds.

Generally, the pulp yield obtained for the woodof the mutant tree was lower than that of the wildtype. MacKayet al. (1999) suggest this could bedue to the difference in kappa number after sodapulping or excessive peeling of polysaccharides be-cause of overpulping. Brightness of the soda pulp

TABLE 10Properties of Soda Pulping for a 12-Year-Old cad Pine Mutant

Characteristics Control cadMutant

26% active alkaliKappa number 53.1 41.6Pulp yield (%) 31.1 26.9Brightness (% ISO) 31.3 28.7

28% active alkaliKappa number 46.3 26.0Pulp yield (%) 33.7 30.0Brightness (% ISO) 33.0 34.0

Data are from MacKayet al. (1999).

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of the pinecad mutant was similar to that of thewild type (Table 10). In contrast, brightness of theKraft pulp of the mutant was slightly lower than thatof the wild type, although kappa values were com-parable (MacKayet al., 1999). Taken together, thedata currently available concerning the potential ofgene engineering for improving pulping suggest thatCAD and CCR suppression affect lignin structurein ways that improve chemical pulping efficiencyby reducing the amount of required chemicals, andthereby reducing the environmental impact of pulpproduction.

E. Mechanical Pulping of Woodfrom Transgenic PoplarsDown-Regulated for COMTand CAD

Preliminary refiner mechanical pulp (RMP) ex-periments, carried out at a pilot scale, have been per-formed with wood of poplar trees down-regulatedfor COMT and CAD grown in both the French andUK field trials mentioned above (Petit-Conilet al.,1999, 2000). The mechanical properties of the RMPpulp were not modified, except for the ASCOMTline ASB10B which presented higher tensile andtear strength. For the wood of the ASCAD lineASCAD21, the brightness of the unbleached pulpsand the pulp bleachability were lower than those ofthe control. These results are possibly caused by amodification in the chromophore nature caused bythe alteration of CAD expression (Petit-Conilet al.,2000).

VI. CONCLUSIONS ANDPERSPECTIVES

Our understanding of the lignin biosyntheticpathway is constantly and rapidly changing. Thecombination of classical biochemical approaches,allied with the use of transgenic plants to investi-gate the pathwayin vivo, and the interest in im-proving wood and plantfibers for industrial appli-cations, have contributed to recent progress in thisresearch area. A new avenue for studying the molec-ular biology of plant cell walls is to characterize de-velopmental processes, such as wood formation, atthe level of the transcriptome and the metabolome(Hertzberget al., 2001; Trethewayet al., 1999), andcompare these processes in wild-type and transgenicplants modified in cell wall biosynthesis. Such anal-

yses should provide a comprehensive and holisticview on cell wall assembly at a level that was notpossible just a few years ago. In addition to theidentification of novel genes involved in wood for-mation, whose function can be further studied byreverse genetics, applications of these techniqueswill shed light onto the interrelations betweenthe biochemical pathways leading to the biosyn-thesis of the different cell wall macromoleculesand onto their relation with plant growth anddevelopment.

Despite our currently poor understanding of thedetailed biochemistry of plant cell walls, it is clearthat we can modify this biochemistry for useful pur-poses. We already have evidence that genetic engi-neering of lignin biosynthesis has potential for im-proving both the efficiency and the environmentalimpact of pulp and papermaking. A major objec-tive for the future will be to learn how to optimizelignin profiles for ease of pulping. Similarly, lignin-modified plants will need to be grown, experimen-tally at least, on a scale useful for assessing their im-pact for industrial pulping. Results obtained to dateconcern mainly model plants, such as tobacco andpoplar. In the next step, the technology needs to betransferred to other species and to elite clones suit-able to clonal forestry plantations. Transformationprotocols for most of the commercially importanttree species, including gymnosperms, have been es-tablished over the past years.

It is important to appreciate that tree breed-ing is only in the third or fourth generation of se-lection and, therefore, significant genetic improve-ments can be expected through conventional breed-ing alone. However, the genetic improvement byclassical breeding is inevitably slow because of thelong generation times typical of trees, and the factthat many traits can only be scored at rotation age.Because the current level of wood consumption ishigher than the amount of wood that natural forestscan sustainably produce, more wood will need tobe grown in high-yielding plantations (Food andAgriculture Organization, 2001b). Molecular biol-ogy and genetic engineering can be of great valuein advancing progress in this sector, for example,by allowing genes of interest to be identified andintroduced into elite genotypes or by altering theexpression of native genes. It is conceivable thatfuture agro-forestry will utilize genetically engi-neered trees that produce wood of better qualityand with higher yields, either for pulping or fortimber use. In that context, plantation of trans-genic trees could increase wood production and helpthe efforts to reduce the pressure on native forests

