Lignin genetic engineering for improvement of wood quality: Applications in paper and textile industries, fodder and bioenergy production

South African Journal of Botany 91 (2014) 107–125

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South African Journal of Botany

j ourna l homepage: www.e lsev ie r .com/ locate /sa jb

Review

Lignin genetic engineering for improvement of wood quality:Applications in paper and textile industries, fodder andbioenergy production

Smita Rastogi Verma a,⁎, U.N. Dwivedi b

a Department of Biotechnology, Delhi Technological University, Delhi 110042, Indiab Department of Biochemistry, Lucknow University, Lucknow 226007, India

⁎ Corresponding author. Tel.: +91 9560546679.E-mail addresses: [email protected] (S.R. V

[email protected] (U.N. Dwivedi).

0254-6299/$ – see front matter © 2014 SAAB. Published bhttp://dx.doi.org/10.1016/j.sajb.2014.01.002

a b s t r a c t
a r t i c l e i n f o
Article history:Received 16 May 2013Received in revised form 30 December 2013Accepted 3 January 2014Available online 31 January 2014

Edited by E Balazs

Keywords:BiofiberBiofuelForageLignin genetic engineeringPaper industryTextile industryTransgenic plants

Lignin, a complex racemic phenolic heteropolymer present in plant cell walls, plays crucial role in the adaptivestrategies of vascular plants. But from agroindustrial perspective, lignin exerts a negative impact on the utilizationof plant biomass in pulp and paper industry, textile industry, forage digestibility and production of biofuel. In thisdirection, ligninmanipulation by genetic engineering approaches serves as a promising strategy. The researches onlignin biosynthesis, especially monolignol biosynthesis, have demonstrated that alteration of lignin content andcomposition can be attained to acquire economic and environmental benefits. Thus, transgenic plants withmodified lignin content and composition can cope with large shifts in p-hydroxyphenyl/guaiacyl/syringyl ligninratios and modified lignin can serve as improved feedstock for production of paper, biofibers, biofuels and forage.This review provides an overview of lignin genetic engineering in plants to yield new insights into the ligninbiosynthetic pathway and quality amelioration of wood for efficient pulping, ease of forage digestibility, and pro-duction of biofiber and biofuel.

© 2014 SAAB. Published by Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1072. Approaches for genetic manipulation of lignin in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

2.1. Application of lignin manipulation in pulp and paper industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1152.2. Application of lignin manipulation in biofiber production for textile industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1162.3. Application of lignin manipulation in forage production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1162.4. Application of lignin manipulation in bioenergy/biofuel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

3. Impacts of lignin genetic engineering on plants and environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1184. Conclusion and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

1. Introduction

Lignin, a complex aromatic heteropolymer, is a major constituent ofthe secondary cell walls of all vascular plants. It plays essential roles instrengthening and impermeabilization of the cell walls and constitutesmechanical and chemical barriers against pathogens (Rastogi and

erma),

y Elsevier B.V. All rights reserved.

Dwivedi, 2003, 2008; Vinardell et al., 2008). Lignification of plant cellwall is developmentally regulated; however, its deposition is alsoinduced by various abiotic and biotic stresses, such as metabolic stress,wounding, pathogen infection as well as changes in cell wall structure(Caño-Delgado et al., 2003; Tronchet et al., 2010). It is primarily synthe-sized anddeposited in the secondary cellwalls of xylemvessels, trachearyelements, phloem fibers and periderm (Lewis et al., 1999; Rogers andCampbell, 2004).

Lignin is derived by oxidative polymerization of three cinnamylalcohols, i.e. monolignols (namely, p-coumaryl, coniferyl and sinapyl

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108 S.R. Verma, U.N. Dwivedi / South African Journal of Botany 91 (2014) 107–125

alcohols), which differ in their degree of methoxylation and formp-hydroxy phenyl (H),monomethylated guaiacyl (G) and dimethylatedsyringyl (S) units, respectively (Boerjan et al., 2003; Boudet, 1998;Cesarino et al., 2012; Chen et al., 2006; Chiang, 2005, 2006; Dixonand Reddy, 2003; Dixon et al., 2001; Douglas, 1996; Goujon et al.,2003b; Harakava, 2005; Harris and DeBolt, 2010; Heitner et al., 2010;Humphreys and Chapple, 2002; Humphreys et al., 1999; JinHua et al.,2007; Li and Chapple, 2010; Liu, 2012; Morreel et al., 2004; Ralph et al.,2004; Ralph and Brunow, 2006; Rastogi and Dwivedi, 2008; Sederoffet al., 1994; Vanholme et al., 2010a; Weisshar and Jenkins, 1998;Whetten and Sederoff, 1991, 1995; Whetten et al., 1998). Monolignolbiosynthesis occurs in cytoplasm involving a series of steps forminga ‘metabolic grid’, in which some enzymes display broad substratespecificities and some are multifunctional (Fig. 1). In brief, thephenylpropanoid pathway for the biosynthesis of monolignolscommences with the deamination of phenylalanine derivedfrom the shikimate biosynthetic pathway occurring in the plastid.Then several reactions including side chain reduction, aromatic ringhydroxylation/methoxylation and conversion of the side chain carboxylto an alcohol group occur (Liu, 2012). Genome sequencing, genomearray expression profiling and transcriptome profiling data have

OH

HCT

CO-SCoA

4-Coumarate

4CL

OH

C4H

COOH

COOH

NH

PAL

2

COOH

C3H?

CA

CC

4C

OH

OR O

4-Coumaryl shikimic acid /4-Coumaryl quinic acid

OH

OR O

OH

Caffeoylshikimic acid /Caffeoylquinic acid

C3H HCT

Caffeoyl

Caffeoyl

Caffeo

CH

OH

CH

OH

OH

CO-

Caffeat

COO

OH

4-Coumaryl alcohol

4-Coumaraldehyde

OH

p-Hydroxyphenyl (H unit)

CAD / SAD

CH OH2

OH

Phenylalanine

Cinnamate

CCR

4-Coumaryl CoA

CHO

?

?

Fig. 1.Biosynthetic route towards three monolignols, namely, p-coumaryl, coniferyl and siC3H: p-Coumarate 3-hydroxylase or p-Coumaroyl CoA 3-hydroxylase or p-Coumaroycinnamoyl-CoA: shikimate/quinate p-hydroxy cinnamoyl transferase; CCoAOMT: CaCoumarate: coenzymeA ligase; CCR: Cinnamoyl coenzymeA reductase; Cald5H: Coniferaldehydalcohol dehydrogenase; CAD, Cinnamyl alcohol dehydrogenase;?: Yet unconfirmed reactions].

provided useful information concerning different gene familiesencoding lignin biosynthesis pathway enzymes (Bonawitz andChapple, 2010; Bosch et al., 2011; Casu et al., 2007; Courtial et al.,2013; Ko andHan, 2004;Minic et al., 2009). Through combination of se-quence homology or specific transcript-profiling techniques, it is nowpossible to isolate genes that are more specific for secondary cell wallformation (Bedon and Legay, 2011). Three enzymes of the monolignolbiosynthesis pathway, namely, C4H, C3H and F5H, are membrane-bound cytochrome P450 enzymes and are active at the cytosolic sideof endoplasmic reticulum. The monolignols, synthesized in the cyto-plasm, are then translocated to cell wall by a still unclear mechanism(Alejandro et al., 2012; Kaneda et al., 2011; Liu et al., 2011; Miao andLiu, 2010; Vanholme et al., 2010a; Wang et al., 2012; Whetten andSederoff, 1995). According to onemodel,monolignol glucosides, formedby UDP-glucosyltransferase, are transported to plant cell wall andhydrolyzed by coniferin β-glucosidase. According to the second model,monolignols are transported to the plasma membrane by Golgi-derived vesicles. However, significant evidences for these two modelsare lacking and at present the roles of ATP-binding cassette-like (ABC)transporters or unaided diffusion of monolignols across the plasmamembrane are being demonstrated. In the cell wall, polymerization of

OCH3CH O3OCHHOOCHOH

5-Hydroxyguaiacyl

OMT / AldOMT

5-Hydroxyconiferyl

5-Hydroxyconiferaldehyde

CH OH

OCH

Coniferyl alcohol

Coniferaldehyde

CH OH

OCH

OH

(G unit)Guaiacyl

2

OH

OMT

D / SAD

2

OH

CAD / SAD

OH

(5-OHG unit)

alcohol

3 HO

F5H / Cald5H

3

2

CAD / SAD

OH

Sinapyl alcohol

Sinapaldehyde

CH OH

OCH

OH

(S unit)Syringyl

CH O33

3

2

CAD / SAD

OH

3

5-Hydroxyferuloyl CoA

CO-SCoA

OCH

5-Hydroxyferulate

OCH

Feruloyl CoA

CO-SCoA

OCH

OCH

OMT

OH

R

CHO

OH

CCR

CCoAOMT

L

OH

4CL

FerulateOH

F5H

3 HO

CHO

CCR

OH

3 HO

4CL

OH

COOH

OMT F5H

COOH

OCH

CO-SCoA

OCH

Sinapyl CoA

OMT

CH O33

CHO

CCR

OH

CH O33

4CL?

SinapateOH

3

3

OMT

COOH

F5H?COMT

CCoAOMT

COMT

alcohol

aldehyde

yl CoA

OH

O

SCoA

e

H

napyl alcohols [PAL: Phenylalanine ammonia-lyase; C4H: Cinnamate 4-hydroxylase;l shikimate/quinate 3-hydroxylase; OMT: O-methyltransferase; HCT: p-Hydroxyffeoyl coenzyme A O-methyltransferase; F5H: Ferulate 5-hydroxylase; 4CL: 4-e 5-hydroxylase;AldOMT: 5-Hydroxy coniferaldehydeO-methyltransferase; SAD: Sinapyl

109S.R. Verma, U.N. Dwivedi / South African Journal of Botany 91 (2014) 107–125

a large and extensively cross-linked lignin polymer of undefined dimen-sions takes place. Polymerization involves oxidative radicalization/dehydrogenation of phenols, mediated by peroxidases and/or laccases,followed by combinatorial radical coupling, wherein lignin macro-molecule itself acts as template for further lignification (Berthet et al.,2011; Chabanet et al., 1994; Christensen et al., 1998; Fagerstedt et al.,2010; Marjamaa et al., 2009; Morreel et al., 2010; O'Malley et al.,1993; Vanholme et al., 2010a; Zhao et al., 2013a).

The total content and relative abundance of monolignols, theirlinkages, degree of polymerization, number of free phenolic groupsand degree of cross-linking with polysaccharides vary among planttaxa, plant developmental stage, cell types and plant tissue (Chenet al., 2002; Grabber et al., 2004). In general, the lignin in angiospermdicot plants is composed of G and S units and traces of H units, whereasthe lignin in gymnosperms is mostly made up of G units with minoramounts of H units. Lignin in monocots contains relatively higheramount of H units and similar levels of S and G units (Vanholme et al.,2010a). In addition, lignin in grasses contains considerable amountsof the hydroxycinnamates, ferulic acid and p-coumaric acid (Vogel,2008). The S, G and H units may link through various dimer bonding-patterns, such as β-O-4, β-5, β-1, 5-5, 4-O-5 and β-β (Buanafina, 2009;Davin and Lewis, 1992; Terashima et al., 1996). Coupling ofmonolignolsdepends on their chemical characteristics, their speed of delivery to thecell wall and the oxidative environment in the wall (Parijs et al., 2010;Vanholme et al., 2008). The S/G ratio in lignin indicates the degreeand nature of cross-linking. G-rich lignin is highly cross-linked due toa greater proportion of biphenyl and other carbon–carbon bonds,where-as S-rich lignin is less condensed, linked bymore labile ether bonds at the4-hydroxyl position (Ferrer et al., 2008). S-rich lignin is more easilydepolymerized than G-rich lignin, because methoxy groups on a ligninmonomer result in less possible combinations during polymerizationdue to reduced available reactive sites (Ziebell et al., 2010). The incorpo-ration of eachmonomer and lignification of cell wall are developmental-ly programmed both temporally and spatially. Differential distribution oflignin monomers is observed between primary and secondary cell walls,between tissues and also in specific cell types (Grabber et al., 2004;McCann and Carpita, 2008). The content of lignin increases with plantmaturity. H and G units are incorporated at the commencement oflignin deposition, while only a few S units are detected duringearly stages of lignification. Subsequently, coniferyl alcohol and in-creasing amounts of sinapyl alcohol are incorporated to form a mixof G and S units during secondary wall formation (Grabber, 2005).In grasses, hydroxycinnamates are also incorporated during sec-ondary cell wall development and lignification. Ferulic acid is themajor hydroxycinnamic acid derivative in young grass cell walls,while p-coumaric acid is indicative of cell wall maturity, since it ismainly esterified to side chains of S units and its incorporation followsthe same deposition pattern of S units (Riboulet et al., 2009). Inmost dicots, thickening of secondary walls occurs mainly in water-conducting vessels of the xylem, which tend to contain higher levelsof G units, and in structural fibers, which are normally enriched in Sunits (Bonawitz and Chapple, 2010). In grasses, parenchyma, epidermisand hypodermis contain limited but significant amount of lignin, whilexylary tissues and sclerenchyma accumulate high amounts of ligninenriched in S lignins (Grabber et al., 2004).

Monolignols cross-link with other biomolecules such as cellulose,hemicelluloses and proteins. Together with hemicelluloses, ligninforms a complex matrix in which cellulose microfibrils are embedded(Vega-Sanchez and Ronald, 2010). Lignin composition and cell wallstructural organization in grasses are considerably different fromherba-ceous andwoody dicots or gymnosperm lignins (Buanafina, 2009; M. Liet al., 2012). One distinctive feature of the monocot lignins is the sub-stantial incorporation of the p-hydroxycinnamic acids including ferulicand p-coumaric acids. Ferulate monomers and dimers are ester-linkedto glucuronoarabinoxylan and are involved in ether and C–C linkagesto the lignin polymer that act as cross-links between hemicellulose

and lignin polymer chains. Monomers of p-coumaric acid are esterifiedto the lignin polymer at the γ-carbon of the side chain region of β-O-4linked syringyl moieties and to a lesser degree esterified toglucuronoarabinoxylan. Grass lignins are significantly more condensed(i.e., contain more C–C linkages between monolignols) and have higherphenolic hydroxyl contents than the lignins of dicots.

Lignin biosynthesis is controlled by a regulatory cascade of upstreamtranscription factors controlling the formation of secondary walls by ac-tivating several other transcription factors. Some of these transcriptionfactors, for example,MYB, LIM, NAC,WRKY, KNOX, and SHN, then inducethe expression of lignin biosynthetic pathway genes (Ambavaram et al.,2011; Bedon and Legay, 2011; Bhargava et al., 2010; Chavigneau et al.,2012; Demura and Fukuda, 2007; Fornalé et al., 2010; Guillaumie et al.,2010; Goicoechea et al., 2005; Grant et al., 2010; Gray et al., 2012;Grima-Pettenati et al., 2012; Handakumbura and Hazen, 2012; Hisanoet al., 2009; Kawaoka and Ebinuma, 2001; Kim et al., 2012; Ko andHan, 2004; E. Li et al., 2012; Ma et al., 2011; McCarthy et al., 2010;Minic et al., 2009; Ohman et al., 2013; Ohtani et al., 2011; Rahantamalalaet al., 2010; Rushton et al., 2010; Sonbol et al., 2009; Tamagnone et al.,1998; Townsley et al., 2013; Wang et al., 2010; Yamaguchi, 2010; Yanet al., 2013; Zhong and Ye, 2007, 2009; Zhao et al., 2008; Zhong et al.,2008, 2011; Zhao andDixon, 2011; Zhou et al., 2009). The transcriptionalregulation of lignin biosynthesis is thus under the control of the coordi-nated transcriptional systems that regulate the biosynthesis of other sec-ondary wall components such as cellulose and xylan (Zhong and Ye,2009).

The degree of cross-linking of lignin with other biomolecules as wellas the distribution, content and composition of lignin in plants hasprofound industrial, agricultural, environmental and economic implica-tions (Novaes et al., 2010; Rastogi andDwivedi, 2008). It exerts negativeimpacts on the exploitation of plant biomass for pulp production inpaper industry, biofiber production in textile industry, ease in livestockdigestibility, biofuel production from lignocellulose biomass and bio-degradation of lignocellulosic materials (Baucher et al., 1996, 1999;Dwivedi et al., 1994; Kwiatkowska et al., 2007; Rastogi and Dwivedi,2006, 2008; Stephens and Halpin, 2007; Whetten and Sederoff, 1991).Commercial and environmental benefits may thus be attained byintentionally (by either induced mutation or genetic manipulation)targeting lignin biosynthesis pathway genes in plants to manipulatelignin content and composition, however, it is pertinent that no nega-tive consequences occur on plant growth anddevelopment due to ligninmanipulation. This possibility owed to the potential of oxidative radical-ization of many new phenolic units generated by perturbedmonolignolbiosynthetic pathway entering the cell wall and their integration intolignin polymer by coupling or cross-coupling (Derikvand et al., 2008;Grabber et al., 2008; Ralph, 2006; Vanholme et al., 2010a). This has ledto directing the lignin biosynthetic pathway toward the biosynthesisof new phenolic molecules, which upon incorporation into the ligninpolymer improve lignin degradation (Grabber et al., 2008; Vanholmeet al., 2012). Another point of encouragement was the generation ofviable and normal transgenic plants with altered lignin structuresupon influx of various types of monolignols into their cell wall and theease of processing of their biomass (Huntley et al., 2003; Leplé et al.,2007; Pilate et al., 2002; Ralph, 2006; Ralph et al., 2006). The likelihoodto manipulate lignin was also supported by the occurrence of naturalvariations in lignin content and composition due to taxonomic differ-ences, developmental changes and responses to biotic and abiotic stressfactors such as exposure to pathogen, herbicides, light and hormones(Campbell and Sederoff, 1996; Donaldson, 2001; Fengel and Wegener,1984; Grand et al., 1982; Marita et al., 2003b; Sederoff et al., 1999).The prospects for genetically modifying lignin content and compositionwere also suggested by the studies based on mutants, for example,monopteros, lop1, lig1, ifl1, xtc1, xtc2, amp1, rsw1, avb1, irx4 and ref8 inArabidopsis, and bm mutants of maize, sorghum and related grasses(Anterola and Lewis, 2002; Barrière et al., 2013; Bhargava et al., 2010;Bout and Vermerris, 2003; Bucholtz et al., 1980; Chabbert et al., 1994;

Table 1Transgenic plants raised by manipulation of monolignol biosynthetic pathway genes (as single/double/multiple gene constructs or crossing-overs) (n.d. = not determined).

