The Growth Reduction Associated with Repressed LigninBiosynthesis in Arabidopsis thaliana Is Independentof Flavonoids C

Xu Li, Nicholas D. Bonawitz, Jing-Ke Weng,1 and Clint Chapple2

Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907

Defects in phenylpropanoid biosynthesis arising from deficiency in hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl

transferase (HCT) or p-coumaroyl shikimate 39-hydroxylase (C39H) lead to reduced lignin, hyperaccumulation of flavonoids,

and growth inhibition in Arabidopsis thaliana. It was previously reported that flavonoid-mediated inhibition of auxin

transport is responsible for growth reduction in HCT-RNA interference (RNAi) plants. This conclusion was based on the

observation that simultaneous RNAi silencing of HCT and chalcone synthase (CHS), an enzyme essential for flavonoid

biosynthesis, resulted in less severe dwarfing than silencing of HCT alone. In an attempt to extend these results using a

C39H mutant (ref8) and a CHS null mutant (tt4-2), we found that the growth phenotype of the ref8 tt4-2 double mutant, which

lacks flavonoids, is indistinguishable from that of ref8. Moreover, using RNAi, we found that the relationship between HCT

silencing and growth inhibition is identical in both the wild type and tt4-2. We conclude from these results that the growth

inhibition observed in HCT-RNAi plants and the ref8mutant is independent of flavonoids. Finally, we show that expression of

a newly characterized gene bypassing HCT and C39H partially restores both lignin biosynthesis and growth in HCT-RNAi

plants, demonstrating that a biochemical pathway downstream of coniferaldehyde, probably lignification, is essential for

normal plant growth.

INTRODUCTION

In vascular plants, the phenylpropanoid pathway is responsible

for the biosynthesis of a variety of metabolites, including lignin, a

major structural component of secondary cell wall, and flavo-

noids, a diverse collection of compounds involved in pigmenta-

tion, herbivory defense, and plant–microbe interactions (Figure

1). The presence of lignin protects cell wall polysaccharides from

enzymatic and chemical degradation, with substantial negative

impacts on forage quality, pulping efficiency, and cellulosic

biofuel production (Li et al., 2008). Accordingly, a number of

approaches have been employed to identify or engineer plants

with low lignin content. Although such plants have been obtained

in several species, including tobacco (Nicotiana tabacum), pop-

lar (Populus spp), alfalfa (Medicago sativa), and Arabidopsis

thaliana, and do indeed exhibit increased cell wall degradability,

they typically also show reduced growth and decreased biomass,

making them unsuitable for practical applications (Piquemal

et al., 1998; Pincon et al., 2001; Franke et al., 2002a; O’Connell

et al., 2002; Hoffmann et al., 2004; Reddy et al., 2005; Leple et al.,

2007; Shadle et al., 2007).

The growth defects of lignin-deficient plants have previously

been attributed to impaired water transport resulting from col-

lapse of the xylem (Piquemal et al., 1998; Jones et al., 2001;

Franke et al., 2002a), which in wild-type plants is highly lignified

and is subjected to strong negative pressures during water

transport. By contrast, the authors of a recent report argued that

the growth phenotype resulting from downregulation of one

lignin biosynthetic gene is instead due to accumulation of flavo-

noids and inhibition of auxin transport (Besseau et al., 2007). In

this report, the authors used RNA interference (RNAi) to knock

down the expression of the gene encoding hydroxycinnamoyl-

CoA:shikimate hydroxycinnamoyl transferase (HCT), which acts

at a branch point in the phenylpropanoid pathway where

p-coumaroyl CoA can be committed either to lignin production

if acted upon by HCT (Hoffmann et al., 2003, 2004), or to

flavonoid biosynthesis if acted upon by the competing enzyme

chalcone synthase (CHS; Burbulis et al., 1996). It had been

previously shown that plants expressing anHCT-RNAi transgene

accumulate high levels of flavonoids (Hoffmann et al., 2004),

compounds known to have an inhibitory effect on auxin polar

transport (Jacobs and Rubery, 1988; Brown et al., 2001). To test

whether the hyperaccumulation of flavonoids in HCT-RNAi

plants could be the cause for the growth phenotype, Besseau

et al. downregulated the flavonoid biosynthesis in these plants by

RNAi silencing of CHS and were able to show that the dwarf

phenotype could be significantly alleviated. The recovery in

growth was also associated with the restoration of auxin trans-

port, which was greatly inhibited in the HCT-RNAi plants. The

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.www.plantcell.org/cgi/doi/10.1105/tpc.110.074161

The Plant Cell, Vol. 22: 1620–1632, May 2010, www.plantcell.org ã 2010 American Society of Plant Biologists

Figure 1. The Arabidopsis Phenylpropanoid Pathway.

The biosynthetic pathways for flavonoids and lignin are interconnected at p-coumaroyl CoA. Mutations in CHS (tt4) and C39H (ref8) affect the flavonoids

and lignin biosynthesis, respectively. Sinapate esters and syringyl lignin share a common precursor, sinapaldehyde. The F5H gene from S.

moellendorffii (Sm-F5H) has the unique ability to catalyze the hydroxylation of p-coumaraldehyde and p-coumaryl alcohol to generate caffeylaldehyde

Flavonoid-Independent Growth Inhibition 1621

authors thus suggested that the growth phenotype resulting from

downregulation of HCT is due to accumulation of flavonoids and

inhibition of auxin transport, rather than perturbation of lignin

content and composition.

