Manipulation of Guaiacyl and Syringyl MonomerBiosynthesis in an Arabidopsis Cinnamyl AlcoholDehydrogenase Mutant Results in Atypical LigninBiosynthesis and Modified Cell Wall Structure

Nickolas A. Anderson,a,b Yuki Tobimatsu,c,d,e Peter N. Ciesielski,f Eduardo Ximenes,g John Ralph,c,d,h

Bryon S. Donohoe,f Michael Ladisch,g,i and Clint Chapplea,1

a Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907bHeartland Plant Innovations, Manhattan, Kansas 66502cDepartment of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706dDOE Great Lakes Bioenergy Research Center and Wisconsin Energy Institute, University of Wisconsin-Madison, Madison,Wisconsin 53726eResearch Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japanf Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401gDepartment of Agricultural and Biological Engineering and the Laboratory of Renewable Resources Engineering, Purdue University,West Lafayette, Indiana 47907hDepartment of Biological Systems Engineering, University of Wisconsin-Madison, Madison, Wisconsin 53706iWeldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907

ORCID IDs: 0000-0002-6572-8954 (N.A.A.); 0000-0001-9087-0218 (E.X.); 0000-0002-2272-5059 (B.S.D.); 0000-0001-9953-9599 (M.L.);0000-0002-5195-562X (C.C.)

Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production.Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsisthaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignincomposition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacylor syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derivedlignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc caddplants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast,disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in theseplants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd,fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given thatthese CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehydemonomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant ofdigestibility than lignin content.

INTRODUCTION

Lignin is a complex polymer present in the secondary cell wall ofall tracheophytes (Bonawitz and Chapple, 2010; Vanholme et al.,2010a). In angiosperms, these polymers are primarily com-posed of guaiacyl (G) and syringyl (S) subunits with minoramounts of p-hydroxyphenyl (H) subunits, derived from the hy-droxycinnamyl alcohol monomers (or monolignols) coniferyl,sinapyl, and p-coumaryl alcohols (Vanholme et al., 2012).The hydroxycinnamaldehydes and hydroxycinnamic acids

corresponding to some of these alcohols are also incorporatedinto lignin in smaller amounts (Baucher et al., 1996; Sibout et al.,2005; Ralph et al., 2008a). In addition to the predominant mon-olignols, other naturally occurring monomers, such as caffeylalcohol, the flavonoid tricin, hydroxybenzaldehydes, dihydro-hydroxycinnamyl alcohols, and the variously acylated (by acetate,p-coumarate, p-hydroxybenzoate, and ferulate) monolignols, areincorporated into the polymer to varying degrees in certainspecies and in specific tissues (Boerjan et al., 2003; Del Río et al.,2007; Lu and Ralph, 2008; Chen et al., 2012; del Río et al., 2012;Rencoret et al., 2013; Wilkerson et al., 2014; Lu et al., 2015).Lignin polymers interact with wall polysaccharides, adding to

the structural integrity of the cell wall, and are responsible forrendering certain cell types impermeable to water (Boerjan et al.,2003); however, the interactions between lignin and cell wallpolysaccharides and the enzymes that hydrolyze them greatlyimpede the conversion of these polymers for industrial and

1Address correspondence to chapple@purdue.edu.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Clint Chapple (chapple@purdue.edu).www.plantcell.org/cgi/doi/10.1105/tpc.15.00373

The Plant Cell, Vol. 27: 2195–2209, August 2015, www.plantcell.org ã 2015 American Society of Plant Biologists. All rights reserved.

agricultural purposes (Pauly andKeegstra, 2010; Somerville et al.,2010; Carpita, 2012; Jung et al., 2012; Kim et al., 2015; Ko et al.,2015). To overcome this challenge, many strategies to reducelignin content or alter lignin composition and structure have beenimplemented with the overall goal of increasing cell wall de-gradability (Vanholme et al., 2008; Van Acker et al., 2013).

Most of the enzymes that play a role in lignin biosynthesis havebeen identified and are well characterized (Fraser and Chapple,2011). Disruptions of several lignin biosynthetic genes result inplants that accumulate atypical lignin polymers that are either notpresent or are not abundant in wild-type plants. For example,downregulation of p-coumaroylshikimate 3ʹ-hydroxylase orhydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferaseresults in plants with elevated levels of H lignin (Hoffmann et al.,2004; Reddy et al., 2005; Besseau et al., 2007; Shadle et al., 2007;Coleman et al., 2008; Li et al., 2010a; Bonawitz et al., 2014).Downregulation of ferulate 5-hydroxylase (F5H) reduces oreliminates S subunits (Chapple et al., 1992; Reddy et al., 2005);conversely, its overexpression results in plants with a greatlyelevated %S levels (Meyer et al., 1998; Franke et al., 2000;Huntley et al., 2003; Stewart et al., 2009). Reduction in caffeicacid-O-methyltransferase activity leads to the incorporation of5-hydroxyconiferyl alcohol into lignin polymers, even though it is notdetectable in the wild-type polymer (Ralph et al., 2001b; Vanholmeet al., 2010b; Weng et al., 2010). These manipulations have notonly demonstrated the plasticity of lignin biosynthesis but havealso led to the development of plants with superior cell wall di-gestibility characteristics (Ralph et al., 2004; Li et al., 2008;Vanholme et al., 2012; Van Acker et al., 2014).

