Engineering traditional monolignols out of ligninby concomitant up-regulation of F5H1 and down-regulationof COMT in Arabidopsis

Ruben Vanholme1,2, John Ralph3, Takuya Akiyama3, Fachuang Lu3, Jorge Rencoret Pazo3, Hoon Kim3, Jørgen Holst

Christensen1,2, Brecht Van Reusel1,2, Veronique Storme1,2, Riet De Rycke1,2, Antje Rohde1,2, Kris Morreel1,2 and

Wout Boerjan1,2,*

1Department of Plant Systems Biology, VIB, 9052 Gent, Belgium,2Department of Plant Biotechnology and Genetics, Ghent University, 9052 Gent, Belgium, and3Department of Biochemistry and Great Lakes Bioenergy Research Center, University of Wisconsin, Madison, WI 53706, USA

Received 14 June 2010; revised 20 August 2010; accepted 26 August 2010; published online 15 October 2010.*For correspondence (fax +32 9 3313809; e-mail wout.boerjan@psb.vib-ugent.be).

SUMMARY

Lignin engineering is a promising strategy to optimize lignocellulosic plant biomass for use as a renewable

feedstock for agro-industrial applications. Current efforts focus on engineering lignin with monomers that are

not normally incorporated into wild-type lignins. Here we describe an Arabidopsis line in which the lignin is

derived to a major extent from a non-traditional monomer. The combination of mutation in the gene encoding

caffeic acidO-methyltransferase (comt) with over-expression of ferulate 5-hydroxylase under the control of the

cinnamate 4-hydroxylase promoter (C4H:F5H1) resulted in plants with a unique lignin comprising almost 92%

benzodioxane units. In addition to biosynthesis of this particular lignin, the comt C4H:F5H1 plants revealed

massive shifts in phenolic metabolism compared to the wild type. The structures of 38 metabolites that

accumulated in comt C4H:F51 plants were resolved by mass spectral analyses, and were shown to derive from

5-hydroxy-substituted phenylpropanoids. These metabolites probably originate from passive metabolism via

existing biochemical routes normally used for 5-methoxylated and 5-unsubstituted phenylpropanoids and

from active detoxification by hexosylation. Transcripts of the phenylpropanoid biosynthesis pathway were

highly up-regulated in comt C4H:F5H1 plants, indicating feedback regulation within the pathway. To

investigate the role of flavonoids in the abnormal growth of comt C4H:F5H1 plants, a mutation in a gene

encoding chalcone synthase (chs) was crossed in. The resulting comt C4H:F5H1 chs plants showed partial

restoration of growth. However, a causal connection between flavonoid deficiency and this restoration of

growth was not demonstrated; instead, genetic interactions between phenylpropanoid and flavonoid

biosynthesis could explain the partial restoration. These genetic interactions must be taken into account in

future cell-wall engineering strategies.

Keywords: cell wall, benzodioxane, NMR, phenolic profiling, monolignol, irx.

INTRODUCTION

Lignins are aromatic heteropolymers that are predominantlydeposited in secondarily thickened plant cell walls, wherethey provide strength and imperviousness to the wall, andprotect the cellulose microfibrils from microbial attack. Atthe same time, lignins limit the efficiency of several indus-trial processes that use plant cell walls as a feedstock.Engineering of lignin amount and structure through bio-technology is a promising strategy to improve the industrialprocessing of lignocellulosic plant biomass. During the lasttwo decades, many details of the biochemical pathway

producing coniferyl and sinapyl alcohol, the primarymonomers from which polymeric lignins derive, have beenestablished. The genes for each of the steps have beenidentified, and the activities of the derived enzymes havebeen reasonably well characterized (Boerjan et al., 2003;Ralph et al., 2004; Vanholme et al., 2010).

Many of the recent advances in lignin research have comefrom examining the consequences on overall lignin struc-ture of up- and down-regulating the various genes of thepathway. Such studies have also revealed the rather striking

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The Plant Journal (2010) 64, 885–897 doi: 10.1111/j.1365-313X.2010.04353.x

metabolic malleability of the lignification process. Massivecompositional changes (i.e. changes in the ratio of guaiacyl(G) and syringyl (S) moieties resulting from incorporation ofconiferyl and sinapyl alcohol monomers, respectively) inangiosperm lignins can result from mis-regulation of singlegenes in the pathway. For example, lignins consist largely(or almost entirely) of G units when F5H, the gene encodingferulate 5-hydroxylase, is down-regulated (Figure 1) (Meyeret al., 1998; Marita et al., 1999). Conversely, up-regulation ofF5H using a xylem-specific promoter in Arabidopsis, poplar(Populus tremula · alba) or tobacco (Nicotiana tabacum)leads to lignins with extraordinarily high S:G ratios (Meyeret al., 1998; Marita et al., 1999; Franke et al., 2000; Stewartet al., 2009). Despite containing essentially linear and appar-ently low-molecular-weight lignins, poplar plants over-expressing F5H displayed no visible growth phenotypes(Stewart et al., 2009). However, the wood was markedlyeasier to pulp than that of the wild type (Huntley et al., 2003),demonstrating that altering lignin composition can be aviable approach towards improved processing.

In addition to the compositional shifts in S:G ratios, it hasbecome apparent that lignification may tolerate the (partial)substitution of monolignols by various other phenolicprecursors. Such substitution is compatible with the currenttheory of lignification, in which the polymer derives viachemical radical coupling reactions, primarily coupling of amonomerwith the growing polymer; any phenolic precursorwithin the lignification zone may co-polymerize with otheravailable phenolic precursors to the extent allowed bychemical compatibility (Ralph et al., 2004). Less obvious,but apparently possible, is that phenolic precursors, whichare often formed in cells with perturbed phenylpropanoidbiosynthesis, can be transported to the wall for lignification.For example, angiosperms deficient in caffeic acid O-meth-yltransferase (COMT) incorporate 5-hydroxyconiferyl alco-hol into their lignins, forming benzodioxane units that arereadily apparent by NMR, thioacidolysis and phenolic profil-ing of lignin oligomers (Atanassova et al., 1995; Van Doorss-elaere et al., 1995; Jouanin et al., 2000; Guo et al., 2001;Ralph et al., 2001a,b; Marita et al., 2003; Morreel et al.,2004b; Do et al., 2007; Lu et al., 2010).

The goal of the current study was to develop plants inwhich the lignin composition approaches the state wherebynone of its constituents are derived from traditional mono-lignols. Despite the already overwhelming evidence thatvarious non-traditional monolignols are readily incorpo-rated into the lignin polymer in a variety of natural andtransgenic plants, a plant with a minor complement oftraditional monolignols would provide insight into how farand in which directions lignin might be redesigned. Onesimple strategy to produce non-traditional lignins consistsof up-regulating F5H while blocking COMT expression(Figure 1). If there is no feedback inhibition on F5H specif-ically, or within the phenylpropanoid pathway in general, it

is anticipated that the cells will primarily produce 5-hy-droxyconiferyl alcohol for lignification. Substantial levels of5-hydroxyconiferyl alcohol can apparently be tolerated inlignification without obvious adverse effects on plantgrowth and development (Figure S1). Here we describecomt C4H:F5H1 plants in which the lignin comprises approx-imately 92% benzodioxane units, far more than everreported previously and how plants respond to such amassive replacement of traditional monolignols with5-hydroxyconiferyl alcohol.