After its synthesis by HCT, p-coumaroyl shikimate is subse-

quently hydroxylated at the 3-position of the hydroxycinnamoyl

moiety byp-coumaroyl shikimate 39-hydroxylase (C39H; the prod-uct of the REF8 gene in Arabidopsis) to yield caffeoyl shikimate

(Figure 1; Schoch et al., 2001; Franke et al., 2002b; Nair et al.,

2002). Like HCT-deficient plants, C39H-deficient ref8mutants are

also dwarf and accumulate high levels of flavonoids. However, as

we show here, blocking flavonoid biosynthesis with a CHS null

mutation (tt4-2; Burbulis et al., 1996) has no effect on the ref8

growth phenotype. Unexpectedly, we also found that the pheno-

type of HCT-RNAi plants is similarly unaffected by tt4-2. By

contrast, we found that rerouting lignin biosynthesis with a heter-

ologously expressed gene to bypass HCT and C39H in HCT-RNAi

plants substantially rescues their growth phenotype, consistent

with the previously held notion that lignin deficiency is the cause of

growth reduction and dwarfing in phenylpropanoid mutants.

RESULTS

Elimination of Flavonoid Biosynthesis by Disruption of CHS

Does Not Rescue the Growth Phenotype of ref8Mutants

Arabidopsis ref8mutants exhibit growth reduction and increased

accumulation of flavonoids when compared with wild-type con-

trols (Figure 2; Franke et al., 2002a; Abdulrazzak et al., 2006).

Based on the previous report that flavonoid accumulation is the

cause of the reduced growth of HCT-RNAi plants (Besseau et al.,

2007), we sought to test if the growth inhibition observed in the

ref8 mutant could also be due to the hyperaccumulation of

flavonoids. To this end, we crossed the ref8 mutant with the

CHS null mutant tt4-2, which is devoid of flavonoids (Burbulis

et al., 1996). Initial genotyping of the F2 progeny from this cross

identified a few homozygous ref8 tt4-2 double mutants, all of

which were small and sterile. Further detailed analysis was per-

formed using F3 progeny of an F2 plant with a REF8/ref8 tt4-2/

tt4-2 genotype. Of 27 F3 progeny analyzed, 21 plants showed

wild-type growth, whereas six plants exhibited reduced rosette

size and dwarfing indistinguishable from that seen in the ref8

mutant (Figure 2).Genotypingconfirmed that the six smaller plants

were all homozygous for both the ref8 and tt4-2 mutations,

whereas none of the F3 plants exhibiting wild-type growth were

homozygous for the mutant ref8 allele. HPLC analysis of meth-

anolic leaf extracts confirmed that all F3 progeny were devoid of

flavonoids (Figure 2), as expected from their lack of a functional

CHS. Thus, C39Hdeficiency leads to severe growth reduction and

dwarfing even in the absence of flavonoids.

Silencing of HCT Has Identical Growth Effects in Both

Wild-Type and tt4-2Mutant Backgrounds

Given the positions of HCT and C39H in the phenylpropanoid

pathway and the similar phenotypes of ref8mutants and HCT-

RNAi plants, we were initially surprised that blocking flavonoid

biosynthesis failed to rescue the growth phenotype of the ref8

mutant. This observation led us to further investigate the

relationship between flavonoid hyperaccumulation and the

dwarf phenotype in HCT-deficient plants. If the reduced-

growth phenotype exhibited by HCT-RNAi plants is dependent

on the hyperaccumulation of flavonoids as reported, then

silencing of HCT in the flavonoid-lacking mutant tt4-2 should

have no effect on growth. To test this, we generated an intron-

containing hairpin RNAi construct targeting Arabidopsis HCT

and subsequently introduced it into both wild-type and tt4-2

mutant plants. Consistent with previous reports (Hoffmann

et al., 2004; Besseau et al., 2007), RNAi downregulation of HCT

in wild-type plants led to reduced growth and increased

flavonoid accumulation, as revealed by analysis of indepen-

dent lines exhibiting phenotypes of varying severity (Figures

3A and 3G). The suppression of HCT in these lines was evident

at both themRNA level and the level of enzyme activity (Figures

3C and 3D). Surprisingly, however, HCT-RNAi lines generated

in the tt4-2 mutant background and analyzed in parallel

also exhibited the reduced growth phenotype (Figures 3B,

3E, and 3F), despite the fact that they lacked flavonoids (Figure

3H). It is noteworthy that in moderately dwarfed HCT-RNAi

plants, levels of HCT activity only 2% of that found in wild-type

plants are sufficient to prevent the extreme dwarfing seen

in more severely affected plants grown under the same

conditions.

To investigate the relationship between HCT silencing and

plant growth reduction in the above two genetic backgrounds

and to eliminate the remote possibility that T-DNA insertion of

the above described HCT-RNAi construct had disrupted a

growth-regulating gene, the inactivation of which could not be

rescued by flavonoid depletion, we analyzed HCT expression

and growth in multiple T2 transgenic lines derived from inde-

pendent T1 transformants. We measured plant height at ma-

turity, after inflorescence stems had ceased elongating, to

eliminate any possible confounding effects due to differences in

rates of development. When the HCT mRNA levels in these

plants were compared with their heights, it was apparent that

HCT silencing was strongly correlated with plant growth reduc-

tion in both wild-type and tt4-2 backgrounds (Figure 4). Multiple

regression analysis showed that HCT expression has a signif-

icant effect on plant height (P < 0.001), and this effect is not

affected by these two different genetic backgrounds (test

for interaction of HCT expression and genetic background,

P = 0.6).