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(Fenning and Gershenzon, 2002). Several forestrycompanies and Universities currently managefieldtrials of transgenic trees with potentially improvedtraits (for a list, see Information Systems forBiotechnology http://www.isb.vt.edu/) and thesewill hopefully pave the way to commercialization.Alternatively, when the function of agronomicallyimportant genes has been demonstrated in trans-genic plants, a more efficient exploitation of the ex-isting genetic diversity within a given species willbe possible by combining favorable alleles for thesegenes by marker-assisted breeding, again accelerat-ing the domestication of trees.

Potential risks of large-scale production of en-gineered trees have obviously to be taken into ac-count, but these must be balanced with the clearpotential benefits of cultivating genetically mod-ified clones (Strauss, 2003). At present, the im-pact of transgenes on the environment is evaluatedin controlled and environmentally safefield trials(Hancock & Hokanson, 2001; Daleet al., 2002).However, a variety of research strategies aimedat minimizing the possible escape of introducedgenes are being investigated, such as the modifi-cation offlowering and sterility (Weigel & Nilsson,1995; Rottmannet al., 2000; Pena & Seguin, 2001;Daniell, 2002), and the use of transformation meth-ods to produce plants harboring only the target gene,devoid of selectable marker genes conferring antibi-otic resistance (Ebinumaet al., 1997; Hare & Chua,2002; Eriksonet al., 2003). Because of the impor-tance of high yielding plantation forests to sustain-ably supply wood and reduce destruction of nat-ural forests, regulatory costs and hurdles forfieldtrials involving certain classes of genetically mod-ified trees, such as those that are transformed withspecies-own genes, should be reduced andfield-trialing promoted (Fenning and Gershenzon, 2002;Strauss, 2003).

A critical point to consider is whether the geneengineering approach to tree improvement is eco-nomically attractive in comparison with the cost oftraditional breeding methods. The traits introducedshould have major economic value, as it is the casefor lignin content or quality. In addition, the ge-netic engineering should confer improvement noteasily obtainable by other means and should notadversely affect growth or survival of the plants.Important in this respect is the fact that current ge-netic engineering technology allows the stacking ofseveral transgenes simultaneously in elite genotypesby co-transformation (Halpin & Boerjan, 2003; Liet al., 2003), further speeding-up the domesticationof trees, and improving the cost effectivity of the

transgenic approach. In conclusion, biotechnologyand genetic engineering hold promise for the ge-netic improvement of trees and are important strate-gies to be considered to accelerate current breedingprograms.

ACKNOWLEDGMENTS

The authors thank Martine De Cock for helpin preparing the manuscript. This work was car-ried out in the framework of the European UnionResearch Programs AGRE-0021-C, FAIR-CT95-0424, and AIR2-CT93-1661 and by funding to CHfrom the UK Biotechnology and Biological Sci-ences Research Council (BBSRC). M.B. is a Re-search Associate of the Belgian“Fonds National dela Recherche Scientifique” (FNRS).

GLOSSARY

aAcetyl bromide extraction—Method toquantify lignin in which lignin is extracted by acetylbromide and then quantified spectrophotometricallyby measuring the absorbance at 280 nm (Iiyama &Wallis, 1988).

Active alkali—Percentage of active ingredi-ents in the pulping liquor (NaOH+ Na2S in theKraft pulping process; NaOH in the soda pulpingprocess).

bAlkaline nitrobenzene oxidation—Methodto determine lignin composition. Lignin is oxi-dized with nitrobenzene in alkali and the degra-dation products are measured after HPLC separa-tion or gas chromatography. This method cleavesthe β-O-4 and the Cα-Cβ linkages in the poly-mer. The compounds recovered do not representan absolute value for phenylpropanoid units inlignin and may also include esterified cell wallphenolics. Nitrobenzene oxidation yields vanillin(degradation product of coniferyl alcohol and fer-ulic acid [Van]), syringaldehyde (degradation prod-uct of sinapyl alcohol and sinapic acid [Syr]) andp-hydroxybenzaldehyde (degradation product ofp-coumaryl alcohol andp-coumaric acid). Themonomeric composition of lignin is generally ex-pressed by the Syr/Van ratio.

Basis weight (g/m2) or grammage—Weightof paper per ream (specified area of paper or paper-board). The TAPPI Standard ream is 1 m2 of paperor board.