RESULTS

comt C4H:F5H1 plants have an altered development

In order to design lignins that are derived predominantlyfrom non-traditional 5-hydroxyconiferyl alcohol monomers,Arabidopsis plants over-expressing F5H1 under the controlof the cinnamate 4-hydroxylase (C4H) promoter (C4H:F5H1plants) were crossed with Arabidopsis comtmutants. Plantshomozygous for the comt mutation and the C4H:F5H1 con-struct (comt C4H:F5H1 plants) were identified in the F2progeny. Although the comt mutant grew normally and theC4H:F5H1 over-expressing line showed only moderategrowth effects, plant development in comt C4H:F5H1 plantswas severely affected. When grown on agar medium, leavesof the comt C4H:F5H1 seedlings were smaller compared tothose of thewild type (Figure 2a). The inflorescence stems ofagar-grown comt C4H:F5H1 plants remained small, with amaximal observed final length of 8 cm, whereas wild-typestems were over 30 cm at maturity. Extensive phenotypicvariation was observed for the length, number and color ofthe inflorescence stems (Figure 2b–d), with the color rang-ing from green to purple red. In many cases, additionalinflorescences developed, resulting in a bushy phenotype(Figure 2d). The total stem biomass of senesced soil-growncomt C4H:F5H1 plants was only a fraction of that of the wildtype (Table 1). Although comt C4H:F5H1 plants developedflowers, they did not set seeds.

comt C4H:F5H1 plants have an irregular xylem phenotype

To reveal the morphological consequences of F5H1 over-expression in the comt mutant background at the tissuelevel, sections of the inflorescence stem were analyzed bylight microscopy. Toluidine blue-stained sections of comtC4H:F5H1 plants showed collapsed xylem vessels, a phe-notype typically known as the irregular xylem (irx) pheno-type and observed in a range of mutants with perturbedsecondary cell-wall development (Figure 2f,g) (Turner andSomerville, 1997; Jones et al., 2001; Franke et al., 2002;Goujon et al., 2003; Hoffmann et al., 2004; Brown et al.,2005; Besseau et al., 2007; Mir Derikvand et al., 2008;Schilmiller et al., 2009; Li et al., 2010). No obvious differ-ences were noted in the other stem tissues. In contrast to thewild type, Maule staining, which is indicative of S units in

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Figure 1. Biosynthetic pathway for coniferyl and sinapyl alcohol (green box) and the metabolites that accumulate during concomitant up-regulation of F5H1 anddown-regulation of COMT (red boxes).The black route is expected to be the favored route; the gray routes may also be followed depending on the genetic background and growth conditions. Dashedarrow: route suggested by Mir Derikvand et al. (2008), Leple et al. (2007) and Nair et al. (2004); ?, conversion not demonstrated. Possible routes towards the fourclasses of compounds are indicated by thin red arrows. CCR, cinnamoyl CoA reductase; COMT, caffeic acidO-methyltransferase; HCALDH, hydroxycinnamaldehydedehydrogenase; F5H, ferulate 5-hydroxylase. For convenience,metabolites are classified in four classes based on their chemical identity: class I, oligolignols; class II,hexosylated oligolignols; class III, 5-hydroxyferulic acid-containing dimers; class IV, derivatives of 5-hydroxylated phenylpropanoid monomers (see Table 3).

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lignin, generally did not result in red coloration of the sec-ondary cell wall, indicating a strong reduction in, or absenceof, S units in the comt C4H:F5H1 plants (Figure 2e). How-ever, a slight red coloration was observed in a few of theseplants, suggesting the presence of residual COMT.

Transmission electron microscopy (TEM) of the cell wallsrevealed multiple concentric cell-wall sub-layers in the comtC4H:F5H1 plants (Figure 2i,k). This stratification appeared tobe restricted to the S2 region of the cell wall, and was moreobvious in interfascicular fibers than in xylem vessels.However, a wide variability in the extent of cell-wall strat-

ification, ranging from absent to extensive, was observedin the fibers. This remarkable phenotype, already describedfor ccr1 Arabidopsis mutants and CCR down-regulatedpoplars, has been suggested to be caused by an alteredassembly of cellulose microfibrils (Goujon et al., 2003; Lepleet al., 2007).

NMR analyses reveal massive incorporation of 5-hydroxy-

coniferyl alcohol into lignin

Although acetylbromide lignin levels in the senesced inflo-rescence stems of either comt or C4H:F5H1 plants were notsignificantly different from those of the wild-type, ligninamounts in comt C4H:F5H1 plants were reduced to approx-imately 56% of wild-type levels (Table 2). Cellulolyticenzyme lignins (CEL) were isolated from senescedinflorescence stems and analyzed by NMR. The aromaticregions of the 2D 13C–1H correlation (heteronuclear singlequantum coherence, HSQC) spectra highlight differences inthe distribution of guaiacyl, 5-hydroxyguaiacyl and syringyl(G, 5H and S) units in the lignins. The resulting spectra(Figures 3 and S2) clearly show that G signals from comtC4H:F5H1 plants are low, representing approximately 13.8%of the total aromatic content (based on volume integration),compared to 82.7% in the wild type. The dominant signalsare from 5H units. It is not immediately clear that S units aredepleted, because the S2/6 contours overlap with those from

(a)

(c) (d)

(e)

(b) (f) (h) (j)

(g) (i) (k)

(l)

Figure 2. Phenotype and inflorescence stem morphology of comt C4H:F5H1 and comt C4H:F5H1 chs plants.(a) Fourteen-day-old seedlings, segregating from a comt/COMT C4H:F5H1/C4H:F5H1 parental line. The double homozygous comt C4H:F5H1 plant is indicated withan arrow. (b–d) comt C4H:F5H1 plants with a wide phenotypic diversity: (b,c) 13-week-old and (d) 24-week-old plants.(e) Maule staining of comt C4H:F5H1 plant grown for 28 weeks in vitro. A section of a greenhouse-grown wild-type plant is visible in the upper left corner.(f, g) Light microscopy of toluidine blue-stained sections of wild-type and comt C4H:F5H1 tissue culture-grown plants, respectively. Arrows indicate xylem cells.Note the collapsed xylem vessels in the comt C4H:F5H1 plants.(h–k) TEMs of cell walls stained with uranyl acetate, showing interfascicular fibers of the wild type (h), interfascicular fibers of comt C4H:F5H1 (i), the xylem region ofthe wild type (j), and the xylem region of comt C4H:F5H1 (k).(l) Partially recovered phenotype of a comt C4H:F5H1 chs plant (see Table 1 for biomass data).

Table 1 Inflorescence height and the total inflorescence stem dryweight, as a measure for biomass, of the various lines

Line

Height (cm) Dry weight (mg)

Mean ! SD n Mean ! SD n

Wild type 47.7 ! 3.5 29 104.9 ! 32.0 10comt 44.2 ! 4.9 30 125.4 ! 27.1 10C4H:F5H1 29.4 ! 4.7* 29 77.6 ! 21.8 8chs 48.1 ! 4.8 27 130.4 ! 58.0 10comt C4H:F5H1 1.1 ! 1.4* 14 1.4 ! 3.4* 10comt C4H:F5H1 chs 11.0 ! 2.1* 50 10.9 ! 3.0* 12

n, number of plants analyzed.*Significantly different from the wild type (P < 0.001).

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5H6. However, the level of the 5H2 contours suggests thatmost of the 5H6/S2/6 contour region must belong to 5H6.The S2/6 volume integral can be estimated by subtractingthe volume integral of the 5H2 region from that of the5H6+S2/6 region; the volume integrals for 5H2 and 5H6

regions should be equal. This assumption results in a G:5H:Sratio of approximately 13.8:71.0:15.2; i.e. we estimatethat the lignin is derived from approximately 71% of5-hydroxylated monomers.

