Biochemistry and molecular biology of lignification

New Phytol. (1995), 129, 203-236

Tansley Review No. 80Biochemistry and molecular biology oflignification

BY A. M. BOUDETi*, C. LAPIERRE^ AND J. GRIMA-PETTENATP

^Centre de Biologie et Physiologie Vegetales, URA CNRS 1941, Universite PaulSabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France^ Laboratoire de Chimie Biologique, INRA-Grignon, 78850 Thiverval-Grignon, France

{Accepted 15 October 1994)

CONTENTS

SummaryIntroductionI.

II.

III.

Main structural features of lignins1. Problems with the analysis of enigmatic

polymers2. Three complementary tools for probing

lignin structure: principle andperformance criteria(a) Determination of the molecular

structure and dynamics of lignin bysolution- and solid-state "C NMRspectroscopy

(6) Microstructural screening oflignocellulosics by analyticalpyrolysis

(c) Molecular fingerprint of lignin unitsand interunit bonds by thioacidolysis

3. Three case studies of molecularunravelling of lignin{a) Structural peculiarities of normal

and mutant grass lignins{b) Evaluation of ' lignin-like' material

in suberized tissues(c) Lignification in cell suspension

culturesLignification and cell wall differentiation:spatio-temporal deposition of lignins andinter-relations with other wall components1. Deposition of lignin during cell wall

differentiation

2. Covalent cross-links in lignified walls 213203 3. Proteins and wall components associated204 with deposition of lignin in vascular205 elements 214

IV. Enzymes and genes involved in the205 biosynthesis and polymerization of

monolignols 2161. The common phenylpropanoid pathway 216

206 {a) General discussion 216{b) Phenylalanine ammonia-lyase 216(c) Cinnamate 4-hydroxylase 218{d) Coumarate 3-hydroxylase and

206 ferulate hydroxylase 218(e) O-Methyl transferases 218(/) 4-Coumarate CoA ligase 220

207 2. The lignin branch pathway 221(a) Cinnamoyl-CoA-reductase 221

207 {b) Cinnamyl alcohol dehydrogenase 2213. The polymerization stage: peroxidases

208 and/or laccases? 224{a) Enzymes involved 224

208 {b) Peroxidases 225(c) Laccases 225

211 V. Lignin mutants as a way to improve plantbiomass and to explore lignin biochemistry

211 and metabolism 226VI. Concluding remarks 229

Acknowledgements 230213 References 230

213

SUMMARY

Lignins, which result from the dehydrogenative polymerization of cinnamyl alcohols, are complex heteropolymersdeposited in the walls of specific cells of higher plants. Lignins have probably been associated to land colonizationby plants but several aspects concerning their biosynthesis, structure and function are still only partiallyunderstood. This review focuses on the modern physico-chemical methods of structural analysis of lignins, andon the new approaches of molecular biology and genetic engineering applied to lignification.

The principles, advantages and limitations of three important analytical tools for studying lignin structure are

To whom correspondence should be addressed.

204 A. M. Boudet, C. Lapierre and J. Grima-Pettenati

presented. They include carbon f 3 nuclear magnetic resonance, analytical pyrolysis and thioacidolysis. The useof these methods is illustrated by several examples concerning the characterizatiot-i of grass fignins, ' lignin-like'materials in protection barriers of plants and fignins produced by cell suspension cultures.

Our present limited knowledge of the spatio temporal deposition of lignins during cell wall difTerentiationincluding the nature of the wall components associated to lignin deposition and of the cross-links between thedifferent wall polymers is briefly reviewed.

Emphasis is placed on the phenylpropanoid pathway enzymes and their corresponding genes which aredescribed in relation to their potential rofes in the quantitative and qualitative control of lignification. Recentfindings concerning the promoter sequence elements responsible for the vascular expression of some of these genesare presented.

A section is devoted to the enzymes specifically involved in the synthesis of monolignols; cinnamoyl CoAreductase and cinnamyl alcohol dehydrogenase. The recent characterization of the corresponding cDNAs/genesoffers new possibilities for a better understanding of the regulation of lignification.

Finally, at the level of the synthesis, the potential involvement of peroxidases and laccases in the polymerizationof monofignols is critically discussed.

In addition to previously characterized naturally occurring lignin mutants, induced lignin mutants have beenobtained during the last years through genetic engineering. Some examples include plants transformed by O-methyltransferase and cinnamyl alcohol dehydrogenase antisense constructs which exhibit modified lignins.

Such strategies offer promising perspectives in gaining a better understanding of lignin metabolism andfunctions and represent a realistic way to improve plant biomass.

Key words: Lignins, lignin analysis, cell wall, lignin synthesis genes, genetic engineering.

I. INTRODUCTION

There is a striking contrast between the easyidentification of lignin (from the Latin lignum: wood)in plant tissues, which has been performed for morethan a century through histochemical staining, andour present limited knowledge of lignification.

Lignin is a complex phenolic heteropolymer,resulting from the dehydrogenative polymerizationof three different cinnamyl alcohols, the monolignols

.(see Fig. 8 below), and which is deposited in thewalls of specific cells at the last stages of theirdifferentiation. Lignin reinforces and waterproofsthe walls of specialized cells and plays a fundamentalrole ill the strategies of mechanical support, soluteconductance and also disease protection of higherplants. In this way, lignin is, from an evolutionarypoint of view, probably associated to land coloniz-ation by plants and pteridophytes seem to representthe first plants able to synthesize true lignins (Lewis& Davin, 1994). Over the course of evolution, it isinteresting to observe that the chemical complexityof lignin has increased from pteridophytes andgymnosperm lignins to the grasses, the most de-veloped plants, whose lignins appear to present themost complex composition.

There are several explanations for the gaps in ourknowledge of lignification, all related to the com-plexity of the phenomenon and the complexity oflignin structure.

At the biochemical level, the synthesis of ligninrepresents a long sequence of reactions from in-termediate metabolism (unlike cellulose, anothermajor cell wall component) through the shikimatepathway, the common phenylpropanoid pathwayand the specific lignin pathway. The potential sitesof regulation from the qualitative and quantitative

points of view could consequently be very numerousalong these pathways.

At the cellular level, the synthesis of ligninprecursors and lignins occurs at least in two majorcompartments; the protoplast and the wall. Theprecise nature of the molecules transported to thecell wall and the mechanisms of this transport are atpresent unknown.

At the chemical level, lignin is an ill-definedpolymer (here again compare to the well definedstructure of cellulose) whose monomeric compo-sition and nature of inter-unit linkages vary fromplant to plant and even in the same plant from cell tocell. In this context, it is preferable to use theexpression ' lignins' rather than ' lignin' since thereis a potentially great diversity of chemical structuresbetween the various lignins. In grasses, the oc-currence of hydroxycinnamic acids in the wallsrepresents an additional complexity and raises thequestion of their involvement as lignin components.

Another limitation in the study of the regulationand the physiology of lignification is the scarcity ofappropriate experimental systems. When lignifica-tion is induced in model systems, it usually takes along time after the application of a stimulus and thesynthesis of lignins concerns only a portion of thecells under study. When cell suspension cultures areused, the synthesized lignins seem to differ from the'normal' lignins.

At the present time, the clearest aspects oflignification are the association of lignins withspecific tissues involved in conduction (xylem) orsupport (sclerenchyma), the cell wall location of thepolymer, the biosynthetic pathway of the threemonomeric units of lignins and the nature of somechemical bonds between the different monomericunits. Apart from these basic characteristics, many

Biochemistry and molecular biology of lignification 205

postulates and much preliminary evidence concern-ing lignification and lignin structure remain to beconfirmed.

It is not the intent of this review to cover allaspects of lignification. During the last decadeexcellent reviews have been published which haveconsidered specific aspects of lignification in greaterdepth, such as the role of cinnamyl alcohol glucosidesin lignification (Grisebach, 1981), the enzymaticcontrol of the monomeric composition of lignins(Higuchi, 1985), the heterogeneity of lignins(Monties, 1985), the chemical and structural aspectsof lignification (Lewis & Yamamoto, 1990) and theextracellular enzymes potentially involved in thepolymerization of lignins (Dean & Eriksson, 1992).General aspects of lignification have been reviewedby Campbell (1993) and more specifically the geneticregulation of lignin biosynthesis by Sederoff et al.(1994). The role of lignification in disease resistance,which will not be considered in the present reviewhas also been recently dealt with by Nicholson &Hammerschmidt (1992) and Walter (1992).

It is the intent of this review to focus on recentadvances in two different areas which should allow abetter understanding of lignification and its control:the modern physico-chemical methods of structuralanalysis of lignins and the new approaches ofmolecular biology and genetic engineering applied tothe field of lignification.

In addition to a fundamental interest for thecomplex process of lignification, which represents atypical example of cell differentiation, applied andbiotechnological objectives have emerged duringrecent years. Indeed, lignins represent more than25 % of the terrestrial biomass and thus play animportant role in the carbon cycle. However, theirpresence in plants has a negative impact on biomassutilization, particularly in forage digestibility andpaper making. An improved knowledge of lignifica-tion, through the use of combined approaches,should also help to select and redesign plants moreadapted for human purposes.

II. MAIN STRUCTURAL FEATURES OF LIGNINS

1. Problcjns zvith the attalysis of enigmatic polymers

Even though the story of deciphering lignin structurebegan five decades ago (reviewed in Erdtman, 1957;Freudenberg & Neish, 1968) there is still muchspeculation about these enigmatic polymers. Thistrouble is derived directly from analytical difficulties.Obviously, a critical aspect of lignin research is thatof structural analysis. Whether one is investigatingthe involvement of lignins in plant defence reactionsor determining the changes in lignification patternassociated with natural or induced mutations, thechoice of appropriate and informative analyticalmethods is critical to achieve progress.

In contrast to other biopolymers, the ultimate

analysis of lignin structure has not been accom-plished. No method can provide a complete schemefor lignins and it is unlikely that such a method willever be devised. This situation is stimulating for theexperimentalist, as there is a large scope for innova-tive design of analytical strategies. Our sketchyknowledge on lignin structure arises from manymethods based on different principles (reviewed inDence & Lin, 1992). Instead of their exhaustivepresentation, we have chosen to focus this section onthree analytical tools which recently made greatheadway in the field of lignin research. These arecarbon-13 nuclear magnetic resonance ("C NMR)spectroscopy, analytical pyrolysis, and thioacido-lysis. After a survey of their principles, advantagesand limitations, the structural information gained bythese procedures will be illustrated with three casestudies.

At this point, we need to introduce the mainstructural features of lignins and their specificterminology. Lignins are cross-linked polymerscomposed of /)-hydroxyphenyl (H), guaiacyl (G),and syringyl (S) building units, in various ratiosaccording to their origin. Gymnosperm ligninsessentially consist of G units. Angiosperm ligninsare mainly a mixture of G and S units. Grass ligninsare composed of H, G, and S units (as well asattached hydroxycinnamic acids). These CgCg unitsare interconnected by a series of ether andcarbon-carbon linkages (Higuchi, 1990), in variousbonding patterns (Fig. 1). The most frequentinterunit /J-O-4 bonds are the targets of lignindepolymerization processes. In contrast, the other/?-5, ^ - 1 , /^-/?, 5-5, and 4-O-5 interunit bonds are moreresistant to degradation. Their relative amounts arenot yet clearly established. For example, controversystill rages over whether the /i-1 substructures aresubstantial lignin bonding patterns or trace com-ponents.

When the analytical method requires a solublelignin-derived preparation, a serious problem arises:there is no method for isolating lignins in their intactstate. Soluble lignin preparations, such as milledwood, dioxane, and kraft lignins can only be obtainedby vigorous cell wall treatments and cannot beconsidered as representative of native lignins, eitherqualitatively or quantitatively.

Besides the insoluble and cross-linked lignins thatprovide strength and durability to lignocellulosics,non-lignin phenolics occur in plant cell walls. Thisfurther complication is responsible for interferencesassociated with unspecific lignin evaluations, and forterminology confusion. For example, a typicalfeature of grass cell walls is the presence of p-couniaric (PC) and ferulic (FE) acids, attached to thelignin and polysaccharide polymers through labileester and/or ether bonds (Jung & Deetz, 1993). Theterm 'non-core' lignin has been introduced todescribe these labile PC and FE units, whereas


Phenylcoumaran /?-5

- C -

Biphenyl - Q _5-5

Biphenyl ether 4-0-5

Diarylpropane /?-1

Resinol /?-/?

Figure 1. Principal bonding patterns of native lignin units. The/)-hydroxyphenyl (R = H), guaiacyl (R at C-3 = OMe; R at C-5 = H), and syringyl (R = OMe) units are outlined with their usual carbon numbering andsymbol.

'core' lignin is the high molecular weight polymerthat resists mild hydrolysis. This terminology makessense with respect to nutritional aspects, as bothcomponents detrimentally affect forage digestibility.It is however unsound with regard to the differentstructure of lignin units and of PC and FE acids. Thefirst reported case study in this review is theproblematic characterization of grass lignins, in thepresence of these potentially interfering PC and FEacids.

A similarly cotnplicated situation concerns theprotective barriers involved in plant defence re-actions. These barriers typically comprise polymers,namely suberins and/or lignins, together with lowmolecular weight phenolics, such as FE esters and/oramides (Kolattukudy, 1984; Davin & Lewis, 1992).The term ' lignin-Iike material' is frequently used todescribe a fairly resistant phenolic fraction thatmimics lignins with regard to unspecific histo-chemical stainings (Lewis & Yamamoto, 1990) orsevere oxidations. The second case study willillustrate to what extent the reported tools giveevidence for typical lignin structures in 'lignin-likematerial'.

The last case study concerns the lignins producedby cell suspension cultures subjected to low levels orswitching of growth regulators (Simola, Lem-metyinen & Santanen, 1992; Brunow et al., 1993;Eberhardt et al., 1993). Such polymers constituteunique in vivo models for comparing the structure ofstress and normal lignins.

2. Three complementary tools for probing ligninstructure: principle and performance criteria

{a) Determination of the molecular structure anddynamics of lignin by solution- and solid-state ^^CNMR spectroscopy. Since the pioneering work ofNimz and coworkers in the 70s, solution state^ C NMR spectroscopy of lignins has made greatprogress. To date, a high field one-dimensional(1 D) "C NMR spectrum obtained from a solublelignin preparation may contain more than 50 re-solved lines ascribable to the various carbons of thelignin skeleton. This represents both an immenseamount of molecular information and a real challengefor spectral assignment. To sum up, the ligninspectrum, assigned with a myriad of monomer anddimer model compourids, can be divided in threemain segments: the first (200-165 pptn) representsthe carbonyl area; the second (165—lOOppm) isassignable to aromatic and olefinic carbons, and thethird (100—lOppm) represents the aliphatic carbonarea (Robert, 1992). A major upgrading of thetechnique was afforded by the quantitative use of thespectra when recorded with special conditions. Morerecently, one-dimensional (1 D)- and two dimen-sional (2 D)-NMR experiments applied to ligninsminimized the problem of peak overlap and/orspectral assignments (Robert, 1992). The mainavenues opened by ^ C NMR of lignin solutions arethe characterization of the major lignin units, thedetermination of key functional groups such as the

Biochemistry and molecular biology of ligttification 207

methoxyl and the phenolic or alcoholic hydroxylgroups, and the tracing out of the main bondingpatterns. The main limitations are the low sen-sitivity, which involves a high sample demand andlong recording time, the problem of peak overlap andthat of spectral interferences between lignins andnon lignin phenolics. For example, in the 1 D-"C NMR spectra, most of the aromatic signals of PCand FE acids are overlapped by those of H and Gunits, respectively. These problems notwithstand-ing, the use of sophisticated solution state ^ C NMRstrategies will certainly open new frontiers tounderstanding lignin structure and relationshipswith other cell wall components, as exemplified withthe case study of grass lignins. The stumbling blockof this technique still remains the extent to whichsoluble lignin fractions are representative of nativelignins. In this respect, solid state '''C NMR oflignins (Maciel et al., 1985; Leary & Newmann,1992) seems more suitable. However, the structuralinformation recovered from solid state ''C NMRspectra is restricted by the inherently broad signalsand the resulting severe peak overlap. The widerange of proton and carbon relaxation parameters,which makes the quantitation of solid state spectracomplicated, can be the source of unique informationabout the molecular dynamic of cell wall components(Gerazimowicz, Hicks & Pfeffer, 1984; Stark &Garbow, 1992). This kind of study seems to be avery promising development of solid state '''C NMRapplied to lignocellulosics.