Downregulation of (hydroxy)cinnamyl alcohol dehydrogenase(CAD) genes is another promising strategy to increase cell walldigestibility (Halpin et al., 1994; Vailhé et al., 1998; Baucher et al.,1999; Chen et al., 2003; Jouanin et al., 2004; Saathoff et al., 2011).CAD is required for the reduction of hydroxycinnamaldehydes tohydroxycinnamyl alcohols, and downregulation of CAD genesgenerates unusual lignin polymers partially derived from hy-droxycinnamaldehyde subunits (Kim et al., 2003; Kim et al., 2004).In Arabidopsis thaliana, two CADs (CADC and CADD) are thoseprimarily involved in lignin monomer synthesis and, as in othersystems, cadc cadd plants deposit aldehyde-enriched lignins(Sibout et al., 2005), although the structural details of themodified lignins remain elusive. The characterization of CAD-deficient plants has been conducted not only in model plants likeArabidopsis, but also in agriculturally relevant plants includingpoplar (Populus sp), alfalfa (Medicago sativa), tobacco (Nicotianatabacum), maize (Zea mays), sorghum (Sorghum bicolor), rice(Oryza sativa), switchgrass (Panicum virgatum), and tall fescue(Festuca arundinacea). In addition to the increase in cell wall di-gestibility, CAD-deficient mutants and transgenics display red-pigmentedstemsand, in thecaseofArabidopsis, a limpfloral stem(Pillonel etal., 1991;Baucheretal., 1996;Siboutetal., 2005;Zhanget al., 2006). Although the disruption of CAD activity results inamajor shift in lignin composition, this modification has aminimaleffect on the overall plant yield under most environmental con-ditions (Kaur et al., 2012; Zhao et al., 2013). Although unalteredgrowth appears to be the general case for CAD-deficient plants,other manipulations of lignin biosynthesis have resulted indwarfed and, in some severe cases, infertile plants (Franke et al.,

2002; Mir Derikvand et al., 2008; Schilmiller et al., 2009; Huanget al., 2010; Li et al., 2010a).Here, we used a combinatorial approach to generate plants

containing two unusual lignin polymers enriched in aldehydes bydisrupting twoArabidopsisCADgenes,CADC andCADD, inG/S-modified lignin backgrounds. NMR resolved themajor shifts in thelignin composition and structures in the series of cad mutants.These data further support the observation that CADC and CADDare partially functionally redundant; however, these data alsosuggest that either CADD has an important role in catalyzing thereduction of sinapaldehyde or is themajor formof CADexpressedin cells that deposit S lignin. In addition, these aldehyde ligninsubunit compositionalmanipulations lead to increased enzymaticcell wall digestibility even though total lignin content was con-served. Taken together, these results show that lignin composi-tional changes can increase cell wall digestibility compared withmanipulating lignin content alone and bolster the contention thatviable plants can result from lignins derived from essentially notraditional monolignols.

RESULTS

Stacking Lignin Modification Strategies Generates Plantswith Unexpected Dwarf Growth Phenotypes

The biosynthesis of lignin, one of the main end-products of thephenylpropanoid pathway, is metabolically plastic and dependsheavily on subunit availability to determine composition (Chappleet al., 1992;Meyer et al., 1998; Sederoff et al., 1999; Boerjan et al.,2003; Vanholme et al., 2008, 2012; Ralph et al., 2004, 2008b;Ralph, 2010; Anderson andChapple, 2014;Wilkerson et al., 2014;Bonawitz et al., 2014). Togenerateplants enriched in units derivedfrom either coniferaldehyde (Gʹ) or sinapaldehyde (Sʹ), we crossedthe previously studied Arabidopsis cadc and cadd mutations(Sibout et al., 2003, 2005) into the F5H-deficient (high G) ferulicacid hydroxylase1 (fah1) mutant and a transgenic line in which theF5H gene is overexpressed under the control of the cinnamate4-hydroxylase (C4H) promoter (high S) (C4H-F5H), respectively(Figure 1).The fah1,cadc, andcaddsinglemutantsaswell as theC4H-F5H

line display wild-type growth (Figures 2 and 3). Similarly, cadccadd, fah1 cadc cadd, and cadc C4H-F5H plants obtain a wild-type height even though they display the limp floral stem phe-notype previously reported for cadc cadd (Sibout et al., 2005).Surprisingly, cadd C4H-F5H is dwarfed, with a significant re-duction in both rosette size and stem length. The most drasticchange in morphology is observed in cadc cadd C4H-F5H, whichbolts but is severely dwarfed and is infertile.Dwarfism in plants with disrupted lignin biosynthesis is not

uncommon (Hoffmann et al., 2004; Mir Derikvand et al., 2008;Stout et al., 2008; Schilmiller et al., 2009), but themechanisms thatlead to dwarfism are still in contention andmay bemultifaceted innature (Patten et al., 2005; Laskar et al., 2006; Bonawitz andChapple, 2013; Bonawitz et al., 2014). To test if the dwarfism weobserved is associated with a deficiency in lignin, we quantifiedlignin using the acetyl bromidemethod (Chang et al., 2008). In ouranalysis, we found a 30% total lignin decrease in cadc cadd

2196 The Plant Cell

comparedwith thewild type andplants disrupted in eitherCADCorCADD alone (Figure 3), as has been described previously (Siboutet al., 2003, 2005), and not surprisingly also in fah1 cadc cadd. Asimilar reduction in lignin content was unexpectedly observed forcadcC4H-F5Handfor thedwarfedcaddC4H-F5Hplants (Figure3).Theseresultssuggest that thedwarfismobservedincaddC4H-F5Hplants is not directly or solely the result of reduced lignin contentgiven that they deposit levels of lignin comparable to other CAD-disrupted plants that are wild-type in growth.

Hydroxycinnamaldehydes Can Be Incorporated intoLignin Polymers

To determine how disruption of monolignol biosynthesis alterslignin structure, we performedNMRanalysis on cell walls from theseriesofplantsdescribedabove (Figure4).Gel-stateNMRspectrawere acquired with samples prepared by directly swelling the

whole stem cell wall fractions, after fine milling, in DMSO-d6/pyridine-d5 (4:1, v/v) (Kim and Ralph, 2010; Mansfield et al.,2012a). Although we were able to generate the cadc cadd C4H-F5H line, we were not able to obtain sufficient material to conductthe NMR analysis as it displays such a severe dwarf phenotype(Figure 2).Changes in lignin monomer composition are most readily vi-

sualized from thearomatic regions of the two-dimensional 1H–13Ccorrelation (heteronuclear single quantum coherence [HSQC])spectra (Figure 4). The spectra of the wild type displayed signalsfrom G-rich G/S-type lignins derived from polymerization of bothconiferyl and sinapyl alcohols, as is typical for most angiospermlignins. The lignin alterations seen in the fah1 and C4H-F5H linesare consistent with the outcomes of the gene manipulationsdescribed in the earlier studies (Chapple et al., 1992; Meyer et al.,1998), with the former showing G lignins derived exclusively frompolymerization of coniferyl alcohol and the latter S lignins from

Figure 1. The Angiosperm Phenylpropanoid Pathway.

