RESEARCH ARTICLES

Convergent Evolution of Syringyl Lignin Biosynthesis viaDistinct Pathways in the Lycophyte Selaginella andFlowering Plants C W

Jing-Ke Weng,a,1 Takuya Akiyama,b Nicholas D. Bonawitz,a, Xu Li,a John Ralph,b,c and Clint Chapplea,2

a Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907b U.S. Dairy Forage Research Center, U.S. Department of Agriculture–Agricultural Research Service, Madison, Wisconsin 53706c Department of Biochemistry (Enzyme Institute) and Department of Energy Great Lakes Bioenergy Research Center, University

of Wisconsin, Madison, Wisconsin 53726

Phenotypic convergence in unrelated lineages arises when different organisms adapt similarly under comparable selective

pressures. In an apparent example of this process, syringyl lignin, a fundamental building block of plant cell walls, occurs in

two major plant lineages, lycophytes and angiosperms, which diverged from one another more than 400 million years ago.

Here, we show that this convergence resulted from independent recruitment of lignin biosynthetic cytochrome P450-

dependent monooxygenases that route cell wall monomers through related but distinct pathways in the two lineages. In

contrast with angiosperms, in which syringyl lignin biosynthesis requires two phenylpropanoid meta-hydroxylases C39H and

F5H, the lycophyte Selaginella employs one phenylpropanoid dual meta-hydroxylase to bypass several steps of the

canonical lignin biosynthetic pathway. Transgenic expression of the Selaginella hydroxylase in Arabidopsis thaliana

dramatically reroutes its endogenous lignin biosynthetic pathway, yielding a novel lignin composition not previously

identified in nature. Our findings demonstrate a unique case of convergent evolution via distinct biochemical strategies and

suggest a new way to genetically reconstruct lignin biosynthesis in higher plants.

INTRODUCTION

In response to environmental pressures, phylogenetically unre-

lated species sometimes arrive at similar adaptive solutions

through independent mechanisms (Tanaka et al., 2009). Al-

though convergent evolution happens rarely in nature, it repre-

sents an important evolutionary phenomenon, which has been

documented in action at multiple levels of biological processes

(Conant and Wagner, 2003). For example, birds and bats inde-

pendently evolved wings for powered flight (Hedenstrom et al.,

2007), both higher plants and fungi developed the ability to

synthesize growth regulator gibberellins via nonorthologous

pathways (Hedden et al., 2001), and mammals and fungi re-

cruited two distinct families of proteins to methylate Lys residues

on histone tails (Cheng et al., 2005). Elucidation of the molecular

mechanisms underlying phenotypic convergence is key to un-

derstanding how this process contributes to evolution.

Vascular plants arose in the Late Silurian period (;420 million

years ago). They diversified rapidly during the Early Devonian

period (416 to 398 million years ago), when an early split in the

history of land plant evolution gave rise to twomajor lineages: the

lycophytes andeuphyllophytes (Kenrick andCrane, 1997) (Figure

1A). These two lineages are united as a monophyletic group by

the presence of specialized water-conducting tracheary ele-

ments, the cell walls of which are physically reinforced by the

phenolic lignin heteropolymer (Kenrick and Crane, 1997). Lignin

endowsvascular plantswith the rigidity to standupright, prevents

their tracheids and vessel elements from collapsing during long-

distance water transport, and has greatly contributed to the

dominance of these plants in terrestrial environments (Boerjan

et al., 2003). Although lignin appears to be fundamental to the

biochemistry of all vascular plants, its monomer composition

exhibits an intriguing distribution pattern across the major line-

ages. Among the euphyllophytes, ferns and gymnosperms gen-

erally contain p-hydroxyphenyl (H) and guaiacyl (G) lignin units,

derived from p-coumaryl alcohol and coniferyl alcohol, respec-

tively, and angiosperms additionally contain syringyl (S) units

derived from sinapyl alcohol (Boerjan et al., 2003) (Figure 1). This

observation has led to the notion that S lignin is a recent invention

restricted primarily to angiosperms. In fact, S lignin has also been

found in some lycophyte species belonging to the order of

Selaginellales but is absent from species belonging to its extant

sister order Lycopodiales and is thought to have been absent

from its extinct sister order Lepidodendrales, suggesting that

S lignin might have arisen independently in lycophytes and

1Current address: Howard Hughes Medical Institute, Jack H. SkirballCenter for Chemical Biology and Proteomics, The Salk Institute forBiological Studies, La Jolla, CA 92037.2 Address correspondence to chapple@purdue.edu.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Clint Chapple(chapple@purdue.edu).CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.109.073528

The Plant Cell, Vol. 22: 1033–1045, April 2010, www.plantcell.org ã 2010 American Society of Plant Biologists

euphyllophytes by evolutionary convergence (Towers and

Gibbs, 1953; White and Towers, 1967; Logan and Thomas,

1985, 1987; Jin et al., 2005; Weng et al., 2008c) (Figure 1A).

Lignin monomer biosynthesis in angiosperms is largely de-

pendent on the expression of three cytochrome P450 monoox-

ygenases (P450s; Figure 1B) (Boerjan et al., 2003). Cinnamate

4-hydroxylase (C4H) catalyzes the phenylpropanoid ring para-

hydroxylation reaction that is required for the formation of all

three types of lignin units (Teutsch et al., 1993), but two distinct

P450s carry out each of the two subsequent hydroxylations

required for S lignin synthesis. p-Coumaroyl shikimate 39-hydroxylase (C39H) catalyzes the first ring meta-hydroxylation

reaction, necessary for the biosynthesis of both G and S lignin

units (Franke et al., 2002a), and ferulate 5-hydroxylase (F5H)

catalyzes the second ringmeta-hydroxylation reaction leading to

the biosynthesis of S lignin units from G-substituted precursors

(Meyer et al., 1998).

We recently isolated from the lycophyte Selaginella moellen-

dorffii a novel P450 (CYP788A1) that, like angiosperm F5Hs, can

divert G-substituted intermediates toward S lignin synthesis

(Weng et al., 2008c). Selaginella F5H (Sm F5H) shares only 37%

amino acid sequence identity with its angiosperm counterparts, a

level of similarity that can be expected from any of two random

plant P450 enzymes from families with unrelated functions,

suggesting that the similar catalytic activities of F5Hs in the two

lineages were derived from convergent evolution (Weng et al.,

2008c). Here, we report the discovery of a novel activity of SmF5H

that reveals an unexpected metabolic distinction between Selag-

inella and angiosperms and suggests that S lignin is elaborated via

distinct biosynthetic routes in these two groups of plants.