{b) Microstructural screening of lignocellulosics byanalytical pyrolysis. Analytical pyrolysis consists ofthermal fiash degradation of non-volatile materialinto a volatile degradation mixture, in the absence ofoxygen. This mixture is either directly introducedinto a mass spectrometer to recover a complex massspectrum (Py-MS), or flushed by helium into acapillary gas chromatography column that ensuresthe separation of the various degradation products,before their mass spectral analysis (Py-GC-MS).The Py-MS mass spectra or the Py-GC-MS profilesare molecular fingerprints of the sample, as di-agnostic ion markers or diagnostic peaks of cell wallcomponents have been identified (recently reviewedin Boon, 1989; Ralph & Hatfield, 1991; Meier &Faix, 1992). In contrast with "C NMR, the majorattribute of analytical pyrolysis is its screeningcapability, as it is completed within the range ofminutes (Py-MS) to 1-5 h (Py-GC-MS) and frommicrogram amount of cell walls. The Py-MS spectraof lignocellulosics mainly comprise the sugar ionmarkers, below m/z 120, and those of cell wallphenolics, from m/z 120-210 for the monomer ionsto m/z 272-418 for a few dimeric ions (Niemann etal., 1992; Logan, Boon & Eglinton, 1993). Whereasthe screening capabilities of Py-MS are invaluable,its main limitation is the somewhat equivocal origin

of the various ions. For example, among the ligninion markers, the ion at m/z 180 may be assigned toconiferyl alcohol and/or to vinylsyringol. In con-trast, Py-GC-MS provides chromatograms with aset of diagnostic GC peaks identified from theircomplete mass spectra (Ralph & Hatfield, 1991;Meier & Faix, 1992). However, Py-GC-MS sufferssome interference between lignin units and non-lignin phenolics. This interference phenomenon isparticularly severe in the case of grass lignins as bothH units and PC esters give rise to vinylphenol, whensubjected to pyrolysis. The main applications of Py-GC-MS of lignocellulosics are the compositionalanalysis of plant tissue samples and the deter-mination of lignin monomer composition. Duringthermal depolymerization of lignins, which essen-tially proceeds by cleavage of interunit ether bonds,side reactions occur, such as dehydration or short-ening of lignin sidechains, demethylation of methoxylgroups. Hence, little detailed information aboutlignin bonding patterns and functionality can berecovered from the determination of the variouspyrolysis products. The lignin-derived productswere estimated to represent c. 20 % of the polymer(Faix & Meier, 1989). This limited degradationyields for lignins is however higher than that ofpolysaccharides, which makes the method valuablefor the detection of trace lignin amounts in poly-saccharidic samples. Future improvements of thisunequalled screening method applicable to tissuefractions issued from micromanipulation will con-cern a better basic knowledge of pyrolytic mech-anisms and the introduction of new techniques suchas Py-HPLC-MS or Py-MS-MS.

(c) Molecular fingerprint of lignin units and interunitbonds by thioacidolysis. The traditional way ofanalyzing polymers is by chemical degradationsleading to low molecular weight products. Theamount and relative distribution of such productsprovide structural information about the originalpolymer. In the case of lignins and due to resistantinterunit bonds, the limitation of this strategy is themoderate degradation yield. In addition, the lignin-derived phenolics are prone to side reactions whichfurther decrease their recovery yield or createartifacts. Nonetheless, degradative methods werepivotal tools for the elaboration of our currentknowledge on lignin structure. Even though thesemethods happened to be severely criticized for theirlow yields and/or potential artifact formation, themost complete descriptions of lignin bonding pat-terns stem from the thioacetolysis and hydrogeno-lysis of lignins, worked out by Nimz (1974) andSakakibara (1980), respectively. However, for prac-tical reasons, these informative procedures are notsuited for routine analysis. In contrast, lignin alkalineoxidation, with nitrobenzene or cupric oxide, is themost frequently used procedure (Chen, 1992). This


OHErythro/threo, 50/50

Figure 2. Thioacidolysis pivotal reaction. The p-hydroxypbenyl (R = H), guaiacyl (R at C-3 = OMe; R atC-5 = H), and syringyl (R = OMe) units only involved in/?-O-4 bonds specifically give rise to H, G, and Sthioethylated monomers, respectively and with a c. 80%reaction yield.

method has a fairly high degradation yield, rangingfrom 30 to 50% for native lignins, and is supportedwith much literature data. Its main limitation is dueto the oxidative cleavage of the benzylic bonds,which definitely prevents the recovery of structuralinformation about sidechain arrangements and po-tentially causes interference between lignin units andother phenolics such as PC, FE, tyramine, andtyrosine, which are degraded to the same benz-aldehydes as H and G lignin units.

To better examine lignins, we thus developed anoriginal acid solvolysis, thioacidolysis, capable toprovide a definite fingerprint of lignin structures,with high reaction yield and routine capabilities(reviewed in Lapierre, 1993). The key reaction ofthioacidolysis (solvolysis in dioxane/ethanethiol,9./1, v/v, 0-2 M boron trifluoride etherate) is depictedin Fig. 2. The target structures are the H, G and/orS lignin units only involved in labile /?-O-4 bondingpattern. These units, which are the most typicalstructures of native lignins, specifically give rise to

thioethylated H, G, and/or S monomers with a highreaction yield. On this basis, the recovery of thesemonomers from a plant material may be viewed as adiagnostic test for the presence of lignins whereother histochemical, degradative, or spectroscopicmethods fail for lack of specificity and/or sensitivity.As thioacidolysis degrades lignins to compoundswhich retain features of sidechain functionality, thedetermination of a wide range of minor lignin-derived monomers provides further information, asoutlined in Table 1. This table also stresses that PCand FE units give rise to degradation productsdistinct from the lignin-derived ones, thereby rulingout the risk of interference. Finally, a recentdevelopment of thioacidolysis, involving a furtherdesulfurization step after the depolymerization one,significantly upgraded the method through therecovery of a series of lignin-derived dimers rep-resentative of the main condensed bonding patterns(Fig. 3). The two-steps protocol, applicable to a fewmilligrams of cell walls, thus provides a detailed andquantitative profiling of lignin units and interunitbonds. This characterization concerns about 40-50and 70% of the softwood and hardwood nativelignins, respectively. Such substantial appraisal ofnative lignins cannot be obtained for lignins withlower content of fi-O-A- target structures, such asheavily degraded lignins. In this case, thioacidolysisshould be used in combination with other methodsof lignin analysis, not exclusively aimed at the /?-O-4 linked units, such as NMR spectroscopy.

3. Three case studies of molecular unravelling oflignin

(a) Structural peculiarities of normal and mutantgrass lignins. Grass lignins have been studied less

Table 1. Specific monomers obtained by thioacidolysis of various phenolicstructures occurring in the plant cell wall. R = p-hydroxyphenyl, guaiacylor syringyl rings {only involved in P-O-4 linkages {Fig. 1) and not incarbon-carbon or diarylether linkages). It is worthy to note that all theoutlined guaiacyl structures and ferulic acid would provide vanillin onnitrobenzene oxidation whereas thioacidolysis is capable to specificallydiscriminate them

Initial structure Recovered monomers

Lignin structuresCgC, arylglycerol-^-aryl etherR-CHOH-CHOAr-CH.,OH

CgC., /?-arylether with aCOCinnamaldehyde end groupsR-CH=CH-CHO

Cinnamyl alcohol end groups2

Benzaldehyde end groups, R-CHONon lignin constituents

Cinnamic acid units, ester or etherlinked R-CH=CH-COOH

R-CHSEt-CHSEt-CH^SEterythro/threo, 50/50.

R-CSEt=CHSEt, Z and E

R-CHSEt-CHj-CHjSEt and

R-CH(SEt).,

R-CH=CH-COOH andR-CHSEt-CHj-COOH


OMe

MeO OMe

OH

OMe

Figure 3. Main dimers recovered from the thioacidolysis-I-desulfurization of native lignins. Angiospermlignins yield guaiacyl-guaiacyl (G), mixed guaiacyl-syringyl (M), and syringyl-syringyl (S) dimers. S resinolstructures (Fig. 1) are converted to the outlined ft-fi tetralin dimers provided with an unusual Ca-C6 bondformed during tbioacidolysis. Gymnosperm lignins essentially give rise to G dimers.

Table 2. Yield of the main H, G, and S monomers issued from thethioacidolysis of extractive-free cell walls {in mM g~^ of Klason lignin); tr :traces ; nd: not detected

Sample

WoodsSprucePine

Compression woodOpposite wood

PoplarBirchOakNothofagiis dombeyiLaurelia phillipiana

StrawsWbeatTriticaleRyeCorn

• Rice

Yield(H + G-t-S)

1230

1140102023902460197023551860

104016101670610630

Relative distribution(H:G:S)

2:98:tr

18:82:tr2:98:tr

nd:41:59nd:24:76nd:32:68nd:14:86nd:66:44

5:49:463:42:552:44:544:35:61

15:45:40

than wood lignins. However, structural knowledgeon these polymers is a prerequisite to a better designof the strategies applicable to annual crops in orderto optimize their industrial and feed uses. Thestructural features examined herein are the monomercomposition of grass lignins, the proportions ofthe main bonding patterns, and the relationshipsbetween grass lignins and the attached hydroxy-cinnamic acids. In addition, the structural efTect ofthe brown midrib (bm) mutation will be reported inthe case of corti lignins.

As previously mentioned, neither ^ C NMR, noranalytical pyrolysis allows the unequivocal estima-tion of H and G units in grass lignins, due to theinterference of PC and FE units, respectively.Accordingly, this was performed by thioacidolysis.The results shown in Table 2 indicate that from thelabile moiety of grass lignins, H thioacidolysismonomers are systematically recovered, but inrelatively low amounts. S and G ones are obtained insimilar yields. The total yields of thioacidolysis

monomers indicate that lignins of woody angiospermcontain twice as many labile units than lignins fromgrass straws or from woody gymnosperms. As regardto the determination of H lignin units, it is a widelyheld, although unsubstantiated, belief that theyspecifically occur within the more resistant moiety ofthe polymer, on the rationale that the 3- and 5-aromatic positions are available for carbon-carbonlinkages. If so, one should expect a higher relativedistribution of H units in the thioacidolysis dimersrepresentative of such linkages. However, only traceamount of H-H and H-G dimers were observed,mainly of the biphenyl type (Table 3). Interestingly,the absence of detectable amoutits of mixed H-Sdimers is consistent with the results of Terashima etal. (1993) who demonstrated that the deposition of Hand S lignin units are distinct temporal and/orspatial events, H units being deposited at the initialstage of lignification within middle lamella, and Sones later in the secondary wall. Although the data ofTable 3 are to be confirmed with other samples


Table 3. Relative frequencies of the main dimersrecovered from angiosperm lignins (% molar)

Inter-unit linkage types

/?-l G, M, S/?-/? from syringaresinols/i-5 G, M4-O-5 G, M5-5 G

Poplarwood

3129171013

Wheatstraw*

2925191512

* Only low amount of H-G dimers.

PPm 150 100 50

Figure 4. Carbon 13 NMR spectra of lignin fractionsisolated from the corn internodes of (A) mutant bm and(B) normal lines. Main quoted peaks correspond to G:guaiacyl and S: syringyl aromatic carbons; Ca, C/?, Cd:aliphatic carbons in ji-O-A linked sidechains (Fig, 1); PC:/)-coumaric esters; X: xylose units (adapted from Lapierre,1993).

(Lapierre & Pollet, to be published), they indicatethat the relative proportions of the main dimers frompoplar and wheat lignins are very similar, therebysuggesting a similar distribution of carbon-carbonbonding patterns in grass and woody angiosperm.

The last point about grass lignins focuses on thebrown midrib bmr corn mutants which have receivedrepeated attention since the first studies of Kuc &Nelson (1964) and which will be described in moredetails in section V of this review. In these cornmutants, a depressed lignin content (compared to thenormal line) was found associated with a lower

catechol-O-methyltransferase activity (Grand et al.,19856). We have investigated the structure of ligninsfrom mature internodes of brown midrib cornmutant and compared them to the analogous normalline lignins by '''C NMR spectroscopy and thio-acidolysis (Lapierre, 1993).

The examination of the '^C NMR spectra of ligninfractions isolated from the mutant and normal corninternodes (Fig. 4) reveals that both lignins areassociated with non-cellulosic polysaccharides, asevidenced from the signals of xylose units (quotedX). The presence of PC esters associated with thislignin sample is evidenced by their specific and sharpsignals. The relative importance of the S and Gsignals suggests that mutant corn lignins are richerin G units, in accordance with the results of Kuc &Nelson (1964). All this structural information isobtained in a non-destructive way and concerns thewhole soluble lignin samples. However, as pre-viously emphasized, these soluble lignin samplesmay have undergone significant structural changeduring the ball milling step used for their isolation,compared to native cell wall lignins.

The analysis of the thioacidolysis monomersreleased from mutant and normal corn lignins,analyzed in situ, revealed novel information. Whilethe lower content of syringyl units in mutant cornlignins was confirmed, the occurrence of additional5-hydroxyguaiacyl (5-OH G) units was unequi-vocally demonstrated from the GC-MS analyses ofthe main lignin-derived monomers (Fig. 5). Interest-ingly, the occurrence of 3,4-dihydroxyphenyl unitscould not be demonstrated, which' clearly showsthat the methylation step affected by this bmrmutation is the converting of 5-OH ferulic acid intosinapic acid. The incorporation of additional mono-mer units in mutant corn lignins has been suggestedby Kuc & Nelson (1964), without experimentalsupport as O-diphenolic rings do not survive thenitrobenzene oxidation used by these authors. Theselabile structures could not be detected by analyticalpyrolysis either: while Py-MS mapping of bmr corncell walls confirmed their lower lignin content,compared to other lines, it could not reveal theoccurrence of 5-OH G units in the abnormal bmrlignin (Boon, 1989). This may be related to the severeconditions of pyrolytic depolymerization, whichprevents the recovery of native and labile ortho-diphenolic rings.

The same degradative strategies were employed toinvestigate the structural changes induced by amutation affecting the cinnamyl alcohol dehydro-genase (CAD), in bmr sorghum mutants. In thiscase, not only thioacidolysis (Chabbert, Tollier &Monties, 1993) but also Py-MS (Pillonel et al., 1991)analyses showed the substantial incorporation ofconiferaldehyde sidechains in mutant lignins, fromthe determination of their specific degradationmonomer.


|X16

S '

73

MeOs^

MeO

299 (i)QHRTHR CH R

26511

M^^OMeOTMS

x16

M©433 448

5-OHG

73 ^

751 8,9

ITMSO.