The highlighted gray region outlines the strategy for lignin-incorporated coniferaldehyde enrichment that is achieved by blocking at F5H and CAD. Thestrategy for lignin-incorporated sinapaldehyde enrichment expands on the coniferaldehyde enrichment strategy (gray, and the additional area outlined ingreen) and includes the combination of F5H overexpression and CAD disruption.

Aldehyde-Enriched Lignins 2197

sinapyl alcohol. Thenatural presenceof thealdehydeunits (G’andS’) in wild-type and fah1 plants was minimal and typical; G’2 andS’2/6 signals are overestimates of hydroxycinnamaldehyde in-corporation as they also occur due to the presence of benzal-dehyde units and oxidized b-aryl ether units with a-carbonylcarbons, some of which, particularly the syringyl units S’, areproduced during ball-milling (Kim and Ralph, 2010; Zhao et al.,2013). On the other hand, as expected, levels of aldehyde-derivedunits are significantly elevated in the CAD mutants when bothCADCandCADDgenesaredownregulated.OurNMRdataclearlyshow that the lignins produced in the cadc cadd line are strikingG’/S’ lignins derived exclusively from polymerization of coni-feraldehyde and sinapaldehyde. In addition, the fah1 cadc caddtriplemutant producedG’ lignins solely fromconiferaldehyde. Thetypical alcohol-derived lignin units (G and S) are below the level ofdetection in these CAD-deficient mutants. Loss of function ineither only CADC or CADD did not eliminate lignin units derivedfrom coniferyl and/or sinapyl alcohols. TheCADCmutation hardly

affects the monomer composition of the lignins, which is con-sistent with the previous observation in these Arabidopsismutants (Sibout et al., 2005). Also consistent with previous ob-servations, our NMR data showed that disruption of CADD couldonly modestly elevate S’ lignin unit levels in the cell walls (Siboutet al., 2003). Strikingly, cadd C4H-F5H produced considerablyS’-rich S/S’ lignins, mainly derived from polymerization of sina-paldehyde (;70% based on the HSQC signal intensities). Asnotedabove, and regrettably,wewereunable toconduct theNMRanalysis on the cadc cadd C4H-F5H line that displays a severedwarf phenotype (Figure 2); we anticipated that the lignin in thisline would be entirely sinapaldehyde-derived.The aldehyde regions of the HSQC spectra (Figure 4) determine

the nature of the various aldehyde units in the polymers, whereasaliphatic regions (Supplemental Figure 1) show the distributionsof the intermonomeric linkages in the lignins. Consistent withthe above aromatic compositional data, the characteristic andaldehyde-derived 8–O–4-units (I9G and I9S) that are diagnostic for

Figure 2. Representative Pictures of F5H- and CAD-Manipulated Plants at 2 Months of Age.

Bars = 1 cm.

2198 The Plant Cell

the (cross-)coupling of hydroxycinnamaldehydes onto a growinglignin chain (Kim et al., 2000, 2003; Zhao et al., 2013) are clearlyseen in the mutant lines producing aldehyde-rich lignins (cadccadd, fah1 cadc cadd, and cadd C4H-F5H), whereas these al-dehyde signals, except for the naturally occurring cinnamalde-hyde (X2) and benzaldehyde (X3) end-units, are not detectable inthe spectra from the wild type and other mutant lines producingtypical alcohol-derived lignins (fah1, cadc, cadd, and cadc C4H-F5H) (Figure 4). In addition, typical lignin units, such as b-aryl

ethers (IG and IS), phenylcoumarans (II), resinols (III), and cinnamylalcohol end-groups (X1), are practically absent or significantly de-pleted in the mutant lines (cadc cadd, fah1 cadc cadd, and caddC4H-F5H) producing ligninsmainly fromhydroxycinnamaldehydes,in comparison to the wild type and other mutant lines (fah1, cadc,cadd, and cadc C4H-F5H) producing normal lignins fromp-hydroxycinnamyl alcohols (Supplemental Figure 1). We alsoanalyzed cell wall polysaccharide unit profiles by HSQC NMR(Kim and Ralph, 2010; Kim and Ralph, 2014). The signal distri-bution patterns in sugar anomeric regions appear similar amongthe wild type and all of themutants analyzed here (SupplementalFigure 2), suggesting that alteration of lignin biosynthesis inthese plants does not dramatically affect the composition of cellwall polysaccharides.

Major Soluble and Wall-Bound Metabolites Are OnlyModestly Affected by CAD Disruption

In addition to lignin, the phenylpropanoid pathway is required forthe synthesis of cell wall-bound hydroxycinnamic acids andsoluble hydroxycinnamate esters (HCEs). To assess how thepathwaymanipulationsdescribedaboveaffect thesemetabolites,we analyzed leaf HCE content (Figure 5) and the phenolics re-leased after alkaline hydrolysis of inflorescence cell wall tissue(Figure 5). In these experiments, we observed a modest yet sig-nificant increase in total HCEs (measured primarily as sina-poylmalate) in cadc cadd, fah1 cadc cadd, cadc C4H-F5H, caddC4H-F5H, and cadc cadd C4H-F5H (Figure 5). These data sug-gest that, in HCE-producing cells, reduced CAD activity permitsenhanced conversion of coniferaldehyde and sinapaldehyde,presumably by the hydroxycinnamaldehyde dehydrogenase en-coded by REDUCED EPIDERMAL FLUORESCENCE1 (REF1), toferulic acid and sinapic acid, respectively, which are subsequentlyavailable for enhanced HCE synthesis.Unlike the relatively minor changes in soluble leaf metabolite

biosynthesis, in all CAD-disrupted lines, dramatic changes areobserved in phenolic compounds released from inflorescencetissue by alkaline hydrolysis (Figure 5). We observed about a 10-fold increase in cell wall-bound ferulic acid in cadc cadd and fah1cadc cadd comparedwith thewild type and fah1, respectively, butno increase inp-coumaric acid. In addition, we observed a 30-foldincrease in vanillin release from both cadc cadd and fah1 cadccadd, which is most likely the result of degradation of aldehyde-rich lignins during the saponification treatment. Another likelylignin degradation product, syringaldehyde, increased 6-fold incadc C4H-F5H samples and 60-fold in cadd C4H-F5H hydro-lysates compared with C4H-F5H. These data indicate that, as inleaves, reductions in CAD activity results in redirection of carbonflux toward hydroxycinnamic acid biosynthesis and enhancedesterification of these metabolites by cell wall components.