Figure 1. S Lignin Biosynthesis via Distinct Biosynthetic Routes in Angiosperms and the Lycophyte Selaginella.

(A) Plant phylogenetic tree showing the distribution of lignin monomer composition across major land plant lineages. Open circle, no lignin; yellow circle,

presence of H and G lignin; red circle, presence of S lignin in addition to H and G lignin. †Lignin composition indicated by the analysis of fossil samples of

extinct Sigillaria species (Logan and Thomas, 1987).

(B) Alternative pathways toward S lignin biosynthesis in Selaginella and angiosperms. The shared routes between Selaginella and angiosperms are

shaded in purple, whereas the divergent branches specific to Selaginella or angiosperms are shaded in pink or light blue, respectively. The two aromatic

ring meta-hydroxylation steps are catalyzed by one bifunctional enzyme, Sm F5H (circled in red) in Selaginella, instead of two enzymes, C39H and F5H

(circled in blue), in angiosperms. At, Arabidopsis thaliana; Sm, Selaginella moellendorffii. PAL, phenylalanine ammonia-lyase; 4CL, 4-hydroxycinnamoyl-

CoA ligase; CCoAOMT, caffeoyl-CoA O-methyl transferase; HCALDH, hydroxycinnamaldehyde dehydrogenase; COMT, caffeic acid O-methyltrans-

ferase; CAD, (hydroxy)cinnamyl alcohol dehydrogenase.

1034 The Plant Cell

RESULTS

Sm F5H Is a Phenylpropanoid DualMeta-Hydroxylase

in Vitro

In our previous study, we demonstrated that like angiosperm

F5Hs, Sm F5H uses coniferaldehyde and coniferyl alcohol as

substrates in preference to ferulic acid (Humphreys et al., 1999;

Weng et al., 2008c). To test whether Sm F5H can use phenyl-

propanoids other than those G-substituted intermediates as

substrates, we performed a series of kinetic assays using Sm

F5H against a wider range of phenylpropanoid pathway inter-

mediates. Parallel comparative assays were conducted using

Arabidopsis thaliana F5H (At F5H) recombinant protein.

Surprisingly, we found that although both At F5H and Sm F5H

can catalyze 5-hydroxylation reactions on G-substituted inter-

mediates equally well, Sm F5H can also efficiently catalyze the

3-hydroxylation of p-coumaraldehyde and p-coumaryl alcohol

(Table 1; see Supplemental Figure 1 online). In contrast with

Sm F5H, At F5H shows little activity toward p-coumaraldehyde

and p-coumaryl alcohol with Km values so high that these

activities are not likely to be relevant in vivo (Table 1; see

Supplemental Figure 1 online). The similarly ring-substituted

acid, p-coumaric acid, is not an optimal substrate for Sm F5H,

consistent with the high Km value observed for ferulic acid.

Neither Sm F5H nor At F5H showed any detectable activity

toward cinnamic acid, cinnamaldehyde, and cinnamyl alco-

hol, indicating the ring para-hydroxyl is required for the meta-

hydroxylase activity. When p-coumaroyl shikimic acid, the

substrate for angiosperm C39H (Schoch et al., 2001), was tested,

no activity was detected in either Sm F5H or At F5H assays,

suggesting that Sm F5H does not display promiscuous activity

toward any given p-coumaroyl derivatives. We also detected no

activity of Sm F5H toward caffeic acid, caffealdehyde, or caffeyl

alcohol, suggesting that the 3-hydroxylO-methylation is required

for the subsequent 5-hydroxylase activity of Sm F5H. Collec-

tively, the kinetic data imply that Selaginellamay have a pathway

for S lignin biosynthesis via the H-substituted aldehyde and

alcohol, a route that is thought to be absent in angiosperms

(Figure 1B).

Sm F5H Partially Rescues the Growth Defects of the

Arabidopsis C39H-Deficient Mutants

In Arabidopsis, mutants defective in each of the two phen-

ylpropanoid meta-hydroxylases, C39H and F5H, have been

isolated and characterized. Whereas various alleles of the

C39H-deficient reduced epidermal fluorescence8 (ref8) mutants

exhibit severe dwarfism, female sterility, greatly reduced soluble

sinapate ester and total lignin content, and a lignin composed of

almost pure H units (Franke et al., 2002a, 2002b; Abdulrazzak

et al., 2006), the F5H-deficient fah1-2 mutant shows a total loss

of sinapate esters and S lignin but normal growth (Chapple et al.,

1992). We have shown that the Sm F5H transgene can rescue

fah1-2mutant biochemical phenotypes (Weng et al., 2008c), but

our revised kinetic analysis of the enzyme’s substrate specificity

suggested that this complementation experiment may have

exploited only a portion of the catalytic repertoire of Sm F5H.

We postulated that if the phenylpropanoid dualmeta-hydroxylase

activity of Sm F5H observed in vitro is relevant in vivo, transgenic

expression of Sm F5Hwould also be able to rescue ref8 because

the p-coumaraldehyde and p-coumaryl alcohol that the mutant

employs for H lignin synthesis would be available as substrates

for the enzyme.

To test this hypothesis, we generated Sm F5H transgenics in

ref8 fah1-2 double mutant backgrounds by crossing plants

carrying one of two ref8 alleles, ref8-1 (a slightly leaky allele

that carries a point mutation as described by Franke et al.