MeO

)VCHR-CHR-CH2R

323

' ' ©9"^" ix16

JTMSO^OMe \ 4M©506

91 1

5-OHG

Figure 5. Partial GC chromatograms of the thioacidolysis main monomers recovered from corn internodes of(A) mutant bm and (B) normal lines. Tbe mass spectra of the guaiacyl (G), syringyl (S), and 5-hydroxyguaiacyl(5-OH G) monomers (analyzed as their trimethylsilylated derivatives) allow tbeir identification (R = SEt). Thethioacidofysis G/S/S-OH G yields for the mutant and normal lines are 305/118/32 and 214/369/trace,respectively, expressed in micromoles per gram of Klason lignin (adapted from Lapierre, 1993).

{b) Evaluation of 'lignin-like' material in suberizedtissues. A common response of plants towardswounding or pathogen infection is the production of'lignin-like' compounds which form a protectivebarrier together with suberin. Few attempts havebeen made to examine the structure of the phenolicmoiety of suberin, in order to appreciate to whichextent it resembles cell wall lignins (Kolattukudy,1984; Borg-Olivier & Monties, 1993).

Erom quantitative solid state ^ C NMR spectra,the suberized potato periderm was estimated to becomposed of 50 % polysaccharides, even after re-moval of cellulose and pectin by standard enzymicmethods. Also, the aromatic and olefinic carbonswere shown to outnumber fatty metbylene groups2:1 (Garbow, Ferantello & Stark, 1989). A similarlybigh relative importance of the aromatic moiety incork suberin was recently determined by Py-GC-MS analyses (Marques et al, 1994). On the basis ofdifferent relaxation parameters, solid state '^C NMRof potato suberin further demonstrated the oc-currence of two phenolic pools with high and lowmobility. During the process of suberin deposition,the phenolic population with restricted mobilityincreased, relative to the highly mobile pool, whichsuggests the further anchoring of aromatics to thecell wall (Stark & Garbow, 1992).

Erom the data of nitrobenzene oxidation,Kolattukudy (1984) proposed a tentative model forthe aromatic moiety of suberin where H, G, and Sunits were interconnected by typical lignin /?-O-4bonds and also associated with ferulic esters. In thecase of potato suberin, tbis model was recentlyreassessed by thioacidolysis: whereas the main Gand S thioacidolysis monomers confirmed the oc-currence of G and S units involved in /?-O-4 bondingpattern, no H homologous compounds could bedetected, suggesting the absence of /J-O-4 linked Hunits in suberin. The formation of /)-hydroxy-

benzaldehyde by nitrobenzene oxidation of suberinwas thus ascribable to non lignin phenolics, such astyramine and tyrosine (Borg-Olivier & Monties,1993). We further investigated the structure ofpotato suberin with the analysis of the dimersrecovered after thioacidolysis and Raney nickeldesulfurization (Lapierre, Pollet & Negrel, to bepublished). The lignin-derived dimers involving Gand S units interconnected through various bondingpatterns have been found (Eig. 3). Their relativedistribution was however diflferent from that ofangiosperm cell wall lignins. This result shows thatthe phenolic moiety of suberin has both structuralsimilarities to and differences from cell wall lignins.Besides tbe various monomers and dimers recoveredfrom potato suberin, we obtained substantialamounts of phenolamide derivatives released by thethioacidolysis and desulfurization steps (Lapierre,Pollet & Negrel, to be published).

Taken together, the ^ C solid state NMR and thethioacidolysis data outline the supramolecular organ-ization of suberized tissues, involving G and S unitsengaged in typical lignin bonding patterns, togetberwith fatty and polysaccharidic components, possiblycross-linked by pbenolamide bridges.

(c) Lignification in cell suspension cultures. Callus andcell suspension cultures originating from variousgymnosperm and angiosperm samples have beenused to study the xylogenesis and lignificationprocesses (Venverloo, 1969; Carceller et al., 1971;Robert, MoUard & Barnoud, 1989). The last casestudy presented herein deals with the structuralinvestigation of lignins formed by gymnosperm cellcultures subjected to cbanges in growth regulators(Brunow et al., 1990, 1993; Davin & Lewis, 1992;Simola et al, 1992; Eberhardt et al, 1993). Suchinduced lignification in cell suspension cultures wasalso obtained by other methods such as fungal


elicitation (Lapierre et al, 1993). Interestingly, thesecell culture lignins are both released into the nutrientmedium of the suspension cell cultures and producedas an integral part of the cell wall. The structuralinvestigation on these extracellular and cell wallassociated lignins, compared to normal cell walllignins, may thus provide new insights for under-standing the matrix effect on lignin structure and theelicited lignification process.

Lewis and co-workers recently characterized thestructure of cell wall associated lignins in Pinus taedacell suspension cultures, as a model system wherebylignification can be investigated in the cell wall(Davin & Lewis, 1992; Eberhardt et al, 1993).Induced lignification was caused by switchinggrowth regulators. Lignified cell walls were re-covered from cultures after administration andmetabolism of [l- '^C], [2-^-*C], or [3-"C] phenyl-alanine, and lignins were examined in the intacttissue in situ by solid state '''C NMR spectroscopy.The main bonding environments of the ligninsidechains were traced out from the Ca, C/?, and Cylarge '''C enriched resonances, corresponding to theincorporation into cell wall lignins of the [1-"C], [2-'^C], or [3-"C] phenylalanine, respectively. Al-though the spectra were not recorded in quantitativeconditions, the authors concluded from the relativeimportance of some specific signals that the majorlinkages involving the sidechains of these cell-wallassociated cell culture lignins were the y3-O-4linkages, immediately followed with the /?-^ resinolbonding pattern (Fig. 1). In contrast, the /?-5 and /?-1 linkages corresponding to overlapping signals wereestimated to correspond to minor bonding patterns.

The lignins released into the medium fromsuspension-cultured conifer cells are unique models;(i) to characterize carbohydrate-free lignins withoutdenaturing isolation techniques and (ii) to study invivo the matrix eflfect on lignin structure. Theseextracellular lignins were produced in high amountby Picea abies cell cultures grown in a medium withlow levels of growth regulators (Simola et al, 1992).Solution-state quantitative 1 D '^C NMR spectro-scopy of released suspension cell culture lignin(RSCL) produced by Picea abies revealed majordifferences between RSCL and a milled wood ligninMWL sample, isolated from spruce wood by ballmilling and solvent extraction. '''C NMR thus dem-onstrated that the methoxyl content per Cj-Cg unit islower in RSCL than in MWL (0-7 versus 0-9),which suggests the incorporation of non-methoxylated units in RSCL lignin. Besides, itfurther indicated that 40-50 % of phenolic groups inRSCL were free, versus 10-20% in MWL (Brunowet al, 1990). Other substantial differences wereelucidated from the 2 D ^H-'H NMR spectra: crosspeaks assigned to coniferyl alcohol sidechains and to(i-P structures (such as in resinol. Fig. 1) weresubstantial in RSCL, whereas they were very weak

Table 4. Relative frequencies of the main dimersrecovered from spruce cell wall lignins OWL, sprucereleased suspension cell culture lignin RSGL and froma synthetic guaiacyl lignin {dehydropolymer DHP)prepared by pcroxidasic polymerization of coniferylalcohol (% molar)

Inter-unit linkage types

5-S

/?-/? from resinols

4-O-5

CWL

3331Traces26

6-5

RSCL

332821135

DHP

17 '5322

63

and absent in MWL, respectively (Ede et al, 1990).Accordingly, these NMR results demonstrate thatextracellular lignins from Picea abies definitely donot resemble lignins isolated from spruce wood. Asthe isolation procedure may affect lignin structure,thioacidolysis performed without the lignin isolationstep was carried out on native spruce lignin andRSCL (Brunow et al, 1993). The analysis of thelignin-derived monomers demonstrated that extra-cellular lignins were enriched in /?-O-4 linked Hunits, thereby confirming the low methoxyl contentevidenced from the '^C NMR data. The relativedistribution of the lignin-derived dimers fromRSCL, native spruce lignins, and guaiacyl syntheticlignins prepared by peroxidasic polymerization ofconiferyl alcohol was very differenf (Table 4). InRSCL and in synthetic lignins, there was a sub-stantial proportion oi fi-jf resinol structures, whereasthe dimer specifically recovered from resinol struc-tures was recovered as a trace component fromspruce cell wall lignins. This result suggests thatresinol structures are actually minor components ofgymnosperm wood lignins. Besides, they6'-l bondingpattern was found to be another discriminatingstructure between normal wood lignins and RSCLor synthetic lignins (Table 4). When the extracellularRSCL were produced by Picea abies culturessubjected to fungal elicitation, instead of changes ingrowth regulators, their structure was found to bevery similar to that outlined in Table 4 (Lapierre etal, 1993). To sum up, guaiacyl lignins polymerizedoutside a cell wall matrix, seem to be typified by ahigh content in ji-fi resinol structures and a lowcontent in /^-l bonding pattern. In a more com-prehensive study, aimed at mimicking the lig-nification process in synthetic lignins prepared invarious conditions, it was repeatedly found that theavoidance of excessive ff-fi reticulation via resinolstructures and the formation of significant amount ofP-\ bonding pattern were the most difficult steps tocontrol, which still prevents synthetic lignins pre-pared so far to resemble closely cell wall lignins


{ToWieretal, 1991; Terashima, Lapierre & Monties,unpublished results).

ni. LIGNIFICATION AND CELL WALL

DIFFERENTIATION: SPATIO-TEMPORAL

DEPOSITION OF LIGNINS AND INTER-

RELATIONS WITH OTHER WALL COMPONENTS

1. Deposition of lignin during cell walldifferentiation

The deposition of lignin in situ can be investigatedby direct examination of protolignin in the cell wallthrough non-destructive methods. The group ofTerashima (Terashima, Fukushima & Takabe, 1986;Terashima & Fukushima, 1988) has used micro-autoradiographic methods combined with selectiveradio-labelling of a specific structural unit in lignin(specific monolignol glucosides). These methodshave been developed to visualize the growth pro-cesses of the protolignin macromolecule in a specificmorphological region of differentiating xylem ofgymnosperms and angiosperms.

Lignification occurs in three distinct stages, pre-ceded by deposition of carbohydrates at the differentlayers of the secondary cell wall (SI, S2, S3)(Terashima et al., 1993). The first stage of lig-nification oceurs at the cell corners atid middlelamella after the deposition of pectic substances hasfinished and SI formation has started. The second isa slow lignifieation stage associated with cellulosemierofibrils and mannan and xylan deposition in S2.The main lignification occurs in the third stage afterthe deposition of cellulose mierofibrils in S3 hasstarted.

Microautoradiograms of differentiatitig xylemtissues indicated that three kinds of monolignol unitsare iticorporated at differetit stages of cell wallformation iti the order: /)-hydroxyphenyl (H), guai-acyl (G) and syringyl (S) units (Terashima &Fukushima, 1989). Thus, in different species, the Hunits would be preferentially deposited iti the earlierstages and in the middle lamella and cell corners.These observatiotis of a sequential deposition oflignin monomeric units are in accordance with thosefound by UV microscopic speetrophotometry(Musha & Goring, 1975).

Finally, examination of protoligniti in the sec-ondary wall by Raman microscopic speetrophoto-metry suggested that the aromatic ring is orientedparallel to the surface of the cell wall (Agarwal &Atalla, 1986). All these different data stronglysupport the idea that lignin deposition in individualwalls is a highly organized process. Iti additioti, theseresults underline that lignin heterogeneity is pri-marily related to the nature of each individual cellsince the process of lignification is fundamentallycontrolled by each individual eell. However, it is tiotelear at the moment if lignifying cells are autonomousfor the synthesis of their monolignols or if these

nionotneric units are synthesized in and transportedfrom adjacent cells. Recent results concerning thecell specific expression of the cinnamyl alcoholdehydrogetiase gene which will be discussed later inthis review support the second hypothesis eventhough both possibilities may exist for one and thesame cell.

Several groups have recently tried to use immuno-cytochemical studies to trace lignin precursors andto study the in situ ultrastructural location anddistribution of the different chemical types of ligninsin plant tissues. The respective distribution of the H,G atid S lignins into the various types of cells froma maturing internode of corn was studied intratismission electron microscopy by the use of novelimmunoeytochemieal probes, specific for each typeof lignin (Ruel & Joseleau, personal communication).For that purpose, antibodies were prepared againstsynthetic lignins resulting from the polymerizationof /)-coumaryl alcohol (H) or coniferyl and sinapylalcohol (GS). A typical result obtained was the lateappearance of H lignin and its predominant locationin mature tissues with secondary thicketiings (vesselsand thiek-walled fibres). The most abundant GSlignin was observed at the earliest stage of lig-nification in most tissues.

These results are in contradictioti with those ofTerashima's group but the synthetic lignins pre-pared from H units, which served for the preparationof antibodies were obtained in a way which favoursthe formation of uncondensed products and may notadequately represent the structure of native lignins.

In the future, such immunoeytochemieal probescould however provide a promising approach for thestudy of the qualitative distribution of lignin duringthe lignification process, provided that the structureof the natural or synthetic lignin antigen is knownand/or controlled. In addition, antibodies againstmonolignols would be of prime importance forstudying the transport mechanisms of lignin mono-mers through the plasma membrane.

2. Covalent cross-links in lignified walls

Iiyama, Lam & Stone (1994) recently reviewed thepotential cross links which can occur between ligninsand other cell wall polymers. Three types of directcross-links between lignin and polysaccharides canoccur: (1) ester linkages between uronic acids andhydroxyl groups of lignin monolignols on ligninsurfaces; (2) ether linkages between polysaccharidesand lignins involving glucose or mannose residues;(3) glycosidic linkages betweeti carbohydrates andterminal phenolic or side chain hydroxyls in lignins.

In addition to these direct linkages, polysac-charides and lignins can be associated throughhydroxycinnamic acid ester-ether bridges. Ferulicacid is esterified to polysaccharides in grass cell wallsand in addition it can also be etherified to lignin


Tyrosine

Tyrosineammonia lyase(TAL)

Trans-cinnamic acid

Cinnamate-4-hydroxylase^ (C4H)

Phenylalanineammonia iyase (PAL)

Phenylalanine

4-Coumarate-3-hydroxylase

OH

p-Coumaric acid

OH

Caffeic acid

HydroxycinnamateCoA ligase (4CL)

S —CoA

OH

p-CoumaroyI CoA

OCH.,OH

Ferulic acid

OH

5-Hvdroxyferulic acid

OH

Sinapic acid

I HydroxycinnamateCoA ligase (4CL)

S —CoA

OCH3OH

FeruloyI CoA

Figure 6. The common phenylpropanoid pathway.

HydroxycinnamateCoA ligase (4CL)

S —CoA

OCH.,OH

SInapoyI CoA

(Scalbertef a/., 1985; Lam, Iiyama & Stone, 1992a).Dehydroferulic acid diesterified to polysaccharidesmay also be etherified to lignins.

Finally there is evidence that lignins are associatedwith cell wall proteins such as hydroxyproline andglycine-rich proteins (Iiyama et al., 1993).

These cross links between lignins and poly-saccharides and proteins have been demonstratedfrom indirect evidence but have never been con-firmed by the isolation and characterization of thechemical fragments associating lignin and non-lignincomponents through covalent linkages.

3. Proteins and wall components associated withdeposition of lignin in vascular elements

Tracheary elements (TEs), the major conductingcells of the xylem, are characterized by their lignifiedsecondary wall thickenings. When the lignin de-position is complete the protoplast degeneratesleaving a dead cell protected against collapse by itsstrengthened walls. Each plant species contains TEswith a specifically patterned secondary cell wall andit is possible to identify most trees by the structure oftheir xylem (Preston, 1988). Fukuda & Komamine(1980) have established an elegant experimentalsystem in which single Zinnia mesophyll cells underthe control of an appropriate hormonal balancedifferentiate rapidly and synchronously into TEs.