The inter-unit linkage distribution in the lignin fraction canbe determined from the aliphatic side-chain region of theHSQC spectra (Table 2 and Figure S3). The correlationcontours in the spectra (Figure S3), such as those for b-arylether (b–O–4) units (A), phenylcoumaran (b–5) units (B),resinol (b–b) units (C), dibenzodioxocin (5–5/b–O–4) units (D)and benzodioxane units (J), can be readily assigned bycomparison with model data and from lignin and cell-wall

spectra obtained previously (Ralph et al., 1999; Ralph andLanducci, 2010). As for the aromatic regions in Figures 3 andS2, the side-chain region of the comt C4H:F5H1 plants(Figure S3e) is strikingly different from that of the wild type(Figure S3a). Even though they are plotted at a lowercontour level (closer to noise level, to show the weakresidual signals from the traditional lignin structures), itis evident that proportions of the various ‘normal’ ligninstructures (A–C) are minor. The obvious major difference isthe overwhelming presence of benzodioxane units (J) in thecomt C4H:F5H1 plants, for which the a- and b-correlationsare well resolved. No such correlations exist in the wild-typespectrum. The volume integrals of the correlation contoursprovide a measure of the relative contribution of each unittype in the lignin polymer (Table 2). In the comt C4H:F5H1plants, benzodioxanes account for approximately 92% of theinter-unit linkage integrals measured. As noted, the 5H level

Table 2 Lignin composition as measured by NMR and total acetyl bromide lignin content

Line

Monomeric composition (%) Unit types (%) Acetyl bromide lignin(%)

G 5H S A B C D J Mean ! SD n

Wild type 82.48 0.00 17.52 75.88 17.21 6.18 0.73 0.00 12.0 ! 1.7 7comt 94.79 1.28 3.92 63.59 17.67 3.94 2.92 11.87 10.5 ! 1.0 4C4H:F5H1 6.55 1.91 91.55 83.84 0.00 3.50 0.00 12.66 9.4 ! 1.7 4chs 84.20 0.00 15.80 73.70 18.40 6.70 1.30 0.00 11.7 ! 0.9 5comt C4H:F5H1 13.78 70.99 15.23 6.44 0.85 0.79 0.00 91.93 6.8 ! 0.8*** 5comt C4H:F5H1 chs 33.54 20.02 46.44 59.07 1.42 1.50 0.00 38.02 8.8 ! 1.1** 8

A, b-aryl ether; B, phenylcoumaran; C, resinol; D, dibenzodioxocin; J, benzodioxane (see Figure S3). n, number of plants analyzed.Statistical tests relative to the wild type were performed for total lignin only. Asterisks indicate values that are significantly different from the wildtype at **0.001 < P < 0.01 and ***P < 0.001, respectively.

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

Figure 3. Partial short-range 13C–1H (HSQC) spectra (aromatic regions) of acetylated enzyme lignins.See Figure S2 for spectra of the complete set of transgenic lines and controls.

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is approximately 71%; benzodioxane levels are higherbecause they may result from addition of coniferyl orsinapyl alcohol, as well as 5-hydroxyconiferyl alcohol, tothe 5-hydroxyguaiacyl end of the growing polymer, i.e. theycan be S, 5H or G benzodioxanes (see also Figure S1).

Phenolic profiling reveals benzodioxane-rich soluble

phenolics

To investigate whether perturbation of the monolignol bio-synthetic pathway resulted in metabolites derived from5-hydroxyconiferyl alcohol, a semi-quantitative comparisonof the profiles of methanol-soluble phenolics from comtC4H:F5H1 and wild-type inflorescence stems was per-formed. The profiles of wild-type stems were dominated bysinapate esters and three flavonol glycosides (Figure S4). Incontrast, the comt C4H:F5H1 plants had lower levels ofsinapate esters and higher levels of flavonol glycosides, andaccumulated a large number of phenolic molecules thatwere below the detection limit in wild-type plants (Fig-ure S4). Elucidating their identities by MS2 spectral analyses(Figure S5) (Morreel et al., 2004a,b; 2010a,b) revealed thatthese metabolites were 5-hydroxylated phenylpropanoids.Based on their chemical identity, they were grouped intofour main classes (Figure 1 and Table 3). Class I consists ofoligolignols with at least one and up to four 5H units (1–11).Class II compounds (12–18) consist of hexosylated di- andtrilignols with at least one 5H unit. Class III containsheterodimers of 5-hydroxyferulic acid with coniferyl,5-hydroxyconiferyl or sinapyl alcohol, and their hexoseand malate derivatives (19–28), and class IV compounds(29–38) are a series of derivatized 5-hydroxylated phenyl-propanoids.

Chalcone synthase deficiency reduces C4H-driven gene

expression in comt C4H:F5H1 plants

Although the parental comt and C4H:F5H1 lines had a heal-thy appearance, growth of the comt C4H:F5H1 plants wasseverely affected. This phenotype is reminiscent of that ofhydroxycinnamoyl CoA:shikimate/quinate hydroxycinna-moyl transferase (HCT) down-regulated Arabidopsis plants,whose growth retardation was thought to result from inhi-bition of auxin transport by the high levels of flavonoids inthese mutants (Besseau et al., 2007). Recent findings, how-ever, disproved the existence of this link between flavonoidsand growth retardation in HCT down-regulated Arabidopsisplants (Li et al., 2010). To test whether the increased levels offlavonol glycosides (Figure S4) were responsible for growthretardation in the comt C4H:F5H1 plants, a chalcone syn-thase (chs) mutation was crossed into the comt C4H:F5H1background. Soil-grown comt C4H:F5H1 chs mutants grewsignificantly taller than comt C4H:F5H1 plants (Figure 2l andTable 1), and produced viable seeds. However, such resto-ration of the phenotype could also be caused by negativefeedback of the chs mutation on the C4H promoter and thus

on C4H:F5H1 expression. To investigate this, quantitativeRT-PCR was performed on inflorescence stems. Expressionof C4H and F5H1was significantly reduced in comt C4H:F5H1chs plants compared to C4H:F5H1 and comt C4H:F5H1 plants(Figure 4a). The benzodioxane level in the lignin of comtC4H:F5H1 chs plants was reduced to 38%,much less than the92% found in comt C4H:F5H1 plants (Figure S3j andTable 2). Furthermore, comt C4H:F5H1 chs plants had higherlignin amounts compared to comt C4H:F5H1 plants, but lessthan the wild-type (Table 2). Thus, the partial restoration ingrowth can be explained by inhibition of C4H:F5H1 expres-sion.

To further explore possible feedback regulation within thephenylpropanoid pathway in comt C4H:F5H1 and comtC4H:F5H1 chs plants, we measured the expression ofphenylpropanoid biosynthetic genes in 14-day-old seedlingleaves. At this stage, the visual phenotype is much lesspronounced (Figure 2a). Strikingly, the expression of manygeneral phenylpropanoid biosynthetic genes was higher inC4H:F5H1, comt C4H:F5H1 and comt C4H:F5H1 chs plantscompared to the wild type, suggesting a higher flux throughthe pathway (Figure 4b). In contrast to inflorescence stems,however, C4H gene expression was similar between comtC4H:F5H1 and comt C4H:F5H1 chs in the seedling leaves,indicating developmental differences in feedback regula-tion.

DISCUSSION

Lignins primarily consist of benzodioxane structures

Plants deficient in COMT and plants over-expressing F5Hwere previously shown to incorporate small amounts of5-hydroxyconiferyl alcohol into their lignins, demonstratingthat 5-hydroxyconiferyl alcohol can be transported, single-electron-oxidized (‘radicalized’), and co-polymerized intolignins (Atanassova et al., 1995; Van Doorsselaere et al.,1995; Jouanin et al., 2000; Guo et al., 2001; Ralph et al.,2001a,b; Sibout et al., 2002; Marita et al., 2003; Morreelet al., 2004a,b; Do et al., 2007; Lu et al., 2010). Its incorpo-ration gives rise to specific units, i.e. benzodioxanes. Abenzodioxane results from coupling of a monolignol at itsb-position with a 5-hydroxyguaiacyl unit at its 4-O position,followed by internal trapping of the resulting quinone met-hide by the 5-OH (Ralph et al., 2001a,b). Although thesebenzodioxane units are the result of typical b–O–4 couplingreactions that also take place in natural G/S-type lignins, theless flexible benzodioxane ring structures alter the macro-molecular configuration of the lignin polymer. Strikingly,when Arabidopsis F5H1 is over-expressed in a comt mutantbackground, the lignin almost exclusively comprises ben-zodioxane structures, accounting for approximately 92% ofthe units (Figure S3 and Table 2). This study shows thatlignin can be composed almost entirely from units that areundetectable in wild-type plants.