Transgenic plants and genes targeted Alterations in lignin content (% decrease/increase with respect to control)and lignin composition; Production of other metabolites;Changes in plant characteristics (if any)

References

Tobacco(PAL cosuppression)

Decreased lignin content; unusual phenotypes; altered leaf shape and texture;stunted growth; reduced pollen viability; altered flower morphologyand pigmentation

Bate et al. (1994);Elkind et al., 1990

Tobacco(PAL sense suppression)

Increased lignin content; unchanged lignin composition Howles et al. (1996)

Tobacco(PAL cosuppression)

Decreased (43%) lignin content; increased S:G due to decreasedG; increased methoxyl group content

Sewalt et al. (1997a, 1997b)

Tobacco(PAL sense suppression)

Decreased lignin content; decreased S:G Korth et al. (2001)

Alfalfa(C3H antisense)

Decreased lignin content; lignin rich in H units; improved digestibility Reddy et al. (2005)


Decreased lignin content; normal G and S units;increased p-hydroxyphenyl units; higher levelsof phenyl coumarans and resinols

Ralph et al. (2006)

Poplar(C3H RNAi)

Decreased (56%) lignin content; generation of p-hydroxyphenyl unitsat the expense of G units while the proportionof S moieties remained constant; accumulation of substantialpools of 1-O-p-coumaroyl-β-D-glucoside and otherphenylpropanoid glycosides, and p-coumaroyl shikimate

Coleman et al. (2008)

Alfalfa(C3H)

Significant structural changes had occurred tothe alfalfa ball-milled lignins;increased p-hydroxyphenyl units;decreased guaiacyl and syringyl units; decreased methoxylcontent (~55–58% in the ball-milled lignin);decreased β-O-4 linkage contents; abundance ofphenylcoumaran and resinol in the ball-milled lignins

Pu et al. (2009)

Tobacco(C4H antisense)

Decreased (20%) lignin content; decreased S:G Sewalt et al. (1997a)

Tobacco(C4H sense suppression)

Decreased (15%) lignin content; decreased methoxyl group content Sewalt et al. (1997a)

Tobacco(C4H over-expression)

Unchanged lignin content and composition Sewalt et al. (1997a)

Tobacco(C4H antisense)

Decreased (30%) lignin content; decreased S:G Blee et al. (2001)


Decreased lignin content; no significant impacton lignin composition; Improved digestibility

Reddy et al. (2005)

Tobacco(OMT antisense)

Decreased (12%) lignin content; decreased S Dwivedi et al. (1994)

Arabidopsis(OMT antisense)

Decreased (10-38%) lignin content; vine like growth Dwivedi and Campbell (1995)

Arabidopsis(OMT cosuppression)

Decreased (20-36%) lignin content; vine like growth Dwivedi and Campbell (1995)


Decreased (15-58%) lignin content; unchanged lignin composition Ni et al. (1994)


Unchanged lignin content; decreased S; new 5-OH G Atanassova et al. (1995)

Tobacco(OMT sense)

Unchanged lignin content; unchanged lignin composition Atanassova et al. (1995)

Poplar(OMT antisense)

Unchanged lignin content; decreased S; new 5-OH G; pale rose xylem Doorssalaere et al. (1995)

Tobacco(OMT sense)

Unchanged lignin content; decreased S; decreased hemicellulose content Vailhe et al. (1996)


Unchanged lignin content; decreased S; decreased hemicellulose content Vailhe et al. (1996)


Decreased (35%) lignin content; G more reduced than S Sewalt et al. (1997a)

Aspen(OMT cosuppression)

Unchanged lignin content; decreased S; red brown coloration Tsai et al. (1998)

2 year old poplar(Populus tremula × Populus alba)(OMT antisense)

Unchanged lignin content; increased G; pale rose coloration Lapierre et al. (1999)

Poplar(OMT sense)

Decreased (17%) lignin content; near elimination of S;increased cellulose; new 5-OH G; brownish coloration

Jouanin et al. (2000)


Decreased (62%) lignin content Ogras et al. (2000)

Alfalfa(OMT antisense)

Decreased (30%) lignin content; decreased G; new 5-OH G Guo et al. (2001a)

Poplar(OMT antisense)

Unchanged lignin content; decreased S:G Pilate et al. (2002)

Maize(OMT antisense)

Decreased (25-30%) lignin content; decreased S;lower p-coumaric acid content; new 5-OH G

Piquemal et al. (2002)


Decreased (24%) lignin content; decreased S; new 5-OH G Huayan et al. (2002)


Table 1 (continued)


References

Alfalfa(OMT antisense)

Incorporation of 5-hydroxyconiferyl alcohol; new 5-OH G Marita et al. (2003a)

Maize(OMT antisense)

Decreased (20% in stems, 12% in leaves, average 17% in whole plantwith the greatest reduction of 31%) lignin content; improved digestibility(2% in leaves, 7% in stems, from 72 to 76% mean whole-plant digestibility)

He et al. (2003)

Leucaena leucocephala(OMT antisense)

Decreased (28%) lignin content; decreased S; Increased cellulose (9%);increased methanol soluble phenolics;increased alkali soluble phenolics;increased pulp extractability

Rastogi and Dwivedi (2006)

4 year old poplar (OMT antisense) Trees healthy; growth normal; no unexpected biological or ecological impacts;interactions with leaf-feeding insects, microbial pathogens and soil organismswere unaltered; short-term decomposition of transgenicroots was slightly enhanced

Halpin et al. (2007)

Switchgrass(OMT antisense)

Modest decrease in lignin content; reduced recalcitrance;decreased S:G; phenotypically normal plants; improved forage quality;increased ethanol yield (up to 38% using conventional biomass fermentationprocesses); requirement of less severe pretreatment and 300–400%lower cellulase dosages for equivalent product yields usingsimultaneous saccharification and fermentation with yeast;fermentation of diluted acid pretreated transgenic switchgrassusing Clostridium thermocellum added enzymes showed betterproduct yields than obtained with unmodified switchgrass

Fu et al. (2011)

Arabidopsis (F5H/CALD5H over-expression) Only S Meyer et al. (1998)Tobacco(F5H/CALD5H over-expression)

Decreased (25%) lignin content; increased S Franke et al. (2000)

Poplar(F5H/CALD5H over-expression)

Decreased (25%) lignin content; increased S Franke et al. (2000)

Aspen(F5H/CALD5H sense)

Unchanged lignin content; increased S:G (three-fold) Y. Li et al. (2003),L. Li et al. (2003)

Poplar (Populus tremula × Populus alba)(F5H over-expression)

No significant modification in total lignin content; alteration of S lignin;significant increases in chemical (kraft) pulping efficiency fromgreenhouse-grown trees (decreases of 23 kappa units and increasesof N20 ISO brightness units as compared to wild-type wood);no observed phenotypic differences

Huntley et al. (2003)

Alfalfa(F5H/CALD5H antisense)

Decreased lignin content; reduced S;lignin rich in G units; improved digestibility

Reddy et al. (2005)

Alfalfa(F5H/CALD5H antisense)

Decreased lignin content; reduced accumulation ofsyringyl lignin in fiber and parenchyma cells,but not in vascular elements

Nakashima et al. (2008)

Populus tremula × P. alba(F5H over-expression)

97.5% S lignin (68% in control); lignin more linear,displayed lower degree of polymerization,higher resinol (β-β) and spirodienone (β-1) contents,virtually no phenylcoumarans (β-5); p-hydroxybenzoates,acylating the γ-positions of lignin side chains, were reduced by N50%,suggesting consequent impacts on related pathways

Stewart et al. (2009)

Arabidopsis(HCT RNAi)

Dwarf phenotype; changes in lignin composition Hoffmann et al. (2004)

Nicotiana benthamiana(HCT RNAi)

Decreased S units; increased p-hydroxyphenyl units Hoffmann et al. (2004)

Pinus radiata(HCT RNAi)

Decreased (42%) lignin content; predominant deposition of4-hydroxyphenyl units (31% of wild-type); increased resinols;reduced dibenzodioxocins; presence of glycerol end groups

Wagner et al. (2007)

Alfalfa(HCT)

Decreased lignin content;predominant deposition of 4-hydroxyphenyl units;strong reductions of neutral- and acid-detergent fiber;increased (20%) dry matter forage digestibility;stunted with reduction in biomass; impaired vascular structure;delayed flowering

Shadle et al. (2007)

Alfalfa(HCT)

Significant structural changes had occurred to thealfalfa ball-milled lignins; increased p-hydroxyphenyl units;decreased guaiacyl and syringyl units;decreased methoxyl content (~73% in the ball-milled lignin);decreased β-O-4 linkage contents; abundance ofphenylcoumaran and resinol in the ball-milled lignins

Pu et al. (2009)

Alfalfa(HCT)

Reduced cell wall recalcitrance; increased saccharification;changes in the extractability of hemicellulosicand pectic polysaccharides in cell walls; xylan andpectic polysaccharides more readily solubilizedfrom the cell walls; alterations inlignin-polysaccharide interactions

Pattathil et al. (2012)

Tobacco(CCoAOMT antisense)

Decreased (47%) lignin content;G more reduced than S

Zhong et al. (1997, 1998)

Poplar(CCoAOMT antisense)

Decreased (12%) lignin content; increased S:G Meyermans et al. (2000)

(continued on next page)


Table 1 (continued)


References

Alfalfa(CCoAOMT antisense)

Decreased (47%) lignin content; decreased G; No reduction of S Guo et al. (2001a, 2001b)


Decreased (15%) lignin content; unchanged S Pinchon et al. (2001a, 2001b)


Decreased (30%) lignin content; decreased S and G;G more decreased than S; increased S:G

Huayan et al. (2002)

Alfalfa(CCoAOMT antisense)

Decreased (20%) lignin content;no change in lignin composition; increased cellulose (10%) content;increased cellulose: lignin ratio

Marita et al. (2003a)

3 year old field-grown poplar(Populus tremula × Populus alba)(CCoAOMT antisense)

Decreased (13%) lignin content; slight increment in S:G;less benzene-ethanol extractive wood; no significantdifferences were found in contents of ash, cold water extractive,hot water extractive, 1% NaOH extractive, holocellulose,pentosans and cellulose; Improved the fiber quality;REMARKABLY improved pulp quality and increased pulp yield

Wei et al. (2008)

Linum usitatissimum (flax)(CCoAOMT antisense)

Decreased lignin content; modified S:G ratio;altered xylem organization; reduced cell-wall thickness;appearance of an irregular xylem (irx) phenotype;changes in xylem cell-wall structure

Day et al. (2009)

Arabidopsis(4CL antisense)

Decreased (30%) lignin content; decreased G Lee et al. (1995)

Tobacco(4CL sense)

Decreased (44%) lignin content; decreased S Kajita et al. (1996)

Tobacco(4CL antisense)

Decreased (44%) lignin content; decreased S Kajita et al. (1996)

Tobacco(4CL antisense)

Unchanged lignin content; decreased S Kajita et al. (1997)

Aspen(4CL antisense)

Decreased (45%) lignin content; increased cellulose content Hu et al. (1999)

Aspen(4CL antisense)

Decreased (40%) lignin content; increased (14%) cellulose L. Li et al. (2003)

Chinese white poplar(Populus tomentosa) (4CL antisense)

Decreased (42%) lignin content;unchanged holocellulose content; red brown coloration

Caihong et al. (2004)

Populus tremula × Populus alba(4CL antisense)

Decreased (50%) lignin content; reddish-brown discolored wood;twice the extractive content as compared to control;capacity for lignin reduction is limited such that belowa threshold, large changes in wood chemistry and plant metabolismwere observed that adversely affected productivity and potential ethanol yield

Voelker et al. (2010)

Tobacco(CCR antisense)

Decreased (47%) lignin content;increased S:G due to decrease in both S and G

Piquemal et al. (1998)

Tobacco(CCR antisense)

Decreased lignin content; decreased G;significant amount of tyramine ferulate; markedly reduced vigor

Ralph et al. (1998)

Tobacco(CCR partial sense suppression)

Decreased lignin content; increased S:G due to decreasein S and G; improved paper pulp

O'Connell et al. (2002)

Arabidopsis thaliana (CCR antisense) Decreased lignin content; S:G decreased orincreased depending on culture conditions

Goujon et al. (2003a)

Solanum lycopersicon(CCR RNAi)

Decreased (20-37%) lignin content;Changes in soluble phenolic pool

Rest et al. (2006)

Populus tremula × Populus alba(CCR antisense)

Decreased (50%) lignin content; S more reduced than G lignin;ferulic acid incorporated into the ligninleading to orange-brown, often patchy, colorationof the outer xylem; reduced biosynthesis and increasedbreakdown or remodeling of noncellulosic cell wall polymers;Reduced hemicellulose content; Increased cellulose content;growth affected in all 5 year old field growing transgenic lines

Leplé et al. (2007)

Alfalfa(CCR antisense)

Decreased lignin content; increased in vitro digestibility;50–60% improvement in saccharification efficiency

Jackson et al. (2008)

5 year old Norway spruce(Picea abies [L.] Karst)(CCR antisense)

Decreased (8%) lignin content; contribution of H-lignin to thenon-condensed fraction of lignin decreased (34%); normal phenotypebut smaller stem widths; Kappa number of small-scaleKraft pulps reduced (3.5%)

Wadenbäck et al. (2008)

Nicotiana tabaccum transformedwith Leucaena leucocephala CCR(CCR antisense)

Decreased (24.7%) lignin content; S:G increased; accumulationof unusual phenolics like ferulic and sinapic acids in cell wall;Reduction in aldehyde units; stunted growth and development,wrinkled leaves, delayed senescence; orange-brown colorationof debarked stems; thin walled, elongated xylem fibers,collapsed vessels, drastic reduction of secondary xylem;increased thickness of secondary wall layers andpoor lignification of S2 and S3 wall layers;increase holocellulose content (15%)

Prashant et al. (2011)

Nicotiana tabaccum transformedwith Leucaena leucocephala CCR(CCR sense up-regulation)

Increased (15.6%) lignin content;robust growth, development; orange-brown colorationof debarked stems

Prashant et al. (2011)

Tobacco(CAD antisense)

Unchanged lignin content; decreased S Halpin et al. (1994)


Table 1 (continued)


References


Unchanged lignin content; more aldehydes Hibino et al. (1995);Higuchi et al. (1994)


Unchanged lignin content; more aldehydes Stewart et al. (1997)


Unchanged lignin content; decreasedS:G; more cinnamaldehyde

Yahiaoui et al. (1998)

2 year old poplar(Populus tremula × Populus alba)(CAD antisense)

Unchanged lignin content; increased cinnamaldehyde Lapierre et al. (1999)

Poplar(CAD antisense)

Unchanged lignin content; increased aldehyde Chapple and Carpita (1998)


Decreased G and S units;increased benzaldehydes and cinnamaldehydes;Increased pulp extractability

Baucher et al. (1996)


Unchanged lignin content; decreased S Pinchon et al. (2001a, 2001b)

Field grown poplar(CAD antisense)

Lignin more easily extractable from wood;wood suitable for kraft pulping

Boerjan et al. (2001)


Unchanged lignin content; decreased S Vailhe et al. (1998)

Poplar(CAD antisense)

Decreased (13%) lignin content; increased S Lapierre et al. (1999)

Alfalfa(CAD antisense)

Unchanged lignin content; decreased S Baucher et al. (1999)


Decreased lignin content; increased S:G; improved paper pulp O'Connell et al. (2002)

Flax(CAD RNAi)

Decreased lignin content; changed lignin composition;reduction in the pectin and hemicellulose contents;improved mechanical properties;increased Young's modulus (75% higher);reduced (two-fold) resistance of thetransgenic lines to Fusarium oxysporum

Kwiatkowska et al. (2007)

4 year old poplar(CAD antisense)

Trees healthy; growth normal;no unexpected biological or ecological impacts;interactions with leaf-feeding insects, microbial pathogensand soil organisms were unaltered; short-termdecomposition of transgenic rootswas slightly enhanced

Halpin et al. (2007)

Alfalfa(CAD antisense)

Decreased lignin content; increased in vitro digestibility;improved saccharification efficiency

Jackson et al. (2008)

Maize(CAD RNAi)

Unchanged lignin content, slightly decreased S:G ratio andincreased cellulose and arabinoxylan contents in stems;reduced lignin content and cell wall polysaccharides in midrib;midribs and stems more degradablethan wild-type plants;Field-grown transgenic plants presented a wild-typephenotype and produced higher amounts of dry biomass;biomass produced higher levels of ethanol comparedto wild-type, making CAD a good target to improveboth the nutritional and energetic values ofmaize lignocellulosic biomass

Fornalé et al. (2012)

Arabidopsis(OMT C4H: F5H1)[combination of mutationin omt (down-regulation) withover-expression of F5H under thecontrol of the C4H promoter(C4H: F5H1 construct)]

Lignin comprising ~92% benzodioxane units;massive shifts in phenolic metabolism;38 metabolites derived from 5-hydroxy-substitutedphenylpropanoids accumulated

Vanholme et al. (2010b)

Tobacco(CCR × CAD antisense; cross pollinated)

Decreased (50%) lignin content;decreased S:G; CCR silencing predominatedover that of cad; plantsshowed behaviormuch like that of CCR down-regulated plants

Chabannes et al. (2001a, 2001b)

Tobacco(OMT × CCoAOMT antisense)

Decreased (41–66%) lignin content;decreased S:G; plants bore normal flowersand grew to heights similar to wild-type plants;deformed vessel walls

Zhong et al. (1998)

Poplar(OMT × CAD antisense;CAD down-regulated transgenicsretransformed with OMTantisense gene)

Decreased (15%) lignin content;increased G; red brown coloration,typical of CAD down-regulated plants

Lapierre et al. (1999)


Decreased (35%) lignin content; decreased S Huayan et al. (2002)

(continued on next page)


Table 1 (continued)


References

Alfalfa(OMT × CCoAOMT antisense)

Decreased (13%) lignin content; decreased G Guo et al. (2001a)

Tobacco(OMT × CCR antisense; cross pollinated)

Decreased (50%) lignin content; decreased S;substantial enrichment in condensed bonds;Intermediate phenotype; pale red coloration

Pinchon et al. (2001a)


Decreased (48%) lignin content; decreased S;new 5 OH-G; decreased and β-O-4 bond contents;morphological and developmental alterations suchas shorter mean stem internode length,narrower leaves and decreased stamen size

Pinchon et al. (2001b)

Tobacco(OMT × CAD antisense;sexually crossed; OMT-male;CAD-female)

Unchanged lignin content and composition Abbott et al. (2002)

Tobacco(OMT × CAD sense; single chimeric transgene;prepared by fusion of partial sense sequencesof these two non-homologous genes, OMT and CAD)

Decreased (20–30%) lignin content; decreased S Abbott et al. (2002)

Tobacco(OMT × CCR × CAD sense;multigene cotransformation)

Decreased (8–18%) lignin content; decreased S;wood bronze colored; few transformants exhibitedstunted phenotype and irregular vessel walls,characteristic of CCR down-regulation

Abbott et al. (2002)

Aspen(4CL × CALD5H antisense 4CLand sense CALD5H;multigene cotransformation)

Decreased (52%) lignin content; increased S:G (64%);increased cellulose (30%); accelerated cellmaturation in stem secondary xylem

L. Li et al. (2003)


Chapple et al., 1992; Chen et al., 2012; Cherney et al., 1990, 1991; Dean,2005; Derikvand et al., 2008; Franke et al., 2002; Gill et al., 2003;Goto et al., 1994; Grenet and Barry, 1991; Guillaumie et al., 2007;Halpin et al., 1998; Lapierre et al., 2000; Marita et al., 1999; Pattenet al., 2010; Ralph et al., 1997; Rohde et al., 2004; Saballos et al., 2008,2009, 2012; Sattler et al., 2009; Stasolla et al., 2003; Tamasloukhtet al., 2011; Thévenin et al., 2011; Thorstensson et al., 1992; Townsleyet al., 2013; Vanholme et al., 2013; Vermerris and Boon, 2001;Vermerris et al., 2002, 2010; Vignols et al., 1995; Wu et al., 1999; Zhaoet al., 2013a). Presently, genomics, proteomics and metabolomics arebeing used to improve our understanding of lignin biosynthesis pathwayand the ability to manipulate the genes involved. The genes involved inlignin biosynthesis pathway as well as transcription factors regulatinglignin biosynthesis have also been cloned (Bout and Vermerris, 2003;Bugos et al., 1991; Cao et al., 2012; Dharmawardhana et al., 1995;Doorssalaere et al., 1995; Dumas et al., 1992; Gupta, 2008; Heath et al.,1998; Jones et al., 2001; Kristensen et al., 1999; LaFayette et al., 1999;Laskar et al., 2006; Lee et al., 1995; Ma et al., 2011; Martz et al., 1998;Prashant et al., 2011; Ranocha et al., 1999; Selman-Housein et al., 1999;Teutsch et al., 1993; Wang et al., 2012; Whetten and Sederoff, 1992).

This review gives an elaborative account of various biotechnologicalapproaches employed for altering lignin content and composition ofplants and their impact on pulp production, textile industry, foragedigestibility and biofuel/bioenergy production (Baucher et al., 1998;Bedon and Legay, 2011; Boerjan et al., 1996; Bonawitz and Chapple,2010; Boudet, 1998, 2000, 2007; Boudet and Grima-Pettenati, 1996;Boudet et al., 1995, 2003; Carpita and McCann, 2008; Cesarino et al.,2012; Chen et al., 2003a, 2003b; Dixon et al., 1994, 1996; Grima-Pettenati and Goffner, 1999; Hisano et al., 2009; JinHua et al., 2007;Jung and Ni, 1998; Jung et al., 2012a; Nieminen et al., 2012; Palit et al.,2001; Rastogi and Dwivedi, 2003, 2008; Sederoff, 1999; Sederoff et al.,1994; Sticklen, 2008; Vanholme et al., 2008, 2010c; Vanholme et al.,2012; Weng et al., 2008; Whetten and Sederoff, 1991).

2. Approaches for genetic manipulation of lignin in plants

The strategies for alteration of lignin content and composition areselected depending on the end utility. In recent times, themanipulation

of lignin in plants is widely done by technologies including expressionof antisense RNA, sense expression, cosuppression, ribozyme and RNAinterference (RNAi) and mutations. As the plants can tolerate largevariations in lignin composition, oftenwithout apparent adverse effects,substitution of some fraction of the traditional monolignols by alterna-tive monomers through genetic engineering is a promising strategy toengineer lignin in plants for suitability in paper and textile industriesas well as biofuel and easily digestible forage production (Vanholmeet al., 2012).

