Downregulation of Cinnamoyl-Coenzyme A Reductase
in Poplar: Multiple-Level Phenotyping Reveals Effects on
Cell Wall Polymer Metabolism and Structure
Jean-Charles Leple ´,a,b,c,1Rebecca Dauwe,a,b,1Kris Morreel,a,b,1Ve ´ronique Storme,a,bCatherine Lapierre,d
Brigitte Pollet,dAnnette Naumann,eKyu-Young Kang,f,2Hoon Kim,gKatia Ruel,hAndre ´e Lefe `bvre,h
Jean-Paul Joseleau,hJacqueline Grima-Pettenati,iRiet De Rycke,a,bSara Andersson-Gunnera ˚s,j
Alexander Erban,kInes Fehrle,kMichel Petit-Conil,lJoachim Kopka,kAndrea Polle,eEric Messens,a,b
Bjo ¨rn Sundberg,jShawn D. Mansfield,fJohn Ralph,mGilles Pilate,cand Wout Boerjana,b,3
aDepartment of Plant Systems Biology, Flanders Institute for Biotechnology, 9052 Gent, Belgium
bDepartment of Molecular Genetics, Ghent University, 9052 Gent, Belgium
cUnite ´ Ame ´lioration Ge ´ne ´tique et Physiologie Forestie `res, Institut National de la Recherche Agronomique, 45166 Olivet cedex,
dUnite ´ de Chimie Biologique, Unite ´ Mixte de Recherche 206 AgroParisTech/Institut National de la Recherche Agronomique,
AgroParisTech Centre de Grignon, 78850 Thiverval-Grignon, France
eInstitut fur Forstbotanik, Universita ¨t Go ¨ttingen, 37077 Go ¨ttingen, Germany
fDepartment of Wood Science, University of British Columbia, Vancouver, BC, Canada V6T 1Z4
gU.S. Dairy Forage Research Center, Agricultural Research Service, Department of Horticulture, University of Wisconsin,
Madison, Wisconsin 53706
hCentre de Recherche sur les Macromole ´cules Ve ´ge ´tales, Unite ´ Propre de Recherche 5301, Centre National de la Recherche
Scientifique, 38041 Grenoble Cedex 09, France
iPo ˆle de Biotechnologies Ve ´ge ´tales, Unite ´ Mixte de Recherche/Unite ´ Propre de Service 5546, Centre National de la Recherche
Scientifique, 31326 Castanet Tolosan, France
jDepartment of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 90183 Umea ˚, Sweden
kMax-Planck Institute of Molecular Plant Physiology, 14476 Golm-Potsdam, Germany
lCentre Technique du Papier, 38044 Grenoble Cedex 9, France
mU.S. Dairy Forage Research Center, Agricultural Research Service, U.S. Department of Agriculture and Department of Biological
Systems Engineering, University of Wisconsin, Madison, Wisconsin 53706
Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of
CCR in transgenic poplar (Populus tremula 3 Populus alba) was associated with up to 50% reduced lignin content and an
orange-brown, often patchy, coloration of the outer xylem. Thioacidolysis, nuclear magnetic resonance (NMR), immunocy-
tochemistry of lignin epitopes, and oligolignol profiling indicated that lignin was relatively more reduced in syringyl than in
A synthetic lignin incorporating ferulic acid had a red-brown coloration, suggesting that the xylem coloration was due to the
metabolite profiling were used as comprehensive phenotyping tools to investigate how CCR downregulation impacted
metabolism and the biosynthesis of other cell wall polymers. Both methods suggested reduced biosynthesis and increased
breakdown or remodeling of noncellulosic cell wall polymers, which was further supported by Fourier transform infrared
spectroscopy and wet chemistry analysis. The reduced levels of lignin and hemicellulose were associated with an increased
improved pulping characteristics, but growth was affected in all transgenic lines tested.
Lignins are defined as complex, heterogeneous polymers of
2004, 2007a). They are present mainly in the walls of secondary-
thickened cells of vascular plants and represent ;20 to 30% of
1These authors contributed equally to this work.
2Current address: Department of Forest Resources, Dongguk Univer-
sity, Seoul, Korea.
3Address correspondence to email@example.com.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Wout Boerjan
WThe online version of this article contains Web-only data.
The Plant Cell, Vol. 19: 3669–3691, November 2007, www.plantcell.org ª 2007 American Society of Plant Biologists
the dry weight of wood. Lignins confer rigidity to the cell wall for
structural support and impermeability for transport of water and
nutrients over large distances. The intrinsic properties of the
lignin polymers have been essential for plants to adapt to a
terrestrial habitat, enabling them to grow upward, but are also
crucial in determining the value of plants as raw materials. For
example, lignins are a major concern for the pulp and paper
industry because they need to be extracted from the wood by
et al., 2007). Similarly, they are the main limiting factor in fodder
digestibility and in the conversion of plant biomass to ferment-
able sugars in the process to bioethanol (Chen and Dixon, 2007).
Over the past decade, considerable attention has been focused
on understanding the lignin biosynthetic pathway and on explor-
composition for industrial applications (Baucher et al., 2003;
Boudet et al., 2003).
Although the roles of most genes of the monolignol pathway in
determining lignin amount and composition have been eluci-
dated, our knowledge is still scarce on how monolignol biosyn-
thesis integrates into wider plant metabolism and how plant
metabolism responds to changes in the expression of individual
that enable unbiased transcriptome- and metabolome-wide
analyses, such interactions can now be elucidated. Indeed,
deep phenotyping of transgenic plants defective in monolignol
biosynthesis has revealed far-reaching consequences on gene
2004; Robinson et al., 2005; Abdulrazzak et al., 2006; Shi et al.,
2006; Dauwe etal.,2007). Knowledge of thesebroader effects at
the transcriptome and metabolome levels is essential to fully
comprehend the relationships between gene function and cell
wall properties, how these cell wall properties are elaborated,
and how they relate to the quality of raw material destined for
agroindustrial processes (http://www.epobio.net/).
Cinnamoyl-CoA reductase (CCR; EC 188.8.131.52) catalyzes the
conversion of feruloyl-CoA to coniferaldehyde and is considered
the first enzyme in the monolignol-specific branch of the phenyl-
propanoid pathway (Lacombe et al., 1997). Because downregu-
lation of the CCR gene in annual model plants significantly
reduced lignin content (Piquemal et al., 1998; Chabannes et al.,
2001a, 2001b; Jones et al., 2001; Pinc ¸on et al., 2001; Goujon
et al., 2003), downregulating CCR in a woody perennial was an
interesting potential avenue to improve wood quality for pulping.
Here, we investigated the consequences of altering CCR ex-
pression in transgenic poplar (Populus tremula 3 Populus alba)
at multiple levels.
Generation of Transgenic Poplars Downregulated for CCR
Previously, we cloned a full-length CCR cDNA from a xylem
cDNA library of poplar (Populus trichocarpa cv Trichobel; Leple ´
et al., 1998). BLAST alignments against the P. trichocarpa cv
Nisqually 1 genome sequence (Tuskan et al., 2006) indicated the
presence of a single gene model corresponding to this cDNA,
the poplar genome. TheCCR gene wecloned istheonlyone that
is strongly expressed in developing poplar xylem (Li et al., 2005).
The poplar CCR cDNA sequence was used to design sense
and antisense constructs under the control of the cauliflower
mosaic virus (CaMV) 35S promoter for downregulation of the
CCR expression. Following the introduction of four different
constructs into poplar (P. tremula 3 P. alba), 40 to 60 indepen-
dent transformants were regenerated for each construct. Ap-
proximately 5% of all transformants, either from sense or
antisense lines, was dwarfed. These plants could be maintained
for up to 7 months in tissue culture but died upon in vitro
propagation and acclimation steps. The remainder of the lines
lines that were downregulated for CCR, all transformants were
screened for the presence of an orange-brown coloration of the
xylem after 3 and 7 months of growth in the greenhouse. This
phenotype was previously observed in transgenic tobacco (Ni-
cotiana tabacum) severely depressed for CCR activity (Piquemal
thexylemcoloration wereidentified: two(FAS13andFAS18)and
three (FS3, FS30, and FS40)lines transformed withthe antisense
and the sense construct, respectively (Figure 1A). RT-PCR indi-
cated reduced steady state CCR transcript levels down to 3 to
4% of wild-type levels (see Supplemental Table 1 online). Four of
these transgenic lines (FS3, FS30, FAS13, and FAS18) were
selected in 1999 to be field-grown for up to 8 years (Figure 1D).
Variability of the Orange-Brown Phenotype
The coloration of the debarked stems of the selected green-
house-grown transformants ranged from orange to wine-red
(Figure 1A) and faded soon after peeling the bark. The color was
not always uniformly distributed along the stem. Sometimes, the
pattern of coloration was patchy (Figure 1B). RT-PCR revealed
that CCR was more downregulated in the colored than in the
white zones of such stems (see Supplemental Table 1 online).
For the trees grown in the field trial, the xylem coloration was
consistently absent from the upper side of the branches (tension
wood zone), whereas it was present on the opposite wood side
(Figure 1C). Furthermore, the coloration was generally more
pronounced in the basal part of the branches and the stem,
whereas it became mottled and ultimately disappeared toward
the apical end.
Because the intensity of the xylem coloration often varied
among lines and among ramets of a given line and because the
molecular phenotype correlated with the color intensity (see
below), particular lines were preferred in one experiment and
other lines in other experiments, depending on the xylem color
intensity these trees presented at the harvest time for the
different experiments. For particular experiments, red and white
patches of the same stem were compared for phenotypic con-
sequences of CCR downregulation.
Histochemical and Autofluorescence Changes Associated
with CCR Downregulation
Patchy stems of 6-month-old greenhouse-grown plants and
3670The Plant Cell
and analyzed for overall morphology, altered lignification by
Wiesner (phloroglucinol-HCl) and Ma ¨ule staining, altered cellu-
lose content by Astra Blue staining, and for autofluorescence
see Supplemental Figure 1 online). Phloroglucinol-HCl and
Ma ¨ule staining are considered to stain specifically cinnamalde-
hyde end groups (Adler et al., 1948) and syringyl (S) units in
lignin (Lewis and Yamamoto, 1990), respectively. Under long-
wavelength UV light, at elevated pH (pH 10.3), ferulate esters
fluoresce intensely green (Harris and Hartley, 1976).
In the CCR-downregulated transformants, differences in his-
tochemical staining and autofluorescence were associated with
the colored areas, whereas the noncolored areas of the same
sections were similar to the wild type. Specifically in the colored
zones, vessels appeared irregular, and Wiesner and Ma ¨ule (data
not shown) staining were weaker, whereas staining with Astra
Blue was more intense (blue) in the colored areas (Figure 2B).