Cytological, ultrastructural and biochemicalchanges associated with TE differentiation in thissystem have been recently reviewed by Church(1993). Lignification of secondary wall thickeningsof differentiating Zinnia TEs is a late event occurringseveral hours after cellulose deposition (Ingold,Sugiyama & Komamine, 1988). Expected increasesin phenylalanine ammonia-lyase (PAL), 4-coumarate CoA ligase (4CL) (see Fig. 6) and specificperoxidases have been reported to coincide withlignin deposition (Fukuda & Komamine, 1983;Masuda, Fukuda & Komamine, 1983; Church &Galston, 1988; Lin & Northcote, 1990; Sato et al,1993). More interestingly, early changes in geneexpression preceding lignin deposition and inter-relationships between the location of lignin and thedeposition of other cell wall components have beeninvestigated. The results of such studies couldprovide interesting information on the mechanismsand molecular changes taking place in a lignifyingcell, that are necessary for a normal lignification.Through comparisons of two-dimensional gel elec-trophoresis of''^S labelled proteins in differentiatingand non-differentiating cells, Fukuda & Komamine(1983) identified two proteins of unknown functionthat may be regarded as biochemical markers of TEdifferentiation. More recently two different groups(Demura & Fukuda, 1993; Ye & Varner, 1993)attempted to characterize the genes expressed at the


earlier stages of differentiation prior to morpho-logical changes (at 48 h of culture). Using a sub-tractive hybridization method. Ye & Varner (1993)isolated several cDNA clones associated with theprocess of tracheary element formation. These clonesexhibited homologies with known proteins: adeny-late kinase and papaya proteinase I (Ye & Varner,1993) and also with lignin related enzymes (Varner,personal communication). Demura & Fukuda (1993)characterized other cDNA clones related to TEsformation through differential screening of cDNAlibraries (induced and non-induced cells).

The transcripts corresponding to these cDNAclones, TED2, TED3 and TED4, are preferentiallyexpressed at earlier stages of differentiation after48 h of culture in the induction medium. TED4 wasfound to be similar to the barley aleurone specificclone B l l E (Demura & Fukuda, 1993). TED2deduced polypeptide sequence exhibited homologieswith a guinea pig lens-specific protein and also, forlimited regions, with several alcohol dehydrogenases(Demura & Fukuda, 1994). TED3 contained nu-merous repeats of an Asn-Gly-Tyr motif and waspredicted to be an hydrophilic component of the cellwall.

In situ hybridization of the TED probes withyoutig Zinnia seedlings revealed that expression ofthe three TED genes was restricted to vascular cellsand was regulated in a very specific spatio-temporalmanner (Demura & Fukuda, 1994). From thesetemporal and spatial expression patterns Demura &Fukuda (1994) deduced that TED2 would beitivolved in an early process that is common to alldifferentiating vascular cells, TED4 in a process thatis specific to xylem cells and TED3 in a late processthat is unique to tracheary elements. These cDNAclohes represent novel and efficient markers fordevelopment of the vascular system.

Even though we are far from understanding all themolecular changes resulting from differential geneexpression which are driving cells to lignification,these recent results suggest that the Zinnia systemrepresents a convenient tool to answer these complexquestions. Tracheary element differentiation, whichincludes secondary wall deposition, lignin biosyn-thesis and programmed cell death, may thus rep-resent an interesting model system for plant biolo-gists focused on the molecular approaches of dif-ferentiation.

The technique of differential display (Liang &Pardee, 1992) performed in parallel on Zinnia cells atdifferent stages of differentiation could help tocharacterize differentially expressed cDNA clonescorresponding to low abundant mRNA. Similarstudies on cambium/differentiating xylem cells col-lected from stems (Leinhos & Savidge, 1993) couldalso be interesting since in these materials theititercellular connections which are disrupted iti thepreparation of Zinnia cells would be maintained.

In order to assess the interrelationships betweenthe synthesis and deposition of different cell wallpolymers, a more direct but more limited approachconsists in using chemical inhibitors of wall com-ponent synthesis. Using L-a-aminooxy-/?-phenyl-propionic (AOPP), a potent inhibitor of PAL,Ingold, Sugiyama & Komamine (1990) have shownthat the inhibition of lignification did not causeinhibition of the synthesis of cell wall poly-saccharides nor the differentiation to trachearyelements. Lignification is a late event in walldifferentiation that does not prevent the completionof earlier events. In a very interesting study, Tayloret al. (1992) inhibited cellulose synthesis and,secondarily, xylan deposition, by 2-6-dichloro-benzonitrile and isoxaben in Zinnia differentiatingtracheary elements. The inhibitor-treated TEsexhibited cellulose depleted thickenings and showeddispersed lignins whereas control TEs cotitainedlignins specifieally localized in the secotidary cellwall thickenings. This dispersion of lignin whencellulose synthesis is inhibited provides evidencethat the location of cellulose in secondary wallthickenings directly or indirectly mediates thelocation of lignins and that some molecules of thesecondary cell wall mediate the patterning of others.As discussed by Taylor et al. (1992 and referencestherein), it has been commonly hypothesized that thelocation of lignin is dependent on specific inter-actions of its precursors or enzymes involved in itspolymerization with particular polysaccharides orproteins in the wall matrix. Their study provides thefirst experimental evidence that lignin locationdepends on the presence of particular cell wallpolymers. These authors summarize differentpotential mechanisms by which initiation oflignification through different eell wall eotnponentsor association of lignins to these components (poly-saccharides or proteins) could occur. At the moment,these hypotheses are still unsubstantiated. In thefollowing paragraph, we will describe, as a point incase, the postulated relationships between ligninsand glycine-rich proteins.

Glycine-rich proteins (GRPs) are a class of plantcell wall structural proteins containing a highproportion (60%) of glycine which are involved inthe structural organization of the cell wall. Usingantibodies directed against bean GRP 1-8, Keller,Templeton & Lamb (1989) showed that, in bean, theprotein is closely associated with the vascular systemand particularly with the protoxylem trachearyelements. Transgenic tobacco plants carrying GRPpromoter GUS gene fusions expressed the gene intheir vascular tissues (Keller, Schmid & Lamb,1989) and it was shown that this cell type-specificactivity of the GRP 1-8 promoter was controlled bya complex set of positive and negative interactionsbetween cis-acting regulatory regions (Keller &Baumgartner, 1991).

216 A. M. Boudet, C. Lapierre andj. Grima-Pettenati

These observations and others (Ye & Varner,1991), demonstrating that glycine rich proteins areexpressed in lignifying tissues, initially suggested aclose relationship between GRP 1-8 deposition andlignification. An interesting speculation was thatGRP which is laid down early during cell wallthickening could play the role of scaffold or templatefor lignin deposition and polymerization.

Using a specific inhibitor of PAL, Keller,Nierhaus-Wunderwald & Amrhein (1990) showedthat lignin biosynthesis can be inhibited in vasculartissues without affecting the production of GRP 1-8.However, the possibility remained that lignificationwas dependent on deposition of GRP 1-8. A morerecent careful ultrastructural study using GRP 1-8antibodies (Ryser & Keller, 1992) discarded thishypothesis and showed that GRP 1-8 is locatedmainly in the unlignified primary walls of theprotoxylem. These last results strongly suggest thatdeposition of GRP 1-8 and lignification are mostprobably independent processes. Interestingly, theseauthors propose that GRP 1-8 is produced by xylemparenchyma cells and then secreted into the walls ofneighbouring xylem elements.

IV. ENZYMES AND GENES INVOLVED IN THE

BIOSYNTHESIS AND POLYMERIZATION OF

MONOLIGNOLS

1. The common phenylpropanoid pathway

(a) General discussion. A set of enzymatic reactions,starting from the deamination of phenylalanine andleading to the synthesis of the cinnamoyl-CoAs — theesters of hydroxycinnamic acids and coenzymeA-represents a common core in the synthesis of a widerange of phenolic compounds involved in differentaspects of plant development and defence reactions(see Hahlbrock & Scheel, 1989 for a review).

The enzymes involved in this common phenyl-propanoid pathway (Fig. 6) are phenylalanineammonia-lyase (PAL), cinnamate hydroxylase(C^H), coumarate hydroxylase (C3H), O-methyltransferases (OMT), ferulate hydroxylase(FA 5H) and hydroxycinnamate CoA ligases (4CL).The end products of this common pathway, thehydroxycinnamoyl CoAs, are the precursors of themajor classes of phenolic compounds which ac-cumulate in plant tissues, e.g. flavonoids, lignins,stilbenes, phenolamides, esters. These differentsyntheses occur through specific branch pathwayssuch as the lignin or the flavonoid pathways. Due tothe wide range of phenolic end products derivedfrom the phenylpropanoid pathway, this commonset of reactions may be considered as a constitutivepathway whose level of activity depends on the cellsand the environmental conditions. Indeed in stemand leaf sections of young parsley seedlings, PALwas immunohistochemically detected in all cell types(Hahlbrock & Scheel, 1989).

The enzymes of the common phenylpropanoidpathway, and particularly PAL, have been exten-sively studied and are highly inducible throughincreased gene expression during development andin response to environmental factors.

The common phenylpropanoid pathway is closelyassociated with lignification in that it provides thehydroxycinnamoyl CoAs which are converted intothe monolignols through the specific lignin branchpathway. PAL and other enzymes of the commonpathway are stimulated in tissues and cells whichsynthesize lignins (Haddon & Northcote, 1976;Church & Galston, 1988; Lin & Northcote, 1990;Messner & Boll, 1993), ln addition, inhibition ofPAL activity by chemical compounds (Amrhein etal, 1983) or genetic engineering (Elkind et al., 1990)reduces lignification.

At present, the spatio-temporal regulation of thephenylpropanoid pathway in relation to lignificationis poorly understood. At least three importantquestions relative to the functioning of this commonpathway can be raised:

Is the relative production of the differenthydroxycinnamoyl-CoAs important for the deter-mination of the monomeric composition of lignins ?

Is the overall carbon flux through this commonpathway the limiting factor or one of the limitingfactors in lignin synthesis ?

Is the production of lignin precursors in lignifyingtissues associated with the activity of specificisoenzymes/genes of the phenylpropanoid pathway?In other words, the problem is to know if the stimuliwhich induce lignification activate a common set ofgenes involved in the synthesis of the precursors ofdifferent phenolic end products or more specificallyactivate members of multigene families tightlyrelated to the synthesis of lignins. In both cases, thespecific fate of the phenylpropane units could bedetermined by the coordinated activation of theenzymes/genes downstream in the specific branchpathway, or through molecular channelling or sub-cellular compartmentation.

In the following paragraphs, we will present themore recent developments in the characterization ofthe genes of the phenylpropanoid pathway and wewill try to answer the above questions.

{b) Phenylalanine ammonia-lyase. PAL (EC4.3.1 .5) occurs in plants in the form of multipleisozymes and its activity is highly regulated duringdevelopment and in response to different environ-mental cues. Studies have been especially performedon simplified model systems (e.g. parsley or bean cellsuspension cultures) which rapidly accumulate speci-fic classes of phenolic compounds in response toabiotic or biotic stimuli (Hahlbrock & Scheel, 1989).

PAL is encoded by a family of three or four genesin bean (Cramer et al, 1989), parsley (Lois et al,1989), rice (Minami et al, 1989), Arabidopsis (Ohl et


al, 1990) and pea (Yamada et al, 1992), and thetranscripts of individual PAL genes show verydifferent patterns of accumulation (Liang et al,1989; Lois et al, 1989). In potato, the organizationof PAL genes is more complex since at least 10 activegenes have been characterized (Joos & Hahlbrock,1992).

Few studies have dealt with PAL in trees wherelignification is particularly important. Several linesof molecular evidence suggest the presence of twoPAL genes in poplar (Subramaniam et al, 1993). InGymnosperms only one isozyme has been charac-terized in Pinus banksiana (Campbell & Ellis, 1992)and one gene cloned in Pinus taeda (Whetten &Sederoff, 1992). The apparently limited number ofPAL genes in woody species is at the present timeunexplained.

One of the most interesting studies concerning thedevelopmental regulation of PAL genes was per-formed by Bevan et al (1989) who analyzed theexpression of bean PAL2 promoter-GUS fusions intransformed potato and tobacco plants. Three PALgenes have been characterized in Phaseolus vulgaris,PALI, PAL2 and PAL3 (Cramer et al 1989), whichare expressed differentially during development andin response to different environmental cues (Liang etal, 1989), a situation which is in part similar to theobserved differential responses of the members ofthe bean chalcone synthase multigene family todifferent stimuli (Ryder et al, 1987). Two of thesegenes, PAL2 and PAL3, have been cloned andsequenced and the extensive sequence divergence inthe 5' and 3' Ranking regions suggested a differentialregulation.

The experiments on the PAL2 gene aimed toevaluate the tissue and cellular expression of thecorresponding promoter and provided two inter-esting findings:

(1) In xylem tissues, GUS activity occurred indeveloping xylem but also in rays of cells presumedto be xylem parenchyma. GUS activity was notdetected in older xylem elements and differentiatedvessels since these cells are dead and have lost theircytoplasm. In agreement with previous observationsof Pickett-Heaps (1968), Bevan et al. (1989) postu-lated that xylem parenchyma cells adjacent to maturevessels may provide precursors for the continuedlignification of the dead mature xylem vessels. Theseobservations are in complete agreement with ourown observations on the activity of the CADpromoter-GUS fusions in the xylem parenchyma ofpoplar wood as it will be discussed later in thisreview. Recent results by Smith et al. (1994) alsoshowed immunolocalization of PAL and C^H inbean parenchyma cells adjacent to metaxylem.

(2) The PAL2 promoter was active in differentorgans, tissues and cell types which accumulatephenylpropanoid derivatives: fiower, petiole, xylem,epidermis, etc. In addition, the promoter was

activated by mechanical wounding in potato, eventhough lignin deposition was not stimulated. Thesedata suggest that the PAL2 gene is not specificallyassociated with lignification.

Similar data showing a complex pattern of tissue-specific developmental expression have been ob-tained in parallel by Liang et al (1989) in tobaccotransformed by a bean PAL2 promoter-GUS fusion.Thus, the PAL2 promoter transduces a complex setof developmental and environmental cues into anintegrated spatial and temporal program of geneexpression. Ohl et al (1990) drew the same con-clusions in Arabidopsis transformed with an Arabid-opsis PAL promoter-GUS fusion.

In a recent study, Shufflebottom et al. (1993)compared the transcriptional activities of bean PAL2and PAL3 genes by fusing their promoters to theGUS gene and transforming Arabidopsis, tobaccoand potato with these constructs. The PAL2 andPAL3 promoters direct both different and over-lapping patterns of GUS expression. Particularly,the PAL3 gene is not expressed in differentiatingxylem and vascular tissues.

If differential expression of PAL genes depends, atleast in part, on their promoter activities, one canconclude that some PAL genes (PAL2) have a role inproviding phenylpropanoid monomers for poly-merization into lignins, even though it is not theirexclusive function, and that other PAL genes (PAL3)are not related to lignification. It would be interestingin the future to specifically down-regulate theexpression of these different genes in order toevaluate the effects on the synthesis of differentphenylpropanoid derivatives.

Despite the possibility that individual PAL geneswithin multigene families may encode variant pro-ducts with distinct functional specialization, it is notpossible at the moment to unambiguously associateone specific PAL gene with lignification. Thequestion is open now as to whether the diverseenvironmental and developmental stimuli that affectPAL2 gene expression act at different positionswithin the 5' flanking sequences or act via a commonfactor.