Figure 1. (continued).

and caffeyl alcohol, respectively (dashed arrows). PAL, phenylalanine ammonia lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate:CoA

ligase; CHI, chalcone isomerase; CCoAOMT, caffeoyl CoA O-methyltransferase; CCR, cinnamoyl CoA reductase; F5H, ferulic acid 5-hydroxylase;

COMT, caffeic acid O-methyltransferase; CAD, cinnamyl alcohol dehydrogenase; HCALDH, hydroxycinnamaldehyde dehydrogenase.

1622 The Plant Cell

The Ability to Synthesize Flavonoids Has No Effect on the

GrowthPhenotypeofHCT-RNAiPlantsContaining Identical

Transgene Insertions

While the above data strongly suggest that flavonoid accumula-

tion is not themechanism of growth inhibition in HCT-RNAi plants,

a direct comparison of the growth phenotype of the HCT-RNAi

lines generated in different genetic backgrounds is unavoidably

cofounded with potential position effects associated with the

T-DNA insertions in different transgenic plants. To generate wild-

type and tt4-2 mutants with identical HCT-RNAi transgene inser-

tions, we crossed a homozygous T2 HCT-RNAi plant containing

wild-type TT4 with a tt4-2 mutant (scheme shown in Figure 5A).

The resulting F1 plants, which are heterozygous for the tt4-2 allele

and hemizygous for theHCT-RNAi transgene,were again crossed

with the tt4-2mutant. Thirty-eight F1plants from this secondcross

were genotyped and their heights at maturity were measured. As

expected, four genotypes were found at approximately equal

frequency in the F1 population: tt4-2/TT4 (n = 11), tt4-2/tt4-2 (n =

10), tt4-2/TT4 with HCT-RNAi (n = 9), and tt4-2/tt4-2 with HCT-

RNAi (n = 8). The F1 plants with the former two genotypes lacking

the HCT-RNAi transgene have a typical height of;45 cm (Figure

5B). By contrast, plants possessing the HCT-RNAi transgene

exhibit a maximum height of ;25 cm. Critically, no statistically

significant difference in height (Student’s t test, P = 0.57) was

detected between the plants with the latter two genotypes,

Figure 2. The Growth Phenotype of ref8 Is Independent of Flavonoid Accumulation.

Shown are representative pictures of wild-type, tt4-2, ref8, and tt4-2 ref8 plants, along with HPLC profiles of soluble leaf metabolites from these same

plants. Plants were compared 3 weeks after planting. The ref8 and the ref8 tt4-2 double mutants have similar growth inhibition but differ greatly in their

flavonoid accumulation. K1, kaempferol 3-O-[699-O-(rhamnosyl) glucoside] 7-O-rhamnoside; K2, kaempferol 3-O-glucoside 7-O-rhamnoside; K3,

kaempferol 3-O-rhamnoside 7-O-rhamnoside; SM, sinapoylmalate. Bars = 0.5 cm. mAU, milli absorbance unit.

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

Flavonoid-Independent Growth Inhibition 1623

Figure 3. Silencing of HCT in Either the Wild-Type or tt4-2 Background Results in Growth Inhibition.

HCT-RNAi plants showing a moderate (M) or severe (S) growth phenotype with either wild-type TT4 (A) or mutant tt4-2 background (B) were compared

with their cognate controls at 3 weeks after planting. Bars = 1 cm. HCT mRNA levels ([C] and [E]) and enzyme activity ([D] and [F]) as well as HPLC

profiles of soluble phenylpropanoids ([G] and [H]) were determined for the above plants with either wild-type TT4 ([C], [D], and [G]) or mutant tt4-2 ([E],

[F], and [H]) background. The values represent the average of three biological replications and the error bars represent 1 SD. nd, not determined; K1,

kaempferol 3-O-[699-O-(rhamnosyl) glucoside] 7-O-rhamnoside; K2, kaempferol 3-O-glucoside 7-O-rhamnoside; K3, kaempferol 3-O-rhamnoside 7-O-

rhamnoside; SM, sinapoylmalate; mAU, milli absorbance unit; WT, wild type.

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

1624 The Plant Cell

which carry the same HCT-RNAi transgene insertions but differ

in their ability to produce flavonoids.

In a reciprocal approach, we selected a single hemizygous T2

HCT-RNAi plant generated in the tt4-2 background and crossed it

with both tt4-2 and wild-type plants in parallel (scheme in Figure

6A). The first cross resulted in F1 progeny with the genotypes tt4-

2/tt4-2 (n = 20) and tt4-2/tt4-2 HCT-RNAi (n = 16), whereas the

progeny of the second cross had the genotypes tt4-2/TT4 (n =

11) and tt4-2/TT4 HCT-RNAi (n = 14). All plants containing the

HCT-RNAi transgene exhibited a strong dwarf phenotype (aver-

age height;10 cm) regardless of whether they possessed awild-

typeormutant allele ofTT4 (Figure 6B). Taken together, these data

demonstrate that flavonoids have no effect on the reduced-

growth phenotype associated with RNAi silencing of HCT.