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Table 3 Metabolites in comt C4H:F5H1 plants, arbitrarily classified for convenience

Peak number Retention time (min) m/z Molecule number Name

Class I: oligolignols1 18.5 373 1 G(b-O-4)5H2 18.4 403 2 S(b-O-4)5H3 21.6 371 3 G(b-O-4)5H’4 19.0 387 4 5H(b-O-4)5H’5 21.2 401 5 S(b-O-4)5H’6 21.1 567 6 G(b-O-4)5H(b-O-4)5H7 21.4 567 6 G(b-O-4)5H(b-O-4)5H8 24.0 565 7 G(b-O-4)5H(b-O-4)5H’9 24.2 565 7 G(b-O-4)5H(b-O-4)5H’10 21.4 569 8 G(b-O-4)5H(b-O-4)dihydro5H11 21.6 569 8 G(b-O-4)5H(b-O-4)dihydro5H12 22.8 761 9 G(b-O-4)5H(b-O-4)5H(b-O-4)5H13 23.0 761 9 G(b-O-4)5H(b-O-4)5H(b-O-4)5H14 25.4 759 10 G(b-O-4)5H(b-O-4)5H(b-O-4)5H’15 25.8 759 10 G(b-O-4)5H(b-O-4)5H(b-O-4)5H’16 26.4 953 11 G(b-O-4)5H(b-O-4)5H(b-O-4)5H(b-O-4)5H’

Class II: hexosylated oligolignols17 13.6 535 12 G(b-O-4)5H hexosidea

18 14.0 535 12 G(b-O-4)5H hexosidea

19 15.4 535 12 G(b-O-4)5H hexosidea

20 12.7 551 13 5H(b-O-4)5H hexosidea

21 14.0 551 13 5H(b-O-4)5H hexosidea

22 14.4 551 13 5H(b-O-4)5H hexosidea

23 16.6 549 14 5H(b-O-4)5H’ hexoside24 15.4 537 15 G(b-O-4)c-O-hexosyl dihydro5H25 16.3 567 16 S(b-O-4)c-O-hexosyl dihydro5H26 17.2 789 17 G(b-O-4)5H(b-O-4)5H hexoside27 17.5 789 17 G(b-O-4)5H(b-O-4)5H hexoside28 17.5 791 18 G(b-O-4)5H(b-O-4)dihydro5H hexoside29 17.6 791 18 G(b-O-4)5H(b-O-4)dihydro5H hexoside

Class III: 5-hydroxyferulic acid-containing dimers30 19.0 387 19 G(b-O-4)5-hydroxyferulic acid31 15.2 549 20 G(b-O-4)5-hydroxyferuloyl hexose32 15.9 549 20 G(b-O-4)5-hydroxyferuloyl hexose33 9.7 549 21 G(b-O-4)5-hydroxyferulic acid hexosidea

34 16.1 565 22 5H(b-O-4)5-hydroxyferuloyl hexose35 9.9 565 23 5H(b-O-4)5-hydroxyferulic acid hexosidea

36 16.0 579 24 S(b-O-4)5-hydroxyferuloyl hexose37 16.6 579 24 S(b-O-4)5-hydroxyferuloyl hexose38 10.0 579 25 S(b-O-4)5-hydroxyferulic acid hexosidea

39 10.1 503 26 G(b-O-4)5-hydroxyferuloyl malate40 10.7 503 26 G(b-O-4)5-hydroxyferuloyl malate41 8.0 519 27 5H(b-O-4)5-hydroxyferuloyl malate42 8.9 519 27 5H(b-O-4)5-hydroxyferuloyl malate43 9.6 519 27 5H(b-O-4)5-hydroxyferuloyl malate44 10.8 533 28 S(b-O-4)5-hydroxyferuloyl malate

Class IV: derivatives of 5-hydroxylated phenylpropanoid monomers45 2.7 371 29 5-hydroxyferulic acid hexoside46 2.8 371 29 5-hydroxyferulic acid hexoside47 8.3 371 30 5-hydroxyferuloyl hexose48 9.2 371 30 5-hydroxyferuloyl hexose49 10.5 525 31 5-hydroxyferuloyl hexose+155b

50 12.9 525 31 5-hydroxyferuloyl hexose+155b

51 7.7 533 32 5-hydroxyferuloyl hexose hexoside52 18.8 563 33 di-5-hydroxyferuloyl hexose53 3.4 373 34 dihydro 5-hydroxyferuloyl hexose54 7.4 369 35 5-hydroxyscopoletin hexoside55 10.3 355 36 5-hydroxyconiferaldehyde hexoside

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The comt C4H:F5H1 plants accumulate large amounts

of 5-hydroxylated phenylpropanoids and their derivatives

Phenolic profiling of the methanol-soluble fraction revealedmany 5-hydroxylated phenylpropanoids (including alco-hols, aldehydes, acids, and their derivatives) that wereundetectable in the extracts of wild-type plants (Figure 1and Table 3). Class I oligolignols (1–11) are small ligninpolymers with chains of benzodioxane structures, reflect-ing the high level of 5-hydroxyconiferyl alcohol monomersincorporated into the lignin polymer. Although the phe-nolic end unit can be a 5H unit, as seen in compound 4,such units are inherently prone to oxidation to quinonesand beyond. Capping such end units by coupling with atraditional monolignol stabilizes them. Indeed, most of theoligomers identified were found to be capped in this way.Interestingly, except for the cap, all other units in theidentified oligolignols were 5H units, i.e. no mixed oligo-lignols were found with normal G or S units within thechain. End capping was also observed in class II and IIIcompounds. Thus, although not all differential peaks werestructurally resolved, the structures of the most abundantones suggest that 5-hydroxylated monomers do not readilycouple b–O–4 to neither G or S units. Furthermore, it isimportant to note that these small lignin oligomers involveonly b–O–4 binding, because b–5 coupling cannot occurdue to the hydroxylation at C5 in the 5H units. Because ofthe instability of 5H end groups, the only b–b-coupledcompounds likely to be found would be tetramers at least,in which both phenolic ends of a b–b-coupled dimer arecapped.

Classes II, III and IV comprise hexose and malatederivatives of 5-hydroxyconiferyl alcohol, 5-hydroxyconif-eraldehyde or 5-hydroxyferulate, with the latter compoundpotentially derived from 5-hydroxyconiferaldehyde viahydroxycinnamaldehyde dehydrogenase/reduced epidermalfluorescence 1 (HCALDH/REF1) as described for the con-version from coniferaldehyde and sinapaldehyde to ferulicacid and sinapic acid, respectively (Nair et al., 2004). Theclass III molecules (19–28) are dimers of 5-hydroxyferulatewith coniferyl or sinapyl alcohol and their hexose and

malate derivatives. These molecules are 5-hydroxy analogsof the G(8-O-4)feruloyl malate that is found in wild-typeArabidopsis (Rohde et al., 2004). Although the biologicalrole of this compound is unknown, G(8-O-4)feruloyl malatehas been suggested to be involved in the coupling ofhemicellulose to lignin (Rohde et al., 2004). The highabundance of 5-hydroxyferulate esters (30–33) in class IVis also noteworthy. The 5-hydroxyferulate esters mightfunctionally replace the biological role of sinapate estersin vivo, because epidermal fluorescence upon illuminationwith UV (366 nm) is observed in leaves of comt C4H:F5H1plants, indicative of their UV protective role (Ruegger andChapple, 2001). This fluorescence is absent from f5h1(fah1) mutants that produce neither 5-hydroxyferulate norsinapate esters (Ruegger and Chapple, 2001). We deducethat 5-hydroxylated phenylpropanoids and their derivativesoriginate partly from passive metabolism via existingbiochemical routes, and partly from active detoxification.In passive metabolism, 5-hydroxylated phenylpropanoidsare surrogates for their 5-methoxylated and 5-unsubstitut-ed analogs. For example, the presence of di-5-hydroxyfer-uloyl hexose (33) suggests that the enzymes involved indisinapoyl hexose biosynthesis also recognize and metab-olize 5-hydroxylated analogs (Fraser et al., 2007). On theother hand, UDP-glucose-dependent glycosyltransferases(UGTs) have been shown to be involved in the detoxifica-tion of xenobiotics (Welinder et al., 2002; Lim and Bowles,2004; Brazier-Hicks et al., 2007). It is thus anticipated thatthey will also detoxify the accumulating 5-hydroxylatedphenolics, thereby giving rise to the hexosylated com-pounds of classes II, III and IV.