With regard to the involvement of PAL2 inlignification, Levya et al (1992) have determinedwhich regions of the PAL2 promoter are essential forxylem expression. These cis-acting sequences arelocated between nucleotides —289 and —74, relativeto the transcription start site, but are not involved inthe expression of the PAL2 gene in other tissues.This region also contains a negative element thatsuppresses the activity of a cryptic cis-element forphloem expression. These authors conclude thattissue-specific expression of the PAL2 gene in thevascular system involves combinatorial interactionsof different cis-elements. These studies, and thefuture identification of similar regions in other genesencoding enzymes of the phenylpropanoid pathway


or of the specific lignin branch pathway, will allowthe characterization of trans-acting factors that bindto these sequences. As underlined by Levya et al.(1992) such studies provide the basis for theelucidation of the molecular mechanisms governingvascular differentiation by biochemical analysisworking back stepwise from the characterized trans-acting factors and also by genetic approaches inwhich transgenic Arabidopsis plants containingPAL2 promoter-GUS fusions are used to screen formutants altered in the vascular transduetion path-way.

As an example of the recent progress made inidentifying trans-acting factors regulating genesinvolved in the phenylpropanoid pathway, it is worthnoting the work of Sablowski et al. (1994) stronglysuggesting a role for a Myb 305-related protein inthe activation of PAL2 promoter in fiowers. ThisMyb-cognate protein is able to bind a box-P similarto H-box-like elements found in several genes of thephenylpropanoid biosynthesis (Lois et al., 1989) andthought to be implicated in the response to UV lightand to other environmental stimuli. This Myb factoractivates the PAL2 promoter in combination withan other trans-acting factor binding a G-box likesequence. According to Sablowski & Bevan (1994), abZIP protein might be the partner of Myb 305 inthis cooperative activation.

(c) Cinnamate 4-hydroxylase. Cinnamate 4-hydroxyl-ase [C4H; trans-cinnamate, NADPH: oxygenoxidoreductase (4-hydroxylating) EC 1.14.13.11]is a cytochrome P-450 enzyme which catalyses thesecond reaction of the general phenylpropanoidpathway. In contrast to the other major enzymes ofthis pathway, knowledge of this enzyme is verylimited and molecular approaches concerning itsregulation are very recent.

C4H has been purified to homogeneity fromwounded Mn^"^-induced Helianthus tuberosus tubertissues (Gabriac et al., 1991) and highly selectivepolyclonal antibodies have been raised against thepurified protein (Werck-Reichharte; a/., 1993). C^H,as other enzymes of the phenylpropanoid pathway, ishighly inducible by different environmental factorsand preliminary results suggest that changes in geneexpression are responsible for this regulation(Werck-Reichhart et al, 1993).

Recently, a cDNA coding for C4H has beencharacterized from a Jerusalem artichoke cDNAexpression library (Teutsch et al., 1993). Thesequence analysis indicates that this cDNA is amember of a new P450 gene family. In parallel,Fahrendorf & Dixon (1993) have cloned an elicitor-inducible cinnamie acid 4-hydroxylase cytochromeP450 from alfalfa and functionally expressed thecDNA in yeast. These authors have shown thatelicitor induces the transcription of the gene inalfalfa. Mizutani et al (1993) have also cloned a

mung bean P450 possessing C4H activity. Theidentity of the Jerusalem artichoke cDNA was alsounambiguously confirmed through functional ex-pression in yeast (Urban et al, 1994; Pierrel et al,1994). The recombinant enzyme was highly specificfor its natural substrate and did not catalyze theoxygenation of p-coumarate, ferulate, benzoate andother natural phenolic compounds. In contrast, itwas shown to oxygenate five xenobiotics includingthe herbicide chlortoluron, even though cinnamatewas by far the best substrate of this P-450 enzyme.

{d) Coumarate 3-hydroxylase {C^H) and ferulate-5-hydroxylase {F^H). Concerning the conversion ofcoumaric acid into caffeic acid, the mechanisms andthe enzymatic systems involved are still unclear. Thequestion as to whether the hydroxylation of the 4-coumaroyl moiety occurs at the level of the free acidor a suitable conjugate is still open. Some phenolases(EC 1.10.3.1.1,14.18.1) may hydroxylate free 4-coumarate into caffeie acid (Bolwell & Butt, 1983)but these enzymes have not been proven to bespecifically involved in trans-caffeate formation.

In addition, 4-coumaroyl-CoA has been shown tobe a substrate of a FAD dependent monooxygenaseresponsible for the formation of caffeoyl-CoA inSilene dioica (Kamsteeg et al, 1981). A coumaroyl-CoA hydroxylase, initially characterized in parsleycells, is a soluble enzyme present in various othercultured plant cells requiring ascorbate and Zn^^ forits activity (Kneusel, Matern & Nicolay, 1989). Itsacidic optimum pH suggests that the hydroxylase isinactive at the normal cytoplasmic pH, but theenzyme could be activated by the shift in cytoplasmicpH induced by elicitor treatment in parsley cells.Finally, hydroxycinnamoyl esters (shikimate orquinate esters) have been postulated to be transientmetabolic intermediates that are able to serve assubstrates for hydroxylating and methylating en-zymes. This hypothesis was confirmed by the resultsof Heller & Kuhnl (1985) and Kuhnl et al (1987)demonstrating the hydroxylation of trans 5-O-(4-coumaroyl) shikimate into trans 5-O-eaffeoyl shiki-mate by a microsomal preparation of parsley cellsuspension cultures and the hydroxylation of trans5-O-(4-coumaroyl)D-quinate into the caffeoyl estersby a microsomal preparation from carrot cell sus-pension cultures respectively.

Ferulie aeid 5-hydroxylase activity which is anintermediate step in the synthesis of sinapic acid hasbeen characterized in a microsomal fraction frompoplar (Grand, 1984). The enzyme which is cyto-chrome P450 dependent has been poorly studiedfrom that time and seems very difficult to charac-terize (Chappie, personal communication). Recentdata discussed iti section V suggest that a F^H cDNAhas been eloned in Arabidopsis thaliana.

{e) O-Methyl transferases. O-Methyltransferases {S-


adenosyl-L-methionine: o-diphenol-O-methyltrans-ferases, OMT; EC 2.1.1.6) mediate important stepsin lignin precursors synthesis since the}' controlthe degree of methylation of monolignols andpotentially of lignins. In this respect, it is importantto keep in mind that OMTs of conifers andangiosperms have different substrate specificities,which can be correlated with different lignin mono-meric compositions. As pointed out by Higuchi andcoworkers (Shimada, Fushiki & Higuchi, 1973;Kuroda, Shimada & Higuchi, 1975; Kuroda,Shimada & Higuchi, 1981) gymnosperm OMTs aremonofunctional and catalyse 5-adenosyl methionine(SAM) dependent methylation of caffeate, whileangiosperm OMTs are bifunctional and catalyse theSAM dependent methylation of both caffeic and 5-hydroxyferulic acids. O-methyl transferases in-volved in lignification have been purified andcharacterized from several angiosperms: spinachbeet (Poulton & Butt, 1975), alfalfa (Edwards &Dixon, 1991), soybean (Poulton, Hahlbrock &Grisebach, 1976) and tobacco (Hermann et al,1987).

Interestingly, several isoforms of OMT are oftenidentified in higher plants, as illustrated in the caseof tobacco (Hermann et al, 1987). Three OMTs canbe characterized in tobacco OMTI, OMTH,OMTHI, which differ in term of substrate speci-ficity, their immunochemical properties and theirresponses to pathogens. OMTI is the major form inhealthy plants and seems to be particularly related tolignification.

Depending on the species, different relative ac-tivities have been measured towards caffeic acid and5-hydroxyferulic acid, the two main substrates ofbifunctional OMTs. Aspen bi-OMT for examplehas a higher activity with 5-hydroxyferulic acid thanwith caffeic acid (Bugos, Chiang & Campbell, 1991).

Recently cDNA clones encoding OMT have beencharacterized from different plant species. The firstclonings of bi-OMT cDNAs were reported byBugos, Chiang and Campbell (1991) in aspen and byGowri et al. (1991) in alfalfa. Other cDNA cloneshave been successively obtained from poplar (Dumaset al, 1992), tobacco (Jaeck et al, 1992) and eucalypt(Poeydomenge, Boudet & Grima-Pettenati, 1994).Einally, a corn OMT genomic clone has also beencharacterized by Collazo et al. (1992).

The cloning of different OMT genes in tobacco(Pellegrini et al, 1993) confirmed and highlightedthe polymorphism already observed at the proteinlevel. Indeed, OMTI was the major enzyme ofhealthy tobacco leaves and the corresponding genewas shown to be preferentially expressed in stemxylem. OMTH and OMTHI, which are encoded bythe same gene (class II OMT), can only be inducedin leaves during the hypersensitive reaction totobacco mosaic virus (Jaeck et al, 1992; Pellegrini etal, 1993). No significant expression of the class H

OMT gene was observed in lignifying stems andexpression of the OMTI gene was not affected byfungus infection. In addition, the two cDNAs exhibitonly 53 % identity in amino acid sequence. Thus, atleast in this material, it appears that distinct OMTgenes are involved in constitutive lignification anddefence responses. This finding is important in thecontext of genetic manipulation of the OMT genefor lignin modification. Concerning the tissue ex-pression of the OMT gene, mRNA levels of OMThave been observed to be higher in roots and stemsthan in leaves of alfalfa (Gowri et al, 1991) and corn(Collazo et al, 1992).

Sequence homologies between the different clonedOMTI cDNAs have been determined. Taking theeucalypt OMT cDNA clone as a reference we haveshown that the identity is respectively 84-4% withaspen and poplar, 80-8% with alfalfa, 76-4% withtobacco and only 62-6% with corn. As expected, thesequence homology was lower between dicots andmonocots than among different species of dicots. Arelatively high percentage of homology (50%) wasalso found between the bi-functional OMT cDNAfrom eucalypt and a myo-inositol O-methyl trans-ferase induced by osmotic stress in the facultativehalophyte Mesenbryanthemu7n crystallinum (Vernon& Bohnert, 1992). Unfortunately at the moment, nogymnosperm OMT cDNA sequences are availablefor explaining, at the molecular level, the differencesin substrate specificity of mono-functional and bi-functional OMTs.

In parallel to the main synthesis pathway ofcinnamoyl-CoAs, it has been shown that feruloyl-CoA could result from the hydroxylation ofcoumaroyl-CoA into caffeoyl-CoA and the sub-sequent methylation of caffeoyl-CoA into feruloyl-CoA. A specific O-methyltransferase involved in themethylation of caffeoyl-CoA (caffeoyl-CoenzymeA3-O-methyltransferase: CCoAOMT) was initiallycharacterized in parsley cell suspensions (Kneusel etal, 1989) and in carrot cell suspensions (Kuhnl etal, 1989). The reaction product of this enzyme,which exhibits quite specific characteristics (homo-dimer, Zn~+ dependence) not found in OMT is transferuloyl-CoA, an intermediate in lignin synthesisand the question arises as to the involvement of thisbiocatalyst in the lignification pathway. The cDNAencoding CCoAOMT has been characterized inparsley (Schmitt, Pakusch & Matern, 1991). Sur-prisingly enough, no extended homology was foundbetween the sequence of this cDNA and the differentOMT cDNAs confirming that they encode verydifferent proteins.

Following elicitation stimulus, the level ofCCoAOMT and its corresponding mRNA increasesrapidly and transiently (Pakusch, Matern & Schiltz,1991; Schmitt, Pakusch & Matern, 1991). It wassuggested that this methyl transferase mediates theformation of cell wall ferulic esters which are


CoA Ligase

OHp-Coumaric acid

S —CoA

OHp-Coumaroyi CoA

CCoAOMT

S —CoA

OHCaffeoyi CoA

OCH,OH

Feruioyi CoA

Hydroxyiase^

—CoA

CCoAOMT

OCH3OH

5-Hydroxy Feruioyi CoA

CH3O

S —CoA

OCH.,

OHSinapoyi CoA

Figure 7. The alternative methylation pathway (fromMatern et at., 1988 and Ye et at., 1994).

considered to be important in defence reactions.However, as the enzyme and its mRNA are alsopresent in normal development, its involvement inthe synthesis of lignin monomers cannot be dis-carded. Very recently. Ye et al. (1994) have isolateda Zinnia CCoAOMT which exhibits 93 % amino-acid similarity with the parsley CCoAOMT. RNAgel blot analysis showed that the expression ofthe CCoAOMT gene is markedly induced duringtracheary element differentiation from mesophyllcells, and tissue print hybridization showed that theexpression of the gene is associated with the timingof lignification in both xylem and phloem fibres inZinnia organs. These authors propose thatCCoAOMT is involved in an alternative methylationpathway in lignin biosynthesis (Fig. 7) which ispredominant in Zinnia differentiating tracheids.

Ballance & Dixon (personal communication) havealso characterized a CCoAOMT clone from alfalfawhich was functionally expressed in Escherichia coli.The enzyme has no activity versus caffeic acid andexhibited a similar tissue distribution to OMT(principally in vascular tissue). The same groupsuggested that the function of this enzyme couldexplain the lack of lignin reduction in O M Tantisense tobacco plants (Ni, Paiva & Dixon, 1994).

(/) 4-Coumarate-CoA ligase. The coenzyme A estersof 4-coumarate and other cinnamate derivatives are

central intermediates in the synthesis of manyphenylpropanoid derivatives in higher plants (Hahl-brock & Grisebach, 1979). These thiol esters aresynthesized by 4-coumarate CoA ligase (4CL,EC 6.2.1.12), and represent branch points betweenthe general phenylpropanoid metabolism and path-ways leading to the synthesis of various end productsincluding lignins.

In several systems, 4CL is induced by differentstimuli coordinately with PAL and this rise inenzymatic activity is due to transcriptional activation(Hahlbrock et al, 1983).

In different plant materials such as soybean(Knobloch & Hahlbrock, 1975), petunia (Ranjeva,Boudet & Faggion, 1976), pea (Wallis & Rhodes,1977) and poplar (Grand, Boudet & Boudet, 1983),several isoforms of 4CL have been characterizedwhich exhibit different substrate specificities andcatalytic properties. The gymnosperm 4CL andsome angiosperm 4CL do not use sinapic acid as asubstrate. These observations suggested that in-dependent 4CL isoenzymes could play differentialroles in providing precursors for specific phenyl-propanoid derived branch pathways or/and thatdifference in isoform pattern of a tissue couldinfluence its lignin monomeric composition (Grand,Boudet & Boudet, 1983).

Two different genes for 4CL have been charac-terized in parsley cells (Douglas et al, 1987). Bothare activated by elicitor and UV light. Theirorganization into exons and introns was determinedand the deduced sequences of the two corresponding4CL isoenzymes were nearly identical. In contrast toother materials, the two isoenzymes, which areseparable by ion exchatige chromatography, ex-hibited the same substrate specificity as did theproducts of the functional expression of the twogenes in E. coli. In potato, the situation is verysimilar to parsley with two genes coding for twonearly identical isoenzymes (Becker-Andre, Schulze-Lefert & Hahlbrock, 1991).

Thus, the question remains as to whether different4CL isoforms with different substrate affinities andtissue distributions are important in the channellingof phenylpropanoid compounds towards specificbranch pathways.

The parsley 4CL-1 gene exhibits an unconven-tional mode of regulation since exonic sequences arerequired for elicitor and light activation of the gene,but promoter sequences are sufficient for tissuespecific expression (Douglas et al, 1991).

Recently, the promoter domains involved in thevascular specific expression of the 4CL genes weredetermined using deleted promoter fragment-GUSfusions in tobacco plants (Hauffe et al, 1991; Hauffeet al, 1993). Within the region located between— 244 and —78, three separate promoter domainscontaining partially redundant cis-elements directedvascular-specific expression when combined with a


TATA proximal domain. As was shown for theanalysis of the bean PAL2 promoter a negative cis-acting element which represses phloem expressionwas revealed in one of the domains and appears to beresponsible for restricting vascular expressioti to thexylem.