[2002b]) and ref8-2 (a T-DNA insertional null allele), with four

independent transgenic lines previously generated in the fah1-2

background that harbor the Sm F5H transgene under the control

of the Arabidopsis C4H promoter (AtC4H:SmF5H), a promoter

that has been shown to efficiently target expression in vascular

tissue (Weng et al., 2008c). In both cases, visual inspection of the

resulting ref8/fah1-2/AtC4H:SmF5H plants from the F2 genera-

tion indicated a partial but substantial complementation of the

growth phenotype (Figures 2 and 3). Comparedwith ref8 and ref8

Table 1. Kinetic Properties of Sm F5H and At F5H against Phenylpropanoid Intermediates

Substrate

Sm F5H At F5H

Kma (mM) Vmax

a (pkat·mg�1) Vmax/Km Kma (mM) Vmax

a (pkat·mg�1) Vmax/Km

p-Coumaric acid 1.67 3 103 1.67 1.00 3 10�3 N.D.b N.D.b N.D.b

p-Coumaraldehyde 4.50 4.45 0.989 3.71 3 102 0.700 1.89 3 10�3

p-Coumaryl alcohol 5.11 3.58 0.701 5.95 3 102 0.606 1.02 3 10�3

Ferulic acid 1.09 3 103 1.35 1.24 3 10�3 1.85 3 103 2.03 1.10 3 10�3

Coniferaldehyde 2.80 3.40 1.21 3.06 2.18 0.712

Coniferyl alcohol 3.63 2.56 0.705 1.76 1.73 0.983

No activity was detected using either Sm F5H or At F5H against the following substrates at a concentration of 200 mM: cinnamic acid,

cinnamaldehyde, cinnamyl alcohol, p-coumaroyl shikimic acid, caffeic acid, caffealdehyde, and caffeyl alcohol.aExtrapolated using Lineweaver-Burk plots based on triplicate assays. Note that the enzyme assays were performed using yeast microsomes

containing the heterologously expressed catalysts in which the amount of P450 was not quantified. The kinetic constants involving Vmax cannot be

compared between assays conducted using Sm F5H and At F5H.bKinetic constants not determined (N.D.; no detectable activity).

Alternative Route of S Lignin Synthesis 1035

fah1-2 plants, which are severe dwarfs with miniature rosettes,

ref8/fah1-2/AtC4H:SmF5H plants are significantly larger in stat-

ure (Figures 2 and 3). The dark-green/purple color typically

observed in ref8 or ref8 fah1-2 rosette leaves is also greatly

alleviated in the Sm F5H transgenics, indicating a decrease in the

accumulation of anthocyanins. Despite the considerable com-

plementation in growth phenotype, the female sterility pheno-

type of ref8 is not rescued in the Sm F5H transgenics, suggesting

that the alternative phenylpropanoid meta-hydroxylation path-

way mediated by Sm F5H is not sufficient to compensate for the

loss of C39H activity in flower development in ref8 or that the At

C4H promoter does not target Sm F5H expression to the

necessary tissues or cells.

To test whether overexpression of At F5H can rescue ref8,

we generated similar transgenic plants carrying an At C4H

promoter-driven At F5H transgene (AtC4H:AtF5H). None of the

resulting ref8-1/fah1-2/AtC4H:AtF5H transgenic lines showed

any sign of phenotypic complementation (data not shown), which

is consistent with the in vitro observation that At F5H is not

effective as a 3-hydroxylase. The above data suggest that the

complementation of ref8 by Sm F5H is due to its specific

3-hydroxylase activity and not the 5-hydroxylase activity it

shares with At F5H.

Sm F5H Restores S Lignin Biosynthesis in the Arabidopsis

C39H-Deficient Mutants

To examine whether the rescue of the growth phenotype is

associated with a restoration of normal lignin deposition in the

ref8/fah1-2/AtC4H:SmF5H transgenics, we first performed

phloroglucinol-HCl and Maule histochemical staining on stem

cross sections (Figure 4). The phloroglucinol-HCl reagent gives a

red reaction when it reacts with hydroxycinnamaldehyde end

groups in the lignin polymer, whereas the Maule reagent gives a

qualitative indication of lignin monomer composition by staining

G lignin brown and S lignin red. Compared with the wild type and

fah1-2, ref8-1 and the ref8-1 fah1-2 double mutant show much

weaker phloroglucinol-HCl staining and no Maule staining, con-

sistent with decreased total lignin and lack of G and S lignin units

in these mutants. By contrast, the ref8-1/fah1-2/AtC4H:SmF5H

transgenics exhibited strong red Maule staining in both xylem

and interfascicular fiber cells, indicating that S lignin deposition is

restored in these cells. Little phloroglucinol-HCl staining was

detected in sections of these plants, suggesting low quantities of

aldehyde end groups in the transgenic lignin, a character rem-

iniscent of high S lignin as previously reported (Franke et al.,

2000).

We then quantified the total lignin content of the transgenic

plants together with the control plants by Klason analysis (Figure

5). Compared with ref8-1 and the ref8-1 fah1-2 double mutant,

which contain only about one-third of the Klason lignin content

found in the wild type, the ref8-1/fah1-2/AtC4H:SmF5H trans-

genics deposit about three-quarters of the wild-type level of

lignin. Interestingly, the ref8-2/fah1-2/AtC4H:SmF5H trans-

genics also have a Klason total lignin content similar to the

ref8-1/fah1-2/AtC4H:SmF5H transgenics, despite their pheno-

typic difference in growth (Figures 2E and 2H). This observation

is consistent with the previous suggestion that deficiency in

Figure 2. Partial Complementation of the Growth Phenotype of the Arabidopsis ref8 fah1-2 Double Mutant by the Sm F5H Transgene.

(A) Columbia wild type.

(B) fah1-2.

(C) ref8-1.

(D) ref8-1 fah1-2.

(E) A representative line of ref8-1/fah1-2/AtC4H:SmF5H.

(F) ref8-2.

(G) ref8-2 fah1-2.

(H) A representative line of ref8-2/fah1-2/AtC4H:SmF5H.

All plants were photographed at 2 months of age, and all images are shown at an identical scale.

[See online article for color version of this figure.]

1036 The Plant Cell

factors other than lignin (e.g., some unknown growth substance

synthesized via C39H) may contribute to the dwarfism in ref8

mutants (Abdulrazzak et al., 2006).

We further examined the impact of Sm F5H on lignin monomer

composition in the ref8 fah1-2background using the derivatization

followed by reductive cleavage (DFRC) method, a procedure that

specifically releases lignin monomers (as their peracetates) from

b-O-4-linked lignin units (Figure 6, Table 2; see Supplemental

Figure 2 online). Asmentioned previously, compared with the wild

type, which contains both G and S units with traces of H units, S

units in fah1-2 are below detectable limits. The ref8-1 fah1-2 line

resembles ref8-1 in that it deposits essentially only H subunits and

only accumulates low levels of lignin. Both ref8-1/fah1-2/AtC4H:

SmF5H and ref8-2/fah1-2/AtC4H:SmF5H transgenics show a

significant recovery of the total DFRC-releasable lignin monomer

yield. More surprisingly, these plants contain lignin with compa-

rable amounts of releasable H and S units, but very few G units, a

lignin composition not previously identified in nature.