The subcellular location of synthesis of these compoundsremains obscure; oxidative coupling occurs in the cell walland possibly also in the vacuole, given the high number ofperoxidases in this compartment (Welinder et al., 2002).However, glycosylation occurs in the cytoplasm but not inthe vacuole, and malate transferase activity is present in thevacuole. Unraveling the subcellular routes for biosynthesisof the class II–IV compounds requires a new researchapproach that combines subcellular fractionation and phe-nolic profiling.

Table 3 (Continued)

Peak number Retention time (min) m/z Molecule number Name

56 8.6 357 37 5-hydroxyconiferyl alcohol hexoside57 9.2 357 37 5-hydroxyconiferyl alcohol hexoside58 5.9 579 38 hexosyl 5-hydroxyconiferyl alcohol hexoside59 7.6 579 38 hexosyl 5-hydroxyconiferyl alcohol hexoside

The shorthand naming convention for oligolignols is described byMorreel et al. (2004a,b). In short, G, 5H, S and 5H’ are used for units derived fromconiferyl alcohol, 5-hydroxyconiferyl alcohol, sinapyl alcohol and 5-hydroxyconiferaldehyde, respectively. Here, b-O-4 (normally used for lignins)is used instead of 8-O-4 (normally used for metabolites) to ensure consistent nomenclature throughout the text.aThe hexose is not on the c-O position of the b-coupled unit.bThe structure of the 155 Da subunit has not yet been resolved.

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ª 2010 The AuthorsThe Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 885–897

Genetic interactions within phenylpropanoid biosynthesis

Although the comt and C4H:F5H1 parental lines have nor-mal lignin levels, comt C4H:F5H1 plants deposit signifi-cantly less lignin. Moreover, comt C4H:F5H1 plants remainsmall and their xylem vessels have an irx phenotype. Onehypothesis to explain the lower lignin levels is that5-hydroxyconiferyl alcohol is not efficiently translocatedtowards the cell wall and/or not efficiently incorporated intolignin. The reduction in the lignin levels might be sufficientto explain the irx phenotype (Jones et al., 2001; Frankeet al., 2002; Goujon et al., 2003; Hoffmann et al., 2004;Besseau et al., 2007; Mir Derikvand et al., 2008; Schilmilleret al., 2009; Li et al., 2010), but the altered structural andphysico-chemical properties of the 5H lignin may alsocontribute to the irx phenotype. In addition to the collapsedxylem, accumulating phenolic precursors might causeaberrant growth, because they are toxic to cellular

processes. Indeed, the phenolic profile of comt C4H:F5H1plants revealed numerous hexosylated (i.e. detoxified)compounds. Accumulation of these compounds is logicalgiven that the sink, lignin, is reduced. However, we alsoobserved a massive up-regulation of multiple phenylprop-anoid pathway transcripts in the comt C4H:F5H1 plants(Figure 4). It is tempting to believe that the reduction incell-wall integrity, possibly due to the shortage of lignin inthe cell wall, results in a greater demand for biosynthesis ofconiferyl and sinapyl alcohols, and thus a higher fluxthrough the phenylpropanoid pathway (Figure 4). Up-regulation of the phenylpropanoid pathway might thus bepart of the plant’s response to an aberrant cell-wall structure(Cano-Delgado et al., 2003; Rogers and Campbell, 2004).

We also tested whether the growth abnormalities in thecomt C4H:F5H1 plants could have been caused by the higherflavonoid levels, as suggested by Besseau et al. (2007).However, in contrast with their results, and in support of therecent data by Li et al. (2010), our data indicate thatflavonoids are certainly not the sole cause for the growthdefect in comt C4H:F5H1 plants, as crossing in a chsmutation was not able to fully restore the phenotype.Instead, the main reason for the partial recovery in growthobserved in comt C4H:F5H1 chs plants was reduction ofC4H-driven gene expression caused by the chs mutation,resulting in lower over-expression of the F5H1 gene and aconsequent a drop in the frequency of benzodioxane units(resulting from a reduction in the proportion of 5-hydroxy-coniferyl alcohol monomers) and an increase in total lignincontent (Table 2). The reason for reduction of this C4H-driven gene expression may either be negative feedback onthe C4H promoter, or positive feedback on the C4H promoterthat subsequently triggered gene silencing. In either case,the data clearly demonstrate a genetic interaction betweenthe flavonoid and phenylpropanoid pathways. Note that thisinteraction in inflorescence stems was dependent on thegenetic background: the chs mutation reduced C4H-drivengene expression only in comt C4H:F5H1 chs plants, but notin the single chs mutant. In addition, the reduction of C4H-driven gene expression appeared to be dependent on thedevelopmental stage, as it was observed in stems but not inseedling leaves (Figure 4). It is furthermore important tonote that the C4H promoter is one of the promoters of choiceto drive transgene expression in lignifying cells in order tomodify lignin biosynthesis in crop species (Huntley et al.,2003; Stewart et al., 2009). The observation that the expres-sion driven by this promoter is regulated, possibly inresponse to altered concentrations of pathway intermedi-ates, has repercussions on its use in gene engineeringstrategies. Such poorly studied genetic interactions withinthe phenylpropanoid pathway, for which full elucidationrequires a systems approach, need to be taken into accountduring rational design of plant cell walls with improvedproperties for agro-industrial applications.

(a)

(b)

Figure 4. Expression of phenylpropanoid biosynthetic genes in the variousmutants as determined by quantitative RT-PCR.(a) Expression of C4H and F5H1 in inflorescence stems. Error bars arestandard deviations. Significant differences in expression between the wildtype and the mutant (P < 0.05) are indicated by an asterisk.(b) Expression in 14-day-old seedling leaves. Values are the ratio of theexpression in the mutant line versus that in the wild type: a value >1 indicatesup regulation. Standard deviations are given in parentheses. Red and bluebackgrounds indicate higher and lower gene expression relative to the wildtype, respectively. The color intensity reflects the strength of induction andreduction. Values that are significantly different from those of the wild type(P < 0.05) are shown in bold.