The targets for manipulation of lignin content and compositioninclude gene(s) encoding enzyme(s) involved inmonolignol biosynthe-sis, their transport to cell wall, their polymerization to form lignin andalso regulation of lignin biosynthesis. However, till date, majority ofthese approaches are based onmanipulation ofmonolignol biosyntheticpathway genes as single constructs (Baucher et al., 2003; Boerjan et al.,2003; Boudet and Chabannes, 2001; Dean, 2005; Halpin, 2004; Nehraet al., 2005; Rastogi and Dwivedi, 2008; Rogers and Campbell, 2004;Wei et al., 2001). In order to get synergistic effects on lignin contentand composition, two or more genes involved in monolignol biosyn-thetic pathway have also been simultaneously targeted, either asmulti-ple constructs or cross-overs (Abbott et al., 2002; Chabannes et al.,2001a; Guo et al., 2001a; Lapierre et al., 1999; Pinchon et al., 2001a;Zhong et al., 1998). Several studies have indicated the incorporationof unusual structurally related aromatic compounds, for example,cinnamaldehydes, 5-hydroxyconiferyl alcohol, dihydroconiferyl alco-hol, ferulate esters, coniferyl ferulate, feruloyl tyramine, and ferulatemalate, into the cell walls of the transgenic plants (Derikvandet al., 2008; Prashant et al., 2011; Pu et al., 2009; Ralph et al.,1998, 2001; Stewart et al., 2009; Vanholme et al., 2010b; Wagneret al., 2007). A summary of various lignin genetic engineering re-searches pertaining to monolignol biosynthesis is presented inTable 1. Reports are also available on genetic manipulation of genes in-volved in monolignol transport and lignin polymerization; however,these are mostly associated with pleiotropic effects (Blee et al., 2003;Kavousi et al., 2010; Lagrimini, 1993; Lagrimini et al., 1993, 1997; Y. Liet al., 2003; McIntyre et al., 1996; O'Malley et al., 1993; Ranocha et al.,2002; Zhang et al., 2013). Lignin-specific transcription factor genes,for example, MYB genes have also been targeted for altering lignin


content and composition (Ambavaram et al., 2011; Ma et al., 2011;Tamagnone et al., 1998; Yan et al., 2013; Zhang et al., 2013; Zhouet al., 2009). Thus, by engineering the expression of transcription fac-tors, lignification can be altered with fewer adverse effects on plant de-velopment. Moreover, as these transcription factors bind the promotersof multiple genes, the flux through the pathway can be affected in a co-ordinated way. Furthermore, as some transcription factors are specificfor developmental lignification process and not for other processes,such as stress lignin formation, the plant is able to respond to environ-mental factors (Vanholme et al., 2010a).

Recently, a novel O-methyltransferase with a capability to substitutep-hydroxyl group of monolignols has been created (Bhuiya and Liu,2010). As this position is critical for oxidative coupling of phenoxyradicals to form lignin polymers, this modified enzyme exhibited thepotential to manipulate lignin polymerization in planta. Upon planttransformation, the plants accumulated de novo synthesized 4-O-methylated soluble phenolics and wall-bound esters and the lower lig-nin levels of transgenic plants resulted in higher saccharification yields(Zhang et al., 2012). Expressed sequence tags for various monolignolbiosynthetic pathway enzymes have also gained significance as molec-ular markers for manipulation of lignin metabolism (Braz et al., 2001).Further emphasis has also been laid on the alteration of lignificationby spontaneous or induced mutations (Anterola and Lewis, 2002;Bout and Vermerris, 2003; Bucholtz et al., 1980; Chabbert et al., 1994;Chapple et al., 1992; Cherney et al., 1990, 1991; Dean, 2005; Derikvandet al., 2008; Franke et al., 2002; Gill et al., 2003; Goto et al., 1994;Grenet and Barry, 1991; Halpin et al., 1998; Lapierre et al., 2000;Marita et al., 1999; Patten et al., 2010; Ralph et al., 1997; Rohde et al.,2004; Ruel et al., 2009; Stasolla et al., 2003; Thorstensson et al., 1992;Vermerris and Boon, 2001; Vermerris et al., 2002; Vignols et al., 1995;Wu et al., 1999; Zhao et al., 2013b).

2.1. Application of lignin manipulation in pulp and paper industry

Paper industries at present employ chemical delignification process-es for the production of high quality paper with superior characteristicssuch as better brightness stability and no age related reduction inwhiteness. Chemical delignification requires extensive chemicals andenergy, leads to damage of polysaccharide components of wood and isalso associatedwith the release of toxic pollutants into the environment(Dwivedi et al., 1994; Rastogi and Dwivedi, 2006, 2008; Whetten andSederoff, 1991). To circumvent these problems, lignin genetic engineer-ing may provide more economical in planta substitute to chemicaldelignification. Thus, by reducing lignin content and altering lignincomposition in woody plants, the quality and efficiency of pulpingmay be improved with an increase in wood extractability and a reduc-tion in mill effluents. Moreover, more wood products can be producedfrom less land, thus helping in the conservation of natural forests andreducing the environmental effect of processing wood into pulp andpaper.

Different approaches are adopted for genetic manipulation of ligninin plants in order to increase their applicability in pulp and paper indus-try. The first and foremost approach is to raise transgenic plants withreduced lignin content. The second approach targets lignin composition.The studies have revealed that the relative proportions of G and S units(i.e., amounts of methoxy and hydroxyl groups) determine the physicalproperties of lignin with respect to paper pulping (Chiang and Funaoka,1990; Chiang et al., 1988). At large, the efficiency of wood pulping is di-rectly proportional to the amount of S lignin units. In gymnosperms, theefficiency of pulping may be increased by reducing the number of freeC5 positions that may participate in the formation of resistant C5–C5 in-terunit bonds in lignin and make it difficult to delignify. This can beachieved by inducing methoxylation at C5 position, for example, byover-expressing OMT or F5H. On the other hand, in angiosperms, fourmajor strategies may be adopted to improve the efficiency of pulping.The first strategy is based on the increase in S lignin content, for

example, through up-regulation of F5H and F5H-specific OMT. The sec-ond strategy involves partial substitution of methoxy groups with hy-droxyl groups, which have higher hydrophilicity as compared tomethoxy groups and ionize in aqueous media, for example by down-regulation of OMT activity. This improves lignin solubilization inpulping liquor, allowing less intensive chemical treatment to obtain anequivalent pulp grade (Biermann, 1993; Boudet et al., 1995;Doorssalaere et al., 1995; Koehler and Telewski, 2006). The third strate-gy involves incorporation of aldehydes into the lignin leading to animprovement of alkali extractability (alkali catalyzes cleavage of weaklinkages between lignin and polysaccharides and ionization of free phe-nolic functions), for example by down-regulation of CAD. This owes tothe fact that the incorporation of aldehydes at C4 position renders itmore susceptible to ionization in alkali, increases the susceptibility ofα and β carbons to chemical attack by alkali and also reduces the fre-quency of cross-links of γ-terminal alcohol groups with polysaccha-rides. It has been suggested that the down-regulation of CAD activityleads to an increase in the levels of C6–C1 compounds, such as benzal-dehyde, vanillin and syringaldehyde, which aid in alkali extractabilityof lignin. As the kraft pulping method employed in paper industryuses splitting and degradation of lignin, mostly at β-O-4 linkages be-tween monomeric phenylpropane units, the transgenic plants withcinnamaldehyde rich lignin are more responsive to pulping as com-pared to normal lignin, as the aldehyde groups in lignin attract electronmore strongly from these linkages as compared to alcohol groups(Dimmel et al., 2002). The fourth strategy for facilitating cellulose fiberdelignification and utilization could be the incorporation of ester inter-unit linkages into lignin, for example by partial substitution of coniferylalcohol with coniferyl ferulate (Grabber et al., 2008). This enhancesalkaline extractability of lignin and enzymatic hydrolysis of structuralpolysaccharides due to the formation of cell walls containing ligninenriched in ester bonds that are easily breakable at low temperatureand alkaline concentrations and more readily hydrolyzable withfibrolytic enzymes, both with and without alkaline pretreatments.Thus, by bioengineering plants to incorporate coniferyl ferulate intolignin, saccharification of lignocellulosic biomass can be enhancedleading to less energy- and chemical-demanding biomass pretreatmentsto liberate cellulose for paper production (Grabber et al., 2008).

Based on the above facts several lignin genetic engineering studieshave been conducted. The results of such studies that are suitablefor pulp and paper industry are summarized in Table 1. Rastogi andDwivedi (2006) have also reported reduction of lignin content andalteration of lignin composition by down-regulation of OMT activity inwoody tree Leucaena leucocephala. The study involved Agrobacteriumtumefaciensmediated transformation of plants with heterologous geneconstructs (0.47 kb OMT gene construct from aspen) leading to up to60% reduction in OMT activity relative to control. These plants displayeda reduction of 28% in total lignin content as evaluated by the Klasonmethod. A reduction in lignin content, normal xylem development,probable increase in aldehyde levels and a decrease in syringyl unitswere also demonstrated by histochemical analyses of stem sections.Lignin down-regulation was accompanied by an increase in methanolsoluble phenolics to the extent where it had no impact on wood discol-oration and the plants exhibited a normal phenotype. Concurrently, anincrease of up to 9% in cellulose content was also detected. Upon alkaliextraction, modified lignin was more extractable as apparent from in-creased alkali soluble phenolics and reduced Klason lignin in saponifiedresidue. The results are presented in Fig. 2 and tabulated in Table 1. Theresults suggested enhanced applicability of transgenic Leucaena in theproduction of easily extractable wood for applicability in paper industryand easily digestible forage.

To sum up, lignin genetic engineering for suitability in pulp andpaper industry should be aimed at reduction in lignin content, increasein S lignin units, partial substitution of methoxy groups with hydroxylgroups in monolignols, incorporation of aldehydes into lignin andincorporation of coniferyl ferulate into lignin with no detrimental

0

5

10

15

20

25

30

OM

T S

p. A

c.;

KL

;A

SL

; T

L;

MeO

H-S

P;

CE

L

ASL TL MeOH-SP CEL

Plants OMT Sp. Ac. KL

C T1 T2 T3 T4 T5 T6

Fig. 2. Determination of O-Methyltransferase enzyme specific activity (OMT Sp. Ac.; expressed as (nmol14CH3 incorporated/min/mg protein)), Klason Lignin (Acid Insoluble Lignin;KL; expressed as % dried woody tissue), Acid Soluble Lignin (ASL; expressed as % dried woody tissue), Total Lignin (TL; TL = KL + ASL; expressed as % dried woody tissue), MethanolSoluble Phenolics (MeOH-SP; expressed as mg catechol/g dried woody tissue) and Cellulose (CEL; expressed as mg anhydroglucose/g cell wall residue, after multiplying with a factorof 0.9) contents in the stems of control (C) and six transformed plants (T1 to T6) of L. leucocephala. Data represent the mean of three independent measurements ± SD [ C T1T2 T3 T4 T5 T6 Plants].


environmental and ecological effects. Genetic manipulation is thusbeing used successfully in improving alkali extractability and pulpingproperties of wood, for example increased methanol soluble phenolicsin dried woody tissue, increased alkali soluble phenolics in cell wall res-idue, reducedKlason lignin in saponified residue, higher amounts of freeand alkali labile conjugated phenolics, high free phenolics:alkali labileconjugated phenolics ratio, augmented cellulose content, lower kappanumber, enhanced bleachability and increased levels of paper bright-ness (Baucher et al., 1999; Boudet et al., 1998; Halpin et al., 1994;Kajita et al., 2002; Lapierre et al., 1999; O'Connell et al., 1998; Pilateet al., 2002; Rastogi and Dwivedi, 2006, 2008; Wei et al., 2008).

2.2. Application of lignin manipulation in biofiber production fortextile industry

Synthetic fibers used nowadays are non-renewable sources of fiberand their production lead to environmental pollution. Consequently,there is renewed interest in the use of plant fibers, for example, cotton,jute and flax, in textile industry (Jhala et al., 2009; Stephens and Halpin,2007). Flax fibers are lignocellulosic fibers that contain ~70% cellulose,with hemicellulose, pectin and lignin and are routinely used in textileindustry. The presence of lignin gives mechanical strength to the fiberand plant on one hand, but its presence confers poor elastic propertiesto fibers as compared to non-lignocellulosic fibers, for example, cottonfibers. Thus, by producing low-lignin flax, fibers with improved elasticproperties may be obtained. This has been successfully achieved byCAD down-regulation using RNAi technology (Kwiatkowska et al.,2007). Manipulation of CAD gene has been reported to result in a reduc-tion in lignin content in transgenic plants and improved mechanicalproperties with up to 75% higher Young's modulus in the transgenicplants as compared to control plants. A substantial increase in the ligninprecursor contents and a decrease in pectin and hemicellulose contentswere also detected, which improved fiber extractability.

Besides their utility in textile industry, biofibers also find applicationin biomedical science and automotive industry. Biofibers have thepotential to replace petroleum-based synthetic polymers owing totheir low production costs, biodegradable nature, physical propertiesand environmental-friendliness. For such applications, transgenic flaxplants enriched with poly β-hydroxybutyrate have been generated.

Stems of transgenic plants exhibited improved mechanical propertiesowing to an increase in cellulose content, alteration in arrangement ofcellulose, increase in the number of hydrogen bonds and decrease in lig-nin and pectin contents (Wróbel-Kwiatkowska et al., 2009). However,such changes were not detected in fibers and their mechanical proper-ties remained unaltered. It was suggested that these transgenicflax plants producing both components of the flax/β-hydroxybutyratecomposites (i.e., fibers and thermoplastic matrix in the same plantorgan) could serve as a source of an attractive and environmentallysafe material for industry and medicine.

2.3. Application of lignin manipulation in forage production

The intimate association of both core (i.e., highly condensedpolymeric matrices) and non-core lignins (i.e., low molecular weightphenolic monomers) with cell wall polysaccharides, such as celluloseand hemicellulose, through covalent bonding physically separatesdigestive hydrolytic enzymes from cell wall polysaccharides, conse-quently limiting their digestibility by ruminants and decreasing energyyields (Dixon, 2004; Jackson et al., 2008; Jung et al., 2012a; Rastogi andDwivedi, 2008; Shadle et al., 2007; Vailhe et al., 2000). Besides lignincontent, lignin methoxyl content or S:G ratio, p-coumarate:ferulateratio (pCA:FA), ultrastructure and distribution of lignified tissues inforage crops are important determinants of digestibility of cell wallpolysaccharides. Other factors affecting forage digestibility are concen-trations of protein, non-structural carbohydrates, minerals, phenolicacids and fiber components, composition of cell wall polysaccharides,lignin–polysaccharide cross-linkages, maturity level and structuraldifferences limiting microbial and/or enzyme accessibility. The contentof highly condensed G lignin reduces cell wall degradability. Changesin phenolic deposition in parenchyma cells have been suggested tofavor digestion and aid the penetration of digestive enzymes to lessdigestible tissues (Buanafina, 2009). In the cell walls of forage grasses,ferulic acid is esterified to arabinoxylans and participates with ligninmonomers in oxidative coupling pathways to generate ferulate–polysaccharide–lignin complexes that cross-link the cell wall(Buanafina, 2009). Ferulic acid residues are mainly introduced intothe cell wall polysaccharides of grasses via an ester linkage betweenthe carboxylic acid group of ferulic acid and the primary alcohol on


the C5 carbon of the arabinose side chain of arabinoxylans but can alsobe covalently linked to lignin monomers via an ether linkage.Hydroxycinnamic acids are coupled by peroxidase-mediated oxida-tive coupling to form a variety of dehydrodiferulate dimers, cross-linking polysaccharide chains (Brett and Waldron, 1996).Feruloylation of arabinoxylan acts as a nucleating site for the forma-tion of lignin and for the linkage of lignin to the xylan/cellulose networkvia lignin–ferulate–xylan complexes. Bonding between these twoabundant polymers in the lignified walls of grasses contributes tothe low digestibility of most grass species by ruminants and to therecalcitrance of grass tissues to direct degradation by simple mixturesof cellulases and xylanases (Lam et al., 2003). It has been establishedthat ferulic acid esters work as dimerization sites for feruloylatedarabinoxylans and for the formation of ester-ether bridges betweenarabinoxylans and lignin. Larger products defined as oligoferulatesthat may predominate over ferulate dimers have been reported togetherwith the presence of ether-like bonds formed between ferulates andpolysaccharides, which reinforce polysaccharide cross-linking evenfurther. Consequently, bonding between these two abundant polymersin lignified walls of grasses is the main cause for much of the difficultyin digestion of the lignified walls of grasses by ruminants and, in part,to the recalcitrance of grass tissues to direct attack by cellulases andarabinoxylanases (Buanafina, 2009).

Reports based on analysis ofmutants and transgenic plants are avail-able on the improvement of forage digestibility by manipulating lignincontent and composition (Albrecht et al., 1987; Argillier et al., 1996;Baucher et al., 1999; Buxton and Casler, 1993; Buxton and Redfearn,1997; Buxton and Russel, 1988; Casler, 1987; Chen et al., 2002, 2003a,2004; Cherney et al., 1990; Dixon and Guo, 2011; Goujon et al., 2003a;Grabber, 2005; Grabber et al., 1992, 1997, 2004; Grenet and Barry,1991; He et al., 2003; Jung and Deetz, 1993; Jung et al., 1997a, 1997b,1999, 2012a; Lacayo et al., 2013; Lam et al., 2003; Marita et al., 2003b;Reddy et al., 2005; Sewalt et al., 1996, 1997a, 1997b; Thorstenssonet al., 1992; Vailhe et al., 1996, 1998, 2000; Wang and Brummer,2012). Till date, conflicting results have been presented concerningthe relationship of lignin with digestibility. For example, OMT down-regulated transgenic tobacco have been demonstrated to exhibit 5.6%increase in in vitro digestibility with 10-fold decrease in S:G ratio andunaltered lignin content (Vailhe et al., 1996). Brownmidrib 1 (bm1)mu-tants of maize exhibited higher digestibility due to altered pCA:FA ratioresulting from the reduction in the amount of esterified p-coumaric acidwith no change in soluble p-coumarate (Halpin et al., 1998). Arabidopsisf5h mutants showed complete loss of S lignin but no effect cellwall degradability (Jung et al., 1999). Brown midrib mutants of maizeand sorghum exhibited improved digestibility and improved animalperformance. For omt mutant maize brown midrib mutant, bmr3 withdecreased S:G ratio and Klason lignin content, a negative correlationbetween Klason lignin and in vitro neutral detergent fiber (NDF) digest-ibility has been demonstrated (Mechin et al., 2000; Vignols et al., 1995).Moreover, the predicted in vitroNDFdigestibility of three bmr3 lines de-termined by regression analysis exceeded the actual values, suggestingthat each unit of bmr3 lignin inhibited digestibility and that lignin com-position might influence digestibility. In yet another study, Grabberet al. (1997) suggested that lignin composition has little effect on foragedigestibility, at least in vitro. CAD down-regulated transgenic alfalfahave been reported with unaltered Klason lignin content, reduced(50%) S:G ratio and increased forage digestibility in cannulated sheep(Baucher et al., 1999). Guo et al. (2001b) provided better insights intothe relation between lignin content, composition and in situ digestibilityand indicated that both lignin content and composition affect digestibilityof alfalfa forage by down-regulating OMT and CCoAOMT. OMT down-regulated transgenic alfalfa exhibited complete elimination of S ligninwith a significant improvement in in-rumen forage digestibility.Although lignin content was reduced in stem internodes, no significanteffect on lignin levels was observed in the less lignified forage samplesconsisting of upper stem and leaf material. On the contrary, down-

regulation of CCoAOMT, which led to similar reduction in Klason ligninin stem material but an increased S:G ratio, gave a larger increase inin situ digestibility, possibly because of the significant decrease in lignincontent in the forage material. It seemed unlikely that augmented con-densation of lignin (as expected in the OMT down-regulated lines) wasa serious factor affecting forage digestibility. The data suggested that thedown-regulation of CCoAOMT is an effective strategy for improving di-gestibility of alfalfa forage. These improvements appeared to result froma combination of reduced lignin level and increased S:G ratio and prob-ably did not involve significant changes in content and composition ofcell wall polysaccharides or gross changes in vascular morphology.Down-regulation of CCoAOMT in two independent lines led to improve-ment in in vitro true digestibility of up to 4.4% and of in-rumendigestibil-ity of 2.8–6%. An improvement in digestibility of as little as 1% (whichwas attained in all the transgenic lines analyzed) could be highly signif-icant in terms of animal weight gain (Casler and Vogel, 1999). Reddyet al. (2005) targeted C4H, C3H, F5H and generated antisense transgenicalfalfa lines with a range of differences in lignin content and composi-tion. The largest improvements in digestibility were attained in C3Hdown-regulated alfalfa. A strong negative relationship between lignincontent and rumen digestibility was observed. However, no relation-ship between lignin composition and digestibility was observed.Shadle et al. (2007) reported that the antisense expression of HCTunder the control of bean PAL2 promoter led to a strong reduction inlignin content and striking changes in lignin monomer composition,with predominant deposition of 4-hydroxyphenyl units in the lignin.Analysis of forage quality parameters displayed strong reductions ofneutral- and acid-detergent fiber in the down-regulated lines andlarge increases (up to 20%) in dry matter forage digestibility. Recently,CCR and CAD down-regulated alfalfa have also been shown to exhibita reduction in lignin content associated with an increase in in vitrodigestibility (Jackson et al., 2008).