To independently evaluate the novel lignin composition in the

ref8/fah1-2/AtC4H:SmF5H transgenics,we conductedNMRanal-

ysis on whole lignins released from cell walls by treatment with

polysaccharide hydrolases. These data clearly show that the

transgenic expression of Sm F5H can rescue S lignin biosynthesis

inArabidopsis ref8 fah1-2 doublemutants and provide support for

the observation of unique H-S lignins (Figure 7). Contour integra-

tion in two-dimensional 13C–1H-correlated (HSQC) NMR also

allowed better estimates of the H:G:S distribution of the whole

lignin (Ralph et al., 2006; Wagner et al., 2007) (Table 2). The

lignins displayed distinctly different structural attributes result-

ing from the diverse distributions of lignin monomers (see

Figure 3. Photographs of Rosette-Stage Arabidopsis Plants under Visible Light and UV Light.

Blue fluorescence under UV light indicates the presence of sinapoylmalate in the leaf epidermis, whereas red fluorescence indicates its absence and

results from the UV-induced chlorophyll fluorescence. Top, visible light; bottom, UV light.

Figure 4. Impact of Sm F5H Transgenic Expression on Lignin Histochemical Staining in the Arabidopsis ref8 fah1-2 Double Mutant.

Phloroglucinol-HCl (top) and Maule (bottom) histochemical staining of 2-month-old Arabidopsis stem cross sections. The phloroglucinol-HCl reagent

detects aldehyde groups contained in lignin and results in red staining that is generally indicative of the presence of lignin. The Maule reagent is

diagnostic for the presence of S units in lignin, leading to a yellow staining of G lignins and red staining of lignins containing S subunits.

Alternative Route of S Lignin Synthesis 1037

Supplemental Figure 3 online). For example, 5-5–linked struc-

tures (in dibenzodioxocin units) were elevated in the G-rich

fah1-2mutant but greatly reduced in the S-elevated transgenics.

Sm F5H Rescues the Arabidopsis HCT-Deficient Mutant

Taken together, the above results suggest that Sm F5H can

mediate a novel lignin biosynthetic route that bypasses three

enzymes, hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl

transferase (HCT), C39H, and caffeoyl-CoAO-methyl transferase

(CCoAOMT), which catalyze four consecutive steps in the ca-

nonical pathway in angiosperms (Figure 1B). This hypothesis

predicts that Sm F5H should also be able to rescue the pheno-

types exhibited by an Arabidopsis HCT-deficient mutant, just as

it rescues the ref8 mutant. To test this prediction, we generated

AtC4H:SmF5H transgenics in a genetic background where HCT

is downregulated by RNA interference (RNAi). As has been

reported previously, HCT downregulated plants phenocopy

ref8 in that they show severe dwarfism and a dark-green/purple

coloration of their rosette leaves, although in general they are not

female-sterile like ref8 (Figure 8A) (Hoffmann et al., 2004). While

quantitative RT-PCR shows that HCT transcript levels in

HCTRNAi/AtC4H:SmF5H plants are similar to those of HCTRNAi

plants (Figure 8B), the plants are significantly rescued in growth

and exhibit decreased leaf pigmentation (Figure 8A). DFRC lignin

analysis revealed thatHCTRNAiplants deposit trace amounts of H

lignin, whereas the lignin of HCTRNAi/AtC4H:SmF5H plants con-

tains comparable amount of H and S subunits similar to the lignin

of ref8/fah1-2/AtC4H:SmF5H plants (Figure 8C). These obser-

vations further support the rerouted lignin biosynthetic pathway

mediated by Sm F5H, which bifurcates from the canonical

pathway upstream of HCT.

Figure 5. Restoration of Total Acid-Insoluble Lignin Content by Sm F5H

in the Arabidopsis ref8 fah1-2 Double Mutant Determined by Klason

Analysis.

Error bars represent 1 SD of biological triplicates. N.D., not determined

(no inflorescence stems could be harvested from these plants).

Figure 6. Impact of Sm F5H Transgenic Expression on Lignin Compo-

sition in the Arabidopsis ref8 fah1-2 Double Mutant Examined by DFRC–

Gas Chromatography Analysis.

H/G/S are p-hydroxyphenyl/guaiacyl/syringyl lignin-derived hydroxycin-

namyl alcohol peracetates. c/t, cis/trans; IS, internal standard.

[See online article for color version of this figure.]

1038 The Plant Cell

Impact of Sm F5H Transgenic Expression on Soluble

Phenylpropanoid Metabolism in the Arabidopsis

C39H-Deficient Background

We have previously shown that Sm F5H can rescue sinapoyl-

malate accumulation in fah1-2 leaves, thus complementing its ref

phenotype under UV light (Weng et al., 2008c). To test whether

Sm F5H could also rescue the ref phenotype in the ref8 back-

ground, we first inspected the rosette-stage ref8/fah1-2/AtC4H:

SmF5H transgenics under UV light (Figure 3). Compared with

ref8mutants, the blue epidermal fluorescence is increased in the

corresponding Sm F5H transgenics, but clearly not to the level of

the wild type. We further analyzed the leaf soluble phenylpropa-

noid metabolites by HPLC (Figure 9). Consistent with the visual

phenotyping result, we found that ref8-1/fah1-2/AtC4H:SmF5H

plants showed only a modest increase in the sinapoylmalate

levels compared with ref8-1, and ref8-2/fah1-2/AtC4H:SmF5H

plants showed no increase comparedwith ref8-2. HPLC profiling

of leaf soluble phenylpropanoids also revealed a significant

decrease in the level of three major flavonol glycosides in the

Sm F5H transgenics, compared with ref8 and ref8/fah1-2 where

these flavonoids are hyperaccumulated. This observation indi-

cates that either stress induced by the ref8mutation is generally

alleviated by the expression of Sm F5H transgene or that

metabolites that are otherwise channeled into flavonoid biosyn-

thesis when C39H is blocked are redirected into lignin biosyn-

thesis via Sm F5H.

Coexistence of H and S Lignin in Selaginella Stem Cortex

To gain insight into whether the Sm F5H-mediated S lignin

biosynthetic pathway is operating in Selaginella, we took the

advantage of the unique anatomy of Selaginella stems and

separated the stem xylem and cortical tissue for DFRC lignin

analysis (Figure 10A). The results confirmed the presence of a

high percentage of S lignin in cortex and revealed that a signif-

icant amount of H lignin coexists in the same tissue (Figure 10B).