Concomitant F5H1 up-regulation and COMT down-regulation in Arabidopsis 893

ª 2010 The AuthorsThe Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 885–897

EXPERIMENTAL PROCEDURES

Plant material

All experiments were performed with Arabidopsis ecotype Colum-bia (Col-0). The comt mutant (At5g54160, SALK_002373) and chsmutant (At5g13930, SALK_020583) were obtained from the SALKcollection (Alonso et al., 2003). Zygosity was analyzed using prim-ers 1 (5¢-ATGCCAAAGAGTCATGTTTTA-3¢) and 2 (5¢-TGGTG-TCTCTGGAAGTATAC-3¢) to amplify the endogenous COMT gene,and primers 1 and 3 (5¢-GCCCTTTGACGTTGGAGTC-3¢) to amplifythe comt T-DNA left-border insertion region. For CHS, primers 4(5¢-CTCGTCGGTCAGGCTCTTT-3¢) and 5 (5¢-TTCCATACTCGCTCAA-CACG-3¢) were used for the endogenous gene, and primers 3 and 4for the chs T-DNA left-border insertion region. The C4H:F5H1transgenic line (C4H-F5H) in the fah1-2 mutant background (Meyeret al., 1998) was kindly provided by Clint Chapple (Department ofBiochemistry, Purdue University, IN). This line over-expresses F5H1(At4g36220) under the control of the C4H (At2g30490) promoter.The presence of the C4H:F5H1 construct was analyzed usingprimers 5¢-CGTCGTGGGATCTGATAGTT-3¢ and 5¢-CTCCCTCTTA-TAAAATCCTCTC-3¢. To confirm the presence of the fah1-2 pointmutation, primers 5¢-ATGGAGAACTTTGCCTCCTG-3¢ and 5¢-GGA-TAAACAAGCGGCTCGT-3¢ were used. The C4H:F5H1 transgene isnot amplified by these primers. The 907 bp PCR product was furtherdigested using MseI (New England Biolabs Inc., http://www.neb.com), resulting in DNA fragments of 398, 225, 221, 56 and 7 bp forthe wild-type F5H1 and 267, 225, 221, 131, 56 and 7 bp for themutant fah1-2 allele. The comt C4H:F5H1 fah1-2, comt C4H:F5H1and C4H:F5H1 lines were isolated from the F2 progeny of a comt xC4H-F5H1 fah1-2 cross. Because comt C4H:F5H1 fah1-2 andcomt C4H:F5H1 plants are sterile, these genotypes were generatedfrom the parental plants heterozygous for comt and homozygousfor C4H:F5H1 and fah1-2. All experiments describedwere performedon comt C4H:F5H1 plants. However, comt C4H:F5H1 fah1-2 plantshad similar phenotypes.

Plant growth conditions

Agar-grown plants were grown at 23!C, with 16 h of light per day,on the following medium: 0.43% Murashige and Skoog vitaminmedium/basal salt mixture (M0221.0050, Duchefa Biochemie, http://www.duchefa.com), 1% sucrose (177140010, Acros, http://www.acros.com), 0.05% MES (M1503.0100, Duchefa Biochemie), pH 5.7,0.725% agar (A1296, Sigma, http://www.sigmaaldrich.com/) and0.1% 1000·Murashige and Skoog vitamin solution (M3900, Sigma).Soil-grown plants were germinated directly in Saniflor soil (VanIsrael; http://vanisrael.testvds.com) supplemented with 10% v/vvermiculite (16 h light, 22!C, 55% humidity).

Biomass

For biomass measurements, plants were grown in soil until senes-cence. Cauline leaves and siliques were removed from the inflo-rescence before weighing. Statistical analysis was performed usingANOVA followed by the Scheffe post hoc test.

Microscopy

Electron microscopy and lignin staining were performed essentiallyas described previously (Rohde et al., 2004). Stem segments werecut into small pieces and fixed with 4% paraformaldehyde and 2.5%glutaraldehyde, and post-fixed in 1% OsO4 with 1.5% K3Fe(CN)6 in0.1 M sodium cacodylate buffer, pH 7.2. Samples were dehydratedthrough a graded ethanol series, including bulk staining with 2%uranyl acetate at the 50% ethanol step, followed by embedding inSpurr’s resin. For tissue morphology, semi-thin sections (0.5 lm)

were cut using an Ultracut E microtome (Reichert-Jung, http://www.leica.com), stained in a 1% toluidine blue and 2% boratesolution in distilled water, and viewed using a Leica DM LB micro-scope (http://www.leica.com/).

For electron microscopy, ultra-thin sections of a gold interferencecolor were produced on an Ultracut E microtome (Reichert-Jung),and then post-stained in an ultrastainer (Leica) using uranyl acetateand lead citrate. They were viewed using a JEOL 1010 transmissionelectron microscope (JEOL, http://www.jeol.com).

Lignin histochemistry was performed by Maule staining aspreviously described (Rohde et al., 2004). Briefly, agar-imbeddedstem segments were sectioned with a vibroslicer, incubated in 4%KMnO4 for 5 min, rinsed with water, incubated in 37% HCl/H2O (1:1)for 2 min, and observed after addition of a drop of aqueous NH3.Sections were viewed under a binocular microscope (Leica MZ16).

Phenolic profiling and MS analysis

Single inflorescences, lacking cauline leaves, flowers and siliques,were sampled and immediately frozen in liquid nitrogen. Ten wholetissue-culture grown stems between 2.2 and 6.0 cm long wereobtained from comt C4H:F5H1 plants and compared with five wild-type whole stems of approximately 8.0 cm, as well as five apical andfive basal stem segments of 5.0 cm from approximately 20 cm tallwild-type plants. Samples were transferred to 1.5 ml Eppendorftubes. After grinding, the samples were extracted in 600 ll THF/isopropanol/methanol (15:15:70). The extract (550 ll) was freeze-dried in a SAVANT Speedvac (SC21A SpeedVac Concentrator,Thermo, http://www.thermo.com), and subjected to extractionusing water/cyclohexane (100 ll of each). Each individual sample(60 ll) was injected by means of a SpectraSystem AS1000 auto-sampler (Thermo Separation Products) onto a reverse-phase LunaC18(2) column (150 · 2.1 mm, 3 lm; Phenomenex, http://www.phenomenex.com).

Gradient separation was performed on a SpectraSystemP1000XR HPLC pump (Thermo Separation Products), using agradient of two buffers: buffer A (water/acetonitrile (ACN)/aceticacid, 100:1:0.1, pH 2) and buffer B (ACN/water/acetic acid, 100:1:0.1,pH 2). A flow rate of 0.25 ml min)1 was used, with a buffercomposition starting at 5% B, linearly increasing to 17% at 1 min,77% at 33 min, and finally 100% at 36 min. A SpectraSystemUV6000LP detector (Thermo Separation Products) was used tomeasure UV/vis absorption between 200 and 450 nm with a scanrate of 2 Hz. Atmospheric pressure chemical ionization (APCI)operated in the negative-ion mode, was used as an ion source tocouple HPLC to LCQ Classic MS instrument (ThermoQuest) (vapor-izer temperature 350!C, capillary temperature 140!C, source current5 mA, sheath gas flow set at 27, auxiliary gas flow set at 3,m/z range140–1000, normalized collision energy for MS2 35).

NMR sample preparation

NMR samples were prepared essentially as described previously(Weng et al., 2010). Senesced inflorescence stems of Arabidopsis(approximately 200 mg) from tissue culture- and soil-grown plants(Figures S1 and S2) were pre-ground in a Retsch MM400 mixer mill(http://retsch.com), equipped with a 10 ml ZrO2 vessel and two ZrO2

ball-bearings (12 mm diameter), for 1 min at 30 Hz. The groundmaterial was extracted three times with water and subsequentlythree times with 80% ethanol by sonicating in an ultrasonic bath for30 min each time. The freeze-dried extractive-free Arabidopsissamples (approximately 100 mg) were ball-milled using a RetschPM 100 planetary ball mill in a 50 ml ZrO2 vessel with 10 ZrO2 ball-bearings (10 mm diameter) at 600 rpm, with 5 min breaks afterevery 5 min of milling. The total ball-milling time for the sampleswas 16.5 min. Cellulolytic enzyme lignins (CEL) were then isolated