Blue light excitation of stem sections revealed intense autofluo-
rescence in the colored areas of CCR-downregulated lines
(Figure 2A). This blue-excited autofluorescence was particularly
intense in vessel cell walls and in the S1 layer of fibers and/or the
middle lamella. Autofluorescence induced by long-wavelength
UV excitation was intensely green in the colored xylem areas
of the transformants, with the highest intensity in the vessels
(see Supplemental Figure 1C online). Upon alkali treatment (5 N
NaOH, 2 min), the green autofluorescence disappeared and
and noncolored xylem (see Supplemental Figures 1A, 1B and 1D
Together, these data suggest that CCR downregulation re-
duces the level of hydroxycinnamaldehydes and S units in lignin,
increases the level of ferulate esters in lignin, and increases
cellulose content or its accessibility by Astra Blue. Furthermore,
the blue light autofluorescence in colored zones suggests the
presence of metabolites or cell wall structures that are unde-
tectable in noncolored areas.
Cell Wall Ultrastructural Morphology
Transmission electron microscopy (TEM) of cell walls stained
with uranyl acetate depicts the macromolecular arrangement of
the cell walls. The pattern of differential staining underscores the
subdivision of the secondarily thickened walls inthree sublayers,
S1, S2, and S3. In poplar fibers, the S3 sublayer is generally not
distinguishable. Figure 3 shows that in the colored xylem of the
S2) and, more rarely, of the vessels (sublayers S2 and S3) dis-
from absent to extensive, and in some extreme cases, the cell
wall appeared disorganized (Figures 3C to 3E). The disorgani-
zation wasthe strongestin the newlyformed inner sideof the cell
more obvious and more frequently observed in fibers than in
vessels. Notably, the S1 layer of both fibers and vessels did not
show any sign of altered ultrastructure. The sublayering pheno-
type was observed neither in the walls of neighboring colorless
areas of the transformants nor in those of wild-type cells.
Patterns of Lignification in CCR-Downregulated Plants as
Revealed by Immunogold Labeling
To examine the effects of CCR downregulation on lignin struc-
ture, xylem sections were immunolabeled with antibodies
directed toward lignin epitopes. Dicotyledonous lignins are pri-
marily built by the combinatorial coupling of coniferyl and sinapyl
alcohol monomers with the growing polymer. This oxidative
coupling gives rise to structural units with different interunit
linkages, some of which can be recognized by specific anti-
bodies. The antibodies used for topochemical visualization of
lignin had been previously made against synthetic lignin gener-
ated by polymerizing coniferyl alcohol (Gzl antibody) or sinapyl
alcohol (S antibody) and their specificity assessed by affinity
tests (Joseleau and Ruel, 1997; Joseleau et al., 2004a, 200b).
alcohol are enriched in condensed units involving (C–C) linkages
Figure 1. Phenotype of CCR-Downregulated Plants.
(A) Basal part of debarked stems of 4-month-old wild-type and CCR-
downregulated poplars (FAS13 and FS3).
(B) Occasional orange-brown coloration in patches along the stem.
(C) Cross section through a branch of a 7-year-old field-grown CCR-
downregulated poplar transformant (FAS13). The orange-brown colora-
tion is absent in tension wood. TW, tension wood; OW, opposite wood.
(D) Field trial of CCR-downregulated and wild-type poplars.
Downregulation of CCR in Poplar 3671
and syringyl (S) polymers preferentially make noncondensed
units connected by (b–O–4) linkages, immunolabeling of the cell
walls with these antibodies provides insight into the local lignin
structure within individual cell walls. In both wild-type and
transgenic lines, S labeling was weaker in vessels than in fibers,
(Baucher et al., 1998) (Figures 3F and 3G). In fibers of wild-type
plants, labeling with the S antibody revealed more abundant S
epitopes in the inner part of the S2 sublayer. In the fibers of the
colored areas of the FS3 line, S labeling was homogeneously
distributed over the entire S2 sublayer but more weakly than that
in the inner part of the S2 sublayer in wild-type fibers. The
distribution pattern of the G subunits obtained with the Gzl
antibody was similar between FS3 and the wild type (data not
shown). In summary, CCR downregulation seemingly reduced
the S epitopes in the S2 secondary wall sublayer of fibers and
had no or limited effects on the G epitopes.
Altered Lignin Content in CCR-Downregulated Poplars
To analyze whether CCR downregulation reduces lignin content,
as suggested by phloroglucinol-HCl and Ma ¨ule staining and cell
wall structure analysis, branches were collected from 2-year-old
field-grown poplars. Xylem fractions were scraped from the
young developing xylem of the colored areas of the transgenic
poplars and from the corresponding zones of wild-type plants.
Acid-insoluble Klason lignin content in xylem samples from the
lines FS3, FAS13, and FAS18 was significantly (probability of the
8%, respectively, but not from FS30 (see Supplemental Table 2A
Figure 2. Histochemical Changes Associated with the Orange-Brown Xylem of CCR-Downregulated Poplar.
(A) Blue-excited autofluorescence of a stem section of a 6-month-old greenhouse-grown CCR-downregulated poplar (FAS13) with patchy orange-
brown xylem coloration. Blue-excited autofluorescence (450 to 490 nm) shown for details ([a] and [c]) of the cross section (b). The autofluorescence
was increased preferentially in the vessels and the middle lamella and/or S1 layers of fibers in the orange-brown xylem zone. The whitish xylem areas of
the transformants had no or weak blue-excited autofluorescence, reminiscent of the wild type (data not shown). Exposure time was 1 s.
(B) Astra blue and phloroglucinol staining. The cross section from (A) (panel [b]) was stained with phloroglucinol (b) or with both phloroglucinol and Astra
Blue ([a] and [c]). (d) and (e) are cross sections through a wild-type branch (d) and an orange-brown zone in a FAS13 branch (e) from field-grown
poplars, stained with phloroglucinol. Astra Blue staining was more intense, whereas phloroglucinol staining was less intense in the orange-brown zones
of the transformants compared with the wild type.
3672 The Plant Cell
xylem phenotype, whereas only a light patchy phenotype was
observed in the samples of FS30. Additionally, acid-insoluble
Klason lignin contents were measured from noncolored and
colored stem xylem areas of 1-year-old greenhouse-grown FS3
was associated with the coloration (see Supplemental Tables 2
and 3 online).
Altered Lignin Structure in CCR-Downregulated Poplars
The lignins of the FS3 and wild-type branch xylem samples and
of the FS3 and FS40 stem xylem samples, described above,
were analyzed structurally by thioacidolysis (Lapierre et al.,
1999). Thioacidolysis selectively cleaves the b–O–4-ether bonds
in the lignin polymer. The b–O–4-linked G and S lignin subunits
give rise to specific monomeric degradation products that are
quantified and reflect the proportion of G and S units linked by
b–O–4-ether bonds. The lignin fraction that is released by
thioacidolysis is referred to as the noncondensed fraction, in
contrast with the units that involvecondensed bonds and are not
released as monomers by thioacidolysis. The total yield (SþG)
and relative proportion (S/G) of the released thioacidolysis mono-
mers are presented in Supplemental Table 2A online. Lignins
from the colored area of the FS3 branches systematically
released fewer thioacidolysis S and G monomers per gram
of Klason lignin than wild-type lignins (P ¼ 0.046), which is
indicative of a higher frequency of condensed bonds in CCR-
downregulated lines. The S/G ratio based on thioacidolysis data
did not differ significantly from that of the wild type. In addition to
the main S and G monomers, lignins from the colored zoneof the
FS3 branches released 10- to 20-fold higher amounts of a new
thioacidolysis product than the control lignins (P < 0.001), in
which this compound was recovered only in minor amounts (see
by electron-impact mass spectroscopy (MS) indicated that this
Figure 3. TEM of Xylem Sections of Wild-Type and CCR-Downregulated
Poplars and Immunocytochemical Localization of Lignin Epitopes.
(A) and (B) The wild type.
(C) and (D) FAS18.
(A) to (E) The sections are stained with uranyl acetate. Fibers and vessels
of the wild type have a smooth appearance, and the layers S1 and S2 of
fibers and S1, S2, and S3 of vessels are delineated ([A] and [B]). In fibers,
S2 is often divided in a dark outer layer and a lighter inner layer (B). In the
orange-brown area of CCR transformants, concentric sublayers are
visible in the S2 layer of fibers ([C] to [E]) and in the S2 and S3 layers of
vessels (D). Similar results were seen in sections of lines FS3 and FS30.
The ultrastructure of the cell walls was most severely affected in the
orange-brown zones of line FAS13, where the stratification was often
accompanied by a loss of compactness (E).
(F) and (G) Immunolabeling of syringyl epitopes with S antibody,
performed on stem cross sections of wild-type (F) and FS3 (G) poplars,
grown under controlled conditions. S epitopes concentrated in the inner
part of S2 of fibers in the wild type, whereas a weaker and more
homogeneous distribution of S epitopes was observed in S2 of fibers
from orange-brown areas of the transformant. Note: differences in the
size of gold particles are due to uneven silver enhancement that modified
the diameter of the gold particles, but not their number.
F, fibers; V, vessels; R, ray cell. Bars ¼ 1 mm.
Downregulation of CCR in Poplar3673
new product consisted of an aromatic G ring with a two-carbon
side chain in which the a and b carbons are involved in a single
This compound was then designated as G-CHR-CHR2. The
corresponding S analog could also be observed but to a much
lower extent (data not shown). Thioacidolysis released also
increased amounts of ferulic acid from the FS3 colored branch
samples. In the patchy stems of 1-year-old greenhouse-grown
released by thioacidolysis from the colored than from the non-
colored areas, indicating that it was associated with the color-
ation. These changes in the release of thioacidolysis monomers
were also confirmed in extractive-free xylem and the corre-
sponding milled-wood extracted lignins (MWELs) of 1-year-old
greenhouse-grown FS3 compared with the wild type (see Sup-
plemental Table 2B online). In addition, analysis of the dimers
released by thioacidolysis from these extractive-free xylem and
MWEL fractions indicated a substantially lower yield of the
syringaresinol-derived dimer (see Supplemental Table 2B on-
line), as confirmed by nuclear magnetic resonance (NMR) anal-
twofold more p-hydroxybenzoate, vanillic acid, and ferulic acid
upon alkaline hydrolysis (means 6 SE based on technical repli-
cates were 40 6 2, 1.7 6 0.1, and 0.6 6 0.1 and 61 6 0.7, 4.8 6
respectively). Alkaline hydrolysis breaks mainly ester linkages.
The release of ferulate is in agreement with the green autofluo-
rescence of stem sections induced by long-wavelength UV exci-
tation, which has been described to be caused by ferulate esters
(see Supplemental Figure 1 online; Harris and Hartley, 1976).
Whereas the S/G ratio of thioacidolysis monomers reflects
only the etherified units that can release monomeric products,
NMR potentially measures the S/G of the entire lignin. The S/G
ratio of FAS13 and FS40 lignin, calculated from NMR data, was
significantly lower than that of the wild type (see Supplemental
Tables 2C and 3 and Supplemental Figure 2 online). These data
are supported by the interunit linkage distributions (see below).