By contrast, xylem tissue is dominated by G lignin with only trace

amounts of S lignin and no detectable level of H lignin (Figure

10B). Consistent with what we have observed in the Arabidopsis

transgenics, the coexistence of H and S lignin in Selaginella stem

Table 2. Lignin Monomer Composition of Arabidopsis Plants Inferred from DFRC Lignin Analysis and the Contour Integration in HSQC NMR Analysis

Sample

DFRC Releasable Monomer (mmol g�1 Cell Wall 6 SD)DFRC Inferred

H:G:S (Mol %)

NMR Inferred

H:G:S (Mol %)H G S

Columbia wild type 3.1 6 1.1 49 6 3.8 20 6 0.62 4.4:68.2:27.4 0.1:81.5:18.4

fah1-2 3.2 6 0.52 64 6 1.2 N.D. 4.8:95.2:0.0 0.1:99.9:0.0

ref8-1 4.0 6 0.50 N.D. N.D. 100.0:0.0:0.0 98.9:0.0:1.1

ref8-1 ah1-2 4.3 6 0.33 N.D. N.D. 100.0:0.0:0.0 N.A.

ref8-1/fah1-2/AtC4H:SmF5H 22 6 1.2 3.7 6 0.11 47 6 1.6 29.9:5.1:65.0 23.7:2.1:74.2

ref8-2/fah1-2/AtC4H:SmF5H 22 6 1.6 2.5 6 0.52 22 6 1.2 46.8:5.4:47.8 48.7:3.8:47.5

Standard deviation was calculated based on biological triplicates. N.D., not detectable; N.A., not analyzed.

Figure 7. Impact of Sm F5H Transgenic Expression on Lignin Composition in the Arabidopsis ref8 fah1-2 Double Mutant Examined by Partial (Aromatic

Region) HSQC NMR Spectra.

Correlations from the aromatic rings can be categorized by the type of lignin units: H (red), G (blue), and S (green).

Alternative Route of S Lignin Synthesis 1039

cortex suggests that the novel S lignin biosynthetic pathway

derived from H lignin precursors may be active in Selaginella.

DISCUSSION

F5HMediates a C39H-Independent S Lignin Biosynthetic

Pathway in Selaginella

We have provided both in vitro and in vivo evidence to show that

the convergent evolution of S lignin in lycophytes and angio-

sperms is the result of the elaboration of alternative biochemical

pathways. Whereas in angiosperms two phenylpropanoid meta-

hydroxylases, C39HandF5H, are required for S ligninbiosynthesis

from para-hydroxylated intermediates, a unique phenylpropanoid

dualmeta-hydroxylase, Sm F5H, evolved in the lycophyte Selag-

inella where it mediates S lignin biosynthesis via a different

pathway (Figure 1B). Our data suggest that G and S lignin can

be biosynthesized independently of one another through two

distinct sets of enzymes in Selaginella, in comparison to in

angiosperms where S lignin subunits are derived from G lignin

precursors.

We postulate that the C39H-independent S lignin biosynthetic

pathway in Selaginella requires two independent or one bifunc-

tional COMT to catalyze themethylation step after the addition of

each ring meta-hydroxyl by Sm F5H (Figure 1B). Bifunctional

COMTs with activities toward 3- and 5-hydroxylated phenyl-

propanoid aldehydes and alcohols are known in angiosperms

(Parvathi et al., 2001; Do et al., 2007). Such bifunctional COMTs

could be ubiquitous in vascular plants or, like the phenylpropa-

noid 5-hydroxylases, may have emerged independently in Se-

laginella and angiosperms.

It is also important to note that the in vitro kinetics data

showed that SmF5Hdoes not produce 3,4,5-trihydroxycinnamyl

aldehyde/alcohol from p-coumaryl- or caffeyl-substituted

substrates. These data suggest that after the first meta-

hydroxylation step, the dihydroxy-substituted product is

released from the active site of Sm F5H, and subsequent 3-O-

methylation is required for the reentry of the guaiacyl-substituted

substrate into the enzyme active site for the 5-hydroxylation.

These biochemical characteristics resemble those of the re-

cently characterized Arabidopsis CYP98A8, a phenylpropanoid

dual meta-hydroxylase involved in the biosynthesis of N1,N5-

di(hydroxyferuloyl)-N10-sinapoylspermidine (Matsuno et al.,

2009). Interestingly, the phenylpropanoid dualmeta-hydroxylase

activities of Sm F5H and CYP98A8 are distinct from that of the

petunia flavonoid 39,59-hydroxylase, which is capable of cata-

lyzing 39- and 59-hydroxylations of 49-hydroxylated flavonone

substrates without meta-O-methylation after the addition of the

first meta-hydroxyl group (Menting et al., 1994).

The C39H-independent S lignin biosynthetic pathway in

Selaginella also requires a form of hydroxycinnamoyl-CoA

Figure 8. Complementation of the Arabidopsis HCTRNAi Plant by Sm F5H.

(A) Four-week-old Arabidopsis plants showing the partial rescue of growth phenotype of an HCTRNAi plant by the Sm F5H transgene.

(B) Relative HCT transcript levels in plants quantified by quantitative RT-PCR. Error bars represent standard deviations based on assays of biological

duplicates.

(C) DFRC–gas chromatography analysis of lignin monomer composition. H/G/S are p-hydroxyphenyl/guaiacyl/syringyl lignin-derived hydroxycinnamyl

alcohol peracetates. c/t, cis/trans; IS, internal standard.

[See online article for color version of this figure.]

1040 The Plant Cell

reductase (CCR) that can efficiently convert p-coumaroyl-CoA to

p-coumaraldehyde. It is possible that, like Sm F5H, this enzyme

is also specifically expressed in the cortex (Weng et al., 2008c),

where it provides H-substituted precursors for Sm F5H, some of

which escape hydroxylation and lead to the deposition of H lignin

in this tissue.

Unlike Selaginella, angiosperms usually contain only trace

amount of H lignin (Boerjan et al., 2003), an observation that may

be explained by previous studies that have shown that for a

number of angiospermCCR isoforms,p-coumaroyl-CoA is a less

favorable substrate than the G lignin precursor, feruloyl-CoA

(Wengenmayer et al., 1976; Sarni et al., 1984; Baltas et al., 2005).