HCT-RNAi Plants Contain Higher Concentrations of

Flavonoids Than DoWild-Type Plants, but the Total

Amount of Flavonoids per Rosette Is Similar in Both

Genetic Backgrounds

At 3 weeks of age, HCT-RNAi plants are dwarfed and contain a

higher concentration of flavonoids than do wild-type plants.

However, the concentration of sinapoylmalate in HCT-RNAi

plants is approximately the same as that in wild-type plants, in

apparent conflict with the established role of HCT in sinapate

ester biosynthesis (Figures 3G and 3H; Hoffmann et al., 2003,

2004). However, we reasoned that both flavonoid and sinapoyl-

malate measurements could be confounded in HCT-RNAi plants

by the fact that their rosette leaves expand substantially less than

those of the wild type. To gain more insight into the dynamics of

soluble phenylpropanoid accumulation, we quantified sinapoyl-

malate and flavonoids in whole rosettes of soil-grown wild-type

and HCT-RNAi plants at five time points from 14 to 22 d after

planting (Figure 7). Wild-type and HCT-RNAi plants begin to

diverge from one another phenotypically ;16 d after planting,

when rosettes of wild-type, but not HCT-RNAi, plants continue to

grow and expand exponentially (Figures 7A and 7B). After this

point, the concentration of flavonoids, expressed in nmol per mg

freshweight, was significantly higher (Student’s t test, P < 0.05) in

HCT-RNAi plants than in wild-type plants (Figure 7C). However,

the total amount of flavonoids in these plants, expressed in nmol

Figure 4. Plant Height Is Negatively Correlated with HCT Silencing in

Both Wild-Type and tt4-2 Backgrounds.

Height at maturity and HCT mRNA level in 3-week-old rosettes were

measured for HCT-RNAi plants with either wild-type TT4 (A) or mutant

tt4-2 (B) background. For each genetic background, two T2 progeny of

each of five independent T1 transformants were analyzed. Open boxes

indicate untransformed wild-type or tt4-2 plants.

Figure 5. Elimination of Flavonoids from HCT-RNAi Plants Does Not

Rescue Their Growth Phenotype.

(A) Schematic illustration of the crosses made to generate the F1

population composed of four genotypes. The number of plants for

each genotype is indicated in brackets.

(B) Height at maturity of the four types of F1 plants shown in (A). The

averaged values are presented, and the error bars represent 1 SD.

Flavonoid-Independent Growth Inhibition 1625

per rosette, was similar at all time points (Figure 7D). Sinapoyl-

malate measurements followed a similar but distinct trend.

Whereas sinapoylmalate concentration (nmol per mg) was ap-

parently unaffected by the presence of the HCT-RNAi transgene

(Figure 7E), the total amount of sinapoylmalate per rosette was

clearly lower in HCT-RNAi plants than in wild-type plants after

day 18 (Figure 7F). These data are consistent with a role for HCT

in sinapoylmalate biosynthesis and suggest that the high con-

centration of flavonoids and sinapoylmalate in HCT-RNAi plants

may be a result of reduced leaf expansion.

Silencing of HCT in tt4-2 Plants Decreases Lignin Content

and Alters Lignin Composition

As shown above, RNAi silencing of HCT has identical effects on

the growth phenotype of wild-type and tt4-2 plants, despite the

fact that they differ in their ability to synthesize flavonoids. To

investigate if this growth defect is associated with common

changes in lignin, we analyzed lignin content and composition of

HCT-RNAi and tt4-2 HCT-RNAi plants using the derivatization

followed by reductive cleavage (DFRC) method (Lu and Ralph,

1997). The RNAi lines with a moderate growth phenotype were

used in this experiment to allowcollectionof enough inflorescence

stemmaterial for lignin analysis. These analyses showed thatwild-

type and tt4-2 plants have similar total lignin content and lignin

compositions, with a majority of guaiacyl (G) and syringyl (S) lignin

units and trace amount of p-hydroxyphenyl (H) lignin (Figure 8).

Silencing of HCT in either background led to an;80% decrease

in total lignin and to an increase in the proportion of H lignin

monomers, which do not require HCT for their synthesis, relative

to the HCT-dependent G and S monomers (Figure 8). The pres-

ence or absence of the wild-type TT4 allele had no effect on total

lignin content or composition in plants carrying the HCT-RNAi

transgene or in control plants lacking the transgene. These results

demonstrate that the HCT RNAi transgene was highly effective in

altering lignin and that this effect was independent of flavonoids.

Rerouting Lignin Biosynthesis with a Newly Described

Selaginella Enzyme Rescues the Phenotype of HCT-RNAi

Plants Independent of Flavonoids

We recently reported the cloning and characterization of a gene

encoding a cytochrome P450 monooxygenase from the lyco-

phyte Selaginella moellendorffii (Weng et al., 2008). This enzyme,

which we have named F5H, cannot only hydroxylate the

5-position of coniferaldehyde and coniferyl alcohol, as does

Arabidopsis ferulic acid 5-hydroxylase (At-F5H), but also can

hydroxylate the 3-position ofp-coumaraldehyde and p-coumaryl

alcohol (Weng et al., 2010). Expression of Sm-F5H inArabidopsis

thus provides an alternate biosynthetic route from p-coumarate

to coniferaldehyde and downstream phenylpropanoids that

bypasses both HCT and C39H (Figure 1). We have shown that

transformation of HCT- or C39H-deficient plants with an Sm-F5H

expression construct leads to alleviation of their dwarf pheno-

type as well as to an increase in both total lignin and the fraction

of lignin made up by syringyl monomers (Weng et al., 2010). We

concluded from these data that the increase in lignin in HCT-

RNAi plants wasmost likely responsible for the alleviation of their

dwarf phenotypes; however, given the report by Besseau et al.