CAD-Manipulated Plants Have Typical, NoncollapsedVasculature Despite Having Altered Cell Wall Compositionand Structure

To assess the effects lignin compositional changes have onvascular anatomy, we first conducted several histochemicalanalyses on sectioned inflorescence tissue (Figure 6). Previous

Figure 3. The Dwarf Phenotype of cadd C4H-F5HDoes Not Appear to BeCaused Solely by a Decrease in Total Lignin Content.

(A) Height of the primary inflorescence was measured in 2-month-oldplants.(B) Lignin content of 8-week-old cell wall-extracted tissue from in-florescence stems was determined by the acetyl bromide method.Error bars represent the SD of biological triplicates. Asterisks indicate thesignificant difference between the plants with modified lignin compositioncompared with the wild type (*P < 0.05).

Aldehyde-Enriched Lignins 2199

characterization of CAD-deficient plants revealed a reddish-orange coloration in the lignifying cells (Ralph et al., 1997; Siboutet al., 2005; Sirisha et al., 2012). For wild-type and fah1 plants,disruption of bothCADC andCADDwere required to observe thisphenotype; however, lignified cells in cadc C4H-F5H are pig-mented, and this coloration is even more intense in cadd C4H-F5H. These results indicate that disruption of only one CAD isrequired togenerate this phenotype inahighS-lignin background,consistentwith theNMRresults that indicatedenhancedaldehydecontent incaddC4H-F5H. Phloroglucinol-HCl is known to interactwith coniferaldehyde and sinapaldehyde end-groups and is fre-quently used to stain lignifying cell walls (Black et al., 1953; Pomaret al., 2002). All lines stained positively for phloroglucinol-HCl;however, the stainingofcadccaddand fah1cadc cadd is adeeperred than that observed in other plants, which correlates with theincreased aldehyde content in the lignin in these plants. In ad-dition, plants overexpressing F5H exhibit phloroglucinol stainingprimarily in the xylemwith less staining in the interfascicular fibers,a pattern that has been observed previously (Franke et al., 2000;

Weng et al., 2010). It is tempting to overinterpret the reduction infiber staining to suggest that the observed increased aldehydecontent (Figure4) is restricted to thexylemand that lignin in thecellwalls of fibers is not decorated with aldehyde end-units in plantsoverexpressing F5H. However, as has been shown previously,the staining cannot be used as a reliable indicator of hydroxy-cinnamaldehyde incorporation as the fully incorporated units,i.e., those units incorporated into the chain of the polymer asopposed to the (terminal) end-groups, are not colored and do notstain (Kim et al., 2002). We also conducted Mäule staining, whichstains cell walls high in S lignin red and walls high in G lignin tan.Staining of the wild type, fah1, and C4H-F5H gave results con-sistent with these expectations as has been observed previously(Weng et al., 2010). In contrast, the cell walls of plants with thehighest aldehyde content, cadc cadd, fah1 cadc cadd, and caddC4H-F5H, stained black, which is similar to what has been re-ported previously for CAD-disrupted mutants (Jourdes et al.,2007). This difference in staining obviously reflects the structuraldifferences in the lignins between these lines, but as themolecular

Figure 4. HSQC Spectra Showing the Impact That CAD and F5H Manipulation Have on Lignin Subunit Composition.

Expanded aromatic (A) and aldehyde (B) subregions of HSQC NMR spectra of ball-milled whole cell walls from Arabidopsis manipulated lines.

2200 The Plant Cell

mechanism behind this stain is poorly understood, any furtherinterpretation would be only speculative.

The structure of the vascular tissue of these plants was nextexamined by confocal laser scanning microscopy (Figure 7).Vascular bundles in the fah1 and C4H-F5H transgenics appearlargely similar to those of the wild type. The xylem tissue fromthe CAD-disrupted plants display fewer tracheary elementscomparedwith thewild type and the fah1 andC4H-F5H lines. This

Figure 5. Soluble Metabolites Are Modestly Affected by CAD Disruption,Whereas Cell Wall-Bound Metabolite Levels Are Increased.

Soluble (A) and cell wall-bound (B) metabolites of 3-week-old whole ro-settes and 2-month-old inflorescence stems, respectively. Error barsrepresent the SD of biological triplicates. Asterisks indicate the significantdifference between the plants with modified lignin composition comparedwith either the wild type or fah1 (*P < 0.05).

Figure 6. Histochemical Staining of 2-Month-Old Inflorescence StemSections from F5H- and CAD-Disrupted Plants.

Sections were either left unstained or stained with phloroglucinol-HCl orMäule. Bars = 100 mm.

Aldehyde-Enriched Lignins 2201

phenotypewasmostevident in thecaddC4H-F5H tissue, inwhichthe semicontinuous secondary cell walls typical of metaxylemcells are virtually absent from the vascular bundles. Instead, thexylem tissues of the cadd C4H-F5H samples display secondarycell wall thickenings typical of the sparse helical morphologyfound in protoxylem. This pattern of sparse and incompletesecondary cell walls is markedly different from the collapsed andoccluded vasculature recently reported in the lignin-modifieddwarf reduced epidermal fluorescence8 mutant (Bonawitz et al.,2014; Kim et al., 2014), but is likely still an impediment to effectivetransport of water and nutrients throughout the plant. A lesscontinuous lignified secondary wall is likely to allow more lateralwater transport at the expense of efficient axial transport.