Breaking length (km)—Measure of tensilestrength obtained by calculating the length at whicha strip of paper would break under its own weight

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when suspended by one end (NF EN ISO 1924-2).The breaking length of most papers varies from 2.5to 12 km.

Brightness—Measure of the whiteness of thepulp or paper. It is the percentage of diffuse reflectedblue light (457 nm) from the paper, relative to anMgO standard (NF Q 03039).

Bulk (cm3/g)—Volume per unit weight (g) of apaper sheet (NF EN ISO 536-NF EN 20534). Bulkis the reciprocal of density (g/cm3).

Burst index (kPam2/g)—Burst strength is theresistance of a paper sheet to rupture by a stretchingforce perpendicular to the sheet (NF Q 03053). Theburst strength measures the hydrostatic pressure re-quired to rupture a piece of paper.

Canadian Standard Freeness (CSF)—Measure of pulp particle size and ability of a pulp todrain water. Water drainage is a crucial parameterin paper manufacture because it determines theconsistency of paper products. A high drainagerate means a high freeness. The CSF test measuresthe drainage of 1 liter of pulp slurry at 0.3% pulpconsistency through a calibrated screen.

Cellulose degree of polymerization (DP)—Number of monomers in the chain of the cellulosepolymer. Cellulose DP is indicated by the mea-sure of cellulose viscosity, which is determinedafter dissolving the pulp in a solvent, such as acupriethylene-diamine solution. A higher viscosityindicates a higher cellulose DP, and is generally cor-related with better pulp and paper properties.

cDerivatization followed by reductivecleavage (DFRC)—Method to determine the com-position of lignin. Theα- andβ-aryl ether linkagesin lignin are cleaved by acetyl bromide and thereleased monomers (cinnamyl acetates) are quan-tified by gas chromatography (Lu & Ralph, 1997).The results obtained are similar to those obtainedby thioacidolysis. However, yields of releasedmonomers are lower than with thioacidolysis.

Drainage index—Degree Schopper Reigler(◦SR) or Canadian Standard Freeness* (ml CSF),both based on a similar concept. Drainage reflectsthe ease of removing water from the pulp by gravityor by mechanical means.

dDiffuse reflectance infrared Fourier trans-form (DRIFT) spectroscopy—See Fourier trans-form infrared (FTIR) spectroscopy

Fiber coarseness (mg/m)—Mass per chain offibers 1 m long. Fiber coarseness reflects the relativethickness of cell walls.

Fibrillation—Loosening of the cross-linkingwithin a fiber wall, allowing partial separation ofconstituentfibrils at thefiber surface.

dFourier transform infrared (FTIR) anddiffuse reflectance infrared Fourier transform(DRIFT) spectroscopy—Techniques based on in-terferometry, allowing the analysis of nanograms ofmaterial. FTIR and DRIFT spectroscopy rely on theabsorption of energy from an illuminating laser. AnFTIR spectrum is generated from radiation absorbedby molecules with polarized bonds or dipole bondsafter excitation with infrared light. The ratio of ab-sorbance intensities at different wavelengths is re-lated to the concentration of different molecules in acell wall sample. The DRIFT technique provides ab-sorption spectra generated with light reflected fromthe surface of opaque materials. DRIFT of cell wallmaterials is non-destructive.

Kappa number—Measure of the residuallignin in the pulp, obtained by measuring the con-sumption of permanganate ions that react with ligninin the pulp. A high kappa number is indicative of ahigh lignin content. As a rough estimate, Klasone

lignin content (%)= 0.15× kappa number.eKlason lignin determination—Method that

quantifies lignin and consists of hydrolyzing thecell wall polysaccharides with sulfuric acid, leav-ing lignin as an insoluble material, which is driedand quantified gravimetrically (Effland, 1977).

Kraft-Pressure bomb and Kraft-microautoclave—Small reactors designed for laboratory-scale simulation of chemical pulping processes.

Liquor—Aqueous solution of chemicals usedto delignify wood. The white liquor is the freshpulping liquor for the Kraft process and consistsmainly of NaOH, Na2S, a small quantity of Na2CO3,and several impurities coming from wood, whenthe white liquor is recycled. Black liquor is thewaste liquor of the Kraft pulping process after com-pletion of pulping. It includes most of the orig-inal cooking chemicals and the dissolved woodsubstances.

fNMR spectroscopy—Technique that pro-vides a compositional and structuralfingerprintof isolated lignin fractions and allows the char-acterization of the major lignin units (H, G, andS units), the determination of functional groups(such as methoxy and hydroxyl), and the main in-terunit bonds. Limitations are overlapping signals,the complexity of assigning signals, and the lowsensitivity.