(2007), we undertook here to test whether alterations in flavonoid

accumulation in the HCT-RNAi plants expressing Sm-F5H could

play a role in the suppression of their growth phenotype.

To test the possible involvement of flavonoids in the rescue of

HCT-RNAi by Sm-F5H, we first compared flavonoid levels in

wild-type and HCT-RNAi plants either containing or lacking the

Sm-F5H expression construct (Figure 9). Consistent with previ-

ous experiments, we observed that HCT-RNAi plants lacking

Sm-F5H contained higher concentrations of flavonoids than did

wild-type plants. Whereas expression of Sm-F5H had no effect

on flavonoid accumulation in wild-type plants, HCT-RNAi plants

expressing Sm-F5H showed slightly lower flavonoid levels than

those lacking Sm-F5H (Student’s t test, P < 0.001). However,

these data are confounded by the fact that expression of

Sm-F5H also results in increased rosette leaf expansion in

HCT-RNAi plants, allowing the possibility that the lower flavonoid

concentration inHCT-RNAi plants is the result of alleviation of the

growth reduction, rather than the cause.

To conclusively test whether flavonoids are responsible for

suppression of the HCT-RNAi phenotype by Sm-F5H, we trans-

formed tt4-2 HCT-RNAi plants with our Sm-F5H expression

construct. These plants lack flavonoids, and thus any phenotypic

rescue resulting from expression of Sm-F5H must be indepen-

dent of flavonoid accumulation. Analysis of a population of 39 T2

progeny from a single T1 transformant revealed a substantial and

Figure 6. Restoration of the Ability to Synthesize Flavonoids Does Not

Aggravate the Growth Phenotype of the tt4-2 HCT-RNAi Plants.

(A) Schematic illustration of the crosses made to generate the two F1

populations. The number of plants for each genotype is indicated in

brackets.

(B) Height at maturity of the four types of F1 plants shown in (A). The

averaged values are presented, and the error bars represent 1 SD.

1626 The Plant Cell

statistically significant difference (Student’s t test, P < 0.05) in

height between the T2 plants containing the Sm-F5H transgene

(13.5 cm, SD = 2.1 cm, n = 30) and their siblings lacking the

transgene (6.5 cm, SD = 4.7 cm, n = 9) (Figure 10). We conclude

from these data that the rescue of the HCT-RNAi phenotype by

Sm-F5H is independent of flavonoids as previously reported and

is due to the restoration of a biosynthetic pathway downstream

of coniferaldehyde.

DISCUSSION

The biosynthetic pathways leading to lignin and flavonoids, two

major classes of phenylpropanoids, diverge at the common

intermediate p-coumaroyl CoA. Perturbation of lignin biosynthe-

sis by silencing of HCT or mutation of the gene encoding C39H

leads to both decreased lignin content and increased flavonoids.

Either or both of these two biochemical changes could poten-

tially contribute to the growth inhibition observed in these plants,

since lignin is important for the water transport function of xylem

(Boyce et al., 2004) and some flavonoids are known to be able to

modulate the polar transport of auxin (Taylor and Grotewold,

2005; Peer and Murphy, 2007). Previously, Besseau et al. (2007)

reported that the reduced-growth phenotype of HCT-RNAi

plants can be rescued by RNAi silencing of CHS, suggesting

that flavonoid accumulation, not lignin deficiency, is responsible

for this growth inhibition. By contrast, we demonstrated here that

Figure 7. Soluble Phenylpropanoids Decrease with Time to a Greater Extent in Wild-Type Than in HCT-RNAi Plants.

The rosette growth was compared between wild-type and HCT-RNAi plants from 14 to 22 d after planting (DAP) by size (A) and fresh weight (B). A

representative flavonoid (kaempferol 3-O-rhamnoside 7-O-rhamnoside) from whole rosettes of wild-type and HCT-RNAi plants at the time points was

quantified on a fresh weight (FW) basis (C) as well as on a per-rosette basis (D). Similarly, sinapoylmalate content of wild-type and HCT-RNAi rosettes

was also quantified either on a fresh weight (E) or per-rosette basis (F). All graphs show the average of at least three biological replicates. Error bars

represent 1 SD. Asterisks indicate the levels between wild-type and HCT-RNAi plants are significantly different (Student’s t test, P < 0.05).

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

Flavonoid-Independent Growth Inhibition 1627

this growth effect is independent of flavonoids by silencing HCT

in a CHS null background.

One possible explanation for the discrepancy between our

data and that of the previous study is that in Besseau et al. (2007),

transcription of both the HCT and CHS RNAi constructs was

driven by the same promoter (35S), which in some cases can

lead to transcriptional inactivation of one or both transgenes

(Fagard and Vaucheret, 2000). Such promoter-homology based

suppression could reduce the silencing of HCT in plants carrying

both transgenes and consequently alleviate their growth defect.