The side chain region only peripherally reflects the changes in
the S/G distribution but is rich in detail regarding the types and
distribution of interunit bonding patterns present in the lignin
fraction. The control lignin heteronuclear single-quantum coher-
ence (HSQC) spectrum (Figure 4A) is typical of a guaiacyl/
syringyl lignin containing some residual polysaccharides (Ralph
et al., 1999). The lignin was rich in b-aryl ether units A, with
of spirodienone S units, typical for angiosperm lignins. Resinols
C arise usually from sinapyl alcohol dimerization and hence are
Finally, the cinnamyl alcohol end groups X1, like the resinols C,
arise from monomer-monomer coupling (often involving G units)
and are therefore relatively minor.
Figure 4. Side Chain Regions of HSQC Spectra from Enzyme Lignins
Illustrating Lignin Structural Changes.
(A) to (C) HSQC spectra from lignins isolated from the wild type (A) and
FAS13 (B). The contour colors in (A) and (B) correspond with the lignin
units presented in (C).
(D) Difference spectrum (FAS13 – wild type) showing, primarily, the
decreased resinol C (negative, blue) levels.
3674 The Plant Cell
The spectra of the lignins derived from CCR-deficient poplars
revealed differences (Figures 4B and 4D) that were assessed
from the volume integral data of the two-dimensional spectrum
(see Supplemental Table 2C online). The major difference was
that the resinol C content (b–b in Supplemental Table 2C online)
was considerably lower in the FAS13 and FS40 transgenic lines,
which is consistent with the lower amounts of syringaresinol
released by thioacidolysis and a logical consequence of the
reduced S levels documented from analysis of the aromatic
regions, but the magnitude of the reduction suggests that
relatively less dimerization (and more endwise coupling) oc-
curred in the CCR-downregulated lines. This observation is
further validated by the relatively high b-aryl ether levels in the
transgenic lines (see Supplemental Table 2C online).
Elucidating the Derivation of the G-CHR-CHR2
The G-CHR-CHR2thioacidolysis product could not be derived
from any of the normal monolignol coupling products in the lignin
polymer because the involvement of the b carbon in two thio-
ethers revealed that this b carbon was at the oxidation level of an
the lignin, as found by thioacidolysis, suggested that G-CHR-
CHR2was a specific coupling product of ferulic acid into the
polymer. To test whether ferulic acid could produce the precur-
sors from which the thioacidolysis marker derives during free-
radical polymerization, low levels (;5%) of [8-13C]-labeled ferulic
acid were introduced into a 50:50 coniferyl alcohol:sinapyl alco-
hol synthetic lignin (dehydrogenation polymer [DHP]) with per-
oxidase/H2O2to generate the required radicals. This DHP had
the same structure as a DHP without incorporation of ferulic acid
but had two small characteristic ferulic acid–derived compo-
nents as seen by their13C8–1H8correlations at 103.5/6.0 and
116/6.3 ppm in HSQC spectra of the acetylated DHP (see
not from the control DHP without ferulic acid (data not shown). In
addition, HSQC correlation peaks matching those in the DHP
were found at low levels in the NMR spectra of lignins isolated
from transgenic poplars but not in those of the control (see
Supplemental Figure 3 online). Therefore, one or both of these
structures in the lignin polymer might be the source of the
thioacidolysis G-CHR-CHR2 marker compound. The ferulic
acid–enriched DHP was orange-brown colored as the xylem of
CCR-suppressed transformants, suggesting that the coloration
is due to the presence of ferulic acid during lignification. A
complete description of the elucidation of the marker structure
and its derivation from ferulic acid–derived structures is reported
elsewhere (Ralph et al., 2007b).
HPLC Analyses of Soluble Phenolics
The increased amount of ferulic acid in the lignin of CCR-
downregulated poplars, as shown by thioacidolysis and NMR,
or esterified, as indicated by alkaline hydrolysis, pointed to an
increased flux through the phenylpropanoid pathway toward
ferulic acid. To study this flux change, the different cinnamic
acids and cinnamaldehydes present in young developing xylem,
downregulated poplars (FS3, FS40, and FAS13), were analyzed
by liquid chromatography–mass spectrometry (LC-MS) with
selectedionmonitoring (Morreel etal., 2004b). The concentrations
(mean 6 SE) of ferulic acid, sinapic acid, coniferaldehyde, and
sinapaldehyde were 75.5 6 16, 197 6 40, 28.3 6 4, and 90.8 6
14 pmole/mg dry weight in the wild-type poplars and 111 6 30,
384 6 140, 29.9 6 12, and 77.3 6 29 pmole/mg dry weight in the
CCR-downregulated lines, respectively. Because of the large
variation, the mean values did not significantly differ between the
transgenic lines and the wild type with a nested analysis of
variance (ANOVA) model. However, when the six ratios of the ion
current signal of these four intermediates were analyzed by the
same statistical model (Morreel et al., 2004b), all four cinnamic
acid:cinnamaldehyde ratios were approximately doubled in
CCR-downregulated lines (Table 1), indicating that the con-
centrations of ferulic and sinapic acids had increased relative
to those of coniferaldehyde and sinapaldehyde in the CCR-
Table 1. Mean Ratios (6 SE) of the Concentrations of Phenylpropanoid Pathway Intermediates in the Xylem of Control and
n ¼ 5
0.36 6 0.07
2.44 6 0.57
6.25 6 1.17
0.63 6 0.14
1.86 6 0.25
0.26 6 0.01
n ¼ 6
0.44 6 0.06
3.08 6 0.72
5.88 6 1.38
1.05 6 0.20
2.49 6 0.42
0.36 6 0.05
n ¼ 8
0.31 6 0.06
2.75 6 1.17
5.88 6 1.04
0.80 6 0.32
2.23 6 0.44
0.31 6 0.04
n ¼ 4
0.44 6 0.14
7.00 6 2.39
12.5 6 3.1
1.82 6 0.56
4.51 6 1.58
0.26 6 0.04
n ¼ 4
0.39 6 0.10
5.42 6 2.20
10.0 6 3.0
2.27 6 0.81
5.29 6 1.24
0.45 6 0.09
n ¼ 6
0.21 6 0.06
5.31 6 2.78
16.7 6 5.6
2.11 6 1.23
8.69 6 3.45
0.37 6 0.03
The mean ratios of the selected ion currents of the pseudomolecular ions, obtained by LC-MS atmospheric pressure chemical ionization in the
negative mode, for ferulic acid (FA), sinapic acid (SA), coniferaldehyde (Cal), and sinapaldehyde (Sal) are given for the control poplars (wild-type and
transgenic lines 35S 17B and 35S 21B) and CCR-downregulated poplars (lines FS3, FS40, and FAS13). Significantly different ratios in the CCR-
downregulated lines relative to the control lines, revealed by applying a nested ANOVA model (Morreel et al., 2004b), are indicated in bold. Data are
mean 6 SE from biological replicates. n, number of biological replicates.
Downregulation of CCR in Poplar 3675
Amore comprehensive picture of the flux changes through the
phenylpropanoid and monolignol pathways was obtained by
reversed phase HPLC-UV/Vis analysis of the methanol-soluble
phenolics present in the same samples. When the heights of the
91 chromatogram peaks that could be quantified were summed
and expressed relative to the dry weight, a 2.8-fold higher value
was obtained in the transgenic lines, indicating prominent shifts
in aromatic metabolism (data not shown). Of the 91 peaks, a
nested or a one-way ANOVA model indicated that 19 differed in
in the CCR-downregulated lines. Of the four elevated peaks, two
were major peaks in CCR-downregulated poplars and had been
and O4-b-D-glucopyranosyl vanillic acid (GVA) in caffeoyl-CoA
O-methyltransferase (CCoAOMT)–deficient poplar (Meyermans
et al., 2000) (see Supplemental Table 4 online). GVA was in-
creased by 24-fold in the CCR-downregulated lines, whereas
GSA was foundexclusively in the transgenic lines and, taking the
detection limit into account, accumulated to at least 1000-fold
higher levels. The abundance of the other two was too low for
structural identification. Of the15compounds thatwerereduced
in abundance, eight had been previously identified as oligo-
purified but all had UV/Vis spectra indicative of oligolignols.
These data indicate that CCR deficiency efficiently reduces the
synthesis of low molecular mass monolignol-coupling products
and that the flux through the phenylpropanoid pathway is shifted
toward a small number of glucosylated phenolics.
The phenotypes of the transgenic lines described above might
merely result from an altered flux through monolignol biosynthe-
sis. Alternatively, at least part of these phenotypes might be
mediated by transcriptional changes in response to CCR defi-
ciency. To reveal such phenotypic effects at the transcript level,
gene expression was compared in the young developing xylem
from the stems of 6-month-old greenhouse-grown wild-type
poplars and transgenic lines FS3 and FS40. Two pools of xylem
material were generated for each line (see Methods) and the
transcriptome analyzed through an all-pairwise comparison de-
sign in duplicate (see Supplemental Figure 4 online) (Glonek and
Solomon, 2004) with a 25K Populus (POP2) microarray (Sterky
et al., 2004; www.populus.db.umu.se).
In total, 52 distinct genes were identified whose transcript
(see Supplemental Table 5 online). In general, the effect on the
transcript levels was stronger in FS3 than in FS40. Strikingly, all
49 genes for which differential transcript levels were revealed in
FS40 displayed similar differential transcript levels in FS3. For
32 genes, the transcript levels were increased, whereas for
16 genes, they were decreased in the xylem of both CCR-
downregulated lines. For only one gene, the expression was
affected in an opposite way in both lines. In FS3, a decreased
expression level was found for three additional genes.
Based on the annotations and the functional classification
according to the public poplar EST database (POPULUSDB;
www.populus.db.umu.se) (Sterky et al., 2004) and additional
manual curation, 38 of the 49 common differentially expressed
genes (78%) could be grouped into 10 functional categories. For
the 11 remaining genes, the function of the most similar Arabi-
dopsis thaliana protein was either unknown (two genes) or no
significant similarity with an Arabidopsis protein was found
(BLAST score < 100) (nine genes). The complete list of genes
with their identification numbers on the microarray, annotation
and functional classification, and relative transcript level is given
in Supplemental Table 5 online.
Within secondary metabolism, the microarray data confirmed
the downregulated expression level of the CCR gene. Further-
more, transcript levels of two distinct genes encoding Phe am-
monia lyase (PAL), the enzyme channeling carbon from primary
into secondary metabolism via the deamination of Phe, were
elevated in the CCR-downregulated poplars. The ammonium
liberated by the PAL reaction is reassimilated by Gln synthetase
(Croteau et al., 2000). Accordingly, the expression of a Gln
synthetase was increased in the transformants.