That carbon flux is not stoichiometrically redirected into H lignin

when G and S lignin is eliminated in the Arabidopsis ref8 mutant

further supports this suggestion (Franke et al., 2002a; Abdulrazzak

et al., 2006). Surprisingly, we found that transgenic expression of

the bifunctional Sm F5H in the ref8 background not only restored

S lignin biosynthesis but also significantly enhanced H lignin

deposition. This result suggests that at least in the lignifying

tissue, Arabidopsis contains CCR isoforms that can support the

biosynthesis of substantial amounts of H-substituted lignin pre-

cursors. Thus, in contrast with what has been thought previously,

it seems that CCR is unlikely to be the factor that limits H lignin

biosynthesis, at least in wild-type Arabidopsis. Although it is

unclear why Sm F5H is more effective in restoring S lignin

synthesis than it is in complementing the sinapoylmalate-

deficient phenotype of the fah1 ref8 double mutant, it is possible

that a CCR that catalyzes the conversion of p-coumaroyl-CoA to

p-coumaraldehyde may be limiting or absent in Arabidopsis leaf

tissue, limiting the availability of substrate for Sm F5H in leaves.

Despite the presence of a C39H-independent S lignin biosyn-

thetic pathway mediated by Sm F5H, the Selaginella genome

contains a C39H ortholog that is >60% identical to angiosperm

C39H at amino acid level (Weng et al., 2008c), which suggests

that Selaginella may also contain a C39H-dependent pathway,

analogous to the one defined in angiosperms (Franke et al.,

2002b). The fact that Selaginella xylem, where Sm F5H is not

highly expressed (Weng et al., 2008c), contains lignin composed

of almost pure G units, is consistent with this notion, although the

exact biochemical function of C39H ortholog in Selaginella is still

to be determined.

SmF5HProvides a Valuable Tool for Genetic Engineering of

Lignin Biosynthesis in Higher Plants

Lignin is a ubiquitous component of the cell wall of vascular

plants and is essential to normal plant development, and plants in

Figure 9. HPLC Quantification of Major Phenylpropanoid Metabolites in

Leaves of 3-Week-Old Arabidopsis.

Sinapoylmalate can be detected in ref8-1 and even the null mutant ref8-

2, which is consistent with a previous report (Abdulrazzak et al., 2006)

and suggests the presence of a C39H independent 3-hydroxylation

pathway in ref8 mutant background. We observed that At F5H pos-

sesses a level of 3-hydroxylase activity toward p-coumaraldehyde and

p-coumaryl alcohol (Table 1). Although the kinetic constants suggest

such reactions cannot take place in vivo, under ref8mutant background,

the levels of relevant substrates might reach local concentrations that

could permit some flux through At F5H. KG1, kaempferol 3-O-b-[b-D-

glucopyranosyl(1/6)- D-glucopyranoside]-7-O-a-L-rhamnopyranoside;

KG2, kaempferol 3-O-b-D-glucopyranoside-7-O-a-L-rhamnopyranoside;

KG3, kaempferol 3-O-a-L-rhamnopyranoside-7-O-a-L-rhamnopyranoside;

SM, sinapoylmalate.

[See online article for color version of this figure.]

Figure 10. Tissue-Specific Lignin Analysis in Selaginella Stems.

(A) A longitudinal section of mature Selaginella stem showing that xylem and cortical tissue can be separated. Bar = 1 mm.

(B) Lignin monomer composition (H, G, and S) in separated stem xylem and cortical tissue quantified by DFRC lignin analysis. N.D., not detectable.

[See online article for color version of this figure.]

Alternative Route of S Lignin Synthesis 1041

which the lignin biosynthetic pathway has been downregulated

often suffer from significantly reduced growth (Weng et al.,

2008b). The discovery of the phenylpropanoid dual meta-

hydroxylase activity of Sm F5H and its role in the unique C39H-independent S lignin biosynthetic pathway suggests it may be a

valuable tool for fundamentally rerouting lignin biosynthesis in

higher plants. In the presence of Sm F5H, several essential lignin

biosynthetic genes in the canonical pathway, including C39H and

HCT as demonstrated in this study, could be downregulated

without causing deleterious effects and resulted in a unique H-S

lignin composition in the transgenic plants, which may not occur

in nature. The mechanism that leads to the unique H-S lignin is

currently unknown but deserves additional investigation in the

future.

S Lignin Could Have Emerged Multiple Times during

Plant Evolution

Some older literature reported that S lignin could be detected in

some fern and gymnosperm species (e.g., cuplet fern, yew plum

pine, sandarac-cypress, and gnetophytes) (reviewed in Weng

et al., 2008a), which suggests that S lignin might have evolved

multiple times during plant evolution, but additional F5H analogs

and/or alternative pathways toward S lignin biosynthesis have

yet to be discovered. The apparently independent occurrence of

S lignin in distantly related plant lineages implies that it may have

an important role in plants’ adaptation to their environment. S

lignin is present in the anatomically analogous fiber cells in stems

of angiosperms and Selaginella, suggesting that S lignin may

function similarly in both lineages for mechanical support and/or

defense against pathogens and herbivores (Li et al., 2001; Weng

et al., 2008c). For example, the evolution of S-lignified fibers in

angiosperms, Selaginella, and some members of the Gnetales

may indicate that the presence of these strengthening cell types

permitted the development of relatively weaker vessel elements

in the vasculature of these plants (Logan and Thomas, 1985;

Carlquist, 1996).

METHODS

Plant Material

Arabidopsis thaliana was grown under a 16-h-light/8-h-dark photoperiod

at 100 mE·m22·s21 at 228C. Columbia-0 was used as the wild type. The

T-DNA insertional ref8-2 null allele was obtained from the ABRCunder the

accession of SALK_036132 (Alonso et al., 2003). Selaginella moellen-

dorffii was obtained from Plant Delights Nursery (Raleigh, NC) and grown

in a local greenhouse under 50% shade cloth.

Transgenic Plants

To generate ref8/fah1-2/AtC4H:SmF5H plants, ref8-1 and ref8-2 hetero-

zygous plants were used as the female parent in a cross with four

independent lines of previously described fah1-2/AtC4H:SmF5H plants

(lines 2, 6, 7, and 8) (Weng et al., 2008c). In the F2 generation, plants with

the genotype REF8/ref8 fah1-2/fah1-2 that were homozygous for the Sm

F5H transgene were selected and allowed to self. All subsequent anal-

yses were performed in the F3 generation on homozygous Sm F5H

transgenics that were genotyped as being ref8 fah1-2 double homozy-

gotes. All the independent transgenic lines showed partial phenotypic

complementation compared with the corresponding ref8 fah1-2 plants.