The effects of CAD disruption on cell wall ultrastructure wereinvestigated by transmission electron microscopy of fiber cellsadjacent to the xylem tissue (Figure 8). Comparedwith cell walls ofwild-type, fah1, and C4H-F5H plants, the secondary cell walls ofthe cadc cadd, fah1 cadc cadd, and cadd C4H-F5H variants appearloose, less dense, and disorganized. In the extreme case of thecadd C4H-F5H plant, secondary cell walls are barely identifiable

as coherent organelles and instead appear as loose assemblies ofcellulose microfibrils. Such a deficiency in properly formed sec-ondary cell walls will weaken the mechanical properties of thevascular tissue and provide little structural support to the plant,whichmaycontribute to thedwarfismof thecaddC4H-F5Hvariantand the limp floral stem phenotype of cadc cadd mutants.

Aldehyde Enrichment in Lignin Increases CellWall Digestibility

To assess how changes in cell wall composition affect cell walldigestibility, we analyzed enzymatic cellulose conversion onuntreated samples. Previous studies in Arabidopsis showed thatmodifications of cell wall composition can have a dramatic effecton cellulose conversion (Li et al., 2010b; Weng et al., 2010;Bonawitz et al., 2014). We observed for the wild type, fah1, andC4H-F5H that there was no significant difference in celluloseconversion, with ;50% conversion of available cellulose to glu-cose after a 48-h incubation. In contrast, we found that cadc caddand fah1 cadc cadd yielded almost twice as much glucose underthe same conditions (Figure 9). This increase was observed toa lesserextent forcaddC4H-F5Handnotobserved (ordiminished)for cadc C4H-F5H. The increased aldehyde lignin content in cadccaddand fah1cadccadd ismost likely responsible for the increasein enzymatic cellulose conversion compared with cadc C4H-F5Hand cadd C4H-F5H, which have lower aldehyde contents.

DISCUSSION

Lignin content and composition are two factors that contribute tothe recalcitrance of the cell wall (Vanholme et al., 2012). Geneticand transgenic approaches to manipulate these parameters haveresulted in varying degrees of success with regard to improvingcell wall digestibility (Chen andDixon, 2007; Van Acker et al., 2013).On the one hand, several plants with disruptions in lignin contentexhibit an increase in cell wall digestibility, although the reducedlignin has been correlated with decreased yield (Bonawitz andChapple, 2013). On the other hand, plants with perturbations thataffect lignin composition show improvements in cell wall degrad-ability; however, this increase is sometimes only detectable afterpretreatment (Li et al., 2010b; Ciesielski et al., 2014). Lignin com-position isplastic andseveral geneticmanipulationshavegeneratedplants with nearly homogeneous lignin subunit profiles or ligninsderived from noncanonical monomers. These include the alterationof syringylmonomercontentbymanipulationofF5Hexpressionandtheperturbationofalcoholandaldehydesubunitcontent throughthedisruptionofCAD(Meyeretal.,1998;Siboutetal.,2005).Westackedthese two manipulation strategies to generate two unusual lignincompositions in anticipation that this approachmight deliver uniquelignins and provide insights into the impact of lignin content andcomposition on plant growth and approaches to overcome cell wallrecalcitrance to enzyme hydrolysis.

CADD Has the Predominant Role in Catalyzing theReduction of Sinapaldehyde

It has been established that both CADC and CADD need to bedisrupted in a wild-type background to increase aldehyde lignin

Figure 7. Confocal Laser Scanning Microscopy of Vascular Bundles of2-Month-Old Inflorescence Sections Stained Using the Fluorescent DyeAcriflavine.

Bars = 20 mm.

2202 The Plant Cell

subunits (Figure 4; Sibout et al., 2005; Jourdes et al., 2007). Here,we show that both CADC and CADD also need to be disrupted ina fah1 background to synthesize a lignin polymer composed al-most exclusively from coniferaldehyde. These results indicatethat, in this genetic background, CADC and CADD function re-dundantly in the conversion of hydroxycinnamaldehydes to hy-droxycinnamyl alcohols in lignin biosynthesis.

The production of monolignols in many species including Arabi-dopsis involves the activity of several CAD isoenzymes (Feuilletet al., 1995; Barakat et al., 2009; Saballos et al., 2009). In Arabi-dopsis, CADC and CADD are thought to be the result of a recentgene duplication event (Guo et al., 2010). Although they both havesimilar catalytic efficiencies towardmost hydroxycinnamaldehydes(Kim et al., 2004), there has been some dispute over whether or notangiosperms have evolved CADs that specifically utilize sina-paldehyde and whether this neofunctionalization explains the oc-currence of S lignin biosynthesis in flowering plants (Li et al., 2001;Peter and Neale, 2004; Bomati and Noel, 2005; Chiang, 2006;Barakate et al., 2011). Although Li et al. (2001) identified a CADhomolog,which they termedsinapyl alcoholdehydrogenase (SAD),fromPopulus tremuloides that hasahighcatalyticefficiency towardsinapaldehyde and found that this enzyme is missing in gymno-sperms, which are deficient in S lignin, Barakate et al. (2011)thoroughlydemonstrated thatS lignin isdependentonCADactivityand not SAD. Even though our data clearly show a differentialcontribution of the two CAD isoforms to S lignin biosynthesis inArabidopsis, neither CADC nor CADD can be considered an or-tholog of the SAD protein discussed by Li et al. (2001).

Surprisingly, we found that disruption of CADD alone in thehigh-S background leads to sinapaldehyde incorporation intolignin (Figure 4), aphenotypenotobserved inCADCmutants in thesame background. This observation indicates that CADD hasa predominant role in catalyzing the reduction of sinapaldehyde,

which is consistent with the in vitro evidence that shows thatCADD has a higher catalytic efficiency toward sinapaldehydecompared with CADC (Kim et al., 2004) and with the finding thatonlyCADDmutantsdisplayamodest increase inSʹ lignin (Figure4;Sibout et al., 2003). This final observation may also indicate thatCADD is more highly expressed than CADC in interfascicular

Figure 8. Transmission Electron Microscopy Imaging of 2-Month-Old Inflorescence Tissue Showing Cell Wall Ultrastructure.