In addition, the transcript levels of three genes encoding
enzymes involved in the metabolism of cell wall matrix polysac-
charides (i.e., a myo-inositol oxygenase-like gene [MIOX] and a
membrane-bound UDP-D-xylose 4-epimerase [MUR4]) were
reduced in the CCR-downregulated lines, whereas that of a
(1-4)-b-mannan endohydrolase was elevated (see Supplemental
Figure 5 online). The transcript levels of two genes encoding
glycosyltransferases, corresponding to the Arabidopsis glyco-
syltransferase PARVUS (At1g19300) (77 and 80% identity for the
two poplar genes), with predicted involvement in pectin biosyn-
thesis (Lao et al., 2003), were decreased in CCR-downregulated
lines. These data pointed to a reduced biosynthesis and an
Related to cell wall organization, the transcript levels of a
putative arabinogalactan protein (AGP) and a lipid transfer pro-
tein were decreased. Furthermore, a Ser carboxypeptidase-like
gene, likely involved in brassinosteroid (BR) signaling and a Leu-
rich repeat receptor-like protein kinase that is similar to the
Arabidopsis SERK2 displayed elevated transcript levels in the
The transcript levels of eight genes whose expression levels
are typically elevated during stress situations were increased in
the CCR-downregulated lines: four genes encoding metallo-
thionein proteins, two genes encoding glutathione S-transferase
(GST) (one phi and one tau class GST), a gene encoding an
NADP-dependent oxidoreductase similar to z-crystallin (ZCr),
and a gene encoding a U-box domain protein, similar to the
fungal elicitor-induced protein CMPG1 of Petroselinum crispum.
Overall, the transcriptome analysis revealed differences in the
metabolism of cell wall constituents (lignin, carbohydrates, and
proteins) and of stress resistance.
Complementary to transcript profiling, metabolome analysis
can provide profound insight into the effects of gene misregu-
lation on plant metabolism and the molecular mechanisms that
provoke a phenotype. Metabolite profiles of young developing
xylem of 3-month-old greenhouse-grown wild-type and CCR-
downregulated poplars (lines FS3, FAS13, and FS40) were
3676 The Plant Cell
obtained by gas chromatography–mass spectrometry (GC-MS)
and subsequently analyzed by principal component analysis and
t tests. Of the 802 analyzed compounds, 159 corresponded to
known metabolites, of which 20 accumulated differentially in the
CCR-downregulated lines compared withthe wildtype (Table 2).
The most prominent change was found for maleate levels,
which were 2.4-fold higher in the FS40 line and 3.9-fold in lines
FAS13 and FS3 than in the wild type. The concentrations of the
2- to 2.5-fold and 1.6- to 3.0-fold, respectively. In addition, two
other Krebs cycle intermediates (i.e., succinate and cis-aconitate)
were moderately, but still significantly, more abundant (1.7- to
2-fold and 1.2- to 1.8-fold) because of CCR downregulation.
The largest fraction of differentially accumulating metabolites
consisted of carbohydrates. In the transgenic poplars, glucose,
mannose, galactose, and myo-inositol and the oligosaccharides
raffinose and melezitose were all reduced to concentrations
0.5- to 0.8-fold lower than those found in the wild type, reflecting
changes in central carbohydrate metabolism. In cell wall poly-
in the transgenic lines were 0.6- to 0.9-fold of those detected in
the wild-type poplars, whereas concentrations of xylose and
rhamnose were increased 1.8- to 2.6-fold and 1.2- to 1.4-fold,
respectively, in the transgenic lines (see Supplemental Figure 5
online). Again, these data point to reduced synthesis and in-
creased breakdown or remodeling of hemicelluloses and/or
pectins, assuming that partial degradation of the nucleotide
sugars is not at play.
In ascorbate metabolism, decreased levels of myo-inositol,
GlcA, and L-gulono-1,4-lactone, a slightly lower concentration of
dehydroascorbate dimer (0.8- to 1.0-fold) and 1.9- to 2.2-fold
higher levels of glycerate, a possible breakdown product of
ascorbate, were detected in the CCR-downregulated lines com-
Overall, the metabolome analysis revealed a substantial differ-
ence in maleate metabolism, modest shifts in carbohydrate
metabolism, and a potential effect on ascorbate metabolism
(Lorence et al., 2004).
Metabolite Correlation Networks
To reveal additional spots of differential regulation in the metab-
olism of the transformants, networks of strong correlations (r >
0.80) between metabolite levels were visualized for the wild type
and each of the transgenic lines FS3, FS40, and FAS13 (see
Supplemental Figure 6 online). In these networks, vertices and
edges represent metabolites and strong correlations, respec-
tively. Hubs are those metabolites (vertices) that are strongly
Table 2. Differentially Accumulating Metabolites in Young Developing Xylem of CCR-Downregulated Poplar, as Identified by GC-MS
n ¼ 8
n ¼ 9
n ¼ 14
P IDPC7PC8 PC9 x-Fold x-Foldx-Fold
Ascorbic Acid Metabolism
Hemicellulose and Pectin Metabolism
0.42 <0.050 0.3
ID, mass spectral identifier (see Methods); PCA, principal component analysis; PC, principal component; P, significance based on t tests; n, number of
biological replicates (wild type: 19 biological replicates). Projection of PC7 and PC8 into a two-dimensional plot allowed to partially separate the wild-
type poplars from the transgenic poplars. A partial distinction between wild-type and transgenic poplars was also obtained by PC9. The loading
factors of the compounds that contributed prominently (see Methods) to PC7, PC8, and/or PC9 are shown.
Downregulation of CCR in Poplar 3677
correlated to many other metabolites and, therefore, whose
synthesis is strongly coregulated with the remainder of metab-
olism. Four compounds (i.e., mannose 6-phosphate [ID 231001],
S-methyl-L-Cys [ID 144002], and two unknown [ID 313003 and
of the transgenic lines than in those of the wild type, indicating
that their synthesis was highly coregulated with many other
(primary) metabolites because of the CCR downregulation (see
Supplemental Figure 6 and Supplemental Table 6 online). This
was most prominent for mannose 6-phosphate that was not
found at all in the correlation network of the wild type.
Fourier Transform Infrared Spectroscopy
Whole cell wall chemical changes induced by CCR down-
regulation were analyzed in stem sections of 6-month-old
greenhouse-grown plants by Fourier transform infrared (FTIR)
spectroscopy. The differences in absorbance of 15 absorption
FTIR spectra (peaks 1 to 15; see Supplemental Figure 7 online)
were registered in colored and noncolored areas of xylem
sections of transformants (FS3, FS40, and FAS13) and in the
wild type. Analyses were done with the ANOVA model 3 that
takes the coloration intensity into account to distinguish differ-
ences between the poplar lines. In general, significant models
were obtained for the intensities of 14 absorption bands (see
Supplemental Table 8 online). In addition to data obtained from
the literature, FTIR spectra of model compounds were recorded
to aid the interpretation of FTIR data from the stem sections.
Cell wall carbohydrates had been previously associated with
absorption bands at 1778 to 1691 cm?1(1), 1397 to 1349 cm?1
(7), 1188 to 1145 cm?1(10), and 1096 to 999 (12) (see Supple-
mental Table 8 online). More specifically, the absorption band (1)
has been related to ester groups of carbohydrate origin, mainly
xylans and pectins in poplar (see Supplemental Table 8 online).
The intensity of all four absorption bands decreased with in-
creasing coloration intensity.
FTIR absorption bands solely ascribed to aromatics in the cell
wall were observed at 1691 to 1612 cm?1(2), 1612 to 1554 cm?1
(3), and 1527 to 1486 cm?1(4). Absorption band (2) is associated
with carbonyl groups conjugated with aromatic rings and ferulic
acid linked to lignin (see Supplemental Table 8 online). The apex
of thisabsorption band (1652cm?1)corresponded to apeak that
occurred in the spectrum recorded for free ferulic acid and not in
those of four distinct ferulic acid ester references (methyl, ethyl,
glucose, and galactose; data not shown). In the spectra of CCR-
deficient transformants, as well as in those of the ferulic acid–
containing DHP, the intensities of absorption bands (2) and (3)
were increased, in agreement with increased ferulic acid incor-
poration in the wall and in the DHP, and fully correlated with the
color intensity of the xylem. Absorption band (4), generally used
to quantify lignin (see Supplemental Table 8 online), decreased
with increasing coloration.
cell wall carbohydrate and, even more, lignin content decreased
withincreasingcoloration intensity.The orange-brown colorwas
associated completely with aromatically conjugated carbonyls.
Cell Wall Carbohydrate Analyses
Because both transcriptome and metabolome analyses indi-
cated changes in cell wall polysaccharide metabolism in the
transformants, the carbohydrate status of the cell walls in young
developing xylem of the stems of 6-month-old greenhouse-
was analyzed (Table 3; see Supplemental Table 7 online). In the
CCR-downregulated transformants, the reduced lignin content
was associated with a significant decrease in hemicellulose
content, whereas the cellulose content was increased. However,
Table 3. Cell Wall Carbohydrate Profile in Young Developing Xylem of 6-Month-Old Wild-Type and CCR-Downregulated Poplars
Acid-insoluble lignin (Klason)
20.65 6 0.22
17.70 6 0.21
2.95 6 2.95
48.22 6 0.69
30.72 6 0.69
0.14 6 0.01
0.51 6 0.01
2.93 6 0.11
0.44 6 0.01
25.08 6 0.68
1.63 6 0.04
0.10 6 0.00
0.30 6 0.00
1.78 6 0.04
0.27 6 0.02
15.40 6 0.25
16.75 6 0.16
14.54 6 0.15
2.21 6 0.05
56.55 6 0.49
23.19 6 0.70
0.12 6 0.01
0.37 6 0.01
1.69 6 0.11
0.29 6 0.01
19.66 6 0.61
1.06 6 0.05
0.10 6 0.00
0.32 6 0.02
1.60 6 0.05
0.28 6 0.02
18.62 6 0.71
16.64 6 0.18
14.24 6 0.18
2.40 6 0.04
57.07 6 0.81
24.10 6 0.44
0.13 6 0.01
0.41 6 0.01
1.91 6 0.13
0.31 6 0.01
20.22 6 0.39
1.11 6 0.04
0.10 6 0.00
0.38 6 0.02
1.72 6 0.09
0.30 6 0.00
18.32 6 0.58
The mean and the SE are given. Lignin, a-cellulose, hemicellulose, and monosaccharide contents are expressed as weight percentage of dry weight
cell wall material. Values that are significantly (P < 0.05) different from the wild type are indicated in bold.
3678The Plant Cell
because equal amounts of dry weight were analyzed and the cell
wall polymers were measured as percentages, less lignin and
hemicellulose will be mass-balanced by cellulose. Hydrolysis of
composition was similar in wild-type and CCR-downregulated
Chemical Pulping of the Different Poplar Lines
To determine how the decreased lignin and hemicellulose con-
tent would affect the kraft pulping characteristics of wood
derived from CCR-downregulated poplars, stems of five
4-year-old, field-grown trees were pulped for each of the four
transgenic lines (FS3, FS30, FAS13, and FAS18) and for the wild
type. Various reaction conditions were used to determine the
optimal alkali charge for delignification of the wood. Pulping
characteristics were based on residual Kappa number, pulp
viscosity, shives content (level of uncooked particles), and
screened pulp yield.