Another implication of lignin down-regulation on forage digestibili-ty is related to concurrent increase in cellulose content (Hu et al., 1999;Rastogi and Dwivedi, 2006). This compensatory regulation of lignin andcellulose is considered to be an adaptation of trees to sustainmechanicalstrength in lignin-deficient xylem. In lignin-deficientwood, partitioningof carbon occurs into cellulose biosynthesis due to its reduced flow intolignin biosynthesis pathway. Another possible explanation is related toincrease in UDP-glucose pool. As the basic event in cellulose biosynthe-sis is a simple polymerization of glucose residues from a substrate suchas UDP-glucose to form the homopolymer β-1,4-D-glucan (cellulose),a reduction in monolignol biosynthesis in cytoplasm and hence itstransport to the cell wall may lead to accumulation of UDP-glucose,which is probably diverted to cellulose biosynthesis. Furthermore, ascellulose microfibrils adsorb lignin precursors and oligomers and influ-ence their freedom to participate in random polymerization reactions(Houtman and Atalla, 1995), increase in cellulose component of theplant cell wall may influence lignin structure by changing the courseof dehydrogenative polymerization of monolignols. Concomitantincrease in cellulose content with a reduction in total lignin contentand S units in the transformants in turn improves the nutritive valueand digestibility of forage.

To summarize, lignin genetic engineering leading to reduced lignincontent, reduced G lignin content, increased S:G ratio, or reduced pCA:FA ratio may improve forage digestibility. As digestibility and intake offorage are key factors in the growth of ruminant animals, improvementof digestibility would benefit the recoverable yield of forage cropsthereby curtailing the overall cost of animal production.

2.4. Application of lignin manipulation in bioenergy/biofuel production

Due to depleting resources of fossil fuel supply and their hikingprices, there is a need to supplement them with renewable energysources, for example, bio-based fuels. The stored solar energy in plantbiomass can be released by burning, thermochemical conversion to


syngas, or biological conversion using microorganisms and/or enzymesto generate biofuels including alcohols and biogas (Johnson et al., 2007;Lin and Tanaka, 2006; Vertès et al., 2008; Vogel and Jung, 2001; Wenget al., 2008; Yuan et al., 2008). An emerging effort has been expendedto increase the use of plant lignocellulosic biomass for the productionof bioethanol (Carroll and Somerville, 2009; Grabber et al., 2010;Lee et al., 2011). Lignocellulose is, however, recalcitrant in nature dueto its molecular structure and heterogeneity, wherein lignin resistsenzymatic degradation to convert plant cell wall polysaccharides intofermentable sugars for biofuel production (M. Li et al., 2012;Vanholme et al., 2010c; Vega-Sanchez and Ronald, 2010; Weng et al.,2008). Consequently, the conversion of biomass to biofuel requires cost-ly and harsh pretreatments to degrade lignin and further allow the ac-cess to polysaccharides for saccharification (Grabber et al., 2010;Simmons et al., 2010). The plants can thus be rendered more amenableto bioprocessing by genetic modification of lignocellulosic matter char-acteristics. The highly photosynthetic-efficient C4 grasses, such asswitchgrass, Miscanthus, sorghum and maize, are expected to provideabundant and sustainable resources of lignocellulosic biomass for theproduction of biofuels (Bosch and Hazen, 2013; Carpita and McCann,2008; Cesarino et al., 2012;Weijde et al., 2013). An improvement in bio-mass conversion efficiencies into fermentable sugars with themodifica-tion of lignin in switchgrass, Miscanthus, sorghum, maize, alfalfa,Arabidopsis, sugarcane, or poplar feedstocks has been reported(Aristidou and Penttila, 2000; Arruda, 2012; Barriere et al., 2004; Boutand Vermerris, 2003; Carroll and Somerville, 2009; Chabbert et al.,1994; Chen and Dixon, 2007; Davis, 2008; Eudes et al., 2012; Farago,2007; Feltus and Vandenbrink, 2012; Fornalé et al., 2012; Halpin et al.,1998; Hisano et al., 2009; Jung et al., 2012b; Lee et al., 2011; Li et al.,2008; Lin and Tanaka, 2006; Lu et al., 2010; Marita et al., 2003b;Nieminen et al., 2012; Piquemal et al., 2002; Ragauskas et al., 2006;Ralph et al., 2006; Sattler and Funnell-Harris, 2013; Shen et al., 2013;Simmons et al., 2010; Vanholme et al., 2010c; Vega-Sanchez andRonald,2010; Vermerris and Boon, 2001; Vignols et al., 1995;Weng et al., 2008;Xu et al., 2011; Yuan et al., 2008). Populus trichocarpa and Brachypodiumdistachyon are also emerging as model systems for energy crops (Liet al., 2008). Upon comparison of cell wall degradability of wild-typeArabidopsis thaliana with a G-rich mutant line and a S-rich geneticallymodified line, it was found that lignin composition affected theenzymatic digestibility of liquid hot water pretreated plant materialand pretreatment was more effective in enhancing the saccharificationof A. thaliana cell walls containing S-rich lignin.

Form these studies, it has been established that increasing the S unitcontent through genetic engineering may be a promising approach toincrease the efficiency and reduce the cost of biomass to biofuel conver-sion (Li et al., 2010). Reducing lignin content below a critical thresholdcan lead to lower recalcitrance and higher saccharification efficiency.Moreover, a more uniform lignin structure facilitates more efficientcell wall degradation for fuel production (Anterola and Lewis,2002; Boerjan, 2005; Chabannes et al., 2001b; Chen and Dixon, 2007;Davison et al., 2006; Grabber et al., 2009; Hu et al., 1999; Meyermanset al., 2000; Ralph et al., 2006; Rest et al., 2006; Sticklen, 2006;Talukder, 2006). Lignin modification is also a promising path forimproving the access of cellulases to degrade cellulose (Rodriguezet al., 2000; Sticklen, 2008).

Additional research has revealed the co-regulation of lignin andcellulose biosynthesis in several studies. Repressing a single lignin bio-synthetic pathway gene, for example, 4-CL or OMT has been shown toresult in a reduction in lignin content with a concomitant increase incellulose content (Hu et al., 1999; Rastogi and Dwivedi, 2006, 2008).Similarly, an Arabidopsis cellulase synthase gene mutant, impaired incellulose biosynthesis, had altered lignin synthesis (Caño-Delgadoet al., 2003). In another study, expression of an Arabidopsis transcrip-tion factor, SHINE (SHN), in rice, a model for the grasses, caused a 34%increase in cellulose and a 45% reduction in lignin content. The riceAtSHN lines also exhibited an altered lignin composition correlated

with improved digestibility, with no compromise in plant strengthand performance. Globalgene expression analysis of rice revealed thatthe SHN (SHINE transcription factor) regulatory network coordinateddown-regulation of lignin biosynthesis and up-regulation of celluloseand other cell wall biosynthesis pathway genes (Ambavaram et al.,2011). The results supported the development of nonfood crops andcrop wastes with increased cellulose and low lignin with good agro-nomic performance that could improve the economic viability of ligno-cellulosic crop utilization for biofuels. In several cases, manipulation oflignin biosynthesis pathway genes has resulted in alteration in lignincomposition and structure rather than alteration in quantity. Becausethe efficiency of biomass conversion depends on hydrolyzing agentsgaining access to plant polysaccharides, alteration of plant cell wallstructure could yield important advantages. For example, when the lig-nin biosynthesis gene CCR was down-regulated in poplar, cellulosecomponent of the plant cell wall wasmore easily digested by the bacte-rium Clostridium cellulolyticum and twice as much sugar was released.

Thus, reduction of lignin and increase in S lignin in biofuel crops bygenetic engineering are effective ways of reducing costs associatedwith pretreatment and hydrolysis of cellulosic feedstocks, however,some potential fitness issues should also be addressed. Furthermore,enzymatic processing may be facilitated by decreasing lignin contentand increasing cellulose content, modifying enzymes to achieve higheractivities, broad substrate specificities, reduced inhibition and develop-ing new high-temperature and ethanol-tolerant microorganisms thatare able to ferment various six-carbon and five-carbon sugars.

3. Impacts of lignin genetic engineering on plants and environment

Recent advances in tree genetics permit modification of lignin con-tent and structure, however, the consequences of lignin modificationson many wood properties are unpredictable. In some studies, ligninmanipulation has exerted diverse and unpredictable impacts on plantphysiology, for example, brown coloration and dwarfing. (Abbottet al., 2002; Caihong et al., 2004; Chabannes et al., 2001a, 2001b; Chenand Dixon, 2007; Dwivedi and Campbell, 1995; Doorssalaere et al.,1995; Elkind et al., 1990; Franke et al., 2002; Higuchi et al., 1994;Hoffmann et al., 2004; Jouanin et al., 2000; Kwiatkowska et al., 2007;Lapierre et al., 1999; L. Li et al., 2003; Maher et al., 1994; Pinchonet al., 2001a, 2001b; Prashant et al., 2011; Shadle et al., 2007; Tsaiet al., 1998; Zhong et al., 1998). The consequences of lignin manipula-tion in various studies are presented in Table 1. This growth defect is adirect consequence of the lignin-deficient xylem that is unable to sup-port water transport and mechanical strength and is considered as aninherent limitation of the lignin reduction approach for biomass im-provement (Li and Chapple, 2010). While investigating geneticallymodified young quaking aspen trees with reduced lignin content and/or increased S:G ratio for the modulus of elasticity in three-point bend-ing and the compression strength parallel to the grain using modifiedmicromechanical tests, it has been indicated that lignin genetic modifi-cation had a negative effect on these mechanical properties (Horvathet al., 2010). The transgenic trees with reduced lignin content showedextensive reduction in modulus of elasticity and compression strengthparallel to the grain. On the contrary, the transgenic treeswith increasedS:G ratio revealed only a slight decrease in these properties comparedwith the wild-type, while simultaneous modification of lignin contentand S:G ratio showed inconsistent results.

To sum up, evaluation of field performance of modified plants is ofimmense importance (Casler et al., 2002; Halpin et al., 2007). Presently,many studies have demonstrated the beneficial effects of lignin manip-ulation on end-use applications in the laboratory, but only a few studieshave conducted field trials (Boerjan, 2005; Halpin et al., 2007; Hopkinset al., 2007; Kawaoka et al., 2006; Lapierre et al., 1999; Leplé et al., 2007;Pilate et al., 2002; Voelker et al., 2010; Wadenbäck et al., 2008; Walteret al., 2010; Wei et al., 2008). In the field, plants are exposed to variousenvironmental conditions and biotic and abiotic stresses as compared


to greenhouse and hence field trials are essential for demonstratingend-use application. The impacts on plant growth and ecologicalimpacts of lignin manipulation should be addressed.

4. Conclusion and future prospects

The intervention of biotechnological approaches (expression of anti-sense RNA, sense suppression, ribozyme, RNAi) for alteration of lignincontent and composition has immense potential in the direction ofoptimal utilization of plant biomass in pulp and paper industry, textileindustry for biofiber production, easily digestible forage production,bioenergy production and biodegradation. The implementation ofthese transgenic strategies underfield conditions, however, requires ap-propriate facilities and equipments, permits for testing and approval bythe government. Finally, to fully exploit the potential of lignin manipu-lation, it is necessary to increase the public acceptance of transgeniccrops and have proper regulatory system for the release of transgenicvarieties. It, however, appears that the use of transgenic approachesto enhance biomass conversion properties may face less public con-cerns, as long as these transgenic varieties do not contaminate thefood chain. But before the technology receives full public acceptance,more research is needed to develop more precise lignin engineeringstrategies. Proper understanding of functioning of the network linkinglignin biosynthetic pathway to other metabolic pathways, taking intoaccount feedback loops, enzyme kinetics and fluxes is also required.Laboratory experiments should be subjected to field trials in order toevaluate the effect of environmental conditions. Future projects shouldbe aimed at changing lignin and/or the non-condensed fraction of ligninby combinatorial modification of multiple lignin traits in plants throughmultigene co-transformation, stacking several lignin transgenes(e.g. siRNA-CAD and/or siRNA-CCR), using wood-specific promoterfor transgene expression and by transcriptional control of ligninbiosynthesis (e.g. MYB-factors).

References

Abbott, J.C., Barakate, A., Pinchon, G., Legrand, M., Lapierre, C., Mila, I., Schuch, W., Halpin,C., 2002. Simultaneous suppression of multiple genes by single transgenes: down-regulation of three unrelated lignin biosynthetic genes in tobacco. Plant Physiology128, 844–853.

Albrecht, K.A., Wedin, W.F., Buxton, D.R., 1987. Cell wall composition and digestibilityof alfalfa stems and leaves. Crop Science 27, 735–741.

Alejandro, S., Lee, Y., Tohge, T., Sudre, D., Osorio, S., Park, J., Bovet, L., Geldner, N., Fernie,A.R., Martinoia, E., 2012. AtABCG29 is a monolignol transporter involved in ligninbiosynthesis. Current Biology 22, 1207–1212.

Ambavaram, M.M.R., Krishnan, A., Trijatmiko, K.R., Pereira, A., 2011. Coordinatedactivation of cellulose and repression of lignin biosynthesis pathways in rice. PlantPhysiology 155, 916–931.

Anterola, A.M., Lewis, N.G., 2002. Trends in lignin modification: a comprehensive analysisof the effects of genetic manipulations/mutations on lignification and vascularintegrity. Phytochemistry 61, 221–294.

Argillier, O., Barriere, Y., Lila, M., Jeanneteau, F., Gelinet, K., Menanteau, V., 1996. Genotypicvariation in phenolic components of cell walls in relation to the digestibility of maizestalks. Agronomie 16, 123–130.

Aristidou, A., Penttila, M., 2000. Metabolic engineering applications to renewable resourceutilization. Current Opinion in Biotechnology 11, 187–198.

Arruda, P., 2012. Geneticallymodified sugarcane for bioenergy generation. Current Opinionin Biotechnology 23, 315–322.

Atanassova, R., Favet, N., Martz, F., Chabbert, B., Tollier, M.T., Monties, B., Fritig, B.,Legrand, M., 1995. Altered lignin composition in transgenic tobacco expressingO-methyltransferase sequences in sense and antisense orientations. The PlantJournal 8, 465–477.

Barriere, Y., Ralph, J., Méchin, V., Guillaumie, S., Grabber, J.H., Argillier, O., Chabbert, B.,Lapierre, C., 2004. Genetic and molecular basis of grass cell wall biosynthesis anddegradability. II. Lessons from brown midrib mutants. Comptes Rendus Biologies327, 847–860.

Barrière, Y., Chavigneau, H., Delaunay, S., Courtial, A., Bosio, M., Derory, J., Lapierre, C.,Méchin, V., Tatout, C., 2013. Different mutations in the ZmCAD2 gene underlie themaize brown-midrib1 (bm1) phenotype with similar effects on lignin characteristicsand have potential interest for bioenergy production. Maydica 58, 6–20.

Bate, N.J., Orr, J., Ni, W., Meromi, A., Nadler-Hassar, T., Doerner, P., Dixon, R.A., Lamb, C.J.,Elkind, Y., 1994. Quantitative relationship between phenylalanine ammonia-lyaselevels and phenylpropanoid accumulation in transgenic tobacco identifies a rate-determining step in natural product synthesis. Proceedings of the National Academyof Sciences of the United States of America 91, 7608–7612.

Baucher, M., Chabbert, B., Pilate, G., Doorssalaere, J.V., Tollier, M.T., Petit-Conil, M., Cornu,D., Monties, B., Montagu, M.V., Inze, D., Jouanin, L., Boerjan, W., 1996. Red xylemand higher lignin extractability by down-regulating a cinnamyl alcohol dehydrogenasein poplar (Populus tremula × P. alba). Plant Physiology 112, 1479–1480.

Baucher, M., Monties, B., Montagu, M.V., Boerjan, W., 1998. Biosynthesis and geneticengineering of lignin. Critical Reviews in Plant Sciences 17, 125–197.

Baucher, M., Vailhe, M.A.B., Chabbert, B., Besle, J.M., Opsomer, C., Montagu, M.V.,Botterman, J., 1999. Down-regulation of cinnamyl alcohol dehydrogenasein transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition anddigestibility. Plant Molecular Biology 39, 437–447.

Baucher, M., Halpin, C., Petit-Conil, M., Boerjan, W., 2003. Lignin: genetic engineering andimpact on pulping. Critical Reviews in Biochemistry and Molecular Biology 38,305–350.

Bedon, F., Legay, S., 2011. Lignin synthesis, transcriptional regulation and potential foruseful modification in plants. CAB Reviews: Perspectives in Agriculture, VeterinaryScience, Nutrition and Natural Resources 6, 1–28.

Berthet, S., Demont-Caulet, N., Pollet, B., Bidzinski, P., Cezard, L., Le Bris, P., Borrega, N.,Herve, J., Blondet, E., Balzergue, S., Lapierre, C., Jouanin, L., 2011. Disruptionof LACCASE4 and 17 results in tissue-specific alterations to lignification ofArabidopsis thaliana stems. Plant Cell 23, 1124–1137.

Bhargava, A., Mansfield, S.D., Hall, H.C., Douglas, C.J., Ellis, B.E., 2010. MYB75 functions inregulation of secondary cell wall formation in the Arabidopsis inflorescence stem.Plant Physiology 154, 1428–1438.

Bhuiya, M.W., Liu, C.J., 2010. Engineeringmonolignol 4-O-methyltransferases to modulatelignin biosynthesis. The Journal of Biological Chemistry 285, 277–285.

Biermann, C.J., 1993. Essentials of Pulping and Paper Making. Academic Press,San Diego.

Blee, K., Choi, J.W., O'Connell, A.P., Jupe, S.C., Schuch, W., Lewis, N.G., Bolwell, G.P., 2001.Antisense and sense expression of cDNA for CYP73A15, a class II cinnamate 4-hydroxylase, leads to a delayed and reduced production of lignin in tobacco.Phytochemistry 57, 1159–1166.

Blee, K.A., Choi, J.W., O'Connell, A.P., Schuch, W., Lewis, N.G., Bolwell, G.P., 2003. A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissuemodification. Phytochemistry 64, 163–176.

Boerjan, W., 2005. Biotechnology and the domestication of forest trees. Current Opinionin Biotechnology 16, 159–166.

Boerjan, W., Meyermans, H., Chen, C., Leple, J.C., Christensen, J.H., Doorssalaere, J.V.,Baucher, M.M., Petit-Conil, B., Chabbert, M.T., Tollier, B., Monties, G., Pilate, D.,Cornu, D., Inze, L., Jouanin, M., Montagu, M.V., 1996. Genetic engineering of ligninbiosynthesis in poplar. In: Ahuja, M.R., Boerjan, W., Neale, D.B. (Eds.), Somatic CellGenetics and Molecular Genetics of Trees. Kluwer Academic Publishers, pp. 81–88.

Boerjan,W., Meyermans, H., Chen, C., Baucher, M., Doorssalaere, J.V., Morreel, K., Messens,E., Lapierre, C., Pollet, B., Jouanin, L., Leplé, J.C., Ralph, J., Marita, J., Guiney, E., Schlich,W., Petit-Conil, M., Pilate, G., 2001. Lignin biosynthesis in poplar: genetic engineeringand effects on kraft pulping. Progress in Biotechnology 18, 187–194.

Boerjan, W., Ralph, J., Baucher, M., 2003. Lignin biosynthesis. Annual Review of Plant Biology54, 519–546.

Bonawitz, N.D., Chapple, C., 2010. The genetics of lignin biosynthesis: connectinggenotype to phenotype. Annual Review of Genetics 44, 337–363.

Bosch, M., Hazen, S.P., 2013. Lignocellulosic feedstocks: research progress and challengesin optimizing biomass quality and yield. Frontiers in Plant Science 4, 474.

Bosch, M., Mayer, C.D., Cookson, A., Donnison, I.S., 2011. Identification of genes involvedin cell wall biogenesis in grasses by differential gene expression profiling ofelongating and non-elongating maize internodes. Journal of Experimental Botany62, 3545–3561.

Boudet, A.M., 1998. A new view of lignification. Trends in Plant Science 3, 67–71.Boudet, A.M., 2000. Lignins and lignification. Plant Physiology and Biochemistry 38,

81–96.Boudet, A.M., 2007. Evolution and current status of research in phenolic compounds.

Phytochemistry 68, 2722–2735.Boudet, A.M., Chabannes, M., 2001. Gains achieved bymolecular approaches in the area of

lignification. Pure and Applied Chemistry 73, 561–566.Boudet, A.M., Grima-Pettenati, J., 1996. Lignin genetic engineering. Molecular Breeding 2,

25–39.Boudet, A.M., Lapierre, C., Grima-Pettenati, J., 1995. Biochemistry andmolecular biology of

lignification. New Phytologist 129, 203–236.Boudet, A.M., Goffner, D., Marque, C., Teulières, C., Grima-Pettenati, J., 1998. Genetic

manipulation of lignin profiles: a realistic challenge towards the qualitative improve-ment of plant biomass. AgBiotech News and Information 10, 295N–304N.

Boudet, A.M., Kajita, S., Grima-Pettenati, J., Goffner, D., 2003. Lignins and lignocellulosics: abetter control of synthesis for new and improved uses. Trends in Plant Science 8,576–581.

Bout, S., Vermerris, W., 2003. A candidate-approach to clone the sorghum brown midribgene encoding caffeic acid O-methyltransferase. Molecular Genetics and Genomics269, 205–214.

Braz, R.R.L., Tovar, F.J., Junqueiva, R.M., Lino, F.B., Martins, S.G., 2001. Sugarcane expressedsequences tags (ESTs) encoding enzymes involved in lignin biosynthesis pathways.Genetics and Molecular Biology 24, 235–241.