Bars = 1 mm.

Figure 9. Cellulose Conversion of Ground Inflorescence Tissue fromAldehyde-Enriched Plants.

Error bars represent the SD of biological triplicates. Asterisks indicate thesignificant difference between the plants with modified cellulose conver-sion compared with the wild type (*P < 0.05).

Aldehyde-Enriched Lignins 2203

fibers, in which S lignin is most abundantly deposited in wild-typestems, although previous promoter-reporter gene fusion data donot strongly support this hypothesis (Sibout et al., 2003).

Previous radiolabeled substrate feeding studies and the in vitrocharacterization of F5H and caffeic acid-O-methyltransferasesuggested that sinapyl alcohol can be synthesized by hydrox-ylation and subsequent O-methylation of coniferyl alcohol, apathway that obviates the requirement for SAD activity (Chenet al., 1999; Humphreys et al., 1999; Tsuji et al., 2004). If thismetabolic route permits plants to bypass the reduction of sina-paldehyde completely, then it should not be possible to generateplants that accumulate Sʹ lignin in the relative absence of Gʹ ligninby disrupting CAD. The accumulation of Sʹ lignin in C4H-F5H,cadd,cadccadd,cadcC4H-F5H, andcaddC4H-F5Hsuggests, atleast for Arabidopsis, that some phenylpropanoid flux proceedsthrough sinapaldehyde in S lignin biosynthesis. However, wecannot differentiate whether or not the residual S lignin in caddC4H-F5H is derived directly from the partial redundancy of CADCor indirectly derived from bypassed flux coming from the hy-droxylation and O-methylation of coniferyl alcohol.

Hydroxycinnamaldehyde Monomers Can Serve asLignin Precursors

Coniferaldehyde and sinapaldehyde incorporation into ligninresulting in lignin end-groups (on the starting end of a chain) hasbeen well established and is the basis for phloroglucinol-HClstaining (Black et al., 1953; Pomar et al., 2002). In fah1 cadc cadd,coniferaldehyde not only acts as an end-group but also as themajor internal subunit component of the lignin polymer through8–O–4-linkages (Figure 4; Kim et al., 2002, 2003; Sibout et al.,2005; Jourdes et al., 2007). Unfortunately, we were only able todocument an enrichment of sinapaldehyde levels to ;70% incaddC4H-F5H, asestimatedbyNMR,asacopolymerwithsinapylalcohol because we were unable to analyze cadc cadd C4H-F5Hplants. Wewould anticipate that their sinapaldehyde levels wouldbe still higher, perhaps even close to 100%. Although it remainsunclear what impact the incorporation of sinapaldehyde units hason the lignin polymer, it may compromise the function of thispolymer when the content becomes too high. A similar phe-nomenon has been observed when lignins greatly enriched in5-hydroxyconiferyl alcohol monomers were engineered intoArabidopsis (Weng et al., 2010; Vanholme et al., 2010b). Un-like conventional monolignols and hydroxycinnamaldehydes,which mostly form rotatable b–O–4 and analogous 8–O–4 bonds,lignification via 5-hydroxyconiferyl alcohol forms cyclic benzo-dioxane units (from b–O–4-coupling of a monolignol with5-hydroxyguaiacyl phenolic end-units) resulting in a linkage thatis rigid (Marita et al., 2001; Ralph et al., 2001a, 2001b; Lu et al.,2010). BothWenget al. (2010) andVanholmeet al. (2010b) speculatethat the enrichment of this unusual linkage affects the physicalproperties of the polymer. In moderation, benzodioxane linkagesare not problematic; however, when 5-hydroxy units rise to thelevel of G subunits, dwarfism of the plant is observed. However,that such benzodioxane-only homopolymers can function incertain roles is demonstrated by the recent observation of caffeylalcohol-only and 5-hydroxyconiferyl alcohol-only lignins in seedcoats of some Cactaceae (Chen et al., 2013). Although this

hypothesis remains untested, lignin polymers derived from si-napaldehyde alone may also have undesirable physical or ar-chitectural properties that negatively affect plant growth. Fromourresults, it appears that plants canmaintainwild-type growthwhenSʹ levels are lower than those of G, S, or Gʹ units; however, severechanges in plant morphology arise when Sʹ units rival the com-bined levels of the other monomers.

Viable Plants Can Generate Lignins Highly Enriched inHydroxycinnamaldehyde-Derived Units

To the casual observer, it might appear that the polymers gen-erated in the most extreme cases here resemble the normalphenolic polymer. However, closer inspection shows that, be-cause of the differences in functional groups, reactivity, couplingpropensities, and in the alteredway inwhich intermediate quinonemethides rearomatize, the lignins are in fact entirely differentmaterials that bear little structural resemblance to the “normal”lignins derived from hydroxycinnamyl alcohols. As noted above,and as most clearly seen in the aromatic regions of the HSQCspectra in Figure 4, the fah1 cadc cadd mutant is derived fromclose to 100% coniferaldehyde. As such it has none of the tra-ditional structural units normally present in lignins (SupplementalFigure 1), although it is still derived from analogous radical cou-pling reactions. Similarly, the cadc cadd mutant is almost solely(>99%) derived from the two hydroxycinnamaldehydes, coni-feraldehyde and sinapaldehyde. These two mutants, both ofwhich form viable and fertile plants, are developmentally relativelynormal, a finding that demonstrates that plants can contend withmajor structural alterations in a crucial and abundant cell wallpolymer. These mutants are even more extreme than the recentlyreported CAD1 mutant of Medicago truncatula, which has only;5% normal monolignols (Zhao et al., 2013). Why plants high insinapaldehyde-derived lignin are developmentally compromised,and whether their dwarfism is even attributable to a poorlyfunctional lignin polymer, remain unclear at this time.