Importantly, both our data and that of Besseau et al. are in

agreement that very slight differences in HCT activity (within 1 to

4% wild-type level) lead to dramatic differences in growth

phenotype. Such small changes cannot be distinguished at the

protein level by the immunoblotting assay that Besseau et al.

used tomonitor HCT expression in the F1 progeny of the crosses

between HCT-RNAi and wild-type or CHS-RNAi plants (see

Figures 2 and 9 of Besseau et al., 2007). Thus, it is possible that

the alleviation in the growth phenotype of their HCT-RNAi 3CHS-RNAi F1 plants was actually due to a suppression of the

HCT-RNAi construct that is undetectable at the protein level but

that is significant phenotypically. This explanation is consistent

with the fact that the HCT-RNAi 3 CHS-RNAi F1 plants showed

more than 14-fold higher lignin content comparedwith their HCT-

RNAi parents, which was not apparent in stem section staining

(see Table 2 and Figure 10B of Besseau et al., 2007). This

problem was avoided in our analysis by the use of a CHS null

mutant instead of RNAi suppression.

There is a well-established role for flavonoids, such as quer-

cetin and kaempferol, in the inhibition of polar auxin transport,

and the increased accumulation of flavonoids resulting from

suppression of HCT in thewild-type background, as observed by

both Besseau et al. and ourselves, might indeed impact auxin

transport. This effect, however, is unrelated to the dwarfism seen

in these plants. By contrast, the observation that sinapoylmalate

levels in HCT-RNAi plants are at least as high as that in wild-type

plants appears to contradict the established role of HCT in the

core phenylpropanoid pathway. Although this has previously

been taken to suggest the existence of an HCT-independent

route for sinapoylmalate biosynthesis (Abdulrazzak et al., 2006;

Besseau et al., 2007), a simple alternative explanation for the

above observations is that the decrease in flavonoids and

sinapoylmalate observed in wild-type plant rosettes with time

is simply due to a diluting effect resulting from leaf expansion and

that the failure of HCT-RNAi leaves to fully expand leads to higher

measurements of soluble phenylpropanoids when expressed on

a fresh weight basis, as is usually done. It is also possible that the

amount of HCT activity present in the leaves of wild-type plants is

in great excess of the amount required for normal sinapoylmalate

biosynthesis and that the RNAi suppression used here, while

sufficient to lead to lignin defects, fails to affect sinapoylmalate

biosynthesis.

In addition to C39H and HCT, perturbation of lignin biosynthe-

sis at other points in the phenylpropanoid pathway also results in

growth reduction and dwarfing. For example, transgenic to-

bacco deficient in Phe ammonia lyase, transgenic alfalfa with

downregulated cinnamic acid 4-hydroxylase (C4H), as well as

Arabidopsis ref3 and irx4 mutants, which are defective in C4H

and cinnamoyl CoA reductase, respectively, all exhibit reduced

Figure 8. Silencing of HCT in Wild-Type or tt4-2 Background Results in

Similar Lignin Changes.

DFRC lignin analysis was performed for the wild type (TT4), HCT-RNAi

with wild-type background (TT4 HCT-RNAi), tt4-2, and HCT-RNAi with

tt4-2 background (tt4-2 HCT-RNAi). The content of p-hydroxyphenyl (H),

guaiacyl (G), and syringyl (S) lignin monomers was quantified. The values

represent the average of three biological replications, and the error bars

represent 1 SD.

Figure 9. Expressing Sm-F5H in HCT-RNAi Plants Suppresses Flavo-

noid Accumulation.

Flavonoid levels (sum of the three major kaempferol-derived flavonoids,

K1, K2, and K3; see Figure 3) in rosette leaves of the wild type (WT, n = 7),

wild-type transformed with Sm-F5H (WT SmF5H, n = 9), HCT-RNAi (n =

17), and HCT-RNAi transformed with Sm-F5H (HCT-RNAi SmF5H, n =

38) were measured by HPLC. Averaged values are shown, and the error

bars represent 1 SD. Values not labeled with same letter are significantly

different (one-way analysis of variance, P < 0.05).

1628 The Plant Cell

size and stunted growth but without hyperaccumulation of, or in

some cases with reductions in, flavonoids (Elkind et al., 1990;

Jones et al., 2001; Reddy et al., 2005; Schilmiller et al., 2009). The

association between growth defects and low lignin content, as

well as the collapsed xylem phenotype observed in these plants,

suggests that the deficiency of xylem lignification may impede

water transport and account for these deleterious growth effects

because it is well known that the growth of leaves and stems

is inhibited during water deficit (Acevedo et al., 1971; Van

Volkenburgh and Boyer, 1985; Nonami and Boyer, 1990; Bartels

and Sunkar, 2005). Indeed, no such growth inhibition was ob-

served in the Arabidopsis nst1 nst3 double mutant, which ex-

hibits ligninification defects in interfascicular fibers but not in

vascular tissue (Mitsuda et al., 2007). In support of thismodel, we

have shown that partially rerouting lignin biosynthesis via ex-

pression of Selaginella F5H leads to significant alleviation of the

growth defects caused by disruption of C39H or silencing of HCT

and that this effect is also independent of flavonoid biosynthesis.