Brett, C.T., Waldron, K., 1996. Cell wall formation. In: Brett, C.T., Waldron, K. (Eds.),Physiology and Biochemistry of Plant Cell Walls. Chapman & Hall, London,pp. 75–111.

Buanafina, M.M.O., 2009. Feruloylation in grasses: current and future perspectives.Molecular Plant 2, 861–872.

Bucholtz, Y., Cantrell, R.P., Axtell, J.D., Lechtenberg, V.L., 1980. Lignin biochemistry ofnormal and bm mutant Sorghum. Journal of Agricultural and Food Chemistry 28,1239–1241.

http://refhub.elsevier.com/S0254-6299(14)00004-0/rf0005
















































































































Bugos, R.C., Chiang, V.L.C., Campbell, W.H., 1991. cDNA cloning, sequence analysisand seasonal expression of lignin bispecific caffeic acid/5-hydroxyferulic acidO-methyltransferase of aspen. Plant Molecular Biology 17, 1203–1215.

Buxton, D.R., Casler, M.D., 1993. Environmental and genetic effects on cell wallcomposition and digestibility. In: Jung, H.J.G., Buxton, D.R., Hatfield, R.D., Ralph, J.(Eds.), Forage Cell Wall Structure and Digestibility. American Society of Agronomy,Madison, WI, pp. 685–714.

Buxton, D.R., Redfearn, D.D., 1997. Plant limitations to fiber digestion and utilization. TheJournal of Nutrition 127, 5814–5818.

Buxton, D.R., Russell, J.R., 1988. Lignin constituents and cell wall digestibility of grass andlegume stems. Crop Science 28, 553–558.

Caihong, J., Huayan, Z., Hongzhi, W., Zhifeng, X., Kejiu, D.U., Yanru, S., Jianhua, W., 2004.Obtaining the transgenic poplars with low lignin content through down-regulationof 4CL. Chinese Science Bulletin 49, 905.909.

Campbell, M.M., Sederoff, R.R., 1996. Variation in lignin content and composition. PlantPhysiology 110, 3–13.

Caño-Delgado, A., Penfield, S., Smith, C., Catley, M., Bevan, M., 2003. Reduced cellulosesynthesis invokes lignification and defense responses in Arabidopsis thaliana. ThePlant Journal 34, 351–362.

Cao, Y., Hu, S., Huang, S., Ren, P., Lu, X., 2012. Molecular cloning, expression pattern,and putative cis-acting elements of a 4-coumarate:CoA ligase gene in bamboo(Neosinocalamus affinis). Electronic Journal of Biotechnology 15.

Carpita, N.C., McCann, M.C., 2008. Maize and Sorghum: Genetic resources for bioenergygrasses. Trends in Plant Science 13, 415–420.

Carroll, A., Somerville, C., 2009. Cellulosic biofuels. Annual Review of Plant Biology 60,165–182.

Casler, M.D., 1987. In vitro digestibility of dry matter and cell wall constituents of smoothbromegrass forage. Crop Science 27, 931–934.

Casler, M.D., Vogel, K.P., 1999. Accomplishments and impact from breeding for increasedforage nutritional value. Crop Science 39, 12–20.

Casler, M.D., Buxton, D.R., Vogel, K.P., 2002. Genetic modification of lignin concentrationaffects fitness of perennial herbaceous plants. Theoretical and Applied Genetics 104,127–131.

Casu, R.E., Jarmey, J.M., Bonnett, G.D., Manners, J.M., 2007. Identification of transcriptsassociated with cell wall metabolism and development in the stem of sugarcane byAffymetrix GeneChip Sugarcane Genome Array expression profiling. Functional andIntegrative Genomics 7, 153–167.

Cesarino, I., Araújo, P., Domingues, A.P., Mazzafera, P., 2012. An overview of lignin metab-olism and its effect on biomass recalcitrance. Brazilian Journal of Botany 35, 4.

Chabanet, Goldberg, R., Catesson, A.M., Quinet-Szely, M., Delaunay, A.M., Faye, L., 1994.Characterization and localization of a phenol oxidase in mung-bean hypocotyl cellwalls. Plant Physiology 106, 1095–1102.

Chabannes, M., Barakate, A., Lapierre, C., Marita, J.M., Ralph, J., Pean,M., Danoun, S., Halpin,C., Grima-Pettanati, J., Boudet, A.M., 2001a. Strong decrease in lignin content withoutsignificant alteration of plant development is induced in simultaneous down-regulation of cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase in to-bacco plants. The Plant Journal 28, 257–270.

Chabannes, M., Ruel, K., Yoshinaga, A., Chabbert, B., Jauneau, A., Joseleau, J.P., Boudet, A.M.,2001b. In situ analysis of lignins in transgenic tobacco reveals a differential impact ofindividual transformations on the spatial patterns of lignin deposition at the cellularand subcellular levels. The Plant Journal 28, 271–282.

Chabbert, B., Tollier, M.T., Monties, B., Barriere, Y., Argillier, O., 1994. Biological variabilityin lignification of maize: expression of the brown midrib bm2mutation. Journal of theScience of Food and Agriculture 64, 455–460.

Chapple, C., Carpita, N., 1998. Plant cell walls as targets for biotechnology. Current Opinionin Plant Biology 1, 179–185.

Chapple, C.C.S., Vogt, T., Ellis, B.E., Somerville, C.R., 1992. An Arabidopsis mutantdefective in the general phenylpropanoid pathway. The Plant Journal 4,1413–1424.

Chavigneau, H., Goué, N., Delaunay, S., Courtial, A., Jouanin, L., Reymond, M., Méchin, V.,Barrière, Y., 2012. QTL for floral stem lignin content and degradability in three recom-binant inbred line (RIL) progenies of Arabidopsis thaliana and search for candidategenes involved in cell wall biosynthesis and degradability. Open Journal of Genetics2, 7–30.

Chen, F., Dixon, R.A., 2007. Lignin modification improves fermentable sugar yields forbiofuel production. Nature Biotechnology 25, 759–761.

Chen, L., Auh, C., Chen, F., Cheng, X.F., Aljoe, H., Dixon, R.A., Wang, Z.Y., 2002. Lignindeposition and associated changes in anatomy, enzyme activity, gene expressionand ruminal degradability in stems of tall fescue at different developmental stages.Journal of Agricultural and Food Chemistry 50, 5558–5565.

Chen, F., Duran, A.L., Blount, J.W., Sumner, L.W., Dixon, R.A., 2003a. Profiling phenolicmetabolites in transgenic alfalfa modified in lignin biosynthesis. Phytochemistry 64,1013–1021.

Chen, L., Auh, C.K., Dowling, P., Bell, J., Chen, F., Hopkins, A., Dixon, R.A., Wang, Z.Y., 2003b.Improved forage digestibility of tall fescue (Festuca arundinacea) by transgenic down-regulation of cinnamyl alcohol dehydrogenase. Plant Biotechnology Journal 1,437–449.

Chen, L., Auh, C.K., Dowling, P., Bell, J., Chen, F., Hopkins, A., Dixon, R.A., Wang, Z.Y., 2004.Improved forage digestibility of tall fescue (Festuca arundinacea) by genetic manipu-lation of lignin biosynthesis. In: Hopkins, A., Wang, Z.Y., Mian, R., Sledge, M., barker,R.E. (Eds.), Molecular Breeding of Forage and Turf. Kluwer Academic Publishers,Netherlands, pp. 181–188.

Chen, F., Reddy, M.S.S., Temple, S., Jackson, L., Shadle, G., Dixon, R.A., 2006. Multi-sitegenetic modulation of monolignol biosynthesis suggests new routes for formationof syringyl lignin and wall-bound ferulic acid of alfalfa (Medicago sativa L.). ThePlant Journal 48, 113–124.

Chen, W., VanOpdorp, N., Fitzl, D., Tewari, J., Friedemann, P., Greene, T., Thompson, S.,Kumpatla, S., Zheng, P., 2012. Transposon insertion in a cinnamyl alcohol dehydroge-nase gene is responsible for a brown midrib1 mutation in maize. Plant Molecular Bi-ology 3, 289–297.

Cherney, D.J.R., Patterson, J.A., Johnson, K.D., 1990. Digestibility and feeding value of pearlmillet as influenced by the brown midrib, low lignin trait. Journal of Animal Science68, 4345–4351.

Cherney, J.H., Cherney, D.J.R., Akin, D.E., Axtell, J.D., 1991. Potential of bm, low ligninmutants for improving forage quality. Advances in Agronomy 46, 157–198.

Chiang, V.L., 2005. Understanding gene function and control in lignin formation in wood.In: Eaglesham, A. (Ed.), Agricultural Biotechnology: Beyond Food and Energy toHealth and the Environment. National Agricultural Biotechnology Council, U.S.,pp. 139–144.

Chiang, V.L., 2006. Monolignol biosynthesis and genetic engineering of lignin in trees,a review. Environmental Chemistry Letters 4, 143–146.

Chiang, V.L., Funaoka, M., 1990. The difference between guaiacyl and guaiacylsyringyl lignins in their responses to kraft delignification. Holzforschung 44,309–313.

Chiang, V.L., Puumala, R.J., Takeuchi, H., Eckert, R.E., 1988. Comparison of soft wood andhard wood kraft pulping. TAPPI Journal 71, 173–176.

Christensen, J.H., Bauw, G., Welinder, K.G., Van Montagu, M., Boerjan, W., 1998.Purification and characterization of peroxidases correlated with lignification inpoplar xylem. Plant Physiology 118, 125–135.

Coleman, H.D., Park, J.Y., Nair, R., Chapple, C., Mansfield, S.D., 2008. RNAi-mediatedsuppression of p-coumaroyl-CoA 3′-hydroxylase in hybrid poplar impacts lignindeposition and soluble secondary metabolism. Proceedings of the National Academyof Sciences of the United States of America 105, 4501–4506.

Courtial, A., Soler, M., Chateigner-Boutin, A.L., Reymond, M., Méchin, V., Wang, H., Grima-Pettenati, J., Barrière, Y., 2013. Breeding grasses for silage feeding value or capacity tobiofuel production: an updated list of genes involved in maize secondary cell wallbiosynthesis and assembly. Maydica 58, 67–102.

Davin, L.B., Lewis, N.G., 1992. Phenylpropanoid metabolism: biosynthesis of monolignols,lignans and neolignans, lignins and suberins. Recent Advances in Phytochemistry 26,325–375.

Davis, J.M., 2008. Genetic improvement of poplar (Populus spp.) as a bioenergy crop. In:Vermerris, W. (Ed.), Genetic Improvement of Bioenergy Crops. Springer, BerlinHeidelberg, New York, pp. 397–419.

Davison, B.H., Drescher, S.R., Tuskan, G.A., Davis, M.F., Nghiem, N.P., 2006. Variation of S:Gratio and lignin content in a Populus family influence the release of xylose by diluteacid hydrolysis. In: McMillan, J.D., Adney, W.S., Mielenz, J.R., Klasson, K.T. (Eds.),Applied Biochemistry and Biotechnology. Humana Press, pp. 427–435.

Day, A., Neutelings, G., Nolin, F., Grec, S., Habrant, A., Crônier, D., Maher, B., Rolando, C.,David, H., Chabbert, B., Hawkins, S., 2009. Caffeoyl coenzyme A O-methyltransferasedown-regulation is associated with modifications in lignin and cell-wall architecturein flax secondary xylem. Plant Physiology and Biochemistry 47, 9–19.

Dean, J.F.D., 2005. Synthesis of lignin in transgenic and mutant plants. In: Steinbüchel, A.,Doi, Y. (Eds.), Biotechnology of Biopolymers: From Synthesis to Patents. Wiley-VCHVerlag GmbH and Co, Weinheim, pp. 3–26.

Demura, T., Fukuda, H., 2007. Transcriptional regulation in wood formation. Trends inPlant Science 12, 64–70.

Derikvand, M.M., Sierra, J.B., Ruel, K., Pollet, B., Do, C.T., Thevenin, J., Buffard, D.,Jouanin, L., Lapierre, C., 2008. Redirection of the phenylpropanoid pathway toferuloyl malate in Arabidopsis mutants deficient for cinnamoyl CoA reductase 1.Planta 227, 943–956.

Dharmawardhana, D.P., Ellis, B.E., Carlson, J.E., 1995. β-Glucosidases fromlodgepole pine xylem specific for the lignin precursor coniferin. Plant Physiolo-gy 107, 331–339.

Dimmel, D.R., MacKay, J.J., Courchene, C.E., Kadla, J.F., Scott, J.T., O'Malley, D.M., McKeand,S.E., 2002. Pulping and bleaching of partially CAD-deficient wood. Journal of WoodChemistry and Technology 22, 235–248.

Dixon, R.A., 2004. Molecular improvement of forages: from genomics to GMOs. In:Hopkins, A., Wang, Z.Y., Mian, R., Sledge, M., Barker, R.E. (Eds.), Molecular Breedingof Forage and Turf. Kluwer Academic Publishers, pp. 1–19.

Dixon, R.A., Guo, D., 2011. Method for modifying lignin composition and increasing in vivodigestibility of forages. US Patent US 7888553 B2.

Dixon, R.A., Reddy, M.S.S., 2003. Biosynthesis of monolignols. Genomic and reversegenetic approaches. Phytochemistry Reviews 2, 289–306.

Dixon, R.A., Maxwell, C.A., Ni, W., Oommen, A., Paiva, N.L., 1994. Genetic manipulationof lignin and phenylpropanoid compounds involved in interactions withmicroorganisms. In: Ellis, E.E., Kuroki, G.W., Stafford, H.A. (Eds.), Recent Advancesin Phytochemistry: Genetic Engineering of Plant Secondary Metabolism, vol. 28.Plenum Press, pp. 153–178.

Dixon, R.A., Lamb, C.J., Masoud, S., Sewalt, V.J.H., Paiva, N.L., 1996. Metabolic engineering:prospects for crop improvement through the geneticmanipulation of phenylpropanoidbiosynthesis and defense responses — a review. Gene 179, 61–71.

Dixon, R.A., Chen, F., Guo, D., Parvathi, K., 2001. The biosynthesis of monolignols: a‘metabolic grid’, or independent pathways to guaiacyl and syringyl units? Phyto-chemistry 57, 1069–1084.

Donaldson, L.A., 2001. Lignification and lignin topochemistry: an ultrastructural view.Phytochemistry 57, 859–873.

Doorssalaere, J.V., Baucher, M., Chognot, E., Chabbert, B., Tollier, M.T., Petit-Conil, M., Leple,J.C., Pilate, G., Cornu, D., Monties, B., Montagu, M.V., Inze, D., Boerjan,W., Jouanin, L.A.,1995. A novel lignin in poplar trees with a reduced caffeic acid/5-hydroxyferulic acidO-methyltransferase activity. The Plant Journal 8, 855–864.

Douglas, C.J., 1996. Phenylpropanoid metabolism and lignin biosynthesis: from weeds totrees. Trends in Plant Science 1, 171–178.





























































































































































Dumas, B., Doorssalaere, J.V., Gielen, J., Legrand, M., Fritig, B., Montagu,M.V., Inze, D., 1992.Nucleotide sequence of a cDNA sequence encoding O-methyltransferase from poplar.Plant Physiology 98, 796–797.

Dwivedi, U.N., Campbell, W.H., 1995. Antisense inhibition and cosuppression oflignin biosynthesis in transgenic Arabidopsis harboring O-methyltransferasegene constructs from aspen. Plant Physiology (Life Science Advances) 14,45–54.

Dwivedi, U.N., Campbell, W.H., Yu, J., Datla, R.S.S., Bugos, R.C., Chiang, V.L., Podila, G.K.,1994. Modification of lignin biosynthesis in transgenic Nicotiana through expressionof an antisense O-methyltransferase gene from Populus. Plant Molecular Biology 26,61–71.

Elkind, Y., Edwards, R., Marandad, M., Hedrick, S.A., Ribak, O., Dixon, R.A., Lamb, C.J., 1990.Abnormal plant development and down-regulation of phenylpropanoid biosynthesisin transgenic tobacco containing a heterologous phenylalanine ammonia-lyase gene.Proceedings of the National Academy of Sciences of the United States of America 87,9057–9061.

Eudes, A., George, A., Mukerjee, P., Kim, J.S., Pollet, B., Benke, P.I., Yang, F., Mitra, P., Sun, L.,Cetinkol, O.P., Chabout, S., Mouille, G., Soubigou-Taconnat, L., Balzergue, S., Singh, S.,Holmes, B.M., Mukhopadhyay, A., Keasling, J.D., Simmons, B.A., Lapierre, C., Ralph, J.,Loqué, D., 2012. Biosynthesis and incorporation of side-chain-truncated lignin mono-mers to reduce lignin polymerization and enhance saccharification. Plant BiotechnologyJournal 10, 609–620.

Fagerstedt, K.V., Kukkola, E.M., Koistinen, V.V., Takahashi, J., Marjamaa, K., 2010. Cellwall lignin is polymerized by class III secretable plant peroxidases in Norway spruce.Journal of Integrative Plant Biology 52, 186–194.

Farago, J., 2007. Biotechnological tools to improve ethanol production from plant biomass.Nova Biotechnologica 7, 63–68.

Feltus, F.A., Vandenbrink, J.P., 2012. Bioenergy grass feedstock: current options and pros-pects for trait improvement using emerging genetic, genomic, and systems biologytoolkits. Biotechnology for Biofuels 5, 80.

Fengel, D., Wegener, G., 1984. Lignin. In: Fengel, D., Wegener, G. (Eds.), Wood: Chemistry,Ultrastructure, Reactions. De Gruyter, Berlin, pp. 132–181.

Ferrer, J.L., Austin, M.B., Stewart, C., Noel, J.P., 2008. Structure and function of enzymesinvolved in the biosynthesis of phenylpropanoids. Plant Physiology and Biochemistry46, 356–370.

Fornalé, S., Shi, X., Chai, C., Encina, A., Irar, S., Capellades, M., Fuguet, E., Torres, J.L., Rovira,P., Puigdomènech, P., Rigau, J., Grotewold, E., Gray, J., Caparrós-Ruiz, D., 2010.ZmMYB31 directly represses maize lignin genes and redirects the phenylpropanoidmetabolic flux. The Plant Journal 64, 633–644.

Fornalé, S., Capellades, M., Encina, A., Wang, K., Irar, S., Lapierre, C., Ruel, K., Joseleau,J.P., Berenguer, J., Puigdomènech, P., Rigau, J., Caparrós-Ruiz, D., 2012. Alteredlignin biosynthesis improves cellulosic bioethanol production in transgenicmaize plants down-regulated for cinnamyl alcohol dehydrogenase. MolecularPlant 5, 817–830.

Franke, R., McMichael, M., Meyer, K., Shirley, A.M., Cusumano, J.C., Chapple, C., 2000.Modified lignin in tobacco and poplar plants over-expressing the Arabidopsis geneencoding ferulate 5-hydroxylase. The Plant Journal 22, 223–234.

Franke, R., Hemm, M.R., Denault, J.W., Ruegger, M.O., Humphreys, J.M., Chapple, C., 2002.Changes in secondary metabolism and deposition of an unusual lignin in the ref8mutant of Arabidopsis. The Plant Journal 30, 47–59.

Fu, C., Mielenz, J.R., Xiao, X., Ge, Y., Hamilton, C.Y., Rodriguez, M., Chen, F., Foston, M.,Ragauskas, A., Bouton, J., Dixon, R.A., Wang, Z.Y., 2011. Genetic manipulation oflignin reduces recalcitrance and improves ethanol production from switchgrass.Proceedings of the National Academy of Sciences of the United States of America108, 3803–3808.

Gill, G.P., Brown, G.R., Neale, D.B., 2003. A sequence mutation in the cinnamyl alcoholdehydrogenase gene associated with altered lignification in loblolly pine. Plant Bio-technology Journal 1, 253–258.

Goicoechea, M., Lacombe, E., Legay, S., Mihaljevic, S., Rech, P., Jauneau, A., Lapierre, C.,Pollet, B., Verhaegen, D., Chaubet-Gigot, N., Grima-Pettenati, J., 2005. EgMYB2, anew transcriptional activator from eucalyptus xylem, regulates secondary cell wallformation and lignin biosynthesis. The Plant Journal 43, 553–567.

Goto, M., Matsuoka, J., Sato, T., Ehara, H., Morita, O., 1994. Brownmidribmutant maize withreduced levels of phenolic acids ether-linked to the cell walls. Animal Feed Science andTechnology 48, 27–38.

Goujon, T., Ferret, V., Mila, I., Pollet, B., Ruel, K., Burlat, V., Joseleau, J.P., Barriere, Y.,Lapierre, C., Jouanin, L., 2003a. Down-regulation of the AtCCR1 gene in Arabidopsisthaliana: effects on phenotype, lignins and cell wall degradability. Planta 217,218–228.

Goujon, T., Sibout, R., Eudes, A., MacKay, J., Jouanin, L., 2003b. Genes involved in thebiosynthesis of lignin precursors in Arabidopsis thaliana. Plant Physiology and Bio-chemistry 41, 677–687.