Opacity (%)—Ability of paper to hide or maska color or object beyond the sheet. The higher theopacity, the better the hiding power (opaque papershave an opacity of 100%).

Pulp consistency—Ratio of dry to humid pulpweight.

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Pulp viscosity—Measure of the average chainlength (degree of polymerization, DP) of cellulose.A high viscosity is indicative of a high cellulose DPand is a sign of a strong pulp or paper. This parameterhas little importance in mechanical pulping becausethe cellulose chains are not significantly degradedby mechanical pulping.

Pulp yield (% weight)—Ratio between theoven-dry weight of the pulp and the initial oven-drywood mass used for pulping.

gPyrolysis gas chromatography massspectrometry (pyrolysis GC-MS)—Direct de-polymerization of lignin by rapid heating followedby mass spectrometry of the breakdown products.The limitation is the presence of overlappingpeaks originating from other compounds and thestructural changes that may occur during pyrolysis.This technique allows evaluation of lignin contentand composition but cannot provide information oninterunit linkages.

Refining—Mechanical treatment of pulpfibers, performed in a beater or a refiner, to developtheir optimal papermaking properties, such asstrength properties. The effects of refining areincreasedflexibility of the fibers, fibrillation* ofcellulose, and delamination of the cell wall. Refinedpulp has a low freeness. Refining is monitored bythe drainage rate of water through the pulp, relatedto the freeness.

S or scattering coefficient (m2/kg)—Describes the ability of a paper sheet to scatteringof light. Defined as S= the limiting fraction oflight energy scattered backward per unit thicknessas the thickness of the layer approaches zero.Alternatively, when the equations are writtendown in terms of basis weight: S= the fractionalscattering loss of light energy per unit basis weightas the weight of the layer approaches zero.

Screening of the pulp—Process that consistsof removing particles, such as rigid and longfibers,shives, andfines fragments, by passing the dilutedpulp suspension through screens under pressure.

SO2 charge (%)—For acidic cooking, theterms total SO2, free SO2, or combined SO2 areused. The total SO2 (free SO2 + combined SO2)charge is the quantity of SO2 determined by iodome-try. The combined SO2 charge represents the weightof SO2 equivalent to the CaO charge contained inbisulfite. The free SO2 charge is the difference be-tween the percentages of total SO2 and combinedSO2.

Sulfidity—Ratio of Na2S to the active alkali(Na2S/(NaOH+ Na2S) × 100%). Typically, a mill

runs at 24–28% sulfidity depending on the woodto be pulped. Sulfidity increases the rate of delig-nification by cleavage of theβ-O-4 linkages andthe methoxy groups. Because lower cooking timesare required at high sulfidity, carbohydrates are lessdegraded.

Tear index—Ratio of the tear strength* by thebasis weight* of the paper sheet and expressed asmNm2/g.

Tear strength—Measure of the energy re-quired to propagate an initial tear through severalsheets of paper over afixed distance (NF EN 21974).

Tensile index—Ratio of tensile strength* to thebasis weight* of the paper sheet (NF EN ISO 1924-2) and expressed as mN/g.

Tensile strength–Resistance of a paper sheetto rupture by a stretching force parallel to the sheetand measured on paper strips using a constant rateof elongation according to TAPPI standard T 494.

hThioglycolic acid extraction—Method toquantify lignin. After its extraction by thioglycolicacid and alkali, lignin is measured spectrophoto-metrically by measuring the absorbance at 280 nm(Campbell & Ellis, 1992).

iThioacidolysis—Method to determine thecomposition of lignin. Thioacidolysis and sub-sequent gas chromatography analysis identifiesmonomers that are released by selectively break-ing of the intermonomericβ-O-4 linkages, the ma-jor linkages in lignin (Lapierre, 1993). This methodis specific for phenylpropanoids and is very sen-sitive. The yield of degradation products is 40–50% for softwoods and approximately 70% forhardwoods (Boudetet al., 1995). The yield ofthe thioacidolysis products (S+G) reflects the fre-quency ofβ-O-4 linkages, which are referred toas “non-condensed” linkages, whereas C-C link-ages are designated“condensed” linkages. An ad-ditional analysis of the released dimeric structuresallows the determination of the different types ofcarbon-carbon and diphenyl ether bonds betweenmonomers (Lapierreet al., 1999).

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