Plant growth and development are complex biological pro-

cesses regulated by both environmental factors and endoge-

nous signaling molecules. Many mutants with reduced-growth

and dwarf phenotypes have been found to be defective in the

biosynthesis or perception of plant hormones, such as gibber-

ellin or brassinosteroids (Fujioka and Yokota, 2003; Thomas and

Sun, 2004). Although impaired xylem function due to lignin

deficiency can readily explain the growth reduction and dwarfing

in phenylpropanoid pathway mutants, we cannot exclude the

possibility that the accumulation or deficiency of another phenyl-

propanoid derivative may directly or indirectly influence plant

growth and development. For example, dehydrodiconiferyl al-

cohol glucosides (DCGs), derivatives of coniferyl alcohol, have

been shown to have cell division–promoting activities in tobacco

tissue cultures (Lynn et al., 1987; Teutonico et al., 1991). It has

been reported that, in tobacco, inhibition of phenylpropanoid

metabolism by overexpression of MYB308 from Antirrhinum

majus resulted in decreased content of DCGs and abnormal

growth, and feeding the cell culture of these transgenic tobaccos

with dehydrodiconiferyl alcohol, the aglycone precursor of

DCGs, significantly restored cell expansion (Tamagnone et al.,

1998). It would be interesting to explore further whether DCGs

and their growth-promoting activities are required for nor-

mal development in other plant species and whether their ab-

sence could be related to the dwarfing seen in phenylpropanoid

pathway-downregulated plants.

Clearly, further investigation will be required to fully under-

stand the mechanism underlying the phenotypic manifestations

resulting from disruption of phenylpropanoid metabolism. Given

the potential impact of lignin reduction on biofuel production, this

knowledge will have a great impact on future genetic engineering

strategies for the development of improved biofuel feedstocks.

METHODS

Plant Material and Growth Conditions

The ref8mutant was generated previously in our laboratory (Franke et al.,

2002a). The tt4-2 mutant (Burbulis et al., 1996) was kindly provided by

Angus Murphy. Plants were grown in Redi-earth Plug and Seedling Mix

(Sun Gro Horticulture) supplied with Scotts Osmocote Plus controlled

release fertilizer (Hummert International) at 228C under a 16-h-light/8-h-

dark photoperiod. The lighting was supplied at an intensity of 100 mEm22

s21 by both incandescent bulbs and fluorescence tubes.

Generating Transgenic Plants

The generation of the HCT-RNAi and the Sm-F5H expression constructs

has been described previously (Weng et al., 2008, 2010). Agrobacterium

tumefaciens–mediated transformation of Arabidopsis thaliana was per-

formed using a floral dip method (Clough and Bent, 1998). The HCT-RNAi

construct was transformed into either wild-type Columbia or tt4-2 plants,

and the transgenic plants were selected by spraying with 1:1000 dilution

of Finale Concentrate (glufosinate ammonium; Farnam Companies). The

Sm-F5H expression construct was transformed into tt4-2 HCT-RNAi

plants, and the transgenic plants were selected on Murashige and Skoog

medium containing 50 mg mL21 kanamycin.

Genotyping

Tissue samples were ground in a 1.5 mL eppendorf tube, and 0.5mL

extraction buffer (0.2 M Tris-HCl, pH 9.0, 0.4 M LiCl, 25 mM EDTA, and

1%SDS) was added. After centrifugation at 16,000g for 5 min, 0.35mL of

supernatant was mixed with 0.35 mL isopropanol in a new tube. The tube

was centrifuged at 16,000g for 10 min, the supernatant was discarded,

and after the pellet was dry, 0.4mLTE buffer (10mMTris-HCl, pH 8.0, and

1 mM EDTA) was added to redissolve the DNA.

Figure 10. Expression of Selaginella F5H Rescues the Growth Pheno-

type of HCT-RNAi Plants Independent of Flavonoid Accumulation.

Shown are representative tt4-2 HCT-RNAi plants either containing

(+SmF5H) or lacking (�SmF5H) a transgene expressing the F5H enzyme

derived from the lycophyte S. moellendorffii. The tt4-2 mutant is shown

for comparison.

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

Flavonoid-Independent Growth Inhibition 1629

A cleaved amplified polymorphic sequence marker (Konieczny and

Ausubel, 1993) that exploits the EcoRV polymorphism caused by the ref8

mutation was used for ref8 genotyping (Franke et al., 2002b). A derived

cleaved amplified polymorphic sequencemarker (Michaels and Amasino,

1998; Neff et al., 1998) was used for tt4-2 genotyping. The forward primer

59-AGCTAACAAGTATTTACTATTCTCA-39 and reverse primer 59-TTA-

GCGATACGGAGGACA-39 were used to amplify a 372-bp fragment from

the CHS locus. The tt4-2 allele can be distinguished from the wild-type

TT4 allele from the DdeI digestion pattern of the amplicon. The forward

primer 59-GACCTAACAGAACTCGCCGTAAAGA-39, which anneals to the

35S promoter in the HCT-RNAi construct, was used with a HCT gene-

specific reverse primer 59-TAAGGGTAGGAGCAAAATCACCAAA-39 to

detect the HCT-RNAi transgene from the transgenic plants.

HPLC Analysis of Soluble Phenylpropanoid Metabolites

Plant extracts were prepared from fresh tissue with 50% (v/v) methanol

(1 mL per 100 mg tissue). Samples were incubated at 658C for 30 min and

centrifuged at 16,000g for 5 min. Ten microliters of the supernatant were

injected onto a Shim-pack XR-ODS column (3.0 3 75 mm, 2.2 mm)

(Shimadzu) and analyzed using a gradient from 5% acetonitrile in 0.1%

formic acid to 25% acetonitrile in 0.1% formic acid at a flow rate of

1 mL min21. Sinapic acid and kaempferol were used as standard for

quantification of sinapoylmalate and kaempferol-derived flavonoids, re-

spectively.