Grabber, J.H., 2005. How do lignin composition, structure and cross-linking affectdegradability? Crop Science 45, 820–831.

Grabber, J.H., Jung, G.A., Abrams, S.M., Howard, D.B., 1992. Digestion kinetics of parenchy-ma and sclerenchyma cell walls isolated from orchard grass and switchgrass. CropScience 32, 806–810.

Grabber, J.H., Ralph, J., Hatfield, R.D., Quideau, S., 1997. p-Hydroxyphenyl, guaiacyland syringyl lignins have similar inhibitory effects on wall degradability. Journal ofAgricultural and Food Chemistry 45, 2530–2532.

Grabber, J.H., Ralph, J., Lapierre, C., Barriere, Y., 2004. Genetic and molecular basisof grass cell wall degradability: lignin–cell wall matrix interactions. Comptes RendusBiologies 327, 455–465.

Grabber, J.H., Hatfield, R.D., Lu, F., Ralph, J., 2008. Coniferyl ferulate incorporation intolignin enhances the alkaline delignification and enzymatic degradation of cell walls.Biomacromolecules 9, 2510–2516.

Grabber, J.H., Mertens, D.R., Kim, H., Funk, C., Lu, F.C., Ralph, J., 2009. Cell wall fermentationkinetics are impactedmore by lignin content and ferulate cross-linking than by lignincomposition. Journal of the Science of Food and Agriculture 89, 122–129.

Grabber, J.H., Schatz, P.F., Kim, H., Lu, F., Ralph, J., 2010. Identifying new ligninbioengineering targets: 1. Monolignol-substitute impacts on lignin formation andcell wall fermentability. BMC Plant Biology 10, 114.

Grand, C., Boudet, A.M., Ranjeva, R., 1982. Natural variations and controlled changes inlignification process. Holzforschung 36, 217–223.

Grant, E.H., Fujino, T., Beers, E.P., Brunner, A.M., 2010. Characterization of NAC domaintranscription factors implicated in control of vascular cell differentiation inArabidopsis and Populus. Planta 232, 337–352.

Gray, J., Caparrós-Ruiz, D., Grotewold, E., 2012. Grass phenylpropanoids: regulate beforeusing! Plant Science 184, 112–120.

Grenet, E., Barry, P., 1991. Microbial degradation of normal maize and bm3 maize in therumen observed by scanning electron microscopy. Journal of the Science of Foodand Agriculture 54, 199–210.

Grima-Pettenati, J., Goffner, D., 1999. Lignin genetic engineering revisited. Plant Science145, 51–65.

Grima-Pettenati, J., Soler, M., Camargo, E., Wang, H., 2012. Transcriptional regulation ofthe lignin biosynthetic pathway revisited: new players and insights. In: Jouanin, L.,Lapierre, C. (Eds.), Advances in Botanical Research, vol. 61. Academic Press,pp. 173–218.

Guillaumie, S., Pichon, M., Martinant, J.P., Bosio, M., Goffner, D., Barrière, Y., 2007.Differential expression of phenylpropanoid and related genes in brown-midrib bm1,bm2, bm3, and bm4 young near-isogenic maize plants. Planta 226, 235–250.

Guillaumie, S., Mzid, R., Méchin, V., Léon, C., Hichri, I., Destrac-Irvine, A., Trossat-Magnin,C., Delrot, S., Lauvergeat, V., 2010. The grapevine transcription factor WRKY2 influ-ences the lignin pathway and xylem development in tobacco. PlantMolecular Biology72, 215–234.

Guo, D., Chen, F., Inoue, K., Blount, J.W., Dixon, R.A., 2001a. Down-regulation of caffeic acid3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase in transgenicalfalfa: impacts on lignin structure and implications for the biosynthesis of G and Slignin. The Plant Cell 13, 73–88.

Guo, D., Chen, F., Wheeler, J., Winder, J., Selman, S., Peterson, M., Dixon, R.A., 2001b.Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation oflignin O-methyltransferases. Transgenic Research 10, 457–464.

Gupta, S.K., 2008. Isolation, Cloning and Characterization of Lignin BiosynthesisPathway Gene(s) 4-coumarate CoA ligase (4CL) from Leucaena leucocephala. PhDthesis National Chemical Laboratory, Pune, India.

Halpin, C., 2004. Advances in Botanical Research Incorporating Advances in PlantPathology. Academic Press, London.

Halpin, C., Knight, M.E., Foxon, G.A., Campbell, M.M., Boudet, A.M., Boon, J.J., Chabbert, B.,Tollier, M.T., Schuch, W., 1994. Manipulation of lignin quality by down-regulation ofcinnamyl alcohol dehydrogenase. The Plant Journal 6, 339–350.

Halpin, C., Holt, K., Chojecki, J., Oliver, D., Chabbert, B., Monties, B., Edwards, K., Barakate,A., Foxon, G.A., 1998. Brown-midribmaize (bm1)— a mutation affecting the cinnamylalcohol dehydrogenase gene. The Plant Journal 14, 545–553.

Halpin, C., Thain, S.C., Tilston, E.L., Guiney, E., Lapierre, C., Hopkins, D.W., 2007. Ecologicalimpacts of trees with modified lignin. Tree Genetics and Genomes 3, 101–110.

Handakumbura, P.P., Hazen, S.P., 2012. Transcriptional regulation of grass secondarycell wall biosynthesis: playing catch-up with Arabidopsis thaliana. Frontiers in PlantScience 3, 74.

Harakava, R., 2005. Genes encoding enzymes of the lignin biosynthesis pathway inEucalyptus. Genetics and Molecular Biology 28, 601–607.

Harris, D., DeBolt, S., 2010. Synthesis, regulation and utilization of lignocellulosic biomass.Plant Biotechnology Journal 8, 244–262.

He, X., Hall, M.B., Gallo-Meagher, M., Smith, R.L., 2003. Improvement of forage quality bydown-regulation of maize O-methyltransferase. Crop Science 43, 2240–2251.

Heath, R., Huxley, H., Stone, B., Spangenberg, G., 1998. cDNA cloning and differentialexpression of three caffeic acid O-methyltransferase homologues from perennialrye grass (Lolium perenne). Journal of Plant Physiology 153, 649–657.

Heitner, C., Dimmel, D., Schmidt, J., 2010. Lignins and Lignans: Advances in Chemistry.Taylor & Francis Ltd., Boca Raton, FL.

Hibino, T., Takabe, K., Kawazu, T., Shibata, D., Higuchi, T., 1995. Increase ofcinnamaldehyde groups in lignin of transgenic tobacco plants carrying an antisensegene for cinnamyl alcohol dehydrogenase. Bioscience, Biotechnology, and Biochemistry59, 929–931.

Higuchi, T., Ito, T., Umezawa, T., Hibino, T., Shibata, D., 1994. Red-brown color of lignifiedtissues of transgenic plants with antisense cad gene: wine red lignin from coniferylaldehyde. Journal of Biotechnology 37, 151–158.

Hisano, H., Nandakumar, R., Wang, Z.Y., 2009. Genetic modification of lignin biosynthesisfor improved biofuel production. In vitro Cellular and Developmental Biology - Plant45, 306–313.

Hoffmann, L., Besseau, S., Geoffroy, P., Ritzenthaler, C., Meyer, D., Lapierre, C., Pollet, B.,Legrand, M., 2004. Silencing of hydroxycinnamoyl-coenzyme A shikimate/quinatehydroxy cinnamoyl transferase affects phenylpropanoid biosynthesis. The Plant Cell16, 1446–1465.

Hopkins, D.W., Webster, E.A., Boerjan, W., Pilate, G., Halpin, C., 2007. Genetically modifiedlignin below ground. Nature Biotechnology 25, 168–169.

Horvath, L., Peszlen, I., Peralta, P., Kasal, B., Li, L., 2010. Mechanical properties of geneticallyengineered young aspen with modified lignin content and/or structure. Wood andFiber Science 42, 310–317.

Houtman, C.J., Atalla, R.H., 1995. Celluloses lignin interactions. Plant Physiology 107,977–984.

Howles, P.A., Sewalt, V.J.H., Paiva, N.L., Elkind, Y., Bate, N.J., Lamb, C., Dixon, R.A., 1996.Over-expression of L-phenylalanine ammonia-lyase in transgenic tobacco plants

























































































































































reveals control points for flux into phenylpropanoid biosynthesis. Plant Physiology112, 1617–1624.

Hu, W.J., Harding, S.A., Lung, J., Chiang, V.L., Ralph, J., Douglas, D.S., Tsai, C.J., Chiang, V.L.,1999. Repression of lignin biosynthesis promotes cellulose accumulation and growthin transgenic trees. Nature Biotechnology 17, 808–812.

Huayan, Z., Jianhua, W., Jinyu, Z., Huirong, L., Tai, W., Yanru, S., 2002. Lignin biosynthesisby suppression of two O-methyltransferases. Chinese Science Bulletin 47, 1092–1095.

Humphreys, J.M., Chapple, C., 2002. Rewriting the lignin roadmap. Current Opinion inPlant Biology 5, 224–229.

Humphreys, J.M., Hemm, M.R., Chapple, C., 1999. New routes for lignin biosynthesisdefined by biochemical characterization of recombinant ferulate 5-hydroxylase,a multifunctional cytochrome P450 dependent monooxygenase. Proceedings of theNational Academy of Sciences of the United States of America 96, 10045–10050.

Huntley, S.K., Ellis, D., Gilbert, M., Chapple, C., Mansfield, S.D., 2003. Significant increasesin pulping efficiency in C4H–F5H-transformed poplars: improved chemical savingsand reduced environmental toxins. Journal of Agricultural and Food Chemistry 51,6178–6183.

Jackson, L.A., Shadle, G.L., Zhou, R., Nakashima, J., Chen, F., Dixon, R.A., 2008. Improvingsaccharification efficiency of alfalfa stems throughmodification of the terminal stagesof monolignol biosynthesis. Bioenergy Research 1, 180–192.

Jhala, A.J., Weselake, R.J., Hall, L.M., 2009. Genetically engineered flax: potential benefits,risks, regulations and mitigation of transgene movement. Crop Science 49,1943–1954.

JinHua, L., QiWen, Z., ZhengTian, N., MengZhu, L., Douglas, C.J., 2007. Advances in study oflignin biosynthesis and genetic engineering modification. World Forestry Research20, 29–37.

Johnson, J.M.F., Coleman, M.D., Gesch, R., Jaradat, A., Mitchell, R., Reicosky, D., Wilhelm,W.W., 2007. Biomass–bioenergy crops in the United States: a changing paradigm.The Americas Journal of Plant Science and Biotechnology 1, 1–28.

Jones, L., Ennos, A.R., Turner, S.R., 2001. Cloning and characterization of irregular xylem4(irx4): a severely lignin deficient mutant of Arabidopsis. Plant Journal 26, 205–216.

Jouanin, L., Goujon, T., deNadai, V., Martin, M.T., Mila, I., Vollet, C., Pollet, B., Yoshinaga, A.,Chabbert, B., Petit-Conil, M., Lapierre, C., 2000. Lignification in transgenic poplarswith extremely reduced caffeic acid O-methyltransferase activity. Plant Physiology123, 1363–1373.

Jung, H.J.G., Deetz, D.A., 1993. Cell wall lignification and degradability. In: Jung, H.J.G.,Buxton, D.R., Hatfield, R.D., Ralph, J. (Eds.), Forage CellWall Structure andDigestibility.American Society of Agronomy, Madison, WI, pp. 315–340.

Jung, H.J.G., Ni, W., 1998. Lignification of plant cell walls: impact of genetic manipulation.Proceedings of the National Academy of Sciences of the United States of America 95,12742–12743.

Jung, H.J.G., Mertens, D.R., Payne, A.J., 1997a. Correlation of acid detergent ligninand Klason lignin with digestibility of forage dry matter and neutral detergentfiber. Journal of Dairy Science 80, 1622–1628.

Jung, H.J.G., Sheaffer, C.C., Barnes, D.K., Halgerson, J.L., 1997b. Forage quality variation inthe US alfalfa core collection. Crop Science 37, 1361–1366.

Jung, H.J.G., Ni, W.T., Chapple, C.C.S., Meyer, K., 1999. Impact of lignin composition oncell wall degradability in an Arabidopsis mutant. Journal of the Science of Food andAgriculture 79, 922–928.

Jung, H.J.G., Fouad, W.M., Vermerris, W., Gallo, M., Altpeter, F., 2012a. RNAi suppressionof lignin biosynthesis in sugarcane reduces recalcitrance for biofuel productionfrom lignocellulosic biomass. Plant Biotechnology Journal 10, 1067–1076.

Jung, H.J., Samac, D.A., Sarath, G., 2012b. Modifying crops to increase cell wall digestibility.Plant Science 185–186, 65–77.

Kajita, S., Katayama, Y., Omori, S., 1996. Alterations in the biosynthesis of lignin intransgenic plants with chimeric genes for 4-coumarate: coenzyme A ligase. Plantand Cell Physiology 37, 957–965.

Kajita, S., Hishiyama, S., Tomimura, Y., Katayama, Y., Omori, S., 1997. Structuralcharacterization of modified lignin in transgenic tobacco plants in which the activityof 4-coumarate: coenzyme A ligase is depressed. Plant Physiology 114, 871–879.

Kajita, S., Ishifuji, M., Ougiya, H., Hara, S.I., Kawabata, H., Morohoshi, N., Katayama, Y.,2002. Improvement in pulping and bleaching properties of xylem from transgenictobacco plants. Journal of the Science of Food and Agriculture 82, 1216–1223.

Kaneda, M., Schuetz, M., Lin, B., Chanis, C., Hamberger, B., Western, T., Ehlting, J., Samuels,A., 2011. ABC transporters coordinately expressed during lignification of Arabidopsisstems include a set of ABCBs associated with auxin transport. Journal of ExperimentalBotany 62, 2063–2077.

Kavousi, B., Daudi, A., Cook, C.M., Joseleau, J.P., Ruel, K., Devoto, A., Bolwell, G.P., Blee, K.A.,2010. Consequences of antisense down-regulation of a lignification-specificperoxidase on leaf and vascular tissue in tobacco lines demonstrating enhancedenzymic saccharification. Phytochemistry 71, 531–542.

Kawaoka, A., Ebinuma, H., 2001. Transcriptional control of lignin biosynthesis by tobaccoLIM protein. Phytochemistry 57, 1149–1157.

Kawaoka, A., Nanto, K., Ishii, K., Ebinuma, H., 2006. Reduction of lignincontent by suppression of expression of the LIM domain transcription factor inEucalyptus camaldulensis. Silvae Genetica 55, 269–277.

Kim, W.C., Ko, J.H., Han, K.H., 2012. Identification of a cis-acting regulatory motifrecognized byMYB46, amaster transcriptional regulator of secondarywall biosynthesis.Plant Molecular Biology 78, 489–501.

Ko, J.H., Han, K.H., 2004. Arabidopsiswhole-transcriptome profiling defines the features ofcoordinated regulations that occur during secondary growth. PlantMolecular Biology55, 433–453.

Koehler, L., Telewski, F.W., 2006. Biomechanics and transgenic wood. American Journal ofBotany 93, 1433–1438.

Korth, K.L., Blount, J.W., Chen, F., Rasmussen, S., Lamb, C., Dixon, R.A., 2001. Changesin phenylpropanoid metabolites associated with homology dependent silencing of

phenylalanine ammonia-lyase and its somatic reversion in tobacco. PhysiologiaPlantarum 111, 137–143.

Kristensen, B., Bloch, H., Rasmussen, S.K., 1999. Barley coleoptile peroxidases:purification, molecular cloning and induction by pathogens. Plant Physiology120, 501–512.

Kwiatkowska, M.W., Starzycki, M., Zebrowski, J., Oszmiański, J., Szopa, J., 2007. Lignindeficiency in transgenic flax resulted in plants with improved mechanical properties.Journal of Biotechnology 128, 919–934.

Lacayo, C.I., Hwang, M.S., Ding, S.Y., Thelen, M.P., 2013. Lignin depletion enhances thedigestibility of cellulose in cultured xylem cells. PLoS ONE 8, e68266.

LaFayette, P.R., Eriksson, K.E.L., Dean, J.F.D., 1999. Characterization andheterologous expression of laccase cDNAs from xylem tissues of yellow-poplar(Liriodendron tulipifera). Plant Molecular Biology 40, 23–35.

Lagrimini, L.M., 1993. Analysis of peroxidase functions in transgenic plants. In: Welinder,K.G. (Ed.), Plant Peroxidase: Biochemistry, Physiology. University of Geneva,Switzerland, pp. 301–306.

Lagrimini, L.M., Vaughn, J., Erb, W., Miller, S.A., 1993. Peroxidase over-production intomato: wound-induced polyphenol deposition and disease resistance. HortScience28, 218–221.

Lagrimini, L.M., Gingas, V., Finger, F., Rothstein, S., Liu, T.T.Y., 1997. Characterization of anti-sense transformed plants deficient in the tobacco anionic peroxidase. Plant Physiology114, 1187–1196.

Lam, T.B.T., Iiyama, K., Stone, B.A., 2003. Hot alkali-labile linkages in the wall of the foragegrass Phalaris aquatica and Lolium perenne and their relation to in vitrowall digestibil-ity. Phytochemistry 64, 603–607.

Lapierre, C., Pollet, B., Petit-Conil, M., Toval, G., Romero, J., Pilate, G., Leple, J.C., Boerjan,W.,Ferrat, V., Nadai, V.D., Jouanin, L., 1999. Structural alterations in lignins in transgenicpoplars with depressed cinnamyl alcohol dehydrogenase or caffeic acid O-methyltransferase activity have an opposite impact on the efficiency of industrialkraft pulping. Plant Physiology 119, 153–163.

Lapierre, C., Pollet, B., MacKay, J.J., Sederoff, R.R., 2000. Lignin structure in a mutantpine deficient in cinnamyl alcohol dehydrogenase. Journal of Agricultural and FoodChemistry 48, 2326–2331.

Laskar, D.D., Jourdes, M., Patten, A.M., Helms, G.L., Davin, L.B., Lewis, N.G., 2006.The Arabidopsis cinnamoyl CoA reductase irx4 mutant has a delayed but coherent(normal) program of lignification. The Plant Journal 48, 674–686.

Lee, D., Ellard, M., Wanner, L.A., Davis, K.R., Douglas, C.J., 1995. The Arabidopsis thaliana 4-coumarate CoA ligase (4CL) gene: stress and developmentally regulated expressionand nucleotide sequence of its cDNA. Plant Molecular Biology 28, 871–884.

Lee, Y., Chen, F., Gallego-Giraldo, L., Dixon, R.A., Voit, E.O., 2011. Integrative analysisof transgenic alfalfa (Medicago sativa L.) suggests newmetabolic control mechanismsfor monolignol biosynthesis. PLoS Computational Biology 7, e1002047.

Leplé, J.C., Dauwe, R., Morreel, K., Storme, V., Lapierre, C., Pollet, B., Naumann, A., Kang,K.Y., Kim, H., Ruel, K., Lefèbvre, A., Joseleau, J.P., Grima-Pettenati, J., Rycke, R.D.,Andersson-Gunnerås, S., Erban, A., Fehrle, I., Petit-Conil, M., Kopka, J., Polle, A.,Messens, E., Sundberg, B., Mansfield, S.D., Ralph, J., Pilate, G., Boerjan, W., 2007.Down-regulation of cinnamoyl coenzyme A reductase in poplar: multiple-levelphenotyping reveals effects on cell wall polymer metabolism and structure. ThePlant Cell 19, 3669–3691.

Lewis, N.G., Davin, L.B., Sarkanen, S., 1999. The nature and function of lignins. In: Barton,D.H.R., Nakanishi, K., Meth-Cohn, O. (Eds.), Comprehensive Natural ProductsChemistry, vol. 3. Elsevier, pp. 617–745.

Li, X., Chapple, C., 2010. Understanding lignification: challenges beyond monolignolbiosynthesis. Plant Physiology 154, 449–452.

Li, Y., Kajita, S., Kawai, S., Katayama, Y., Morohoshi, N., 2003. Down-regulationof an anionic peroxidase in transgenic aspen and its effect on lignin characteristics.Journal of Plant Research 116, 175–182.

Li, L., Zhou, Y., Cheng, X., Sun, J., Marita, J.M., Ralph, J., Chiang, V.L., 2003. Combinatorialmodification of multiple lignin traits in trees through multigene cotransformation.Proceedings of the National Academy of Sciences of the United States of America100, 4939–4944.

Li, X., Weng, J.K., Chapple, C., 2008. Improvement of biomass through lignin modification.The Plant Journal 54, 569–581.

Li, X., Ximenes, E., Kim, Y., Slininger, M., Meilan, R., Ladisch, M., Chapple, C., 2010. Ligninmonomer composition affects Arabidopsis cell wall degradability after liquid hotwater pretreatment. Biotechnology for Biofuels 3, 27–33.