Dwarfism in Lignin Biosynthetic Mutants Is Not the Result ofCollapsed Xylem

Previous studies have highlighted the correlation betweendwarfism and metabolic blocks in lignin biosynthesis that lead todecreased lignin content (Meyer et al., 1998; Besseau et al., 2007;Mir Derikvand et al., 2008; Li et al., 2010a); however, the mech-anism by which growth is impeded in these plants remains un-known (Bonawitz and Chapple, 2013). There are examples ofplants with reduced total lignin content that maintain wild-typegrowth, including the cadc caddmutant itself (Rohde et al., 2004;Sibout et al., 2005), but many dwarfed low-lignin plants havea collapsed xylem phenotype that has been suggested to lead toreduced water transport that is the proximal cause of growthinhibition. These include plants defective in, or downregulated forC4H, p-coumaroylshikimate 3ʹ-hydroxylase, hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase, and cinnamoyl-CoA reductase (Franke et al., 2002; Mir Derikvand et al., 2008;Schilmiller et al., 2009; Li et al., 2010a; Vanholme et al., 2010b).Although cadd C4H-F5H plants are similar in height, or evenshorter than many of these low-lignin plants, we observed no

2204 The Plant Cell

evidence of collapsed xylem, suggesting that dwarfing arises viasome other mechanism(s) in these plants and is even furtherexacerbated in cadc cadd C4H-F5H plants. These mechanismscould include deficiencies in the production of dehydrodiconiferylalcohol glucoside, CAD-dependent compounds suggested tohavea role in cell expansionandgrowth (Binnset al., 1987;Orr andLynn, 1992; Tamagnone et al., 1998a, 1998b), or changes intranscription related to pathway flux perturbations that were re-cently shown to be dependent upon the Med5 subunit of theMediator complex (Bonawitz et al., 2014).

Increasing Aldehyde Content Correlates with IncreasedPotential for Enzymatic Hydrolysis of Cellulose

Reduction in lignin content is known to improve sugar releasefrom biomass upon treatment with cocktails of polysaccharidehydrolases (Studer et al., 2011), and similar beneficial effects haverecently been reported to arise from modifications to lignincomposition (Li et al., 2010b; Mansfield et al., 2012b; Ciesielskiet al., 2014). We observed that of the four variants examined withlow lignin content,cadccaddand fah1 cadccaddhave thehighestenzymatic cellulosic conversion for nonpretreated Arabidopsisinflorescence tissue. This increase in cell wall digestibility in CAD-disrupted plants has been observed before (Chen et al., 2003; Fuet al., 2011;Muguerzaet al., 2014). There is somedisagreement asto whether this increase in digestibility is the result of modifiedlignin content or, as our study suggests, modified lignin com-position (Baucher et al., 1996; Reddy et al., 2005). The effect mayresult from a significant alteration in the intermolecular forcesbetween biopolymers within the cell wall caused by the increasedaldehyde content. More specifically, the strong dipoles contrib-uted by the aldehyde groups may disrupt typical hydrogenbonding, electrostatic, and dispersive forces that facilitate in-tegration of the lignin polymer with other cell wall biopolymers,thereby facilitating enhanced penetration of enzymes and in-creasing accessibility of the carbohydrate components. Thesedata suggest that increased aldehyde content is an importantdeterminant of cell wall digestibility and that synergistic increasesin yield can be achieved by combining changes in both lignincontent and composition. Based on thioacidolysis experimentsfollowing exhaustive methylation (Jouanin et al., 2004), re-searchers have noted that aldehyde units might be more prone toterminate a chain, resulting in lower molecular mass lignins thatwould indeed be expected to protect polysaccharides to a lesserdegree than larger polymers (Lapierre et al., 2004).

METHODS

Plant Material and Growth Conditions

Arabidopsis thaliana Col-0 plants were grown under a 16-h-light/8-h-dark photoperiod at 100 mE m22 s21 in Redi-Earth Plug and SeedlingMixture (SunGroHorticulture) supplementedwith ScottsOsmocotePluscontrolled release fertilizer (Hummert International) at 22°C.Thecadcandcadd mutants (Sibout et al., 2003, 2005) were crossed to either the fah1mutant (Chapple et al., 1992) or the transgenic line in which F5H isoverexpressed (Meyer et al., 1998). The putative triple mutants andtransgenic double mutants were identified and confirmed by genomicPCR analysis.

Lignin Analysis

Eight-week-old inflorescence tissues stripped of leaves and siliques werepooled, ground in liquid N2, washed with 80% ethanol four or five times toremove soluble metabolites, then washed with acetone and dried, as per-formed by Meyer et al. (1998). Acetyl bromide analysis was conductedaccording toChang et al. (2008). Briefly, sampleswere exposed to a 4:1 (v/v)mixtureofacetic acid/acetyl bromideand incubatedat 70°C for 2h.Sampleswere cooled to room temperature and then transferred to 50-mL volumetricflasks containing 2 M NaOH, acetic acid, and 7.5 M hydroxylamine hydro-chloride. Acetic acid was used to bring each sample up to volume and theneach sample was measured for absorbance at 280 nm. Absorbance valueswere converted tomassusing theextinction coefficient of 23.29g21 L cm21.

NMR Spectroscopy

Preparation of cell walls samples for NMR was conducted as describedpreviously (Kim and Ralph, 2010; Mansfield et al., 2012a). In brief, pre-ground dried stemswere extracted with 80% aqueous ethanol (sonication3 3 20 min). Isolated cell wall fractions (;200 mg) were ball-milled (5 3

5 min milling and 5-min cooling cycles) using a Fritsch Planetary MicroPulverisette 7 ball mill at 600 rpm with ZrO2 vessels containing ZrO2 ballbearings. Aliquots of the ball-milled whole cell walls (;60 mg) weretransferred into NMR sample tubes and swollen in DMSO-d6/pyridine-d5

(4:1, v/v, 600mL). NMR spectra were acquired on a Bruker Biospin Avance700-MHz spectrometer fitted with a cryogenically cooled 5-mm TXI gra-dient probewith inverse geometry (proton coils closest to the sample). Thecentral DMSO solvent peakwas used as an internal reference (dC, 49.5; dH,3.49 ppm). Adiabatic HSQC experiments (hsqcetgpsisp2.2) were per-formed using the parameters described previously (Kim and Ralph, 2010;Mansfield et al., 2012a). Processing used typical matched Gaussianapodization in F2 (LB = 20.5, GB = 0.001) and squared cosine-bellapodizationandone level of linear prediction (32 coefficients) inF1. Volumeintegration of contours in HSQC spectra (processed using no linear pre-diction) used Bruker’s TopSpin 3.1 (Mac) software with no correctionfactors; i.e., the data represent volume integrals only. For quantification oflignin aromatic distributions, only the carbon/proton-2 correlations fromG and G’ units and the carbon/proton-2/6 correlations from S and S’ unitswere used, and the G and G’ integrals were logically doubled.