The Kappa number, a measure of the residual lignin content in
the pulp after cooking, was significantly affected by both the
As expected, a decrease in the Kappa number (improved
delignification) was observed with increasing active alkali in the
alkali charges, the Kappa number of the lines FS3 (PLSD¼ 0.012)
and FS30 (PLSD¼ 0.022) was significantly lower than that of the
wild type, whereas that of the FAS13 and FAS18 transgenic lines
did not significantly differ from the wild type for any active alkali
charge (see Supplemental Figure 8A online).
A high pulp viscosity, reflective of cellulose/hemicellulose
degree of polymerization, is generally associated with better
pulp and paper properties and is generally lower under higher
alkali conditions. Pulp viscosity was significantly lower in the
transgenic line FS3 than that in the wild type at all active alkali
conditions (P ¼ 0.001; PLSD¼ 0.004) (see Supplemental Figure
A significant effect of both the active alkali conditions (P <
0.001) and poplar line (P ¼ 0.004) was observed for the propor-
tion of uncooked particles that are complexes of assembled
fibers. High levels of uncooked particles indicate that the chem-
ical cooking conditions were not strong enough to totally dis-
solve the lignin and individualize the fibers. The percentage of
uncooked particles was lower under higher alkali conditions and
was significantly lower in the transgenic lines FS3 (PLSD¼ 0.017)
and FS30 (PLSD¼ 0.014) than in the wild type for all alkali
conditions (see Supplemental Figure 8C online).
Statistical analysis of the screened pulp yield indicated a
significant interaction (P ¼ 0.002) between the active alkali
conditions and the poplar line. For line FS3, the optimal pulp
yield was at 16% (or lower) active alkali, whereas the wild type
and the other transformants required 18% active alkali to reach
maximum pulp yields (see Supplemental Figure 8D online). One-
way ANOVA revealed a significantly lower yield for line FS3 at
18% active alkali (P ¼ 0.04; PLSD¼ 0.003).
In summary, the optimal cooking conditions for the wild type
were reached at 18% alkali charge. At 16%, the wild-type line
had a much higher Kappa number and lower pulp yield than at
18%alkali. Both transgenic lines FS3and FS30 were moreeasily
cooked at 16% active alkali than the wild type, as revealed by
their low percentage of uncooked particles. The two antisense
lines FAS13 and FAS18 were not significantly different from the
Growth of CCR-Downregulated Poplar in the Field
As described above, downregulation of CCR resulted in stunted
plants in 5% of the regenerants, but the lines that were selected
for further experiments (FS3, FS30, FS40, FAS13, and FAS18)
had an apparently normal development under greenhouse con-
ditions. To investigate whether CCR downregulation impacted
the growth characteristics of poplars during further development
and under natural circumstances, growth parameters (i.e., the
height, girth, girth increase, and volume and volume increase)
were determined annually for all trees in the field trial (10
replicates for each of the lines FS3, FS30, FAS13, FAS18, and
the wild type) (Figure 5). These growth parameters were sub-
jected to a three-way ANOVA involving year, line, and block
effects. A three-way interaction was noticed for girth increase,
to model 1 thatstill included asignificant interaction between the
to genotype 3 environment interaction. The PLSDvalues men-
tioned below are based on the two-way ANOVA model involving
the year and line as main factors.
The height of wild-type poplars increased linearly from 660 cm
on average in 2001 to 960 cm on average in 2003 (Figure 5A).
Across the complete duration of the field trial, the height was
significantly lower in both the FAS13 (PLSD¼ 0.02) and FS3
(PLSD< 0.001) lines, which were 4 and 20% smaller than wild-
type trees, respectively.
In wild-type poplars, the girth increased linearly from 150 to
270 mm between 2001 and 2003 (Figure 5B). As for height,
the values for FAS3 and FAS13 were significantly different
(PLSD< 0.001) from those of the wild type, with 16 and 10%
Girth increase was determined in 2002 and 2003 and was on
average 57 mm/year in the wild type (Figure 5C). All transgenic
lines exhibited a growth reduction between 14 and 23%. These
reductions corresponded to 45 mm/year (PLSD¼ 0.004), 48 mm/
year (PLSD¼ 0.04), 44 mm/year (PLSD¼ 0.002), and 48 mm/year
(PLSD ¼ 0.03) for the FAS13, FAS18, FS3, and FS30 lines,
respectively. Although the slope obtained for line FAS18 was
quite different compared withthe other lines, no significant clone
by year interaction for girth increase was found.
Volume changed exponentially across years and rose from
3550 cm3in 2001 up to 16,300 cm3in 2003 in wild-type plants
(Figure 5D). In the FAS13 and FS3 lines, a significantly lower vol-
ume was found (PLSD< 0.001 for both lines) with an average re-
duction of 23 and 43% compared with the wildtype, respectively.
In wild-type poplars, the volume increase augmented from
4300 cm3/year in 2002 to 8400 cm3/year in 2003 (Figure 5E).
Values for this trait were significantly lower in the transgenic lines
whose volume increase was on average 31, 51, and 29% lower,
Downregulation of CCR in Poplar3679
respectively, than in the wild type. In conclusion, all transgenic
lines evaluated had reduced growth when grown in the field.
Ferulic Acid Cross-Couples with Lignin
in the biosynthesis to the monolignols. Downregulation of CCR
expression in poplar resulted in a decreased Klason lignin
content and reduced abundance of a range of low molecular
weight oligolignols, including dimers and trimers of monolignols
(see Supplemental Table 4 online) (Morreel et al., 2004a, 2004b),
which is in accordance with a decreased monolignol supply in
these plants. The reduced lignin content is associated with an
orange-brown coloration of the xylem. Sometimes a patchy
coloration, correlated with variation in CCR downregulation, was
observed along the stem. Patchy phenotypes associated with
several plants, including petunia (Petunia hybrida), where it was
and poplar (Baucher et al., 1996; Tsai et al., 1998; Meyermans
environmental factors seem to contribute to the variability in
phenotype (Pilate et al., 2002; this study). These phenotypic
variations likely reflect the complexity of the various pathways
that trigger and revert gene silencing (Fagard and Vaucheret,
2000; Kanazawa et al., 2007).
It is important to note that the variability in gene silencing can
only be observed easily when downregulation of the target gene
causes a visible phenotype. When no visible phenotype can
be scored, such variability in gene silencing will contribute to
biological variation among samples and may obscure reproduc-
ibility of phenotypic data.
Transgenic plants deficient in cinnamyl alcohol dehydrogen-
ase(CAD)orcaffeic acid3-O-methyltransferase (COMT)arealso
typified by a reddish xylem coloration, whose origin has been
attributed to the hydroxycinnamaldehydes present during poly-
merization (Higuchi et al., 1994; Baucher et al., 1996; Tsai et al.,
1998; Kim et al., 2000). As CCR catalyzes the biosynthesis of
hydroxycinnamaldehydes, the orange-brown coloration in CCR-
downregulated plants is probably not caused by such aldehydes.
Indeed, phloroglucinol staining that reacts with cinnamaldehyde
end-groups in the lignin was strongly reduced in the colored zones,
and structural analysis of lignins of the CCR-downregulated
transformants indicated that the chemical determinant of the
coloration is probably derived from ferulic acid. Indeed, in-
troducing low levels (;5%) of ferulic acid into a 50:50 coniferyl
alcohol:sinapyl alcohol synthetic lignin (DHP) resulted in a similar
coloration of the DHP. It is also noteworthy that semi in vivo in-
corporation of ferulic acid into tobacco stem cross sections
gave a xylem coloration that was similar to the one observed on
Figure 5. Growth Characteristics of Field-Grown CCR-Downregulated Transgenic Poplars.
For wild-type and the CCR-downregulated lines FS3, FS30, FAS13, and FAS18, the mean values for height (A), girth (B), girth increase (C), volume (D),
and volume increase (E) from 2001 to 2003 are presented. Data are means of 10 biological replicates. Lines for which the given growth characteristic
was significantly differential compared with the wild type are marked by an asterisk.
3680 The Plant Cell
More direct proof for the incorporation of ferulic acid into lignin
was obtained from a range of experiments: thioacidolysis re-
leased the G-CHR-CHR2 structure systematically in higher
amounts from lignin of CCR-downregulated poplar than from
wild-type lignin (see Supplemental Table 2 online), and the same
thioacidolysis marker was released from the synthetic DHP
including ferulic acid but not from that without ferulic acid. In
addition, two-dimensional NMR spectra of the DHP showed
correlation peaks (see Supplemental Figure 3B online) that are
characteristic for incorporation of ferulic acid as bis-8–O–
4-ethers and simple 4–O–b ethers (Ralph et al., 2007b) and the
same correlation peaks were found at low levels in NMR spectra
from lignins of the CCR-downregulated transformants but not
in those from the wild-type lignin (see Supplemental Figure 3A
online). Thioacidolysis also revealed increased amounts of
simple 4–O–b-linked ferulic acid, and alkaline hydrolysis indi-
cated an increased amount of esterified ferulic acid, as previ-
ously reported in the lignin of CCR-downregulated tobacco
(Chabannes et al., 2001a). Further support came from FTIR,
absorbances at 1691 to 1612 cm?1and 1612 to 1554 cm?1in
FTIR spectra of the xylem of CCR-downregulated and wild-type
trees were positively correlated with each other and with the
coloration intensity, indicating an increased amount of aromat-
ically conjugated carbonyls, such as ferulic acids with free
carboxyl groups, in the lignin.
downregulated plants, as low levels of G-CHR-CHR2were also
released by thioacidolysis from wild-type poplar (see Supple-
mental Table 2 online), as well as tobacco and Arabidopsis, and
more substantial levels from grasses (Ralph et al., 2007a). By
contrast, a recent study of the Arabidopsis irx4 mutant, deficient
in CCR, did not find any evidence for the incorporation of ferulic
acid into lignin (Laskar et al., 2006). However, the thioacidolysis
and NMR data were not presented in sufficient detail to exclude
the incorporation of ferulic acid into lignin. Thus, ferulic acid
appears to be incorporated into lignins, particularly in these
CCR-deficient transgenic plants and should be considered as
an authentic lignin monomer. A more in-depth chemical study of
ferulic acid incorporation into lignin, the consequences on lignin
structure, and a comparison with the results of Laskar et al.
(2006) are presented in Ralph et al. (2007b).
Lignin Structure in CCR-Downregulated Poplar
The increased incorporation of ferulic acid was not the only
structural change of the lignins in CCR-downregulated poplar.
Indeed, several structural analyses illustrated a relatively stron-
ger effect of CCR downregulation on S units. Thioacidolysis
indicated that the lignin from the colored xylem was more
condensed and yielded fewer syringaresinol-derived dimers
than the wild type (see Supplemental Table 2 online). In agree-
ment with these data, a low S/G ratio and a low frequency of
by NMR (see Supplemental Table 2 online). The thioacidolysis-
associated S/G ratio was not statistically different, but, as
stated in the results, the latter ratio is based on the uncondensed
lignin fraction only, whereas the NMR-associated S/G ratio
potentially reflects the whole lignin. In addition, oligolignol pro-
filing demonstrated that the levels of all seven oligolignols
detected that involved S units were reduced, whereas the
proportions of only one of the five that involved solely G units
were reduced in abundance (see Supplemental Table 4 online).