Similar combinatorial interactions between posi-tive and negative cis-acting elements control thevascular tissue specific expression of two major genesof the general phenylpropanoid pathway (PAL and4CL). It will be interesting in the future to determineif the same mechanisms occur for other genes of thesame pathway and also in the specific lignin pathway.

In conclusion, the carboti flux through the com-mon phenylpropanoid pathway seems to be one ofthe controlling factors of lignification at the quan-titative level. Enzymes (geties) of the phenyl-propanoid and lignin specific pathways are likelyactivated in a coordinate fashion in specialized tissuesat certain stages of development. In addition, it isclear that a specific step (or steps) of the phenyl-propanoid pathway is likely to play a role incontrolling lignin monomeric compositioti. That isparticularly true for the enzymes involved in themethylation of monolignols. However the situationis likely to be more complex since: (1) other enzymesof the lignin specific pathway could also be involvedas we will see in the next section; (2) recent resultssuggest that interconversions can occur at themonolignol level (Matsui et al 1993). Despite theoccurrence of multigene families coding for phenyl-propanoid pathway enzymes, no specific gene withinthese families appears to be exclusively associated tolignification. However, the cis-acting sequences ofthe PAL and 4CL promoters responsible for vascularexpression have been characterized atid these resultsprovide a basis for elucidation of the molecularmechanisms governing vascular differentiation.

2. The lignin branch pathway

{a) Cinnamoyl CoA reductase. In contrast to theconsiderable knowledge of the regulation of thegenes involved in the general phenylpropanoidpathway [particularly phetiylalatiine ammonia-lyase(PAL) and hydroxycinnamate CoA ligase (4CL)],little is known about the enzymatic steps morespecifically involved in lignin monomer biosynthesisi.e. cinnamoyl CoA reductase (CCR) and cinnamylalcohol dehydrogenase (CAD) (Fig. 8). In thissection, recent advances in the biochemistry andmolecular biology of these enzymes will be de-scribed.

The enzyme CCR (EC 1.2.1.44) catalyses theconversion of hydroxycinnanioyl-CoA esters to theircorresponding aldehydes and, as such is the entry-point enzyme into the biosynthesis of hydroxy-einnamyl alcohols and their derivatives, includinglignins. Studies of this enzyme, which may be

considered as a potential control point regulating theflux of phenylpropanoid metabolites towards thebiosynthesis of lignin, have been infrequent. CCRhas been purified and partially characterized fromsoybean cultures (Wegenmayer, Ebel & Grisebach,1976; Luderitz & Grisebach, 1981), spruce cambialsap (Luderitz & Grisebach, 1981) and poplar xylem(Sarni, Grand & Boudet, 1984) but until now littlewas known about the aspects of the molecularregulation of this enzyme. In our laboratory, CCRhas recently been purified to apparent homogeneityfrom differentiating xylem of eucalypt (Goffner etal, 1994). The purified protein had approximatelyequal affinities for the three natural substrates {p-coumaroyl-CoA, feruloyl-CoA and sinapoyl-CoA),and was approximately three times more effective atconverting feruioyi CoA than the other substrates.CCR activity was feedback inhibited by NADP andCoA. Effectors which bound lysine and cysteineresidues also inhibited CCR activity.

Cloning of the cDNA encoding eucalypt CCR wasachieved usitig oligonucleotides derived from thepeptide sequetices obtained after ' in gel' digestion ofCCR by an endoproteinase lysine-C. Oligoscreeningof a eucalypt xylem cDNA library led to the isolationof a full-length cDNA clone (1193 bp) encoding apolypeptide of 335 amino-acids (Lacombe et al,1994). A significant homology was found at theamino-acid level with dihydroflavonol-4-reductaseDFR-(identity 40 %/similarity 20 %), a reductase ofthe flavonoid pathway. Although CCR is related tothe DFRs, it is more distant than the most unrelatedDFRs among themselves. The production of activerecombinant CCR in E. coli was achieved, provingunambiguously the identity of the CCR clone. Thisfirst characterization of a cDNA for CCR will enableus to address the role of CCR in regulating lignindeposition during normal developmetit and in re-sponse to environmental cues. This is currently beingachieved at the messenger level through Northernblot experiments, and at the gene expression levelthrough promoter-GUS fusions. A CCR genomicclone has been isolated atid the promoter region iscurrently being characterized (Van Doorsselaere etal, 1994).

(6) Cinnamyl alcohol dehydrogenase. CAD(EC 1.1.1.195) catalyses the second reductive stepof the lignin committed branch, leaditig to thehydroxyciiinamyl alcohols (/)-coumaryl, coniferyl,sinapyl alcohols). These compounds are the mono-meric precursors of lignins, but may also be requiredfor the synthesis of lignans and other relatedphenolics, e.g. dehydrodiconiferyl alcohol (Lewis &Yamamoto, 1990).

The role of CAD as a control point enzyme inlignin sytithesis has been addressed essentially bystudies of lignification mutants, both brown-midribmutants and genetically engineered antisense CAD

222 A. M. Boudet, C. Lapierre and y. Grima-Pettenati

0 - - S —CoAS —CoA

OH

p-CoumaroyI CoA

S —CoA

OHFeruloyI CoA

OHSinapoyI CoA

CinnamoyI CoAreductase (CCR)


OH

p-Coumaraldehyde

Cinnamyl alcoholdehydrogenase (CAD)

OCH3OH

Coniferaldehyde

Icinnannyl alcoholI dehydrogenase (CAD)


OH

Sinapaldehyde

Cinnamyl alcoholdehydrogenase (CAD)

p-Coumaryl alcohol(H unit)

OCH,

Coniferyl alcohol(G unit)

Peroxidases/ Laccases •

OCH,,

Sinapyl alcohol(S unit)

Lignins

Figure 8. The lignin branch pathway.

mutants (see corresponding section in this review).In the following paragraph, we will focus on thepotential involvement of CAD in the control ofqualitative lignin variations in plants. Does CAD,through its substrate specificity, play a role indictating lignin heterogeneity in terms of monomercomposition 1 Indeed lignin content and type varywith species, tissue, developmental stage and sub-cellular location (Lewis & Yamamoto, 1990). More-over, environmental cues such as wounding orpathogen attacks, can also induce the deposition oflignins with a monomeric composition different fromthat of constitutive lignins.

CAD polymorphism and its potential role in dic-tating lignin heterogeneity. Until fairly recently, CADwas not considered to be a polymorphic enzyme.The situation seems to be clear in gymnospermswhere one single isoform is present, encoded by onesingle gene (O'Malley, Porter & Sederoff, 1992;Galliano et al, 1993 6) although the presence ofallelic variants has been reported in loblolly pine(O'Malley et al, 1992). However, it seems to bemucb more complex in angiosperms. A parallel can

be made with PAL which is encoded by a smallmultigene family in all angiosperms studied and by asingle gene in gymnosperms. The ancient lineage ofgymnosperms (which lack syringyl lignins) mayretain a more primitive organization of the ligni-fication pathway. It is worth mentioning that inrelation to the progressive evolution of lignins froma guaiacyl to a syringyl type (Monties, 1989)sinapaldehyde is a poor substrate for gymnospermCADs in vitro, in contrast to the more recentangiosperms CADs which use all the hydroxy-cinnamaldehydes in vitro with the same efficiency.This difference in substrate specificity supports theidea that CAD may be one of the regulatory enzymeswhich control the formation of guaiacyl and syringyllignins (Kutsuki, Shimada & Higuchi, 1982).

Our knowledge concerning the potential poly-morphism of CAD enzyme and of the correspondinggenes in angiosperms is still incomplete. In 1976,Mansell, Babbel & Zenk undertook a systematicsurvey of CAD isoforms in 89 plant species by starchgel electrophoresis and, with very few exceptions,detected only one form of CAD in most plant species


studied. However, as stated by Goffner et al (1992),the detection of CAD activity in starch or poly-acrylamide gels is limited to abundant and/or highactivity forms. Strong evidence for the existence ofCAD polymorphism in angiosperms was first pro-vided in soybean by Wyrambik & Grisebach (1975).Since theti, the presenee of CAD isoforms differingin terms of substrate specificity, molecular weightand amino acid sequences, has also been reported ineucalypt xylem (Goffner et al, 1992) and periderm(Hawkins & Boudet, 1994), bean (Grima-Pettenati etal, 1994) and wheat (Pillonel, Hunziker & Binder,1992). Beside the existence of structurally andfunctionally different isoforms (CADI, CAD2) de-scribed in the above studies, the occurrence of adifferential combination of the two subunits of theheterodimeric eucalypt CAD2 has been shown byHawkins & Boudet (1994) and suggested to be amechanism to modulate substrate preference (for areview on CAD polymorphism, see Hawkins,Goffner & Boudet 1994). Halpin et al (1992) havecharacterized in tobacco one form of native CADprotein giving rise to two similar but different sizedsubunits of 42'5 and 44 kDa. This result could alsosuggest the occurrence of isoforms in tobacco. Theexistence of CAD isoforms raises the question oftheir physiological significance. Is it possible thatone form may play a direct role in constitutive ligninsynthesis, whereas others may be involved in thesynthesis of other monolignol-derived end productssuch as lignans (Umezawa, Davin & Lewis, 1990)and defence lignins? Lignin biosytithesis is likelycontrolled by two signal-transduction pathways. Oneis involved in the development of vascular tissue, andthe other in plant defence responses (Walter, 1992).Whether the genes regulated by these two trans-duetion pathways are the same is still a totally openquestion due to the lack of studies and molecularprobes available hitherto. CAD induction which hasbeen demonstrated following ozone treatment (Gal-liano, Heller & Sandermann, 1993(7) has beenstudied in relatively few plant defence responses, andin all cases using only coniferyl alcohol as a substratefor measuring cinnamyl alcohol dehydrogenase acti-vity (Grand, Sarni & Lamb, 1987; Moniz de Sa etal, 1992). Studies on CAD induction performed incell suspension systems treated by elicitor prepara-tions (Grand et al, 1987; Dalkin et al, 1990;Campbell & Ellis, 1992; Moniz de Sa et al, 1992;Messner & Boll, 1993) did not lead to consistentconclusiotis concerning the regulatory role of CADin lignin biosynthesis. In cell suspension cultures ofspruce (Messner 8f Boll, 1993), pine (Campbell &Ellis, 1992), alfalfa (Dalkin et al, 1990) and poplar(Moniz de Sa et al, 1992), CAD is only slightlyinduced upon elicitation. As stated by the authors,the basal activity of CAD in untreated spruce andpine cells is already high. Iti an attempt to explainthis apparent lack of induction, Messner & Boll

(1993) suggested the existence of CAD isoforms,thus one lignin-specific activity could be stronglyinduced while the others remains unaffected. How-ever this hypothesis remains to be supported byexperimental evidence. It is also worth noting that,as indicated by Hawkins & Boudet (1994), non-specific alcohol dehydrogenases exhibiting broadsubstrate specificity can also account for detectable'CAD' activity in a crude extract.

Tbe most conclusive investigations on CAD havebeen performed in grasses where lignificationappears to be a major induced structural defencemechanism. Induced lignification associated withthe hypersensitive response of resistant wheatvarieties challenged with stem rust was stronglyinhibited via the use of CAD specific inhibitors(Grand, Sarni & Boudet, 1985 a) showing theimportance of CAD as a regulatory enzyme indefence lignin synthesis (Moerschbacher et al,1990), at least in this species. Very recently, Mitchell,Hall & Barber (1994) examined the substrate-specificinduction of wheat cinnamyl alcohol dehydrogenasein relatioti to its role in regulating the composition ofdefensive lignin (increased syringyl content) inducedat leaf wound margins. They simultaneously fol-lowed /)-coumaryl, coniferyl and sinapyl alcoholdehydrogenase activities in leaves treated with anelicitor (partially acetylated chitosan hydrolysate)and a non pathogenic fungus {Botrytis cinerea).Their results showed that CAD induction waspredominantly attributable to highly localized in-creases in sinapyl alcohol dehydrogenase and hencestrongly support the idea that CAD plays a role inregulating the guaiacyl-syringyl composition ofdefensive lignins by modulating the availability oflignin precursors. Furthermore, these results em-phasize the fact that it might be important to assayCAD activity with the three substrates.

The development of molecular tools capable ofdiscriminating between CAD isoforms will be anecessary step to address questions concerning CADregulation and the role of each isoform not only inlignification but also in other physiological processes.

In the following sections, we will focus on themost studied CAD isoform which appears closelyrelated to constitutive lignin synthesis and for whichthe cDNA has been cloned and characterized.

CAD structure-function relatiofiship. CAD cDNAshave been recently isolated from angiosperms[tobacco (Knight, Halpin & Schuch, 1992), eucalypt(Grima-Pettenati et al, 1993; Feuillet, Boudet &Grima-Pettenati, 1994), Aralia cordata (Hibino etal, 1993), poplar and alfalfa (Van Doorsselaere et al,1995)] as well as from gymnosperms [loblolly pine(O'Malley et al, 1992) atid spruce (Galliano et al,1993 6)]. The eucalypt and spruce cDNAs have beenunambiguously proven to encode CAD by functionalexpression in E. coli (Grima-Pettenati et al, 1993;Galliano et al, 19936). CAD cDNAs share extensive

224 A. M. Boudet, C. Lapierre and jf. Grima-Pettenati

sequence homology (approximately 80 % identityamong all published angiosperm sequences, and70 % between angiosperms and gymnosperms)suggesting that CAD was very well conserved duringevolution.

It is worth noting that in 1992, Kiedrowski et alreported the cloning of a novel plant defence gene,EL13, from Arabidopsis, the predicted product ofwhich shared no homology to known sequences.Since then, CAD sequences became available andELI3 appeared to share an overall homology ofapproximately 74% with CAD (52% identity/22%similarity) at the amino acid level. The ELI3 geneproduct is believed to play a role in establishing theresistance phenotype during incompatible reactionsof Arabidopsis challenged with Pseudomonas syringae(Kiedrowski et al, 1992). Whether ELD is a CADisoform specifically induced in defence response isstill an open question. The production of the ELI3gene product in a prokaryotic system, followed byfunctional tests with the three CAD substrates couldhelp to elucidate this point.

Sequence analysis of CAD showed that it belongsto the long chain zinc-containing alcohol dehydro-genase family. The homology of eucalypt CAD withhorse liver alcohol dehydrogenase (HADH, EC1.1.1.1) was sufficient to allow homology-basedmolecular modelling of CAD upon the crystal-lographic coordinates of HADH (MacKie et al,1993) and hence to identify amino-acid residues ofpotential functional significance. We took advantageof the powerful tool consisting of the overproductionof catalytically active CAD in E. coli, harbouringthe same biochemical properties as native eucalyptCAD, to verify the eflFect of site-directed mutagenesison amino-acids potentially involved in the active site.Recent results obtained in our laboratory showedthat the cofactor affinity for NADP can be changedby a single point mutation: serine at position 212towards aspartic acid (Lauvergeat et al, unpublishedresults). This result confirmed the quality of theCAD homology model and led to experiments aimedat changing the relative specificity of the enzymetowards the different monohgnols. The comparisonof the active site of a gymnosperm CAD for which athree-dimensional model has been recently built

. (Douglas et al, unpublished results), with that of atypical angiosperm CAD should allow us to targetthe amino acids which could be involved in thesubstrate specificity differences between angiospermand gymnosperm CAD.

CAD gene expression.CAD expression at the mRNA level Northern

experiments performed in a few species showed ahigh level of CAD expression in stems (tobacco.Knight et al, 1992; eucalypt, Grima-Pettenati et al,1993 ; Aralia cordata, Hibino et al, 1993 ; alfalfa andpoplar. Van Doorsselaere et al, unpublished results)and especially in tissue undergoing active lignifi-

cation (e.g. xylem, Grima-Pettenati et al, 1993; VanDoorsselaere et al, unpublished results). Galliano etal (19936) reported that treatment of spruce cellcultures with elicitor, and spruce seedlings withozone both markedly increased the CAD mRNAlevel.