Transgenics that derived from fah1-2/AtC4H:SmF5H line 6 were used

for further detailed analysis. To generate ref8-1/fah1-2/AtC4H:AtF5H

plants, a similar approach was adopted as previously described using

the fah1-2/AtC4H:AtF5H transgenic plants as the male parent in the

initial cross.

To generate the HCTRNAi binary vector, a 356-bp HCT cDNA fragment

was PCR amplified by the primer pair 59-GGGGACAAGTTTGTACAA-

AAAAGCAGGCTAACATCAGAGATTCCACCA-39 and 59-GGGGACCA-

CTTTGTACAAGAAAGCTGGGTCAACTTCGGGAATAAGC-39. The resulting

PCR product was recombined with the modified version of pDONR221

(Invitrogen) using BP clonase (Invitrogen) to generate the entry clone. The

entry clone was then recombined with a destination binary vector

(modified from pBI121; Clontech) that harbors a cauliflower mosaic virus

35S promoter-driven Gateway RNAi cassette to generate the final con-

struct. The HCTRNAi construct was introduced into Arabidopsis via

Agrobacterium tumefaciens–mediated transformation (Clough and Bent,

1998). To generate HCTRNAi/AtC4H:SmF5H plants, plants with the geno-

type REF8/ref8 fah1-2/fah1-2 that were homozygous for the Sm F5H

transgene were used as the female parent to cross with the HCTRNAi

plants. In the F1 generation, the plants with the genotype REF8/REF8

FAH1/fah1-2 that were hemizygous for both the Sm F5H transgene and

the HCTRNAi transgene were analyzed. In parallel, HCTRNAi plants were

crossed to fah1-2 to generate plants with the genotype REF8/REF8

FAH1/fah1-2 that were hemizygous for the HCTRNAi transgene in the F1

generation, which were used as control plants.

To genotype ref8-1, the primer pair 59-CGAGCTATCATGGAGGAGC-

ATA-39 and 59-CAACAAGAGCATGAGCAGCAG-39 was used in com-

bination with EcoRV digestion, exploiting the cleaved amplified

polymorphic sequencemarker resulting from the point mutation in ref8-1.

Similarly, fah1-2 was genotyped using the primer pair 59-TGGTGTGTA-

CATATATGGATGAAGAA-39 and 59-TAGCAAGAGTGGTGAATATGT-

GAAGT-39 in combination with MseI digestion. To genotype ref8-2, LP

primer 59-TCGTGGTTTCTAATAGCGGTG-39 and RP primer 59-TGTTAA-

GAAAAACAATTAGGGTTTTTG-39, together with the T-DNA left border

BP primer 59-TGGTTCACGTAGTGGGCCATCG-39 were used according

to the previously described method (Alonso et al., 2003). To genotype the

presence of the Sm F5H transgene, the gene-specific primer pair

59-CAAGGTCCTCCACAAGAAGC-39 and 59-CAGTCGAAGCACTGGAT-

GAA-39was used. To genotype the presence of theHCTRNAi transgene, a

primer to the 35S promoter 59-GACCTAACAGAACTCGCCGTAAAGA-39

and an HCT gene-specific primer 59-TAAGGGTAGGAGCAAAATCAC-

CAAA-39 were used.

Yeast Expression of P450s and Enzyme Assays

The construction of Sm F5H and At F5H yeast expression vectors is

described by Weng et al. (2008c) and Humphreys et al. (1999), respec-

tively. Constructs were transformed into the WAT11 yeast strain, and

yeast growth, induction, and preparation of yeast microsomal extracts

were conducted as previously described (Humphreys et al., 1999).

Enzyme kinetic assays were performed essentially as described

(Humphreys et al., 1999). In brief, a reaction system containing 1 mM

NADP+, 10mMglucose-6-phosphate, and 4 units of glucose-6-phosphate

dehydrogenase was incubated for 5 min at 308C in the presence of

substrate to allow the generation of NADPH. The reaction was incubated

for 20 min, at 308C after adding the yeast microsomal extract, and was

terminated by adding glacial acetic acid. All the assays except those

involving hydroxycinnamyl alcohols were extracted with ethyl acetate,

dried in vacuo, resuspended in 50% methanol, and analyzed by HPLC.

The assays testing hydroxycinnamyl alcohols were analyzed by HPLC

directly. For assays testing Sm F5H against p-coumaric acid, a substrate

concentration series of 10, 14, 18, 25, 50, and 200 mM was used. For

1042 The Plant Cell

assays testing SmF5H against p-coumaraldehyde/p-coumaryl alcohol or

Sm F5H/At F5H against coniferaldehyde/coniferyl alcohol, a substrate

concentration series of 0.7, 0.8, 1, 1.3, 2, and 10mMwas used. For assays

testing Sm F5H/At F5H against ferulic acid, a substrate concentration

series of 40, 50, 70, 100, 200, and 1000 mMwas used. The same amount

of microsomal extract from a single prep was used for each enzyme

toward various substrates.

Histochemical Staining

For phloroglucinol-HCl staining, hand sections of 2-month-old Arabidop-

sis stems were stained with 1% phloroglucinol (w/v) in 12% HCl for 5 min

and observed under light microscope. For Maule staining, hand sections

of 2-month-old Arabidopsis stems were fixed in 4% glutaraldehyde,

rinsed in water, and treated for 10 min with 0.5% KMnO4. Sections were

then rinsed with water, treated for 5 min with 10% HCl, rinsed in water,

mounted in concentrated NH4OH, and examined under the light micro-

scope.

Lignin Analysis

Cell wall samples free of soluble metabolites were prepared as previously

described (Meyer et al., 1998). For Klason lignin analysis, 100 mg of cell

wall sample was swelled with 3 mL of 72% H2SO4 for 30 min at 308C and

then diluted with water to 4%H2SO4 and autoclaved at 1208C for 1 h. The

residue was filtered through a preweighed glass filter. The residue was

dried in an 808C oven on the filter overnight before being weighed at room

temperature. The DFRC lignin analysis was performed essentially as

previously reported (Lu and Ralph, 1998). Briefly, cell wall samples were

dissolved in acetyl bromide/acetic acid solution, containing 4,49-

ethylidenebisphenol as an internal standard. The reaction products

were dried down using nitrogen gas, dissolved in dioxane/acetic acid/

water (5/4/1, v/v/v), reacted with Zn dust, purified with C-18 SPE columns

(SUPELCO), and acetylated with pyridine/acetic anhydride (2/3, v/v). The

lignin derivatives were analyzed by gas chromatography/flame ionization

detection using response factors relative to the internal standard of 1.26

for p-coumaryl alcohol peracetate, 1.30 for coniferyl alcohol peracetate,

and 1.44 for sinapyl alcohol peracetate. The same samples were run

through gas chromatography–mass spectrometry in parallel to confirm

the identity of the derived hydroxycinnamyl alcohol peracetates.