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by enzymatically saccharifying (most of the) polysaccharides asdescribed by Chang et al. (1975). Cellulysin cellulase (EC 3.2.1.4,Calbiochem, http://www.calbiochem.com), a crude cellulase prep-aration containing hemicellulase activities, from Trichoderma viridewas used. Its activity was 10 000 units g)1 dry weight. The ball-milled material was suspended in 20 mM NaOAc buffer (30 ml, pH5.0) in a 50 ml centrifuge tube, 8 mg of Cellulysin was added, andthe reaction slurry was incubated at 35!C for 48 h. The solids werepelleted by centrifugation (1500 g, 21!C, 40 min), and the processwas repeated with fresh buffer and enzyme. CEL was recovered byfiltration through a nylon membrane (0.2 lm). The product waswashed with water and dried under vacuum at room temperature,yielding 16–28 mg CEL, depending on the sample. CEL (12 mg) wasacetylated using 2 ml DMSO, 1 ml N-methylimidazole and 0.6 mlacetic anhydride, stirring for 1.5 h at room temperature, beforequenching the reaction by pouring the solution into 1 L of distilledwater. The precipitated material was recovered by filtration througha nylon membrane (0.2 mm). Acetylated CEL, typically approxi-mately 15–18 mg from 12 mg CEL, was dried under vacuum anddissolved in 0.5 ml CDCl3 for NMR analysis. For the comt C4H:F5H1line, only approximately 3–4 mg of acetylated CEL was obtainedafter filtering out unidentified gelatinous CDCl3-insoluble material.Nevertheless, the 2D HSQC spectrumwas of excellent quality, and itwas even possible to obtain a 3D TOCSY-HSQC spectrum from thissample.

NMR experiments

NMR spectra were acquired on a Bruker-Biospin AVANCE 500 MHzspectrometer (Bruker-Biospin, http://www.bruker-biospin.com)fitted with a cryogenically cooled 5 mmBruker TCI (triple resonancecryoprobe optimized for 1H and 13C observation) gradient probewith inverse geometry (proton coils closest to the sample). The 2D13C–1H correlation spectra were acquired using an adiabatic HSQCpulse program (Bruker standard pulse sequence ‘hsqcetgpsisp2.2¢)(Kupce and Freeman, 2007) and the following parameters: spectrawere acquired from 10 to 0 ppm (5000 Hz) in F2 (1H) using 1000 datapoints for an acquisition time (AQ) of 200 msec, an interscan delay(D1) of 500 msec, and from 196 to )23 ppm (25 154 Hz) in F1 (13C)using 400 increments (F1 acquisition time 8 msec) of 90 scans, for atotal acquisition time of 17 h 17 min. The number of scans can beadjusted depending on the signal-to-noise required from a sample.The 1JCH used was 145 Hz. Processing used typical matchedGaussian apodization in the 1H dimension and squared cosine-bellapodization in the 13C dimension. Prior to Fourier transformation,the data matrices were zero-filled to 1024 points in the 13C dimen-sion. The central chloroform solvent peak at a dC/dH of 77.0/7.26 ppm was used as an internal reference. The 3D TOCSY-HSQCspectrum was acquired and processed exactly as described previ-ously, providing similar evidence for the benzodioxane units(Marita et al., 2001).

G:5H:S integral ratios were obtained by integrating the contoursfrom the 2 or 2,6 positions of each type of aromatic unit (with Sintegrals being logically halved as the 2 and 6 positions are identicalin the symmetrical S units). In samples with 5H correlations, wherethe 5H6 correlations overlap with the S2/6 correlations, the S integralwas calculated by subtracting the 5H2 integral (which should be thesame as the 5H6 integral) from the integral of the overlapping S2/6 +5H6 contours (Figure 3b–e).

Lignin quantification

Cell-wall material was prepared from 5 mg of senescent inflores-cence stems of soil-grown plants, washed sequentially with 0.5 mlwater, ethanol, chloroform and acetone for 30 min each at 98, 76, 59

and 45!C, respectively. Acetyl bromide lignin extraction was per-formed according a downscaled method similar to that describedpreviously (Dence, 1992). Briefly, 0.1 ml acetyl bromide (25% inacetic acid) and 4 ll 60% perchloric acid were added to the dry cellwall and the mixture was incubated at 70!C for 30 min. NaOH (2 M,0.2 ml) and 0.5 ml acetic acid were then added. After centrifugation(15 000 g, room temperature, 15 min), the supernatant was sepa-rated from the pellet. The pellet was further washed by adding0.5 ml acetic acid (vortex, 5 s), followed by centrifugation (15 000 g,room temperature, 15 min). The second supernatant was combinedwith the first. Acetic acid was added to the combined supernatantsto a volume of 2 ml, and absorption was measured at 280 nm usinga NanoDrop ND-1000 spectrophotometer (Nanodrop, http://www.nanodrop.com). An extinction coefficient for lignin at 280 nm of23.35 L g)1 cm)1 was used (Chang et al., 2008). As for every ligninquantification method, the response varies with the composition ofthe lignin; the acetyl bromide protocol gives only an estimate of thetotal lignin amount (Hatfield and Fukushima, 2005) and we used thesame lignin UV280 extinction coefficient for all samples here.Statistical tests were performed as indicated for biomassmeasurements.

RNA extraction, cDNA preparation and quantitative RT-PCR

Total RNA was extracted from frozen soil-grown inflorescencestems and tissue-cultured seedling leaves using a plant RNeasyextraction kit (Qiagen, http://www.qiagen.com/), and quantifiedusing a NanoDrop ND-1000 spectrophotometer. Total RNA (4.5 lg)was reverse-transcribed in a final volume of 190 ll using a Super-script II reverse transcription kit (Invitrogen, http://www.invitro-gen.com/). Quantitative RT-PCRwas performed using a SYBRGreenI master kit (Roche, http://www.roche.com) on a Lightcycler 480(Roche). The primer sequences used for quantitative RT-PCR aregiven in Table S1. Gene expression was normalized on the basis ofACTIN expression. Statistical tests were performed using a procglmnested model with unequal variances (SAS 9.2; SAS Institute Inc.,http://www.sas.com).

ACKNOWLEDGEMENTS

The authors thank Clint Chapple (Department of Biochemistry,Purdue University, West Lafayette, IN) for kindly providing theC4H:F5H1 fah1-2 line, Bart Ivens and David Casini for practicalassistance, and Martine De Cock for help in preparing the manu-script. We gratefully acknowledge partial funding through the Uni-ted States Department of Energy (DOE) Energy Biosciencesprogram (grant number DE-AI02-00ER15067) and the DOE GreatLakes Bioenergy Research Center (DOE Office of Science, grantnumber BER DE-FC02-07ER64494) to J.R., the Research Foundation-Flanders (grant number G.0352.05N), the European Community’s7th Framework Programme (FP7/2007) under grant agreement no.211982 (RENEWALL), the Global Climate and Energy Project (GCEP)(grants to W.B. for ‘Towards New Degradable Lignin Types’ and toJ.R. for ‘Efficient Biomass Conversion: Delineating the Best LigninMonomer Substitutes’), and the Multidisciplinary Research Part-nership ‘Biotechnology for a Sustainable Economy’ (01MRB510W)of Ghent University. Some of the NMR experiments on the BrukerDMX-500 cryoprobe system made use of the National MagneticResonance Facility at Madison (http://www.nmrfam.wisc.edu). R.V.is indebted to the Agency for Innovation by Science and Technologyfor a pre-doctoral fellowship.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:

Concomitant F5H1 up-regulation and COMT down-regulation in Arabidopsis 895

ª 2010 The AuthorsThe Plant Journal ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 885–897

Figure S1. Percentages of 5H and benzodioxane units in lignins ofvarious mutant and transgenic plant lines.Figure S2. Partial short-range 13C–1H (HSQC) spectra of acetylatedenzyme lignins in aromatic regions.Figure S3. Partial short-range 13C–1H (HSQC) spectra of acetylatedenzyme lignins in aliphatic regions.Figure S4. UV/vis absorption chromatogram of inflorescence stemextracts of comt C4H:F5H1 and wild-type plants.Figure S5. MS spectra of 38 5-hydroxylated phenylpropanoids thatwere identified in comt C4H:F5H1 plants.Table S1. Primers used in quantitative RT-PCR experiments.Please note: As a service to our authors and readers, this journalprovides supporting Information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting Information (other than missingfiles) should be addressed to the authors.