Li, E., Bhargava, A., Qiang, W., Friedmann, M.C., Forneris, N., Savidge, R.A., Johnson, L.A.,Mansfield, S.D., Ellis, B.E., Douglas, C.J., 2012. The class II KNOX gene KNAT7negatively regulates secondary wall formation in Arabidopsis and is functionallyconserved in Populus. New Phytologist 194, 102–115.

Li, M., Foster, C., Kelkar, S., Pu, Y., Holmes, D., Ragauskas, A., Saffron, C.M., Hodge, D.B.,2012. Structural characterization of alkaline hydrogen peroxide pretreated grassesexhibiting diverse lignin phenotypes. Biotechnology for Biofuels 5, 38.

Lin, Y., Tanaka, T., 2006. Ethanol fermentation from biomass resources: current state andprospects. Applied Microbiology and Biotechnology 69, 627–642.

Liu, C.J., 2012. Deciphering the enigma of lignification: precursor transport, oxidation, andthe topochemistry of lignin assembly. Molecular Plant 5, 304–317.

Liu, C.J., Miao, Y.C., Zhang, K.W., 2011. Sequestration and transport of lignin monomericprecursors. Molecules 16, 710–727.

Lu, S., Li, L., Zhou, G., 2010. Genetic modification of wood quality for second-generationbiofuel production. GM Crops 1, 230–236.

Ma, Q.H., Wang, C., Zhu, H.H., 2011. TaMYB4 cloned from wheat regulates ligninbiosynthesis through negatively controlling the transcripts of both cinnamyl alcoholdehydrogenase and cinnamoyl CoA reductase genes. Biochimie 93, 1179–1186.

Maher, E.A., Bate, N.J., Ni, W., Elkind, Y., Dixon, R.A., Lamb, C.J., 1994. Increased diseasesusceptibility of transgenic tobacco plants with suppressed levels of preformed
































































































































































phenylpropanoid products. Proceedings of the National Academy of Sciences of theUnited States of America 91, 7802–7806.

Marita, J.M., Ralph, J., Hatfield, R.D., Chapple, C., 1999. NMR characterization of ligninsin Arabidopsis altered in the activity of ferulate 5-hydroxylase. Proceedingsof the National Academy of Sciences of the United States of America 96,12328–12332.

Marita, J.M., Ralph, J., Hatfield, R.D., Guo, D., Chen, R., Dixon, R.A., 2003a. Structuraland compositional modifications in lignin of transgenic alfalfa down-regulated incaffeic acid 3-O-methyltransferase and caffeoyl coenzyme A 3-O-methyltransferase.Phytochemistry 62, 53–65.

Marita, J.M., Vermerris, W., Ralph, J., Hatfield, R.D., 2003b. Variations in the cell wallcomposition of maize brown midrib mutants. Journal of Agricultural and FoodChemistry 51, 1313–1321.

Marjamaa, K., Kukkola, E.M., Fagerstedt, K.V., 2009. The role of xylem class III peroxidasesin lignification. Journal of Experimental Botany 60, 367–376.

Martz, F., Maury, S., Pincon, G., Legrand, M., 1998. cDNA cloning, substrate specificity andexpression study of tobacco CCOAOMT, a lignin biosynthetic enzyme. Plant MolecularBiology 36, 427–437.

McCann, M.C., Carpita, N.C., 2008. Designing the deconstruction of plant cell walls.Current Opinion in Plant Biology 11, 314–320.

McCarthy, R.L., Zhong, R., Fowler, S., Lyskowski, D., Piyasena, H., Carleton, K., Spicer, C., Ye,Z.H., 2010. The poplar MYB transcription factors, PtrMYB3 and PtrMYB20, areinvolved in the regulation of secondary wall biosynthesis. Plant and Cell Physiology51, 1084–1090.

McIntyre, C.L., Bettenay, H.M., Manners, J.M., 1996. Strategies for the suppression ofperoxidase gene expression in tobacco: in vivo suppression of peroxidase activity intransgenic tobacco using ribozyme and antisense constructs. Transgenic Research 5,263–270.

Mechin, V., Argillier, O., Menanteau, V., Barriere, Y., Mila, I., Pollet, B., Lapierre, C., 2000.Relationship of cell wall composition to in vitro cell wall digestibility of maize inbredline stems. Journal of the Science of Food and Agriculture 80, 574–580.

Meyer, K., Shirley, A.M., Cusumano, J.C., Bell-Lelong, D.A., Chapple, C., 1998. Lignin mono-mer composition is determined by the expression of a cytochrome P450-dependentmonooxygenases in Arabidopsis. Proceedings of the National Academy of Sciencesof the United States of America 95, 6619–6623.

Meyermans, H., Morreel, K., Lapierre, C., Pollet, B., Bruyn, D.A., Busson, R., Herdewijn, P.,Devreese, B., Beeumen, V.J., Marita, J.M., Ralph, J., Chen, C., Burggraeve, B., Montagu,V.M., Messens, E., Boerjan, W., 2000. Modifications in lignin and accumulation ofphenolic glucosides in poplar xylem upon down-regulation of caffeoyl coenzyme AO-methyltransferase, an enzyme involved in lignin biosynthesis. Journal of BiologicalChemistry 275, 36899–36909.

Miao, Y.C., Liu, C.J., 2010. ATP-binding cassette-like transporters are involved in the trans-port of lignin precursors across plasma and vacuolar membranes. Proceedings of theNational Academy of Sciences of the United States of America 107, 22728–22733.

Minic, Z., Jamet, E., San-Clemente, H., Pelletier, S., Renou, J.P., Rihouey, C., Okinyo, D.P.,Proux, C., Lerouge, P., Jouanin, L., 2009. Transcriptomic analysis of Arabidopsisdeveloping stems: a close-up on cell wall genes. BMC Plant Biology 9, 6.

Morreel, K., Ralph, J., Kim, H., Lu, F., Goeminne, G., Ralph, S.A., Messens, E., Boerjan, W.,2004. Profiling of oligolignols reveals monolignol coupling conditions in lignifyingpoplar xylem. Plant Physiology 136, 3537–3549.

Morreel, K., Dima, O., Kim, H., Lu, F., Niculaes, C., Vanholme, R., Dauwe, R., Goeminne, G.,Inze, D., Messens, E., Ralph, J., Boerjan,W., 2010. Mass spectrometry-based sequencingof lignin oligomers. Plant Physiology 153, 1464–1478.

Nakashima, J., Chen, F., Jackson, L., Shadle, G., Dixon, R.A., 2008. Multi-site geneticmodification of monolignol biosynthesis in alfalfa (Medicago sativa): effects on lignincomposition in specific cell types. New Phytologist 179, 738–750.

Nehra, N.S., Becwar, M.R., Rottmann, W.H., Pearson, L., Chowdhury, K., Chang, S., Wilde,H.D., Kodrzycki, R.J., Zhang, C., Gause, K.C., Parks, D.W., Hinchee, M.A., 2005.Forest biotechnology: innovative methods, emerging opportunities. In vitro Cellularand Developmental Biology - Plant 41, 701–717.

Ni, W., Paiva, N.L., Dixon, R.A., 1994. Reduced lignin in transgenic plants containing acaffeic acid O-methyltransferase antisense gene. Transgenic Research 3, 120–126.

Nieminen, K., Robischon, M., Immanen, J., Helariutta, Y., 2012. Towards optimizing wooddevelopment in bioenergy trees. New Phytologist 194, 46–53.

Novaes, E., Kirst, M., Chiang, V., Sederoff, H.W., Sederoff, R., 2010. Lignin and biomass:a negative correlation for wood formation and lignin content in trees. Plant Physiology154, 555–561.

O'Connell, A., Lapierre, C., Pollet, B., 1998. Reduction of lignin content and significantchanges to lignin profiles through inhibition of cinnamoyl coenzyme A reductase.Proceedings of 8th International Cell Wall Meeting, Norwich.

O'Connell, A., Holt, K., Piquemal, J., Grima-Pettenati, J., Boudet, A., Pollet, B., Lapierre, C.,Petit-Conil, M., Schuch, W., Halpin, C., 2002. Improved paper pulp from plants withsuppressed cinnamoyl CoA reductase or cinnamyl alcohol dehydrogenase. TransgenicResearch 11, 495–503.

Ogras, T.T., Kazan, K., Gozukirmiri, V., 2000. Reduction of lignin in tobacco through theexpression of an antisense caffeic acid O-methyltransferase. Turkish Journal of Botany24, 221–226.

Ohman, D., Demedts, B., Kumar, M., Gerber, L., Gorzsás, A., Goeminne, G., Hedenström, M.,Ellis, B., Boerjan, W., Sundberg, B., 2013. MYB103 is required for ferulate-5-hydroxylase expression and syringyl lignin biosynthesis in Arabidopsis stems. ThePlant Journal 73, 63–76.

Ohtani, M., Nishikubo, N., Xu, B., Yamaguchi, M., Mitsuda, N., Goue, N., Shi, F., OhmeTakagi, M., Demura, T., 2011. A NAC domain protein family contributing to theregulation of wood formation in poplar. The Plant Journal 67, 499–512.

O'Malley, D.M., Whetten, R.W., Bao, W., Chen, C.L., Sederoff, R.R., 1993. The role of laccasein lignification. The Plant Journal 4, 751–757.

Palit, P., Sengupta, G., Datta, P., Meshram, J., 2001. Lignin and lignification with specialreference to its down-regulation for the improvement of wood and bastwood quality.Indian Journal of Plant Physiology 6, 217–228.

Parijs, V.F.R.D., Morreel, K., Ralph, J., Boerjan, W., Merks, R.M.H., 2010. Modeling ligninpolymerization. I. Simulation model of dehydrogenation polymers. Plant Physiology153, 1332–1344.

Pattathil, S., Saffold, T., Gallego-Giraldo, L., O'Neill, M., York, W.S., Dixon, R.A., Hahn, M.G.,2012. Changes in cell wall carbohydrate extractability are correlated with reducedrecalcitrance of HCT down-regulated alfalfa biomass. Industrial Biotechnology 8,217–221.

Patten, A.M., Jourdes, M., Cardenas, C.L., Laskar, D.D., Nakazawa, Y., Chung, B.Y., Franceschi,V.R., Davin, L.B., Lewis, N.G., 2010. Probing native lignin macromolecular configura-tion in Arabidopsis thaliana in specific cell wall types: further insights into limitedsubstrate degeneracy and assembly of the lignins of ref8, fah 1-2 and C4H:F5H lines.Molecular BioSystems 6, 499–515.

Pilate, G., Guiney, E., Holt, K., Petit-Conil, M., Lapierre, C., Leple, J.C., Pollet, B., Mila, I.,Webster, E.A., Marstorp, H.G., Hopkins, D.W., Jouanin, L., Boerjan, W., Schuch, W.,Cornu, D., Halpin, C., 2002. Field and pulping performances of transgenic trees withaltered lignification. Nature Biotechnology 20, 607–612.

Pinchon, G., Chabannes, M., Lapierre, C., Pollet, B., Ruel, K., Joseleau, J.P., Boudet, A.M.,Legrand, M., 2001a. Simultaneous down-regulation of caffeic/5-hydroxy ferulicacid-O-methyltransferase I and cinnamoyl coenzyme A reductase in the progenyfrom a cross between tobacco lines homozygous for each transgene: consequencesfor plant development and lignin synthesis. Plant Physiology 126, 145–155.

Pinchon, G., Maury, S., Hoffmann, L., Geoffroy, P., Lapierre, C., Pollet, B., Legrand, M., 2001b.Repression of O-methyltransferase genes in transgenic tobacco affects ligninsynthesis and plant growth. Phytochemistry 57, 1167–1176.

Piquemal, J., Lapierre, C., Myton, K., O'Connell, A., Schuch, W., Grima-Pettenati, J., Boudet,A.M., 1998. Down-regulation of cinnamoyl coenzyme A reductase induces significantchanges of lignin profiles in transgenic tobacco plants. The Plant Journal 13, 71–83.

Piquemal, J., Chamayou, S., Nadaud, I., Beckert, M., Barriere, Y., Mila, I., Lapierre, C., Rigau,J., Puigdomenech, P., Jauneau, A., Digonnet, C., Boudet, A., Goffner, D., Pichon, M.,2002. Down-regulation of caffeic acid O-methyltransferase in maize revisited usinga transgenic approach. Plant Physiology 130, 1675–1685.

Prashant, S., Srilakshmi, S.M., Pramod, S., Gupta, R.K., Kumar, A.S., Rao, K.S., Rawal, S.K.,Kavi Kishor, P.B., 2011. Down-regulation of Leucaena leucocephala cinnamoyl CoAreductase (LlCCR) gene induces significant changes in phenotype, soluble phenolicpools and lignin in transgenic tobacco. Plant Cell Reports 30, 2215–2231.

Pu, Y., Chen, F., Ziebell, A., Davison, B.H., Ragauskas, A.J., 2009. NMR characterization ofC3H and HCT down-regulated alfalfa lignin. Bioenergy Research 2, 198–208.

Ragauskas, A.J., Williams, C.K., Davison, B.H., Britovsek, G., Cairney, J., Eckert, C.A.,Frederick Jr., W.J., Hallett, J.P., Leak, D.J., Liotta, C.L., Mielenz, J.R., Murphy, R.,Templer, R., Tschaplinski, T., 2006. The path forward for biofuels and biomaterials.Science 311, 484–489.

Rahantamalala, A., Rech, P., Martinez, Y., Chaubet-Gigot, N., Grima-Pettenati, J., Pacquit, V.,2010. Coordinated transcriptional regulation of two key genes in the lignin branchpathway – CAD and CCR – is mediated through MYB-binding sites. BMC Plant Biology10, 130.

Ralph, J., 2006. What makes a good monolignol substitute? In: Hayashi, T. (Ed.),The Science and Lore of the Plant Cell Wall: Biosynthesis, Structure and Function.Brown Walker Press, Boca Raton, FL, pp. 285–293.

Ralph, J., Brunow, G., 2006. Lignification: are lignins biosynthesized via simple combina-torial chemistry or via proteinaceous control and template replication? PolyphenolsCommunications. XXIII International Conference Polyphenols, Winnipeg, Manitoba,Canada, pp. 27–28.

Ralph, J., Mackay, J.J., Hatfield, R.D., O'Malley, D.M., Whetten, R.W., Sederoff, R.R., 1997.Abnormal lignin in loblolly pine mutant. Science 277, 235–239.

Ralph, J., Hatfield, R.D., Piquemal, J., Yahiaoui, N., Pean, M., Lapierre, C., Boudet, A.M., 1998.NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamyl alcohol dehydrogenase and cinnamoylcoenzyme A reductase. Proceedings of the National Academy of Sciences of theUnited States of America 95, 12803–12808.

Ralph, J., Lapierre, C., Marita, J.M., Kim, H., Lu, F., Hatfield, R.D., Ralph, S., Chapple, C.,Franke, R., Hemm, M.R., Doorssalaere, V.J., Sederoff, R.R., O'Malley, D.M., Scott, J.T.,MacKay, J.J., Yahiaoui, N., Boudet, A., Pean, M., Pilate, G., Jouanin, L., Boerjan, W.,2001. Elucidation of new structures in lignins of CAD- and COMT-deficient plantsby NMR. Phytochemistry 57, 993–1003.

Ralph, J., Lundquist, K., Brunow, G., Lu, F., Kim, H., Schatz, P.F., Marita, J.M., Hatfield, R.D.,Ralph, S.A., Christensen, J.H., Boerjan,W., 2004. Lignins: natural polymers from oxida-tive coupling of 4-hydroxy phenylpropanoids. Phytochemistry Reviews 3, 29–60.

Ralph, J., Akiyama, T., Kim, H., Lu, F., Reddy, M.S.S., Chen, F., Dixon, R.A., 2006. Effects ofcoumarate 3-hydroxylase down-regulation on lignin structure. Journal of BiologicalChemistry 281, 8843–8853.

Ranocha, P., McDougall, G., Hawkins, S., Sterjiades, R., Borderies, G., Stewart, D., Cabanes-Macheteau, M., Boudet, A.M., Goffner, D., 1999. Biochemical characterization,molecular cloning and expression of laccases – a divergent gene family – in poplar.European Journal of Biochemistry 259, 485–495.

Ranocha, P., Chabannes, M., Chamayou, S., Danoun, S., Jauneau, A., Boudet, A.M., Goffner,D., 2002. Laccase down-regulation causes alterations in phenolic metabolism andcell wall structure in poplar. Plant Physiology 129, 145–155.

Rastogi, S., Dwivedi, U.N., 2003. Genetic engineering for modifications of lignin. In: Singh,R.P., Jaiwal, P.K. (Eds.), Plant Genetic Engineering: Applications and Limitations, vol. 1.SciTech Publishing, Houston, USA, pp. 317–360.

Rastogi, S., Dwivedi, U.N., 2006. Down-regulation of lignin biosynthesis in transgenicLeucaena leucocephala harboring O-methyltransferase gene. Biotechnology Progress22, 609–616.













































































































































Rastogi, S., Dwivedi, U.N., 2008. Manipulation of lignin in plants with special reference toO-methyltransferase. Plant Science 174, 264–277.

Reddy, M.S.S., Chen, F., Shadle, G., Jackson, L., Aljoe, H., Dixon, R.A., 2005. Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa(Medicago sativa L.). Proceedings of the National Academy of Sciences of the UnitedStates of America 102, 16573–16578.

Rest, B.V.D., Danoun, S., Boudet, A.M., Rochange, S.F., 2006. Down-regulation of cinnamoylCoA reductase in tomato (Solanum lycopersicum L.) induces dramatic changes insoluble phenolic pools. Journal of Experimental Botany 57, 1399–1411.

Riboulet, C., Guillaumie, S., Mechin, V., Bosio, M., Pichon, M., Goffner, D., Lapierre, C., Pollet,B., Lefevre, B., Martinant, J.P., Barriere, Y., 2009. Kinetics of phenylpropanoid geneexpression in maize growing internodes: relationships with cell wall deposition.Crop Science 49, 211–223.

Rodriguez, M.R., Haseltine, C., Huess-Larossa, K., Clemente, T., Soto, J., Staswick, P., Blum,P., 2000. Autohydrolysis of plant polysaccharides using transgenic hyperthermophilicenzymes. Biotechnology and Bioengineering 70, 151–159.

Rogers, L.A., Campbell, M.M., 2004. The genetic control of lignin deposition during plantgrowth and development. New Phytologist 164, 17–30.

Rohde, A., Morreel, K., Ralph, J., Goeminne, G., Hostyn, V., De Rycke, R., Kushnir, S.,Doorssalaere, J.V., Joseleau, J.P., Vuylsteke, M., Driessche, G.V., Beeumen, J.V.,Messens, E., Boerjan, W., 2004. Molecular phenotyping of the pal1 and pal2 mutantsof Arabidopsis thaliana reveals far-reaching consequences on phenylpropanoid,amino acid and carbohydrate metabolism. The Plant Cell 16, 2749–2771.

Ruel, K., Berrio-Sierra, J., Derikvand, M.M., Pollet, B., Thevenin, J., Lapierre, C., Jouanin, L.,Joseleau, J.P., 2009. Impact of CCR1 silencing on the assembly of lignified secondarywalls in Arabidopsis thaliana. New Phytologist 184, 99–113.

Rushton, P.J., Somssich, I.E., Ringler, P., Shen, Q.J., 2010. WRKY transcription factors.Trends in Plant Science 15, 247–258.

Saballos, A., Vermerris, W., Rivera, L., Ejeta, G., 2008. Allelic association, chemicalcharacterization and saccharification properties of brown midrib mutants of sorghum(Sorghum bicolor (L.) Moench). Bioenergy Research 1, 193–204.

Saballos, A., Ejeta, G., Sanchez, E., Kang, C.H., Vermerris, W., 2009. A genomewide analysisof the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L.)Moench] identifies SbCAD2 as the Brown midrib6 gene. Genetics 181, 783–795.

Saballos, A., Sattler, S.E., Sanchez, E., Foster, T.P., Xin, Z., Kang, C., Pedersen, J.F., Vermerris,W., 2012. Brown midrib2 (Bmr2) encodes the major 4-coumarate: coenzyme A ligaseinvolved in lignin biosynthesis in sorghum (Sorghum bicolor (L.) Moench). The PlantJournal 70, 818–830.

Sattler, S.E., Funnell-Harris, D.L., 2013. Modifying lignin to improve bioenergy feedstocks:strengthening the barrier against pathogens? Frontiers in Plant Science 4, 70.

Sattler, S., Saathoff, A.J., Palmer, N.A., Funnell-Harris, D.L., Sarath, G., Pedersen, J.F., 2009.A nonsense mutation in a cinnamyl alcohol dehydrogenase gene is responsible forthe sorghum brown midrib 6 phenotype. Plant Physiology 150, 584–595.

Sederoff, R.R., 1999. Building better trees with antisense. Nature Biotechnology 17,750–751.

Sederoff, R.R., Campbell, M.M., O'Malley, D., Whetten, R., 1994. Genetic regulation oflignin biosynthesis and potential modification of wood by genetic engineering inloblolly pine. In: Ellis, E.E., Kuroki, G.W., Stafford, H.A. (Eds.), Recent Advances inPhytochemistry: Genetic Engineering of Plant Secondary Metabolism. Plenum Press,pp. 313–355.