Methanol-Soluble Secondary Metabolite Analysis

Hydroxycinnamoylmalate esters from 3-week-old whole rosette leaveswere analyzed usingmethods previously described byHemmet al. (2003).Briefly, tissue extracts were prepared at a concentration of 100 mg freshweight mL21 50%methanol, extracted for 2 h at 65°C (n = 3). Compoundswere quantified using ferulic and sinapic acid as standards and normalizedper fresh plant tissue.

Cell Wall-Bound Hydroxycinnamic Acid Analysis

Ground, 8-week-old inflorescencecellwall tissuewassaponifiedusing1MNaOH and incubated for 24 h at 37°Cwith constant agitation. The solutionwas acidified with 1 M HCl and extracted using ethyl acetate. The organicphase was then dried, redissolved in 50% methanol, and analyzed byHPLC. Compounds were quantified using p-coumaric and ferulic acidstandards obtained from Sigma-Aldrich.

Histochemical Staining

Eight-week-old inflorescence stemswere sectioned using a Leica VT1000S vibrating blade microtome and left either unstained or stained withphloroglucinol-HCl or Mäule reagent as described (Chapple et al., 1992;Franke et al., 2000).

Aldehyde-Enriched Lignins 2205

Transmission Electron Microscopy

Sections from the inflorescence stem were taken just below the fifthelongating silique of the inflorescence and were high-pressure frozen witha Leica EMPact2 high-pressure freezer in 0.2-mm-deep planchets (LeicaMicrosystems) using 0.15 M sucrose as a cryoprotectant. Next, freeze-substitutionwas performed in a Leica AFS2 automated freeze substitutionunit in 1% osmium tetroxide (EMS). The samples were dehydrated bytreating by solution exchange with increasing concentrations of acetone(15%, 30%, 60%, 90%, and 3 3 100% acetone). The samples were theninfiltrated with Eponate 812 (EMS) by incubating at room temperature forseveral hours toovernight in increasingconcentrationsof resin (15%,30%,60%, 90%, 3 3 100% resin, diluted in acetone). The samples weretransferred to capsules and the resin polymerized in an oven at 60°Covernight. Resin-embedded samples were sectioned to ;50 nm witha Diatome diamond knife on a Leica EM UTC ultramicrotome. Sectionswere collected on 200 mesh copper transmission electron microscopygrids (SPI Supplies) and were poststained for 30 s with 1% aqueousKMnO4. Images were taken with a 4-megapixel Gatan UltraScan 1000camera on an FEI Tecnai G2 20 Twin 200-kV LaB6 transmission electronmicroscope.

Confocal Microscopy

Samples were preserved and embedded as described above. The em-bedded samples were sectioned to 250 nm and positioned on glass mi-croscopeslidesandstainedwith0.1%acriflavine. Imageswerecaptured influorescence mode with a Nikon C1 Plus microscope, equipped with theNikon C1 confocal system with a 488-nm excitation laser.

Cellulose Conversion

Cellulose conversion was conducted as described by Li et al. (2010b) on8-week-old hand-ground inflorescence tissue that had been stripped ofleaves and siliques. Cellulose conversion was conducted without pre-treatment and evaluated colorimetrically using the Megazyme D-Glucose(glucose oxidase/peroxidase) assay kit after 48-h incubation.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL li-braries under accession numbers C4H (At2g30490), F5H (At4g36220),CADC (At3g19450), CADD (At4g34239), and REF1 (At3g24503).

Supplemental Data

Supplemental Figure 1. Aliphatic subregions of HSQC NMR spectraof ball-milled whole cell walls from Arabidopsis mutant lines.

Supplemental Figure 2. Polysaccharide anomeric subregions ofHSQC NMR spectra of ball-milled whole cell walls from Arabidopsismanipulated lines.

ACKNOWLEDGMENTS

This work was supported as part of the Center for Direct Catalytic Con-versionofBiomass toBiofuels, anEnergyFrontier ResearchCenter fundedby the U.S. Department of Energy, Office of Science, Basic EnergySciences under Award DE-SC0000997. This material is also baseduponwork supported by theU.S. Department of Energy, Office of Science,Office of Basic Energy Sciences, Chemical Sciences, Geosciences, andBiosciences Division under Award DE-FG02-07ER15905. J.R. and Y.T.were funded by Stanford University’s Global Climate and Energy Project

and the Great Lakes Bioenergy Research Center by the U.S. DOE’sOffice of Science (DE-FC02-07ER64494). Y.T. also acknowledgesa support from The Ministry of Education, Culture, Sports, Science,and Technology (MEXT), Japan (Grant 26892014). E.X. and M.L. weresupported by the U.S. DOE through Grant DE-FG02-06ER64301 toM.L. and C.C. and by the Purdue University Office of AgriculturalResearch Programs.

AUTHOR CONTRIBUTIONS

N.A.A. and C.C. conceived of the study. N.A.A., Y.T., P.N.C., and E.X.performed the research and along with the other authors wrote the article.

Received April 29, 2015; revised July 13, 2015; accepted July 25, 2015;published August 11, 2015.

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Aldehyde-Enriched Lignins 2209

DOI 10.1105/tpc.15.00373; originally published online August 11, 2015; 2015;27;2195-2209Plant Cell

Donohoe, Michael Ladisch and Clint ChappleNickolas A. Anderson, Yuki Tobimatsu, Peter N. Ciesielski, Eduardo Ximenes, John Ralph, Bryon S.

StructureAlcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall

Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl

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