Lignin Preparation for NMR Spectroscopy

Dried Arabidopsis stems (0.5 to 1.5 g) were ground in a Retsch MM301

shaker mill for 3 min at 30 s21 and extracted sequentially with water

(sonication, 20 min, four times), 80%methanol (four times), acetone (two

times), chloroform-acetone (1/1, v/v, two times), and acetone (one time)

again. The obtained isolated cell walls (0.3 to 1 g) were ball-milled for 5 h

per 1 g of sample weight (in 20 min on/10 min off cycles) using a Retsch

PM100 ball mill vibrating at 600 rpm with zirconium dioxide vessels (50

mL) containing ZrO2 ball bearings (10 3 10 mm). Ball milled walls were

transferred to centrifuge tubes (50 mL) and digested at 308C with crude

cellulases (Cellulysin; Calbiochem; 30mgg21 of sample, in pH5.0 sodium

acetate buffer, 33 2 d, fresh buffer and enzyme each time), leaving all of

the lignin and residual polysaccharides totaling 0.202 g (20.1% of the

original cell wall after extractions, wild type), 0.232 g (23.1%, fah1-2),

0.0452 g (15.3%, ref8-1), 0.0749 g (11.9%, ref8-1/fah1-2/AtC4H:SmF5H),

and 0.137 g (16.1%, ref8-2/fah1-2/AtC4H:SmF5H). The polysaccharidase-

digested cell wall fractions (70 mg each except for ref8-1, 45 mg) were

subjected to solubilization in DMSO/N-methylimidazole (2/1, v/v). Fol-

lowing acetic anhydride addition (0.5 mL, 1.5 h), the polysaccharidase-

digested cell walls gave acetylated samples for NMR measurement (Lu

and Ralph, 2003).

NMR Spectroscopy

The NMR spectra were acquired on a Bruker Biospin DMX-500 instru-

ment fitted with a sensitive cryogenically cooled 5-mm DCH 1H/13C

gradient probewith inverse geometry (proton coils closest to the sample).

Acetylated lignin preparations (5 to 80 mg) were dissolved in 0.5 mL

CDCl3; the central chloroform solvent peakwas used as internal reference

(dC 77.0, dH 7.26 ppm). HSQC experimental conditions were as described

previously (Wagner et al., 2007). Volume integration of contours in HSQC

plots was accomplished by Bruker’s TopSpin 2.0 software as described

(Ralph et al., 2006).

Quantitative RT-PCR

Total RNA was extracted from 3-week-old rosette leaves of Arabidopsis

plants using the RNeasy plant mini kit (Qiagen). Single-strand cDNAs

were synthesized via reverse transcription using the High Capacity cDNA

reverse transcription kit (Applied Biosystems). The cDNAs was treated

with RNase and used as template for real-time PCR. Quantitative real-

time PCR was performed on the StepOne Real-Time PCR system

(Applied Biosystems) using the DDCT method with default cycling pro-

gram. HCT was amplified using the primer pair 59-GAATTCCATAC-

GAGGGTTTGTCTT-39 and 59-GGGCAATGGCAACGGATA-39, whereas

At1g13320, as an internal standard (Czechowski et al., 2005), was

amplified using the primer pair 59-TAACGTGGCCAAAATGATGC-39 and

59-GTTCTCCACAACCGCTTGGT-39. Both the primer pairs were tested in

a standard curve analysis beforehand, which showed amplification

efficiency higher than 90%.

Leaf Sinapoylmalate and Flavonoid Analysis

Three-week-old Arabidopsis rosette leaves were harvested, ground in

liquid nitrogen, and extracted with 50%methanol (1 mL per 100 mg fresh

weight) for 2 h at 658C, and the extracts were then analyzed by HPLC.

Metaboliteswere separated on aC18 column, using a gradient from1.5%

acetic acid to 35% acetonitrile in 1.5% acetic acid at a flow rate of 1 mL

min21. Sinapoylmalate content was quantified using sinapic acid as

standard. The identification of the threemajor kaempferol glycosides was

according to a previous report (Veit and Pauli, 1999); quantification used

kaempferol as standard.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome

Initiative or GenBank/EMBL databases under the following accession

numbers: Sm F5H, EU032589; At F5H, At4g36220; andHCT, At5g48930.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Lineweaver-Burk Representation of the

Kinetic Analysis of Phenylpropanoid Meta-Hydroxylation Reactions

Catalyzed by Yeast-Expressed Sm F5H or At F5H.

Supplemental Figure 2. GC-MS Confirmation of the Identity of

Peracetates of Monolignols Derived from the DFRC Lignin Analysis

Shown in Figure 6.

Supplemental Figure 3. Partial (Sidechain Region) HSQC Spectra

Showing the Structural Changes Resulting from Lignification Using

Altered Monolignol Supplies.

ACKNOWLEDGMENTS

This work is funded by the National Science Foundation (Grant IOB-

0450289). Partial funding to J.R. was via the Department of Energy

Alternative Route of S Lignin Synthesis 1043

(DOE) Office of Science (Grant DE-AI02-06ER64299) and the DOE Great

Lakes Bioenergy Research Center (DOE Office of Science BER DE-

FC02-07ER64494). N.D.B. is supported by a fellowship from the Life

Sciences Research Foundation. We thank J.A. Banks for providing

Selaginella moellendorffii plant materials.

Received December 15, 2009; revised March 8, 2010; accepted March

22, 2010; published April 6, 2010.

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Alternative Route of S Lignin Synthesis 1045

DOI 10.1105/tpc.109.073528; originally published online April 6, 2010; 2010;22;1033-1045Plant Cell

Jing-Ke Weng, Takuya Akiyama, Nicholas D. Bonawitz, Xu Li, John Ralph and Clint Chapple and Flowering PlantsSelaginella

Convergent Evolution of Syringyl Lignin Biosynthesis via Distinct Pathways in the Lycophyte

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