Expression of a eucalypt CAD promoter-reportergene fusion. The CAD gene has been characterized intobacco, (Walter, Schaaf & Hess, 1994) Eucalyptusbotryoides (Hibino et al, 1994) and Eucalyptusgunnii(Feuillet et al, 1995). In order to study the spatialand developmental regulation of the eucalypt CADgene, we fused the CAD promoter region to theglucuronidase (GUS) reporter gene and transferredthe construct into poplar via Agrobacterium tume-faciens-mediated transformation (Feuillet et al,1995). Quantitative fluorimetric assays showed thatGUS activity was highest in roots followed by stemsand leaves. Similar findings have been reported intransgenic tobacco plants transformed with tobaccoCAD promoter-GUS fusion (Walter, Schaaf & Hess,1994). It is also interesting to note that high CADmessenger levels were found in alfalfa roots (VanDoorsselaere et al, unpublished results). The re-quirement of CAD in the formation of lignin inroots, may not be surprising considering the role ofroots in supporting the plant, this organ being underconstant mechanical stress and being subjected tosoil pathogen attacks.

In eucalypt, histochemical staining for GUSactivity indicated a strong expression in the vasculartissues of stems, roots, leaves and petioles. At theonset of xylem differentiation, GUS activity wasdetected mainly in parenchyma cells located betweenthe xylem conducting elements. After secondarygrowth has occurred, GUS activity was located inxylem ray cells and in parenchyma cells surroundingthe phloem fibers. This specific cellular pattern ofCAD gene expression provides the first stronghistochemical evidence suggesting the export oflignin precursors from their site of synthesis towardstheir sites of assembly. These findings support theconcept of cell cooperation in lignin biosynthesis(Feuillet et al. 1995),

3. The polymerization stage - peroxidases and/orlaccases ?

(a) Enzymes involved. The mechanisms involved inthe polymerization of monolignols remain unclear. Itis classically admitted that cell wall oxidases convertmonolignols into mesomeric free radicals which thenspontaneously polymerize to give lignins. In ad-dition, to the oxidase mediated formation of freemesomeric radicals non enzymatic processes (chainpropagation) could also be involved.

The nature of the enzyme(s) involved in theoxidation step is still a matter of debate. Peroxidasehad been considered, until very recently, to be the


major enzyme responsible for the oxidation ofmonolignols. During the last three years a renewedinterest for the potential involvement of laccase(s) inthis process has resulted from the work of severallaboratories (Davin et al, 1992; Driouich et al,1992; Katayama, Davin & Lewis 1992; Savidge &Udagama-Randeniya, 1992; and particularly^ Ster-jiades. Dean & Eriksson, 1992; Bao et al, 1993).

(b) Peroxidases. The potential role of several oxi-dases in the enzymatic oxidative polymerization ofcinnamyl alcohols was under discussion for a longtime until Harkin & Obst proposed in 1973 'anexclusive peroxidase participation'. In their reviewin 1990, Lewis & Yamamoto concluded that 'inorder to establish that a specific peroxidase iso-enzyme is involved in lignification, we shoulddetermine substrate specificity with monolignols,primary structure, subcellular location and a tem-poral correlation with active lignification'. From thattime, new correlative evidence supporting the role ofperoxidases in lignification has been provided.However, these experimental data do not fulfil all thedifferent criteria raised by Lewis & Yamamoto(1990). Therefore it is still difificult to unambiguouslyassign a specific role in lignification to a peroxidaseisoenzyme.

In the following paragraphs we will focus on tworecent developments related to the characterizationof HjOj in lignifying tissues and to the in vivomanipulation of peroxidase activity through geneticengineering.

Several groups have simultaneously demonstratedthe presence of endogenous HjOj in lignifying cells.Olson & Varner (1993) have used a simple histo-chemical test to detect hydrogen peroxide productionin cells undergoing lignification. Czaninski, Sachot& Catesson (1993) demonstrated the presence ofHjOj at the electron microscope level in lignifyingcell walls of poplar but, in contrast, hydrogenperoxide was absent from cambial cells. Morerecently, Schopfer (1994) characterized HjOj in thephloem and sclerenchyma but not in the xylem intissue blots of bean. In any case, as stressed by Olson& Varner (1993), the results showing a co-localizationof HjOj and the zones of lignification are correlativeand do not represent direct evidence of H.^02/peroxidase involvement in the lignification process.However Nose et al (1995) using cell suspensioncultures of Pinus taeda have very recently demon-strated that adding an H2O2 scavenger to the culturemedium inhibits lignification.

Another important approach to clarify the role ofperoxidases in lignifieation is related to the obtentionof transgenic plants with modified peroxidase ac-tivity. The group of Lagrimini (Lagrimini, 1991;Chabbert et al, 1992) obtained several transgeniclines of tobacco with significant over expression ordown regulation of a specific anionic peroxidase

supposed to be involved in lignification (Mader &Fussl, 1982). As it will be discussed in more detailsin the section on lignin mutants some plants with ahigher peroxidase level synthesize more lignins butthe plants with a lower peroxidase activity do nothave less lignins than the control plants. Additionalexperiments using the same strategy in differentplants are necessary to improve our knowledge onthe role of specific isoperoxidases in lignification.

(c) Laccases. As two recent comprehensive reviews{O'MaWey etal, 1993 ; Dean & Eriksson, 1994), havebeen published on laccases and lignification we willonly critically analyze the major arguments favour-ing the involvement of laccase in monolignol poly-merization.

Laccase (^-diphenol: O2 oxidoreductase) (EC:1.10.3.2), a blue metallo-proteiii containing fourcopper ions, catalyzes the oxidation of phenolicsubstrates using molecular oxygen as the electronacceptor. The enzyme is usually a highly glycosyl-ated monomeric protein ranging from 52 to morethan 110 kDa. Different criteria concerning substratespecificity, specific inhibitors, structural features,and mechanisms of action distinguish laccase fromother phenol oxidases (O-diphenol: Oj oxido-reductase) (Mayer, 1987).

Laccase was first identified in a tree {Rhusvernicifera) and then in a limited number of higherplants. In parallel, laccase activity was more widelystudied in fungi for which several laccase genes havebeen characterized and sequenced. Surprisingly, thefungal laccases have been generally involved inbiodegradation processes of lignin while the plantenzyme functions in the biosynthesis of the polymer.

Freudenberg was the first to mention that thereexists, in gymnosperm cambium extracts, a laccase-like activity capable of polymerizing lignin pre-cursors in the absence of added hydrogen peroxide(Freudenberg et al, 1958). However, after theseinitial studies, the role of laccase in lignification wasdismissed since on one hand conflicting results wereobtained for the ability of pure laccase to formsynthetic lignins in vitro (Nakamura, 1967), and onthe other hand, evidence was presented favouringperoxidase in polymerization of monolignols indifferentiating xylem tissues (Harkin & Obst, 1973).

More recently, several observations have resur-rected a potential role for laccase(s) in the poly-merization of monolignols. Almost simultaneously,Savidge & Udagama-Randeniya (1992) demon-strated the occurrence of a eoniferyl alcohol oxidaseactivity distinct from peroxidase in conifers, Ster-jiades et al (1992) showed that a laccase isolatedfrom Acer pseudoplatanus polymerizes monolignolsand Davin et al. (1992) found a laccase activity in cellwall preparations. In parallel, localization studiesusing specific antibodies raised against laccase(Driouich et al, 1992) or in situ histochemical


detection of laccase activity (Bao et al, 1993)confirmed the relatively specific occurrence of laccasein differentiating xylem.

Very recently, Liu et al (1994) demonstrated atight correlation between the temporal and spatiallocalization of a laccase-like phenoloxidase and thelignification of secondary cell walls in developingprimary xylem of Zinnia elegans stem tissues. Inaddition, Chabanet et al. (1994) cbaracterized alaccase-like phenoloxidase only in lignifying andlignified cell walls from mung bean.

Based on the differential ability of laccase andperoxidase to drive the oxidation/condensation ofdifferent lignin precursors in vitro, Sterjiades et al(1993) have interestingly suggested that laccases maybe primarily responsible for the initial polymeriz-ation of monolignols into oligolignols, while peroxi-dases would be more likely required to catalyze thereactions leading from oligolignols to highly con-densed macromolecular lignin. This proposal is inagreement with data from Freudenberg (1959, 1965),considering both peroxidase and laccase as potentialcandidates for polymerizing lignin.

Some negative results refuting the role of laccasesin lignin polymerization have been critically quest-ioned in the recent reviews of O'Malley et al (1993)and Dean & Eriksson (1994). In contrast to fungi, forwhich the enzyme is very often excreted in themedium, the strong association of laccase with theplant cell wall may explain its limited characteriz-ation in plants before now.

At the moment, the main arguments for theinvolvement of laceases in lignin polymerization are:(i) their ability to polymerize monolignols in vitro;(ii) their occurrence in the wall of differentiatingxylem. Other indirect evidence for this involvementinclude the decrease in lignin formation underconditions of Cu^^ deprivation even though enzymesother than laccases may be affected by Cu " star-vation (Downes, Ward & Turvey, 1991).

Despite these different arguments, it is stilldifificult to assign a role to specific laccases inlignification. One problem is related to the difificultyto clearly differentiate laccases from other oxidaseactivities. For example, the coniferyl alcohol oxidaseactivity characterized by Savidge & Udagama-Randeniya (1992) did not present the characteristicsof a true laccase (Dean & Eriksson, 1994). Thepolymorphism of laccases represents an additionallevel of complexity. These enzymes, which are highlyglycosylated, likely exist as several isoforms po-tentially encoded by a gene family, as was demon-strated for fungal laccases. At the moment noevidence is available on the role of a specific laccaseand, as underlined by Dean & Eriksson (1994), veryspecific monoclonal antibodies would be necessary todistinguish the occurrence of a particular laccase atthe tissular level.

At present, various research groups are known to

have complete or partial cDNA sequences forlaccases from sycamore maple, yellow poplar andloblolly pine. However it is surprising to observethat the sycamore maple and yellow poplar laccasecDNA coding sequenees do not cross-hybridize withRNA or DNA from the other species (Dean, personalcommunication).

Progress in the biochemical characterization oflaccase isoenzymes and their corresponding geneswill be necessary to definitively correlate, throughmeasurements of gene expression or through con-trolled regulation of these genes (down regulation,over expression), the functions of specific laccases inlignin polymerization.

V. LIGNIN MUTANTS AS A WAY TO IMPROVE

PLANT BIOMASS AND TO EXPLORE LIGNIN

BIOCHEMISTRV AND METABOLISM

Mutants may occur naturally or be produced bychemical mutagenesis or genetic engineering sinceseveral lignification genes are presently available.The analysis of such mutants represents a powerfulapproach for investigating the regulatory role of aspecific enzymatic step in the lignin pathway, andalso allows us to appreciate the degree of chemicalplasticity of the polymer (composition of the polymeritself and nature of its linkages with other cell wallcomponents).

In addition, since modification of lignin contentand composition would be advantageous for anoptimal utilization of crops and trees, lignin mutantscan represent interesting new plant products from aneconomical point of view.

The brown midrib mutants of corn (bm) andsorghum (bmr) which have an altered lignin con-centration and composition (Kuc & Nelson, 1964)have been particularly investigated as illustrated inthe case study of Section I. The brown-midrib traitis typically seen as a reddish pigmentation noticeablein the leaf midrib; the pigmentation is mostnoticeable at the four- to nine-leaf stage and fades inthe exposed areas as the plant matures. Brown-midrib plants exhibit a lower lignin content thannormal genotypes and/or differences in monomericunits composition and in hydroxycinnamic acidsreleased after alkaline hydrolysis (Barriere &Argillier, 1993). These mutations have been seen asa way to modify lignin quality and quantity in afavourable manner (Cherney, 1990). Indeed, Gallaiset al. (1980) examined bm hybrids in terms of theirfeeding value and observed that the dry matterintake was greater with bm hybrids as compared totheir normal counterparts. Digestibility and rate ofdigestion have also been shown to be higher (Barriere& Argillier, 1993). However, the agronomical valueof the brown-midrib genotypes is lower than that oftheir normal isogenic counterparts (Barriere &Argillier, 1993). The bm trait in corn is associated


with reduced forage yield (Weller, Phipps & Cooper,1985). In addition a greater potential for stalk lodginghas been linked to low lignin concentration in somespecies.

In Sorghum, bmr mutants have been divided intwo groups: (1) those with a significant reductionin lignin content (up to 50%); (2) those with nosignificant difference in the lignin content.

The chemical composition and the enzymologicalcharacteristics of bmr mutants have been examinedby different authors. Bucholtz et al (1980) haveshown in the bmr6 mutant of Sorghum that there isan increased proportion of cinnamaldehydes ( x 3) inthe polymer, when compared to normal lignin, andthat the activity of cinnamyl alcohol dehydrogenase(CAD) is depressed. In addition, the bmr6 mutantscontain less lignin than the wild type. These resultshave been recently confirmed by Pillonel et a/. (1991)who have shown that a single locus genetic lesionresulted in a Sorghum bmr mutant with depressedCAD and OMT activities. This, in turn, resulted inthe production of lignin with an increased incor-poration of cinnamaldehydes as analyzed bypyrolysis mass spectrometry. These results suggestthat the bmr mutation may have pleiotropic effectsand that the mutated gene could correspond to aregulatory gene. In addition, they show that thecinnamaldehydes can participate as building units inthe formation of lignins as well as cinnamyl alcohols.Interestingly enough, as we will see later, similarconclusions have been reached by down-regulatingCAD in tobacco through genetic engineering.

Grand et al (19856) have compared the activitiesof six enzymes involved in lignin biosynthesis in thebm3 corn mutant with those of its normal counter-part. The only difference observed was a reductionin OMT activity in the mutant when compared towild type. These results are in line with the analyticaldata of Lapierre et al. (1993), as discussed in Section11.3 (a) of this review. Similar results have beenobtained very recently by controlled genetic ma-nipulation of OMT in tobacco (Legrand et al,1994). However, despite these biochemical contribu-tions to our understanding of brown midrib mutants,the corresponding mutated genes are still unidenti-fied.

Another lignin mutant has been recently charac-terized by Chappie et al. (1992) in Arabidopsisthaliana. The screening of ethylmethane sulfonategenerated mutants for alteration in the accumulationof sinapic acid esters (sinapoyl malate) allowed theauthors to identify a mutant deficient in these sinapicesters, which also exhibits abnormal lignins lackingsyringyl units. The radiotracer feeding experimentswith the mutant/a/z7 were consistent with a geneticlesion resulting in a dysfunctional ferulate-5-hydroxylase. Using the screening procedure pre-viously exploited for the characterization of the fahlArabidopsis mutant. Chappie (personal communi-

cation) was able to identify a T-DNA tagged allele ina collection of Arabidopsis lines. Using 'plasmidrescue' to retrieve the genomic sequences that flankthe T-DNA insertion in the tagged line, he was ableto characterize a clone encoding a protein with a highdegree of homology with other cytochrome P-450dependent monooxygenases. Since the ferulate-5-hydroxylase is a cytochrome P-450 enzyme, thesedata strongly suggest that the fahl mutant isdefective in the structural gene for ferulate-5-hydroxylase and that the characterized clone cor-responds to ferulate-5-hydroxylase. These resultsnot only illustrate the interest of gene tagging for thecharacterization of genes but also show that a blockupstream in the general phenylpropanoid pathwaymay have pleiotropic effects on Hgnin compositionitself and in addition on the accumulation of otherhydroxycinnamic derivatives.