Sederoff, R.R., Mackay, J.J., Ralph, J., Hatfield, R.D., 1999. Unexpected variation in lignin.Current Opinion in Plant Biology 2, 145–152.

Selman-Housein, G., Lopez, M.A., Hernandez, D., Civardi, L., Miranda, F., Rigau, J.,Puigdomenech, P., 1999. Molecular cloning of cDNAs coding for three sugarcaneenzymes involved in lignification. Plant Science 143, 163–171.

Sewalt, V.J.H., Glasser, W.G., Fontenot, J.P., Allen, V.G., 1996. Lignin impact on fiberdegradation. 1. Quinone methide intermediates formed from lignin duringin vitro fermentation of corn stover. Journal of the Science of Food and Agricul-ture 71, 195–203.

Sewalt, V.J.H., Ni, W., Blount, J.W., Jung, H.J.G., Masoud, S.A., Howles, P.A., Lamb, C.J., Dixon,R.A., 1997a. Reduced lignin content and altered lignin composition in transgenictobacco down-regulated in expression of L-phenylalanine ammonia-lyase orcinnamate 4-hydroxylase. Plant Physiology 115, 41–50.

Sewalt, V.J.H., Ni, W., Jung, H.J.G., Dixon, R.A., 1997b. Lignin impact on fiber degradation:increased enzymic digestibility on genetically engineered tobacco (Nicotiana tabaccum)stems reduced in lignin content. Journal of Agricultural and Food Chemistry 45,1977–1983.

Shadle, G., Chen, F., Srinivasa Reddy, M.S., Jackson, L., Nakashima, J., Dixon, R.A., 2007.Down-regulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyltransferase in transgenic alfalfa affects lignification, development and forage quality.Phytochemistry 68, 1521–1529.

Shen, H., Poovaiah, C.R., Ziebell, A., Tschaplinski, T.J., Pattathil, S., Gjersing, E., Engle, N.L.,Katahira, R., Pu, Y., Sykes, R., Chen, F., Ragauskas, A.J., Mielenz, J.R., Hahn, M.G.,Davis, M., Stewart Jr., C.N., Dixon, R.A., 2013. Enhanced characteristics of geneticallymodified switchgrass (Panicum virgatum L.) for high biofuel production. BiotechnolBiofuels 6, 71.

Simmons, B.A., Loque, D., Ralph, J., 2010. Advances in modifying lignin for enhancedbiofuel production. Current Opinion in Plant Biology 13, 313–320.

Sonbol, F.M., Fornale, S., Capellades, M., Encina, A., Tourino, S., Torres, J.L., Rovira, P.,Ruel, K., Puigdomenech, P., Rigau, J., Caparros-Ruiz, D., 2009. The maizeZmMYB42 represses the phenylpropanoid path-way and affects the cell wallstructure, composition and degradability in Arabidopsis thaliana. Plant MolecularBiology 70, 283–296.

Stasolla, Scott, J., Egertsdotter, U., Kadla, J., O'Malley, D., Sederoff, R.R., Zyl, L.V., 2003.Analysis of lignin produced by cinnamyl alcohol dehydrogenase-deficientPinus taeda cultured cells. Plant Physiology and Biochemistry 41, 439–445.

Stephens, J., Halpin, C., 2007. Lignin manipulation for fiber improvement. In: Ranalli, P.(Ed.), Improvement of Crop Plants for Industrial End Uses. Springer, Netherlands,pp. 129–153.

Stewart, D., Yahiaoui, N., McDougall, G.J., Myton, K., Marque, C., Boudet, A.M., Haigh, J.,1997. Fourier-transform infrared and Raman spectroscopic evidence for the incorpora-tion of cinnamaldehydes into the lignin of transgenic tobacco (Nicotiana tabaccum L.)plants with reduced expression of cinnamyl alcohol dehydrogenase. Planta 201,311–318.

Stewart, J.J., Akiyama, T., Chapple, C., Ralph, J., Mansfield, S.D., 2009. The effects on ligninstructure of overexpression of ferulate 5-hydroxylase in hybrid poplar. Plant Physiology150, 621–635.

Sticklen, M.B., 2006. Plant genetic engineering to improve biomass characteristics forbiofuels. Current Opinion in Biotechnology 17, 315–319.

Sticklen, M.B., 2008. Plant genetic engineering for biofuel production: towards affordablecellulosic ethanol. Nature Reviews Genetics 9, 433–443.

Talukder, K., 2006. Low-lignin wood — a case study. Nature Biotechnology 24, 395–396.Tamagnone, L., Merida, A., Parr, A., Mackay, S., Culianez-Macia, F.A., Roberts, K., Martin, C.,

1998. The AmMYB308 and AmMYB330 transcription factors from Antirrhinumregulate phenylpropanoid and lignin biosynthesis in transgenic tobacco. Plant Cell10, 135–154.

Tamasloukht, B., Lam, M.S.-J.W.Q., Martinez, Y., Tozo, K., Barbier, O., Jourda, C., Jauneau, A.,Borderie, G., Balzergue, S., Renou, J.P., Huguet, S., Martinant, J.P., Tatout, C., Lapierre, C.,Barrière, Y., Goffner, D., Pichon, M., 2011. Characterization of a cinnamoyl-CoAreductase 1 (CCR1) mutant in maize: effects on lignification, fibre development, andglobal gene expression. Journal of Experimental Botany 62, 3837–3848.

Terashima, N., Atalla, R.H., Ralph, S.A., Landucci, L.L., Lapierre, C., Monties, B., 1996.New preparations of lignin polymer models under conditions that approximate cellwall lignification. II. Structural characterization of the models by thioacidolysis.Holzforschung 50, 9–14.

Teutsch, H.G., Hasenfratz, M.P., Lesot, A., Stoltz, C., Garnier, J.M., Jeltsch, J.M., Durst, F.,Reichhart, W.D., 1993. Isolation and sequencing of a cDNA encoding the Jerusalemartichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in thegeneral phenylpropanoid pathway. Proceedings of the National Academy of Sciencesof the United States of America 90, 4102–4106.

Thévenin, J., Pollet, B., Letarnec, B., Saulnier, L., Gissot, L., Maia-Grondard, A., Lapierre, C.,Jouanin, L., 2011. The simultaneous repression of CCR and CAD, two enzymes of thelignin biosynthetic pathway, results in sterility and dwarfism in Arabidopsis thaliana.Molecular Plant 4, 70.

Thorstensson, E.M.G., Buxton, D.R., Cherney, J.H., 1992. Apparent inhibition to digestionby lignin in normal and brown midrib stems. Journal of the Science of Food and Agri-culture 59, 183–188.

Townsley, B.T., Sinha, N.R., Kang, J., 2013. KNOX1 genes regulate lignin deposition andcomposition in monocots and dicots. Frontiers in Plant Science 4, 121.

Tronchet, M., Balague, C., Kroj, T., Jouanin, L., Roby, D., 2010. Cinnamyl alcohol dehydroge-nases C and D, key enzymes in lignin biosynthesis, play an essential role in diseaseresistance in Arabidopsis. Molecular Plant Pathology 11, 83–92.

Tsai, C.J., Popko, J.L., Mielke, M.R., Hu, W.J., Podila, G.K., Chiang, V.L., 1998. Suppression ofO-methyltransferase gene by homologous sense transgene in quaking aspen causesred brown wood phenotypes. Plant Physiology 117, 101–112.

Vailhe, M.A.B., Migne, C., Cornu, A., Maillot, M.P., Grenet, E., Besle, J.M., Atanassova, R.,Martz, F., Legrand, M., 1996. Effect of modification of the O-methyltransferase activityon cell wall composition, ultrastructure and degradability of transgenic tobacco.Journal of the Science of Food and Agriculture 72, 385–392.

Vailhe, M.A.B., Besle, J.M., Maillot, M.P., Cornu, A., Halpin, C., Knight, M., 1998. Effect ofdown-regulation of cinnamyl alcohol dehydrogenase on cell wall composition andon degradability of tobacco stems. Journal of the Science of Food and Agriculture76, 505–514.

Vailhe, M.A.B., Provan, G.J., Scobbie, L., Chesson, A., Maillot, M.P., Cornu, A., Besle, J.M.,2000. Effect of phenolic structures on the degradability of cell walls isolatedfrom newly extended apical internode of tall fescue (Festuca arundinacea Schreb.).Journal of Agricultural and Food Chemistry 48, 618–623.

Vanholme, R., Morreel, K., Ralph, J., Boerjan,W., 2008. Lignin engineering. Current Opinionin Plant Biology 11, 278–285.

Vanholme, R., Demedts, B., Morreel, K., Ralph, J., Boerjan, W., 2010a. Lignin biosynthesisand structure. Plant Physiology 153, 895–905.

Vanholme, R., Ralph, J., Akiyama, T., Lu, F., Pazo, J.R., Kim, H., Christensen, J.H., Reusel, V.B.,Storme, V., De Rycke, R., Rohde, A., Morreel, K., Boerjan, W., 2010b. Engineeringtraditional monolignols out of lignin by concomitant up-regulation of F5H1 anddown-regulation of COMT in Arabidopsis. The Plant Journal 64, 885–897.

Vanholme, R., Van Acker, R., Boerjan, W., 2010c. Potential of Arabidopsis systems biologyto advance the biofuel field. Trends in Biotechnology 28, 543–547.

Vanholme, R., Morreel, K., Darrah, C., Oyarce, P., Grabber, J.H., Ralph, J., Boerjan, W., 2012.Metabolic engineering of novel lignin in biomass crops. New Phytologist 196,978–1000.

Vanholme, B., Cesarino, I., Goeminne, G., Kim, H., Marroni, F., Van Acker, R., Vanholme, R.,Morreel, K., Ivens, B., Pinosio, S., Morgante, M., Ralph, J., Bastien, C., Boerjan, W., 2013.Breeding with rare defective alleles (BRDA): a natural Populus nigra HCTmutant withmodified lignin as a case study. New Phytologist 198, 765–776.

Vega-Sanchez, M.E., Ronald, P.C., 2010. Genetic and biotechnological approaches forbiofuel crop improvement. Current Opinion in Biotechnology 21, 218–224.

Vermerris, W., Boon, J.J., 2001. Tissue-specific patterns of lignification are disturbed in thebrown midrib 2 mutant of maize (Zea mays L.). Journal of Agricultural and FoodChemistry 49, 721–728.

Vermerris, W., Thompson, K.J., McIntyre, L.M., 2002. The maize brown midrib 1 locusaffects cell wall composition and plant development in a dose dependent manner.Heredity 88, 450–457.



















































































































































Vermerris, W., Sherman, D.M., McIntyre, L.M., 2010. Phenotypic plasticity in cell walls ofmaize brownmidribmutants is limited by lignin composition. Journal of ExperimentalBotany 61, 2479–2490.

Vertès, A.A., Inui, M., Yukawa, H., 2008. Technological options for biological fuel ethanol.Journal of Molecular Microbiology and Biotechnology 15, 16–30.

Vignols, F., Rigau, J., Torres, M.A., Capellades, M., Puigdomenech, P., 1995. Thebrown midrib 3 (bm3) mutation in maize occurs in the gene encoding caffeic acidO-methyltransferase. The Plant Cell 7, 407–416.

Vinardell, M., Ugartondo, V., Mitjans, M., 2008. Potential applications of antioxidantlignins from different sources. Industrial Crops and Products 27, 220–223.

Voelker, S.L., Lachenbruch, B., Meinzer, F.C., Jourdes, M., Ki, C., Patten, A.M., Davin, L.B.,Lewis, N.G., Tuskan, G.A., Gunter, L., Decker, S.R., Selig, M.J., Sykes, R., Himmel, M.E.,Kitin, P., Shevchenko, O., Strauss, S.H., 2010. Antisense down-regulation of 4CLexpression alters lignification, tree growth and saccharification potential of field-grown poplar. Plant Physiology 154, 874–886.

Vogel, J., 2008. Unique aspects of the grass cell wall. Current Opinion in Plant Biology 11,301–307.

Vogel, K.P., Jung, H.G., 2001. Genetic modification of herbaceous plants for feed and fuel.Critical Reviews in Plant Sciences 20, 15–49.

Wadenbäck, J., von Arnold, S., Egertsdotter, U., Walter, M.H., Grima-Pettenati, J., Goffner,D., Gellerstedt, G., Gullion, T., Clapham, D., 2008. Lignin biosynthesis in transgenicNorway spruce plants harboring an antisense construct for cinnamoyl CoA reductase(CCR). Transgenic Research 17, 379–392.

Wagner, A., Ralph, J., Akiyama, T., Flint, H., Phillips, L., Torr, K., Nanayakkara, B., Kiri, L.T.,2007. Exploring lignification in conifers by silencing hydroxy cinnamoyl-CoA:shikimate hydroxy cinnamoyl transferase in Pinus radiata. Proceedings of theNationalAcademy of Sciences of the United States of America 104, 11856–11861.

Walter, C., Fladung, M., Boerjan, W., 2010. Twenty years of GM tree field testing implieshigh environmental safety. Nature Biotechnology 28, 656–658.

Wang, Z.Y., Brummer, E.C., 2012. Is genetic engineering ever going to take off in forage,turf and bioenergy crop breeding? Annals of Botany 110, 1317–1325.

Wang, H., Avci, U., Nakashima, J., Hahn, M.G., Chen, F., Dixon, R.A., 2010. Mutationof WRKY transcription factors initiates pith secondary wall formation and increasesstem biomass in dicotyledonous plants. Proceedings of the National Academy ofSciences of the United States of America 107, 22338–22343.

Wang, Y.W., Wang,W.C., Jin, S.H., Wang, J., Wang, B., Hou, B.K., 2012. Over-expression of aputative poplar glycosyltransferase gene, PtGT1, in tobacco increases lignin contentand causes early flowering. Journal of Experimental Botany 63, 2799–2808.

Wei, T., Ogbon, J., McCoy, A., 2001. Genetic engineering and lignin biosynthetic regulationin forest tree species. Journal of Forest Research 12, 75–83.

Wei, J., Wang, Y., Wang, H., Li, R., Lin, N., Ma, R., Qu, L., Song, Y., 2008. Pulping performanceof transgenic poplar with depressed caffeoyl-CoA O-methyltransferase. Chinese Sci-ence Bulletin 53, 3553–3558.

Weijde, T.V., Kamei, C.L.A., Torres, A.F., Vermerris, W., Dolstra, O., Visser, R.G.F., Trindade,L.M., 2013. The potential of C4 grasses for cellulosic biofuel production. Frontiers inPlant Science 4, 107.

Weisshar, B., Jenkins, G.I., 1998. Phenylpropanoid biosynthesis and its regulation. CurrentOpinion in Plant Biology 1, 251–257.

Weng, J.K., Li, X., Bonawitz, N.D., Chapple, C., 2008. Emerging strategies of lignin engineering anddegradation for cellulosic biofuel production. Current Opinion in Biotechnology 19, 166–172.

Whetten, R.W., Sederoff, R.R., 1991. Genetic engineering of wood. Forest Ecology andManagement 43, 301–316.

Whetten, R.W., Sederoff, R.R., 1992. Phenylalanine ammonia-lyase from loblolly pine:purification of the enzyme and the isolation of cDNA clones. Plant Physiology 98,380–386.

Whetten, R.W., Sederoff, R.R., 1995. Lignin biosynthesis. The Plant Cell 7, 1001–1013.Whetten, R.W.,Mackay, J.J., Sederoff, R.R., 1998. Recent advances in understanding lignin bio-

synthesis. Annual Review of Plant Physiology and Plant Molecular Biology 49, 585–609.Wróbel-Kwiatkowska, M., Skórkowska-Telichowska, K., Dymińska, L., Maczka, M.,

Hanuza, J., Szopa, J., 2009. Biochemical, mechanical and spectroscopic analyses of

genetically engineered flax fibers producing bioplastic (poly-beta-hydroxybutyrate).Biotechnology Progress 25, 1489–1498.

Wu, R.L., Remington, D.L., MacKay, J.J., McKeand, S.E., O'Malley, D.M., 1999. Average effectof a mutation in lignin biosynthesis in loblolly pine. Theoretical and Applied Genetics99, 705–710.

Xu, B., Escamilla-Treviño, L.L., Sathitsuksanoh, N., Shen, Z., Shen, H., Zhang, Y.H., Dixon,R.A., Zhao, B., 2011. Silencing of 4-coumarate:coenzyme A ligase in switchgrassleads to reduced lignin content and improved fermentable sugar yields for biofuelproduction. New Phytologist 192, 611–625.

Yahiaoui, N., Marque, C., Myton, K.E., Negrel, J., Boudet, A.M., 1998. Impact of differentlevels of cinnamyl alcohol dehydrogenase down-regulation on lignins of transgenictobacco plants. Planta 204, 8–15.

Yamaguchi, M., 2010. Transcriptional regulation of secondary wall formation controlledby NAC domain proteins. Plant Biotechnology 242, 237–242.

Yan, L., Xu, C., Kang, Y., Gu, T., Wang, D., Zhao, S., Xia, G., 2013. The heterologous expres-sion in Arabidopsis thaliana of sorghum transcription factor SbbHLH1 down-regulateslignin synthesis. Journal of Experimental Botany 64, 3021–3032.

Yuan, J.S., Tiller, K.H., Ahmad, H.A., Stewart, N.R., Stewart Jr., C.N., 2008. Plants to power:bioenergy to fuel the future. Trends in Plant Science 13, 421–429.

Zhang, K., Bhuiya, M.W., Pazo, J.R., Miao, Y., Kim, H., Ralph, J., Liu, C.J., 2012. An engineeredmonolignol 4-O-methyltransferase depresses lignin biosynthesis and confers novelmetabolic capability in Arabidopsis. Plant Cell 24, 3135–3152.

Zhang, K., Lu, K., Qu, C., Liang, Y., Wang, R., Chai, Y., Li, J., 2013. Gene silencing of BnTT10family genes causes retarded pigmentation and lignin reduction in the seed coat ofBrassica napus. PLoS ONE 8, e61247.

Zhao, Q., Dixon, R.A., 2011. Transcriptional networks for lignin biosynthesis: morecomplex than we thought? Trends in Plant Science 16, 227–233.

Zhao, C.S., Avci, U., Grant, E.H., Haigler, C.H., Beers, E.P., 2008. XND1, a member of the NACdomain family in Arabidopsis thaliana, negatively regulates lignocellulose synthesisand programmed cell death in xylem. Plant Journal 53, 425–436.

Zhao, Q., Nakashima, J., Chen, F., Yin, Y., Fu, C., Yun, J., Shao, H., Wang, X., Wang, Z.Y., Dixon,R.A., 2013a. Laccase is necessary and nonredundant with peroxidase for ligninpolymerization during vascular development in Arabidopsis. Plant Cell 25,3976–3987.

Zhao, Q., Tobimatsu, Y., Zhou, R., Pattathil, S., Gallego-Giraldo, L., Fu, C., Jackson, L.A., Hahn,M.G., Kim, H., Chen, F., Ralph, J., Dixon, R.A., 2013b. Loss of function of cinnamylalcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect inMedicago truncatula. Proceedings of the National Academyof Sciences of the United States of America 110, 13660–13665.

Zhong, R., Ye, Z.H., 2007. Regulation of cell wall biosynthesis. Current Opinion in PlantBiology 10, 564–572.

Zhong, R., Ye, Z.H., 2009. Transcriptional regulation of lignin biosynthesis. Plant Signalingand Behavior 4, 1028–1034.

Zhong, R., Jennifer, J.J., Ye, Z.H., 1997. Disruption of interfascicular fiber differentiation inan Arabidopsis mutant. The Plant Cell 9, 2159–2170.

Zhong, R., Morrison, W.H., Negrel, J., Ye, Z.H., 1998. Dual methylation pathways in ligninbiosynthesis. The Plant Cell 10, 2033–2046.

Zhong, R., Lee, C., Zhou, J., McCarthy, R.L., Ye, Z.H., 2008. A battery of transcription factorsinvolved in the regulation of secondary cell wall biosynthesis in Arabidopsis. The PlantCell 20, 2763–2782.

Zhong, R., Lee, C., McCarthy, R.L., Reeves, C.K., Jones, E.G., Ye, Z.H., 2011. Transcriptionalactivation of secondary wall biosynthesis by rice and maize NAC and MYB transcrip-tion factors. Plant and Cell Physiology 52, 1856–1871.

Zhou, J., Lee, C., Zhong, R., Ye, Z.H., 2009. MYB58 and MYB63 are transcriptional activatorsof the lignin biosynthetic pathway during secondary cell wall formation inArabidopsis. The Plant Cell 21, 248–266.

Ziebell, A., Gracom, K., Katahira, R., Chen, F., Pu, Y., Ragauskas, A., Dixon, R.A., Davis, M.,2010. Increase in 4-coumaryl alcohol units during lignification in alfalfa(Medicago sativa) alters the extractability and molecular weight of lignin. Journalof Biological Chemistry 285, 38961–38968.















































































































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Lignin genetic engineering for improvement of wood quality: Applications in paper and textile industries, fodder and bioenergy production