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Concomitant F5H1 up-regulation and COMT down-regulation in Arabidopsis 897

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Ruben Vanholme John Ralph Takuya Akiyama Fachuang Lu Jorge Rencoret Pazo Hoon Kim Jørgen Holst ChristensenBrecht Van Reusel Véronique Storme Riet De Rycke Antje Rohde Kris Morreel Wout Boerjan All Authors

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Ruben Vanholme1,2, John Ralph3, TakuyaAkiyama3, Fachuang Lu3, Jorge RencoretPazo3, Hoon Kim3, Jørgen HolstChristensen1,2, Brecht Van Reusel1,2,Véronique Storme1,2, Riet De Rycke1,2,Antje Rohde1,2, Kris Morreel1,2, WoutBoerjan1,2,*

Article first published online: 15 OCT 2010

DOI: 10.1111/j.1365-313X.2010.04353.x

© 2010 The Authors. The Plant Journal © 2010Blackwell Publishing Ltd

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The Plant JournalVolume 64, Issue 6, pages885–897, December 2010

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Engineering traditional monolignols out of lignin by concomitant up-regulation of F5H1 and down-regulation of COMT in Arabidopsis

Figure S1. The %5H- and %benzodioxane-units in lignins of different mutant and transgenic plant lines, reported in literature and in this study. Data from previously reported NMRstudies was re-evaluated here (from uncorrected volume integrals). %5H is 5H/(S+G+5H) and %benzodioxane levels is J/(A+B+C+J) – the relatively minor dibenzodioxocins Dwere neglected here (see Fig. 3 for structures of G, 5H and S and Figure S3 for structures of A, B, C, D and J). Note that these spectra were run over many years on differentinstruments and under different acquisition conditions. The values are nevertheless reasonably comparable and, interestingly, there is a fairly regular (almost linear) correlationbetween %benzodioxanes and %5H units (See graph). Thioacidolysis releases 5H monomers from only a fraction of the benzodioxane units. 1In the paper of Marita et al. (2003)38% benzodioxane inter-unit linkages were noted as structures (d) were also included in the calculation; 2The COMT-silenced poplars are the same lines; 3The C4H:F5H1Arabidopsis plants are the same lines; n.r. not reported.

Figure S2. Partial short-range 13C– 1H (HSQC) spectra (aromatic regions) of acetylated enzyme lignins isolated from agar-grown plants (a–e) and soil-grown plants (f–k). The S-unit integral estimate comes from subtracting the 5H 2 contour integral from the S 2/6+5H 6 integral; the G-unit integral is determined directly from the resolved G 2 component, as inthe WT. For those genotypes that were grown both in soil and on agar, no major differences in lignin composition are seen between the two growth conditions.

Figure S3. Partial short-range 13C– 1H (HSQC) spectra (aliphatic regions) of acetylated enzyme lignins isolated from agar-grown plants (a–e) and soil-grown plants (f–k), withcolor-coded assignments for units A–D and the benzodioxane units J resulting from 5-hydroxyconiferyl alcohol incorporation. For those genotypes that were grown both in soil andon agar, no major differences in lignin composition are seen between the two growth conditions.

Figure S4. UV-Vis absorption chromatogram of inflorescence stem extracts of comt C4H:F5H1 and WT plants. comt C4H:F5H1 plants accumulate large amounts of 5-hydroxylated phenylpropanoids (1–38 ), the identities of which are given in Table 3. Several peaks that are highly abundant in WT are given, cis- and trans-sinapoyl malate (SM),sinapoyl glucose (SG) and flavonol glycosides (FG1, 3-O-Rha(1->2)Glu-7-O-Rha-kaemferol; FG2, 3-O-Glu-7-O-Rha-kaemferol; FG3, 3,7-di-O-Rha-kaemferol; FG4, kaemferolrhamnoside; FG5, 3-O-Rha(1->2)Glu-7-O-Rha-quercitin; FG6, 3-O-Glu- 7-O-Rha-quercitin). Grey, profile of WT; red, profile of comt C4H:F5H1.

Figure S5. MS spectra of thirty eight 5-hydroxylated phenylpropanoids that were identified in comt C4H:F5H1 plants, with the reasoning for structural identification.

Table S1. Primers used in qRT-PCR experiments. 1For ACTIN (ACT) genes, the primers amplify ACT2 (At3g18780) and ACT8 (At1g49240) simultaneously (Charrier et al., 2002).

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S2/6+5H6

G : 5H : S ~ 33.5 : 20.0 : 46.4

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G : 5H : S~ 13.8 : 71.0 : 15.2

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G : 5H : S ~ 6.5 : 1.9 : 91.5

G : 5H : S ~ 0.0 : 2.8 : 97.2

S2/6

G2

G : 5H : S ~ 96.4 : 2.9 : 0.7

G2

G : 5H : S ~ 94.8 : 1.3 : 3.9

S2/6

G : 5H : S ~ 21.1 : 0.0 : 78.9

G2G2

G2

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G : 5H : S ~ : 84.2 : 0.0 : 15.8

G : 5H : S~ 100.0 : 0.0 : 0.0

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Table S1. Primers used in qRT-PCR experiments. 1For ACTIN (ACT) genes, the primers amplify

ACT2 (At3g18780) and ACT8 (At1g49240) simultaneously (Charrier et al., 2002).

Gene AGI code Forward primer Reverse primer Code Sequence Code Sequence PAL2 At3g53260 421 5'-GTTTTGCGAGAAGGACTTGC-3' 422 5'-TCCGTTCCATTCCTTGAGAC-3' C4H At2g30490 472 5'-CACCGGGAAAGGTCAAGATA-3' 473 5'-CCCAACCTTCACGATTCTGT-3' 4CL1 At1g51680 431 5'-GATGCCGTTAATGCCAAGTT-3' 432 5'-ACCGATGAGCAAAGCCTCTA-3' 4CL2 At3g21240 423 5'-AAGTCACGGTGGCTATGGTC-3' 424 5'-CAATGAAAAGCTCGTCGTCA-3' C3H At2g40890 509 5'-TTTCGTTGATGCGTTGCTAA-3' 510 5'-CGGTCAAGTCCAACCACTCT-3' CCoAOMT1 At4g34050 425 5'-GGCAAAATTCTGGCTATGGA-3' 426 5'-TCACCAACAGGGAGCATACA-3' CCR1 At1g15950 417 5'-AGGGGCTCAAATCCTCTCTC-3' 418 5'-GCGATGGAATAAAGGCAAGA-3' CCR2 At1g80820 351 5'-GAACCCAACTGATCCCAAGA-3' 352 5'-ACAATGGCTTGAGTGTCACG-3' F5H1 At4g36220 469 5'-ATGATGGGGATGTTGTCGAT-3' 434 5'-CGTCCATGATGATTGCTTTG-3' COMT At5g54160 470 5'-AGTGACGAACATTGCGTGAA-3' 471 5'-TGTTTGGTTGAGAGGCTTGA-3' CHS At5g13930 344 5'-CTCGTCGGTCAGGCTCTTT-3' 345 5'-TTCCATACTCGCTCAACACG-3' ACT2/81 At3g18780,

At1g49240 465 5'-GGTAACATTGTGCTCAGTGGTGG-3' 466 5'-AACGACCTTAATCTTCATGCTGC-3'

Charrier, B., Champion, A., Henry, Y. and Kreis, M. (2002) Expression profiling of the whole Arabidopsis shaggy-

like kinase multigene family by real-time reverse transcriptase-polymerase chain reaction. Plant Physiol. 130, 577-590.