These chemical analytical results were supported by the immu-
walls of the transgenic lines with the S antibody (Figure 3),
whereas no obvious variation in labeling intensity was observed
with the G antibody (data not shown). The higher frequency of
or syringaresinol structures, and the higher degree of lignin
acylation by p-hydroxybenzoic acid as revealed by alkaline
hydrolysis of the MWEL fraction all are reminiscent of early
developmental stage poplar lignin (Terashima et al., 1979). In
other words, CCR deficiency appears to induce a delay in the
lignification program as suggested for the Arabidopsis irx4
mutant by Laskar et al. (2006), which was also characterized
by a lower S/G value and a more condensed lignin.
Downregulation of CCR Results in the Accumulation of
Phenolic Acid Glucosides
The reduced flux into the monolignol-specific branch of the
phenylpropanoid pathway in the xylem of CCR-downregulated
poplars results in the strong accumulation of GVA and GSA, of
which the latter was not detected at all in wild-type poplars.
Interestingly, GVA and GSA also accumulated in CCoAOMT-
downregulated poplars, in addition to O3-b-D-glucopyranosyl-
caffeic acid (Meyermans et al., 2000; Morreel et al., 2004b). For
the CCoAOMT-downregulated poplars, caffeoyl-CoA, the sub-
strate of CCoAOMT, was hypothesized to be redirected to
caffeic acid by a thioesterase (Guo et al., 2001). Caffeic acid
would then be converted to vanillic and sinapic acid, after which
all three acids would be detoxified by glucosylation (Meyermans
caffeic acid does not accumulate, but feruloyl-CoA, the sub-
strate for CCR, might be similarly hydrolyzed to ferulic acid.
Indeed, as evidenced by thioacidolysis, NMR spectra, and
alkaline hydrolysis, lignin of CCR-downregulated poplars con-
tains increased levels of ferulic acid. Subsequently, ferulic acid
would be converted to vanillic acid and sinapic acid and further
detoxified by glucosylation to GVA and GSA (Meyermans et al.,
2000). The weak phloroglucinol staining of the xylem and the
increased ratios of either ferulic or sinapic acid to either coni-
feraldehyde or sinapaldehyde in CCR-downregulated trans-
formants suggest that the reduced activity of CCR results in
reduced levels of coniferaldehyde and 5-hydroxyconiferalde-
hyde. These aldehydes have an inhibitory effect on the COMT-
and ferulate-5-hydroxylase–catalyzed conversion of ferulic acid
to sinapic acid (Osakabe et al., 1999; Li et al., 2000). Their
reduced levels might mitigate this inhibitory effect in CCR-
downregulated transformants, facilitating the conversion of
ferulic acid to sinapic acid.
In CCR-downregulated tobacco, quinate conjugates of phe-
nylpropanoid intermediates accumulated in addition to glucose
conjugates (Dauwe et al., 2007). In tobacco and Arabidopsis, the
conversion of p-coumaroyl-CoA to caffeoyl-CoA is thought to
occur via quinate ester intermediates (Schoch et al., 2001;
Downregulation of CCR in Poplar 3681
Hoffmann et al., 2003), and the enzymes catalyzing this pathway
might detoxify accumulating phenylpropanoid intermediates in
in xylem of the wild type nor in that of CCR-downregulated
poplars, although the applied HPLC procedure readily allowed
their detection in tobacco xylem (Dauwe et al., 2007). Either the
quinate esters are present in concentrations below the detection
limit or the hexose esters rather than the quinate esters are
involved in the transesterification reactions in phenylpropanoid
biosynthesis in poplar xylem. p-coumaroyl hexose also has a
LC-MS/MS (data not shown).
Taken together, our data indicate that downregulation of CCR
results in a decreased flux of feruloyl-CoA to lignin and an
increased flux toward ferulic acid, which is detoxified by gluco-
sylation as GSA and GVA, or alternatively is exported to the cell
wall where it is cross-coupled with lignin. The increase in ferulic
acid levels might simply be the result of a reduced flux to
coniferaldehyde, causing a buildup of precursors and deriva-
tives, but possibly ferulic acid biosynthesis might also be in-
duced as a response to a defective cell wall.
Downregulation of CCR Induces PAL Expression
catalyzes the entry of Phe into phenylpropanoid metabolism,
were increased in the CCR-downregulated poplar lines. One
possible reason for the increased PALtranscript levels isthat the
decreased synthesis of monolignols may signal a need for in-
creased carbon flux into this pathway. A signal for increased
developmental lignification could be mediated by mechanistic
aspects of the cell wall, resulting from the decreased lignin
content, or by the reduced concentrations of pathway interme-
diates, such as cinnamaldehydes. Alternatively, the disorganiza-
tion of the cell wall might induce signaling pathways that are
typically induced by cell wall damage during wounding or path-
ogen attack and increase PAL expression. For example, the
cellulose synthase mutant cev1 mimics the physiological re-
2002). The closest Arabidopsis homolog of both induced poplar
infection and abiotic stress (Raes et al., 2003). Interestingly, in
elicitor-treated cell cultures, a strong increase of PAL activity is
associated with reinforcement of the cell wall by induced lignin
deposition and increased amounts of wall-bound ferulic acid
(Hano et al., 2006). Thus, elevated PAL expression is consistent
with the hypothesis that ferulic acid deposition in the wall is not
simply the result of sequestration of phenylpropanoid intermedi-
ates that accumulate because of suppressed CCR activity but is
actively induced to strengthen the cell wall.
The suggested wound-like response induced by CCR defi-
ciency is corroborated by the increased abundance of the
transcript levels of scavengers of reactive oxygen species (me-
tallothionein) (Mir et al., 2004; Wong et al., 2004) and of enzymes
that detoxify oxidative stress metabolites (GSTs and z-crystallin-
like protein) (Wilce and Parker, 1994; Dixon et al., 2002; Mano
et al., 2002). Additionally, transcript levels of a gene encoding a
U-box domain protein with a function in ubiquitylation were in-
creased in the transformants, and because this protein is similar
to the fungal elicitor-induced protein CMPG1 of P. crispum, the
induced expression of this gene supports a stress response. In
a wound-like response in the oxidative stress was corroborated
by the accumulation of feruloyl tyramine. This compound is
typically elicited in solanaceous plants upon wounding or path-
ogen attack. Alternatively, the molecular stress response in
CCR-downregulated tobacco was associated with photooxida-
tive stress caused by an increased efficiency of photosystem II
and a concomitantly elevated photorespiration (Dauwe et al.,
2007). In the CCR-downregulated poplar lines, the increased
Gln synthetase similarly support a possible role of photorespir-
atory H2O2in generating an oxidative stress response.
Altered Hemicellulose and Pectin Metabolism in
The differential transcriptome and metabolome in CCR-down-
regulated poplar both pointed toward increased breakdown or
remodeling of noncellulosic cell wall polysaccharides (see Sup-
plemental Figure 5 online). In poplar, the major cross-linking cell
wall polysaccharides in secondary-thickened cell walls are xylan
(18 to 28% of dry weight) and glucomannan (5% of dry weight)
(Mellerowicz et al., 2001). The increased transcript level of
(1,4)-b-mannan endohydrolase in the transformants suggests
an increased breakdown of (gluco)mannan. Furthermore, xylose
and rhamnose levels were increased in the transformants. Bio-
chemically, xylose and rhamnose are only liberated during the
breakdown of hemicellulose and pectin, mainly xylan and rham-
nogalacturonan, respectively. Thus, the accumulation of these
metabolites indicates an increased breakdown and/or remodel-
ing of xylan and pectin. On the other hand, the differential tran-
scriptome and metabolome indicated, in a complementary way,
a decreased hemicellulose and pectin biosynthesis in the trans-
formants (see Supplemental Figure 5 online). UDP-D-glucuronate
(UDP-D-GlcA) is the precursor of most monomers of both pectin
and hemicellulose. The synthesis of UDP-D-GlcA is considered
rate limiting in the biosynthesis of these noncellulosic cell wall
polysaccharides and can occur either via oxidation of UDP-
D-glucose (UDP-D-Glc) by UDP-D-Glc dehydrogenase, which is
considered to be the main pathway, or via UDP derivatization of
GlcA that, in turn, is formed by oxidation of myo-inositol (Seitz
et al., 2000). MIOX (EC 184.108.40.206) is the regulatory enzyme of the
latter pathway (Kanter et al., 2005). It has been proposed that
the myo-inositol–dependent pathway is of minor importance in
maize(Zeamays;Ka ¨rko ¨nenetal.,2005),yetourresultssupporta
possible in vivo role in UDP-D-GlcA production. A decreased flux
through the myo-inositol–dependent biosynthesis pathway of
UDP-D-GlcA in the CCR-downregulated lines was suggested by
the decreased transcript levels of a MIOX-like gene and sup-
ported by the decreased levels of both the substrate (myo-
inositol) and the product (GlcA) of the MIOX-catalyzed reaction.
However, the differential GlcA level should be interpreted with
caution as GlcA is also a cell wall polysaccharide degradation
product and acts additionally as an intermediate in other
3682 The Plant Cell
pathways, such as ascorbate biosynthesis. UDP-D-GlcA is the
direct precursor of UDP-D-xylose (UDP-D-Xyl) that in itself is an
important substrate for the biosynthesis of xylans and can be
converted to UDP-L-arabinose by MUR4 (Burget and Reiter,
1999; Burget et al., 2003). MUR4 transcript levels, as well as the
transcript levels of two glycosyltransferases corresponding to
the Arabidopsis PARVUS gene, were decreased in the trans-
formants. Because UDP-L-arabinose is used by glycosyltrans-
ferases in the arabinosylation of cell wall polysaccharides, mainly
the pectic rhamnogalacturonan of type I (RGI), and because a
transposon-tagged Arabidopsis parvus mutant showed reduced
levels of RGI branching (Lao et al., 2003), pectin biosynthesis
might have decreased in CCR-downregulated transformants.
The decreased expression levels of the glucosyltransferases in
the CCR-downregulated transformants might also affect the
xylan contents because the parvus mutant also had decreased
xylan content (Lao et al., 2003).
sugars GDP-D-Glc and GDP-D-mannose, the latter of which orig-
inates from mannose-6-phosphate (Man-6-P). The higher tran-
script levels of the (1,4)-b-mannan endohydrolase suggested an
increased degradation of glucomannan in the transgenic lines. In
that the biosynthesis of the glucomannans also was reduced,
implying that the flux from Man-6-P toward the (gluco)mannans
would be lower than in the wild type and that Man-6-P is con-
sequently redirected toward intermediary metabolism, resulting
in the tighter correlation of Man-6-P synthesis with (primary) me-
tabolites seen in the metabolite correlation networks (see Sup-
plemental Figure 6 online).