Quantitative RT-PCR

RNAwas isolated fromplant tissue using theRNeasyplantmini kit (Qiagen).

TURBODNase (AppliedBiosystems) treatmentwas performed to eliminate

possible genomic DNA contamination. cDNA synthesis was performed

with a high-capacity cDNA reverse transcription kit (Applied Biosystems)

following the protocol provided by the manufacturer. SYBR green–based

quantitative PCR was performed using the relative quantification method.

The forward primer 59-GAATTCCATACGAGGGTTTGTCTT-39 and reverse

primer 59-GGGCAATGGCAACGGATA-39 were used to detect the HCT

mRNA. We used the At1g13320 gene, which encodes a regulatory subunit

of a Ser/Thr protein phosphatase 2A, as a reference gene using the

previously published primers since its expression has been shown to be

stable over a wide range of conditions (Czechowski et al., 2005).

Plant Height Measurement

To determine whether there were height differences between the wild-

type and any of the mutant and transgenic lines, the length from the base

of rosettes to the tip of primary inflorescence stems was measured at a

mature stage when inflorescence stems had ceased elongation.

Lignin Analysis

Floral stems from 8-week-old Arabidopsis plants were harvested (with

siliques removed) and ground to a powder in liquid nitrogen. Cell wall

preparations from the above material were performed by an initial

extraction with 0.1 M sodium phosphate buffer, pH 7.2, for 30 min at

378C, three subsequent extractions with 70% ethanol at 808C, and a final

acetone extraction. Fifteen milligrams of the dried cell wall material were

used for DFRC analysis, which was performed following the published

protocol (Lu and Ralph, 1998). Briefly, cell wall material was dissolved in

acetyl bromide/acetic acid solution (20:80, v/v) containing 4,49-ethyl-

idenebisphenol as an internal standard. After evaporating the solvent with

N2 gas, dioxane/acetic acid/water (5:4:1, v/v/v) and zinc dust were added

to cleave the solublized lignin. The reaction products were purified with a

C-18 SPE column (SUPELCO) and acetylated with pyridine/acetic anhy-

dride solution (2:3, v/v). The resulting lignin monomer derivatives were

analyzed by gas chromatography/flame ionization detection. The re-

sponse factors used for quantification are 1.26, 1.30, and 1.44 for H, G,

and S monolignol derivatives, respectively.

HCT Activity Assay

Tissue for each assay was prepared by removing cauline leaves and

siliques from a single mature inflorescence stem in the case of wild-type

or tt4-2mutants. In the case of HCT-RNAi or tt4-2 HCT-RNAi plants, two

or three individual inflorescence stems were pooled for each tissue

sample to compensate for their small size. Tissue was flash frozen in

liquid nitrogen, ground with a mortar and pestle, and suspended in 400

mM Tris, pH 7.8, and 2 mM ascorbic acid with an equal amount of wet

polyvinyl polypyrrolidone. Samples were stirred 5 min on ice, and partic-

ulates were removed by filtering through Miracloth (Calbiochem) and

centrifugation. The resulting extract was desalted using a PD-10 column

(GE Healthcare) according to the manufacturer’s instructions. Plant

extract was preincubated with 200 mM p-coumarate, 220 mM CoA, 5

mM ATP, 5 mM MgCl2, and recombinant 4CL to form p-coumaroyl CoA.

Recombinant 4CL enzyme was obtained by cloning the Arabidopsis 4CL

open reading frame (amplified using primers 59-GGAGAGACATAT-

GGCGCCACAAGAACAA-39 and 59-GATACTCGAGCAATCCATTTGCT-

AGTTTTGC-39) into the NdeI and XhoI sites of the pET30a(+) (Novagen)

bacterial expression vector and transforming this construct into BL21

DE3 cells. The log phase cultures were induced with 0.8 mM isopropyl

b-D-thiogalactoside and grown overnight at 188C, followed by lysing in

400 mM Tris, pH 7.8, and 1 mM PMSF by freeze-thaw and desalting the

resulting lysate using a PD-10 column (GE Healthcare). Finally, HCT

activity was determined by adding shikimate to a final concentration of

10 mM and monitoring p-coumaroyl shikimate formation using HPLC.

Accession Numbers

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

Initiative or GenBank/EMBL databases under the following accession

numbers: C39H, At2g40890; HCT, At5g48930; CHS, At5g13930; and

Sm-F5H, EU032589.

ACKNOWLEDGMENTS

We thank Angus Murphy for providing the tt4-2 seeds and Junko Maeda

for technical assistance. This research was supported by the Office of

Science (Biological and Environmental Research), U.S. Department of

Energy (Grant DE-FG02-06ER64301), and the National Science Foun-

dation (Grant IOB-0450289). N.D.B. is funded by a fellowship from the

Life Sciences Research Foundation.

Received January 19, 2010; revised April 20, 2010; accepted May 10,

2010; published May 28, 2010.

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1632 The Plant Cell

DOI 10.1105/tpc.110.074161; originally published online May 28, 2010; 2010;22;1620-1632Plant Cell

Xu Li, Nicholas D. Bonawitz, Jing-Ke Weng and Clint ChappleIndependent of Flavonoids

IsArabidopsis thalianaThe Growth Reduction Associated with Repressed Lignin Biosynthesis in

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