More orientated genetic manipulation of ligninmetabolism has been recently performed by gen-etically transforming plants with lignification genesin the antisense orientation in order to down-regulatethe expression of the corresponding endogenousgenes. OMT, CCR, CAD and lignin specificperoxidase/laccase genes represent potential targetsfor such a manipulation of lignification.

With regard to strategies for changing the degreeof methoxylation in lignins two different approacheshave been envisaged in order to improve theefficiency of chemical wood pulping: (1) to induce ahigher degree of methoxylation in gymnospermlignins (through the transfer of bifunctional OMT);(2) to induce a lower degree of methoxylation inangiosperm lignins (by using OMT antisense con-structs).

These opposing strategies rely on the followingrationale. The gymnosperm lignins, mainly com-prised of G units, have a 5-aromatic positionavailable for carbon-carbon linkages, which makesthem fairly resistant to the pulping depolymerizationprocess. A genetically induced methoxylation at C-5would thus reduce the lignin content in resistantinterunit bonds and would thereby facilitate thepulping of gymnosperm wood. In angiosperm wood,the partial substitution of OCH3 with OH groups,through genetic reduction of OMT activity, couldconsiderably improve the lignin solubilization in thepulping liquor and/or would allow the use of less ormore friendly chemicals to obtain an equivalent pulpgrade.

Ni et al (1994) have introduced alfalfa OMTantisense constructs into tobacco and alfalfa plants.The authors claimed that some of the regeneratedtobacco plants exhibited a significant reduction inlignin content but, unexpectedly, they observed nochanges in the monomeric composition of lignins.Dwivedi et al. (1994) who used an OMT gene fromaspen for the transformation of tobacco reported aslight reduction of both lignin content and syringyl


units in some of tbe transformed plants. However,tbe reduction in O M T activity did not exceed 45 %in tbese experiments and tbe number of control andtransformed plants was too limited to draw clearconclusions, as tbe variability observed witbin apopulation of tbe same cultivar is usually substantial.

More detailed studies performed by Legrand et al.(1994) resulted in somewbat different conclusions.Several antisense tobacco OMT genes (complete orincomplete) were used for transformation of tobaccoand a statistical analysis (Legrand, personal com-munication) revealed that transformed plants show-ing a strong depression in O M T activity alsoexhibited a pronounced reduction in tbe proportionof syringyl units in tbeir lignins, as compared to tbecontrols even though the total lignin content was notaltered. In addition, a new monomeric 5-hydroxyguaiacyl (5 OH-G) unit was presetit in tbetransgenic tobaccos (Monties et al., 1994). Tbeseresults are consistent witb tbe data previouslypresented in tbis review for tbe bm corn mutants.

Similar experiments bave also been performed onpoplar plants witb poplar O M T antisense constructs.In tbis case, despite a 50% reduction of OMTactivity in stems, tbe monomer composition oflignins was only sligbtly modified (Van Doorsselaere,personal communication). Tbese results seem todemonstrate that a strong reduction of O M T activity(greater tban 60%) is necessary in order to observesignificant cbanges in lignin composition. By de-pressing a bifunctional OMT activity, one wouldexpect a decrease in botb ferulic and sinapic acidand, as a consequence of tbe limited availability ofthese building units, a reduction in lignin content.Tbe results obtained so far showing a preferentialdecrease of syringyl type units suggest more complexinteractions tbat will be discussed later. At tbemoment, tbe available results (Legrand et al., 1994)sbow tbat tbe reduction in tbe degree of metbylationof lignins through genetic engineering can betecbnically successful. It is now necessary to de-termine tbe resulting effects on tbe cbaracteristics oftbe transformed material in pulp and paper making.

Otber lignification genes downstream in tbe ligninbiosyntbetic patbway are also potential targets forgenetic engineering experiments aimed at modu-lating tbe lignin content in plants. In tbis respect itis interesting to observe tbe natural variations oflignin content occurring in one and the same species.For example a range from 25-6 to 35-2% in lignincontent was found in Eucalyptusgrandis, E. urophyllaclones and bybrids (Ikemori, personal communi-cation, Aracruz, Bresil). Tbese observations suggestthe range of modification tbat can be easily envisagedthrough genetic engineering even tbougb it is notknown if bigber reductions can be tolerated by tbeplant. Until now, only data on plants transformedwith CAD and peroxidase antisense constructs areavailable.

Witb regard to the down regulation of CADactivity, transformed tobacco antisense plants ex-bibiting a strong decrease in CAD activity (up to90%) do not present, unexpectedly, quantitativecbanges in lignin content (Halpin et al., 1994).However, the composition of lignin is altered and itcontains more cinnamaldebyde units tban controlplants. In addition, tbe xylem tissue of antisenseplants witb very low levels of CAD activity, is redbrown in colour and reminiscent of tbe brown-midrib mutant pbenotype which, at least in tbe caseof sorgbum is associated witb a reduced CAD activity(Pillonel et al, 1991).

Similar observations (Van Doorsselaere &Jouanin, personal communication) bave been madewitb poplar plants transformed witb CAD antisenseconstructs atid results from Tollier et al. (1994) bavesbown that tbe transgenic reddish poplar wood,compared to normal one, released 10-fold morevanillin and syringaldebyde wben subjected to amild alkaline treatment. Tbe origin of the redcolouration is not clearly defined but Tollier et al.(1994) bave recently sbown tbat syntbetic ligninsformed in vitro by peroxidase copolymerization ofconiferyl alcobol and coniferaldehyde were pink, incontrast to the polymerization products of cotiiferylalcobol alone.

Interestingly, the lignins of sucb transformedplants are more easily extractable suggesting achange in lignin structure. Tbe inclusion of aldehydemonomers in place of alcobols in lignin could giverise to a polymer witb altered intermolecular botidingpattern, extractability and degradability.

Tobacco plants bave also been transformed witbsense and antisense constructs corresponding to ananionic peroxidase supposedly involved in lignific-ation (Lagrimini et al., 1987). Overexpression of tbisperoxidase led to a wilting pbenotype at the time offlower-bud itiitiation. A slight decrease in peroxidaseactivity in antisense plants resulted in a decrease intbe strengtb of tbe stem ultrastrueture wbereas astronger decrease resulted in plants that wereepinastic (Rotbstein, Rice & Lagrimini, 1990). Plantswbicb syntbesized bigber levels of tbe anionicperoxidase exhibited a bigber lignin content (Lag-rimini, 1991). In contrast, no significant modificationof tbe lignin content or composition was observed intbe transformed plants witb up to 20-fold lessperoxidase activity (Cbabbert et al., 1992). Tbesecontradictory results leave open the question toknow if tbis specific peroxidase is closely involved inlignification or if oxidases otber than peroxidases arecrucial for tbe polymerization step of lignin form-ation.

Tbese recent results on tbe manipulation of ligninstbrougb tbe techniques of genetic engineering offervery promising perspectives botb from a funda-mental point of view, for tbe understanding of ligninsynthesis and structure, and from an applied point of

Biochemistry atid molecular biology of lignification 229

view for improving the use of crop and industrialplants. At the present time, it is clear that lignincomposition can be manipulated through geneticengineering. It should also be emphasized that suchmanipulation did not apparently induce majorphenotypical differences (growth, architecture, mor-phology,...) in transformed plants when comparedto controls. This last observation reveals that withinthe same plant there is, to quite a large extent, adegree of chemical variability (or plasticity) in thelignin polymer which has no obvious repercussionson the physiology of the plant. Thus the variabilityinduced by genetic engineering can be considered asan extension of the normal chemical heterogeneity oflignins, as revealed in one and the same plantthrough the analysis of different tissues or cell types.

The changes in lignin extractability observed inplants transformed with CAD antisense constructscan be of immediate utility in the genetic im-provement of crops and industrial plants. Forexample, transformed forest trees with more easilyextractable lignins, will be of significant interest forpulp and paper production. Preliminary observ-ations (Petit-Conil, personal communication) haveshown that in tobaccos and poplars transformed withCAD antisense constructs, less chemicals are necess-ary for pulping and bleaching of these materials.While long term field trials of these transformedplants are still necessary, the results already obtainedoffer interesting perspectives for a range of bio-technological applications.

These initial experiments performed with OMTand CAD antisense constructs on tobacco haveprovided some unexpected results. From the data ofLegrand et al (1994), it is clear that a strongdepression of the bifunctional OMT did not sig-nificantly affect the production of guaiacyl units(derived from ferulic acid) nor the lignin content.Such an observation must raise the question as towhether feruloyl-CoA is produced through anotherpathway involving for example caffeoyl-CoA O-methyl transferase which should be able to produceferuloyl-CoA but not sinapoyl-CoA through thealternative pathway described in the Section IV. 1 (^).

Similarly, the results of Halpin et al (1994) showthat einnamaldehydes may be integrated into thelignin polymer and that this change may inducepronounced modifications in lignin structure andextractability. The sinapaidehyde content is pri-marily increased in these CAD down regulatedtobaccos raising the question as to why the conifer-aldehyde content is less modified. The occurrence ofdifferent CAD genes/enzymes could be related withthis observation (Hawkins & Boudet, 1994). Othertransgenic plants with depressed CCR activityshould be available in a few months. Theoretically,since CCR is the first committed step in the mono-lignol pathway and has a low activity level it shouldbe the regulatory enzyme of the branch pathway

specific to lignin synthesis. It will therefore be veryinteresting to see whether a significant depression ofCCR activity is associated with a decrease in lignincontent or if alternative pathways can supply otherlignin building units for the assembly of the ligninpolymer.

In the case of a reduction in lignin content, such aCCR down regulation strategy should also allow usto determine the level of reduction in lignin contentwhich is compatible with normal development. Inparallel, over-expression of CCR could also po-tentially lead to an increase in lignification, and onceagain, the resulting effects on plant developmentwould be interesting to observe.

The results already obtained with geneticallymodified plants transformed with lignification genesand the evoked potential developments underlinehow much genetic engineering will be in the futurea useful tool to improve plants and to explore theregulatory mechanisms of lignin biosynthesis. Fu-ture experiments will give more insights into thechemical variability of the lignins and will likelyprovide valuable information on the complex inter-actions between different branches of phenolicmetabolism.

VI. CONCLUDING REMARKS

The plant cell wall is now receiving increasingattention both for fundamental reasons and appliedpurposes. There is indeed a growing interest for theactive biological role of some of its normal com-ponents (carbohydrates and proteins) and the physio-logical functions of some specific polymers (lignins,cutin, suberin). In addition, the cell walls areimportant in relation with different utilizations ofplant materials (pulp industry, feeding of cattle,fibres, mechanical properties of fruits and veg-etables...) and the possibility of manipulating cellwall composition for a better adaptation of plantproducts is now realistic through plant geneticengineering.

Lignins remain difficult to study structurally andbiosynthetically and there are still many openquestions concerning their physiological roles, theirlarge heterogeneity and their interactions withpolysaccharides in the wall.

As described in this review, the improvement ofadapted analytical tools offers us at the present timea quite convenient range of methods for probinglignin composition, the nature of the interunitlinkages and some of the interactions with thepolysaccharide network. This progress, combinedwith the recent introduction of molecular biologytechniques in the field of lignification is likely togreatly improve our knowledge of lignification in thecoming years. Indeed a careful analysis of plantstransformed with genes (in sense/antisense orien-tation) along the lignin pathway will be invaluable


for probing the chemical plasticity of the polymerand the key regulatory enzymes in the biosynthesisof lignins. It is clear that these combined approacheswill be extensively used in the near future and willhelp to solve several problems related to ligninbiosynthesis (e.g. peroxidases versus laccases) and tooptimize lignin content of plants for biotechnologicalpurposes.

Other important questions which should be ad-dressed by other approaches concern the mech-anisms of lignin deposition and incorporation in thewall - a complex and fascinating topic in itself. Inthis area new concepts have recently emerged andneed to be verified by experimental data.

We have almost no understanding of the cellbiological aspects of lignification including the tissueand cellular sites of synthesis and accumulation ofmonolignols, the transport mechanisms of the mono-lignols through the membrane, the extent of in-volvement of monolignol glucosides as storageforms. The concept of cell cooperation (synthesis oflignin precursors by neighbouring cells and transportto lignifying cells) is substantiated by several ob-servations reported in this review concerning thelocation of lignification enzymes or promoter ac-tivities in xylem parenchyma cells. All these aspectsshould be highlighted in the future particularlythrough the use of in situ hybridization techniquesand of immunolocalization approaches with anti-bodies specific to monolignols and their glucosides.

Concerning lignin incorporation in the cell wall itis more and more suggested that lignin assembly isnot an ill-defined and random dehydrogenativeprocess and that the nature of the polysaccharidetemplate induces a relative organization of thepolymerization process. Thus the microhetero-geneity of lignin monomeric composition differsaccording to the different cell wall layers which havedifferent polysaccharide structure. This observationas well as the long-established effect of poly-saccharide matrices on the structures of syntheticlignins produced in vitro emphasize the role ofpolysaccharide in organizing the primary structureof lignins (Atalla, personal communication). Inducedmodification of the polysaccharide network couldhelp to confirm these hypotheses.

The potential regulatory mechanisms of lignifi-cation are numerous and concern specific stepsoccurring at the protoplast (hydroxycinnamoyl CoAavailability, NADPH availability, enzymes of mono-lignol synthesis...) or at the cell wall (monolignolavailability, co-substrate availability, HjOj, enzymesinvolved in polymerization, adequate polysaccharidenetwork.,,). Here again, coupled studies involvinggenetic manipulation of specific enzymes andmeasurements of fluxes of lignin synthesis couldhelp to delineate the precise role of some importantsteps in the overall process.

However, we have to keep in mind that, in contrast

to polysaccharides, lignins result from the con-densation of the end products of long and complexpathways starting from intermediate metabolism. Inthis way the triggering of lignification and the extentof lignification likely depend on the coordinatedactivation of the shikimate, the common phenyl-propanoid and the lignin specific pathways. Thiscoordination should be explored in the coming yearsas an example of integration in plant metabolism.

In the same way, the basis of carbon partitioningamong the different polymers of the cell wall (ligninsand polysaccharides) is at least partly related to theactivity of pathways upstream in the production ofphenylpropane units (shikimate and commonphenylpropanoid pathways).

Finally, the recent characterization of genes whichare closely associated to lignification (CCR, CAD)and of their promoters offers very interestingperspectives for the identification of the spatio-temporal expression of these genes and the mech-anisms of their activation (transcription factors). Inaddition, the down-regulation or over-expression ofthese genes in transgenic plants will have importantconsequences for a better understanding of ligninfunctions. Lignin is likely to be essential for plantsurvival but it will be interesting to determine towhich extent its proportion can be reduced in thewall as well as to check its role in plant defence ormore speculatively in reduction of cell growth(Whitmore, 1971; Marigo & Boudet, 1980). Thesemolecular biology tools will also enable us to modifyplants genetically as already performed for somespecies in order to modify lignin composition, toincrease lignin utility or to decrease lignin content.

ACKNOWLEDGEMENTS

We xvish to thank different colleagues for providing uswith unpublished material: Drs Chiang, Douglas, Durst,Heller, Matern, Varner and, for critically reading someparts of the manuscript, Drs Chappie, Dean, Dixon,Haigler; special thanks are due to Dr Lexvis for his helpfulsuggestions.

We are grateful to the European Community (AGRE0021-OPLIGE) for supporting our research on lig-nification genes referenced in this review. Finally thecontribution of research workers working in our lab-oratories is gratefully acknowledged (A, Boudet, C.Feuillet, D. GofFner, S. Hawkins and J. Van Doors-selaere). We thank Y, Barbe and A,-M, Viallele for typingthe manuscript.

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