Thus, the combined transcriptome and metabolome analyses
suggested a reduced biosynthesis and increased remodeling of
hemicelluloses. Reduced hemicellulose content, accompanying
the reduced lignin content in CCR-downregulated poplar, was
indeed confirmed by wet carbohydrate analyses of the cell wall
material (Table 3) and by FTIR (see Supplemental Figure 7 online).
Altered Carbohydrate Deposition Might Be Signaled
through Altered Structural and Mechanical
Cell Wall Properties
Similar to PAL induction, the modifications in the metabolism of
hemicellulose and pectin might be induced in response to the
lower lignin content and the associated loose structure of the
tural disorders of the cell wall, alterations in hemicellulose and
pectin metabolism were apparent as well (Dauwe et al., 2007).
Signaling events that coordinate the deposition of the different
components of the cell wall, possibly mediated by alterations in
cell wall integrity, were also apparent in cellulose synthase mu-
the deposition of hemicellulose, pectin, and lignin (Fagard et al.,
2000; Ellis et al., 2002; Can ˜o-Delgado et al., 2003).
The induction of molecular responses to stimuli, generated by
altered mechanical aspects, organization, or composition of the
cell wall, requires contact sitesand signaling events between the
cell wall and the protoplast. AGPs are proposed to be linkers
between the plasma membrane and the cell wall (Kohorn, 2000)
and are presumed to be involved in molecular interactions and
cellular signaling between the wall and the cell (Oxley and Bacic,
1999; Showalter, 2001; Sun et al., 2004). A differentially ex-
pressed AGP encodes a candidate protein for signaling of the
altered mechanistic aspects of the cell wall. On the other hand, a
signal molecule could have mediated an altered transcription of
several genes involved in cell wall carbohydrate metabolism. A
possible role for BR signaling was suggested by the differential
transcript levels of a SERK2-like Leu-rich repeat receptor-like
protein kinase and a Ser carboxypeptidase-like protein. SERK2
has been postulated to act as a coreceptor of the BR receptor
(BRS1) is a secreted and active Ser carboxypeptidase that is
thought to be involved in an early event in the BRI1 signaling
pathway (Li et al., 2001; Zhou and Li, 2005).
Altered General Carbohydrate Metabolism in
An alteration in carbohydrate metabolism was revealed by the
decreased amounts of glucose, mannose, and myo-inositol in
the metabolite pools (Table 2; seeSupplemental Figure 5online).
Glucose and mannose can be readily interconverted via their
6-phosphates, whereas myo-inositol is derived from Glc-6-P.
Several myo-inositol–dependent pathways appear to be down-
regulated in the CCR-downregulated transformants. As men-
tioned above, the transcriptome and metabolome data indicated
a repression of the MIOX-catalyzed conversion of myo-inositol
into GlcA. This reaction is not only important in cell wall poly-
saccharide biosynthesis, for which additional data indicated a
dependent ascorbate biosynthetic pathway (Lorence et al.,
2004). A suppression of that pathway was supported by de-
creased levels of gulonate-1,4-lactone, the direct precursor of
dimer in the CCR-downregulated transformants might reflect a
decreased concentration of ascorbate. The ascorbate level itself
is not reliable because ascorbate is rapidly oxidized during the
extraction and derivatization procedure. On the other hand,
raffinose is derived frommyo-inositol via galactinol. Accordingly,
the levels of raffinose and its breakdown product, galactose,
were decreased in CCR-downregulated transformants.
Taken together, the decreased biosynthesis of pectin, cross-
linking glycans, ascorbate, and raffinose are potentially all linked
to a decrease in carbohydrate levels (glucose, mannose, and
myo-inositol) in the primary metabolism, which, in turn, are pos-
sibly associated with a stress response. It is therefore also
conceivable that the growth defects observed in the greenhouse
additionally to the altered carbohydrate metabolism and stress
CCR Deficiency Results in Sublayering of the Wall
In the CCR-downregulated poplars, TEM microscopy revealed
alternating strongly and weakly uranyl acetate-stained concentric
Downregulation of CCR in Poplar 3683
bands in the inner part of secondary-thickened cell walls of the
colored xylem, indicating microstructural alterations between
these subsequent concentric rings. This was most obvious in S2
layers of fibers, whereas thisphenotype was rarer and much less
pronounced in vessels.
Sublayering of the inner cell wall layers has been described
before for the interfascicular fibers in CCR1-downregulated
transgenic Arabidopsis (Goujon et al., 2003) and, although not
stated as such, can clearly be observed on the TEM pictures of
fiber and vessel walls of tobacco transformants downregulated
for both CCR and CAD (Chabannes et al., 2001b). Interestingly,
multilayered cell walls are not an exclusive feature of lignin-
modified plants. A similar sublayering has been described in
fibers of bamboo (Bambusa sp), banana (Musa sp), coconut
(Cocos nucifera), flax (Linum usitatissimum), and Douglas fir
(Pseudotsuga sp) (Esau, 1965; Mueller and Beckman, 1979;
Parameswaran and Liese, 1981, 1985; Gritsch et al., 2004). In
subsequent layers (Parameswaran and Liese, 1980). Interest-
ingly, association genetics in eucalyptus (Eucalyptus spp) has
revealed that variation in cellulose microfibril angle correlates
with polymorphism in the splicing of the CCR gene (Thumma
et al., 2005). It remains to be determined whether CCR down-
regulation in transgenic poplar affects the microfibrillar angle as
Transgenic Trees with Improved Pulping Characteristics
but Altered Growth
Wood from CCR-downregulated poplar is more amenable to
chemical kraft pulping. Two lines are of particular interest (FS3
charge (16 versus 18%) as shown by the lower Kappa numbers
and the lower levels of uncooked particles. As was observed for
CCR-downregulated tobacco (O’Connell et al., 2002), a de-
crease in Kappa number is associated with CCR suppression.
The highest level of reduction in Kappa number is found in the
two lines FS3 and FS30. The initial lignin content in these
transformants is lower than that in the wild type (see Supple-
mental Figure 8 online), which might cause a decrease in Kappa
number per se, but the lower hemicellulose content might
additionally be partly responsible for the lower Kappa number.
During cooking, alkali induces some polysaccharide hydroly-
sis; thus, viscosity values decrease when active alkali increases.
The significantly lower pulp viscosity obtained for the line FS3 (at
all alkali charges) indicates that cell wall polysaccharides are
more easily degraded than the control. This result contrasts with
previous results obtained for plants modified for their lignin
metabolism and assessed for their pulping characteristics. Even
in CCR-downregulated tobacco, viscosity values were unaf-
fected (O’Connell et al., 2002).
five blocks harvested in the field. Among the pulping character-
istics, the content of uncooked particles presents the highest
determined by the mean standard deviations. The viscosity is
quite homogeneous between the different clonal replicates.
These variations most probably relate to variability in environ-
mental conditions, particularly soil heterogeneity in the nursery,
but also border effects or clonal variability. Clonal variability in
CCR downregulation was indeed evident from the variability in
intensity and pattern of xylem coloration between ramets (Figure
1).FS3 and FS30 are promising for pulping applications because
their easier delignification might result in;12% savings in chem-
icals, which means economic benefits.
In conclusion, the strongest CCR-downregulated transgenic
poplars had several improved pulping properties, such as lower
Kappa number and reduced levels of uncooked particles. Al-
though these lines had growth penalties, CCR remains an
of transgenic lines with an optimal, yet stable, level of down-
regulation will be difficult with gene silencing approaches. How-
ever, Eco-TILLING approaches might be useful in exploiting the
natural variation that is still abundantly present in native poplar
Trees harboring CCR alleles that result in reduced CCR activity
are interesting progenitors for breeding programs. Furthermore,
the lower lignin and hemicellulose levels and associated relative
increase in cellulose suggest that CCR downregulation might be
a good strategy to improve plant biomass for bioethanol pro-
duction. Short-rotation coppice field trials are being established
to evaluate these possibilities.
Construction of Sense and Antisense Constructs
et al., 1989). The binary vector pBIBHYG (Becker, 1990), derived from
pBIN19 (Bevan, 1984), in which the neomycin phosphotransferase gene
is replaced by the hygromycin phosphotransferase gene, was used to
transform poplar (Populus tremula 3 Populus alba) with four different
constructs harboring DNA sequences from a Populus trichocarpa cv
Trichobel full-length CCR cDNA (accession number AJ224986), cloned in
vector pBluescript SK? (Stratagene) (plasmid name: pPOPCCR2.1)
(Leple ´ et al., 1992). This sequence shares 99% nucleotide identitity with
gene model estExt_fgenesh4_kg.C_LG_III0056 of the P. trichocarpa cv
Nisqually genome sequence (Tuskan et al., 2006). The CCR sequences
were fused downstream from the duplicated 250-bp upstream enhancer
of the CaMV 35S RNA promoter (p70) (Kay et al., 1987).
Plasmid pPOPCCR2.1 was digested by SmaI and StuI to isolate a
1032-bp fragment carrying the coding region of the CCR cDNA. This
fragment was cloned in the blunt-ended SmaI site of the vector pLBR19
that is a pUC19-derived vector containing the p70 and the CaMV
terminator sequence. Two derived vectors pLBR52 and pLBR62 were
obtained carrying the 1039-bp fragment either in the sense or antisense
orientation, respectively. Subsequently, the KpnI/XbaI fragments from
pLBR52 and pLBR62, carrying the p70 sense or antisense insert CaMV
terminator, were cloned in the KpnI/XbaI sites of pBIBHYG, resulting in
vectors pFS-CCR (carrying the full sense fragment) and pFAS-CCR
(carrying the full antisense fragment).
Plasmid pPOPCCR2.1 was digested by BamHI to isolate a 526-bp
fragment in the 59 end of the CCR cDNA. This fragment was cloned in the
BamHI site of pLBR19. A restriction map was used to select the clone
carrying the fragment inserted into the antisense orientation. Then, the
KpnI/XbaI fragment was cloned in the KpnI/XbaI sites of pBIBHYG,
resulting in the antisense vector p59 AS-CCR.
Plasmid pPOPCCR2.1 was digested with EcoRI, self-ligated, and
subsequently digested by EcoRI and XhoI to isolate a 373-bp fragment
3684The Plant Cell
XbaI sites of pBIBHYG, resulting in the antisense vector p39AS-CCR.
number 717-1-B4 (P. tremula 3 P. alba), was transformed via an Agro-
bacterium tumefaciens procedure essentially as described by Leple ´ et al.
(1992). Putative transformants were tested for their ability to root in the
presence of hygromycin (20 mg/L) and screened by PCR with primers
either designed for the hygromycin phosphotransferase gene or for the
introduced CCR cDNA fragments.
A field trial was established in Ardon (Orle ´ans, France) with four trans-
genic lines and the wild type. The transgenic lines FS3, FS30, FAS13,
FAS18, and wild-type poplars were micropropagated in vitro and 10
ramets of each were acclimatized in the greenhouse in January 1999. In
June 1999, upon evaluation by the French ‘‘Commission du Ge ´nie
Biomole ´culaire’’ (file B/FR/99.02.15) and authorization (99/023 of the
09.4.1999) from the ‘‘Ministe `re de l’Agriculture,’’ the 6-month-old poplar
plants (1 to 1.5 meter height) were transferred to the field. Two plants for
each line were planted in five different randomized blocks. Trees were
planted at a 1.5 3 3 m interval. A border of control trees surrounded this
area to limit environmental effects on growth and lignification. All trees
were pruned at the beginning of spring 2000 to get an improved vigor of
the new sprout and to homogenize the size of the plants.
Assessment of Growth Characteristics
Height and girth of the trees in the field for the years 2000 to 2003 were
measured in January to February of the years 2001 to 2004, respectively.
Total volume was calculated from the height and girth values [volume ¼
year were defined as the difference between the values for that year and
the corresponding values for the previous year. Growth parameters for
the year 2000 (collected in February 2001) were not included in the
analysis because of the high clonal variation for growth characteristics in
the first year after pruning.
Cell Wall Autofluorescence and Staining
Cross sections of stems and branches (60 or 40 mm thick) were prepared
from fresh samples with either a freeze or nonfreeze microtome. Wiesner
and Ma ¨ule staining reactions were essentially according to Atanassova
et al. (1995). Autofluorescence was observed with a digital module R
(Leica), equipped with a DFC 320 camera, a fluorescence device, and an
LP515 stop filter in conjunction with a 50-W HBO mercury burner. For
blue-excited autofluorescence, sections were viewed in distilled water
and the excitation wavelength was 450 to 490 nm. For long-wavelength
UV light–excited autofluorescence, sections were viewed at pH 10.3
(adjusted using 0.1 N NH4Cl and 0.1 N NaOH), the excitation wavelength
was 355 to 425 nm, and a long-wave pass filter at 470 nm was used.
TEM on stems of greenhouse-grown wild-type and transgenic lines (FS3,
FS30, FAS13, and FAS18) was done according to Rohde et al. (2004).
Fourteen-week-old and 80-cm high poplars (wild type and FS3) grown
under controlled conditions were used. Two internodes (10 and 16 cm)
were selected for microscopic studies, and 4-mm sections were taken
from the middle of the two internodes for fixation and embedded in LR
White resin (Joseleau and Ruel, 1997). Specific polyclonal antibodies
directed against syringyl substructures (S antibody) and homoguaiacyl
substructures (Gzl antibody) were used as antisera (Joseleau and Ruel,
1997; Joseleau et al., 2004b). For TEM, samples were prepared as
described (Joseleau and Ruel, 1997) and immunolabeled on ultra-thin
transverse sections (500 A˚) floating downward in plastic rings passed on
50-mL drops of reagents deposited on parafilm. The successive steps
wereas byJoseleau and Ruel (1997). The best dilutions for the antibodies
were between 1/50 and 1/100, depending on the sample. Observations
were made at 80 kV with a Philips CM 200 cryoelectron microscope.
All comparative immunolabeling experiments were performed in par-
different poplar lines, under the same conditions as those for the im-
The expression of the endogenous CCR gene was assessed by RT-PCR.
Samples of developing xylem were collected on 2-year-old branches
from 7-year-old field-grown poplars. Colored xylem was scraped from
the branches of lines FS3 and FAS13, and equivalent material was
harvested from wild-type poplars. Additionally, colorless xylem adjacent
was performed with 2 mg of total RNA using SuperScript II according to
the manufacturer’s instructions (Invitrogen). The reverse transcription
reaction was diluted 6 times to a final volume of 120 mL, and 5 mL was
used as template for the PCR using Applied qPCR Mastermix Plus for
SYBR green I (Eurogentec). PCR was performed on three biological
replicates using Smart cycler (Applied Biosystems) and the standard
cycling conditions. Each replicate corresponded to a pool of samples
from two branches of the same tree from the field trial, harvested in
June 2006. The following primer pairs were used for PCR (CCR,
TAGACAACCA-39; 18S RNA, 59-CTTCGGGATCGGAGTAATGA-39 and
59-GCGGAGTCCTAGAAGCAACA-39). The raw threshold cycle (Ct) val-
ues were normalized against 18S RNA to obtain normalized DCt values,
which were then used to calculate the difference in expression levels
compared with the wild-type sample.
Duplicate Klason lignin was determined by the standard procedure
(Dence, 1992). Lignin structure was investigated with thioacidolysis as
previously described (Lapierre et al., 1999). Mild alkaline hydrolysis was
performed on the MWEL lignin fractions from the wild type and FS3 with
2 M NaOH, at 378C, overnight.
Analysis of Cellulose and Hemicellulose
Wood samples harvested from 6-month-old greenhouse-grown wild-
type and transgenic lines FS3 and FS40 were ground in a Wiley mill to
pass a 0.4-mm screen (40 mesh) and Soxhlet extracted overnight in hot
acetone to remove extractives. The wood meal was delignified with a
modified version of Browning (1967) with two successive chlorite extrac-
tions by placing 200 mg of wood into 14 mL of buffer (60 mL of glacial
acetic acid þ 1.3 g NaOH/L) and 6 mL of 20% sodium chlorite solution
(NaClO2) in a 50-mL Erlenmeyer flask, capped and sealed with parafilm,
and gently shaken at 508C for 14 h. The reaction solution was decanted
by a 50-mL wash with acetone. After the first extraction, the entire
Downregulation of CCR in Poplar3685
delignification procedure was repeated, and the resulting wood extract
was permitted to dry in a 508C oven overnight. A small portion of the
residual (holocellulose) was tested by micro-Klason analysis (see below)
In a 50-mL Falcon tube, 100 mg of holocellulose was extracted with
10 mLof5%KOH for 120minat 208C withcontinuousshaking.The tubes
were centrifuged at 3000g for 30 min at 208C, and the supernatant was
collected. The remaining pellet was washed by resuspension in 5 mL of
5% KOH by aggressive vortexing. The supernatant was isolated by
centrifugation and again washed in 15 mL of water. The three superna-
tants (supernatant A) were pooled, while the remaining pellet was treated
to the same regime with the exception of a 24% KOH solution being
applied for the extractions and washes, resulting in the pooling of
supernatant B. The final pellet was washed twice with 2.5 mL of acetone,
washed extensively with water, allowed to dry overnight in a 508C oven,
and quantified gravimetrically, while supernatants A and B were com-
bined. The isolated hemicellulose polysaccharides in the pooled super-
natants were acidified to pH 4.0 with glacial acetic acid, precipitated with
excess 95% ethanol, and collected by centrifugation.
The composition (monomer ratios) of the isolated hemicellulose was
determined by HPLC (see below) after treating the hemicellulose with
at 1218C for 60 min.
Wood chemical analyses were conducted according to the micro-
Klason method of Huntley et al. (2003), which is a micromodified version
of the TAPPI standard. Extracted lignocellulosic material in a 15-mL
vial was cooled in an ice bath, reacted with exactly 3 mL of 72% (w/w)
transferred to a water bath maintained at 208C and mixed for 1 min every
serum bottle with 112 mL of deionized water to rinse all residues and
acids from the reaction vial. The serum bottles were sealed and auto-
claved at 1218C for 60 min. Samples were allowed to cool, and the hy-
sintered-glass crucibles. Each sample was washed with 200 mL of warm
(;508C) deionized water to remove residual acids and sugars and dried
overnight at 1058C. The dry crucibles were reweighed to determine
Klason lignin (acid-insoluble lignin) gravimetrically. The filtrate was ana-
lyzed for acid-soluble lignin by absorbance at 205 nm with a UV/Vis
spectrometer (Lambda 45; Perkin-Elmer Instruments) according to the
TAPPI Useful Method UM-250 (1991).
The concentration of neutral cell wall–associated carbohydrates (arab-
with an HPLC system (DX-600; Dionex) equipped with an ion-exchange
PA1 column, a pulsed amperometric detector with a gold electrode, and
a Spectra AS50 autoinjector. Prior to injection, samples were filtered
through 0.45-mm HV filters (Millipore). A 20-mL volume of sample was
loaded containing fucose as an internal standard. The column was equil-
ibrated with 250 mM NaOH and eluted with deionized water at a flow rate
of 1.0 mL/min.
For phenolic profiling, samples of young developing xylem were har-
vested from stems of 3-month-old greenhouse-grown poplars. In addition
to wild-type poplar, poplar transformed with a PCaMV35S-GUS con-
struct (35S 17B and 35S 21B) (Nilsson et al., 1996; Chen et al., 2000) and
transgenicpoplars downregulated forCCR(linesFS3, FS40, and FAS13),
a set of 12 transgenic poplar lines downregulated for various enzymes
involved in phenylpropanoid biosynthesis (CCoAOMT, COMT, CAD,
phenylcoumaran benzylic ether reductase, and COMTþCCR double
transformants) were included to increase the statistical power of the
experiment. Four to eight ramets were analyzed for each poplar line. In
vitro propagation, growth conditions, sampling, extraction, HPLC anal-
ysis of the soluble phenolics from poplar xylem tissue, and LC-MS/MS
analysis of the cinnamic acids and cinnamaldehydes were as previously
described (Morreel et al., 2004a, 2004b; Damiani et al., 2005).
For the oligolignols, the shorthand naming convention was used
(Morreel et al., 2004b); briefly, bold capitals for units formally derived
from coniferyl alcohol (G), sinapyl alcohol (S), coniferaldehyde (G9),
sinapaldehyde (S9), vanillin (V9), and sinapyl p-hydroxybenzoate (SP),
and the interunit bond formed during the coupling reaction specified in
parentheses: (8–O–4), (8–5), or (8–8).
Ten, nine, and ten plants of the lines FS3, FS40, and the wild type,
respectively, were grown for 6 months in the greenhouse. The basal
40 cm was harvested, immediately frozen in liquid nitrogen, and kept at
?808C for RNA extraction. For RNA isolation, the stems were debarked,
and the developing xylem was scraped off with a razor blade. Two pools
of material were generated for each line (five plants per pool). All sampled
xylem tissue from line FS3 was colored. For line FS40, xylem tissue
scraped from three noncolored individuals was incorporated into one of
the two duplicate pools. None of the sampled plants had a patchy phe-
After LiCl precipitation, the RNA was resuspended in 1.8 mL 2:1 lysis
buffer (RLT)/ethanol and further purified with the RNeasy mini kit (Qiagen)
according to the manufacturer’s recommendations. RNA concentrations
2100 bioanalyzer. The RNA was stored at ?808C in 70% ethanol.
RNA was recovered by centrifugation after addition of one-tenth
volume of 3 M Na-acetate, pH 5.2, and resuspended in RNase-free
water.cDNA wassynthesizedasdescribed(Tayloretal.,2005) withslight
modifications: 30 mg of total RNA and 1 mL oligo(dT) anchor (5 mg/mL;
in reverse transcriptase buffer (Invitrogen), 0.6 mL aa-dUTP/dNTP mix
(500 mM of dATP, dCTP, dGTP, 100 mM dTTP, and 400 mM aa-dUTP;
Sigma-Aldrich), 1 mM DTT, 30 units of RNase inhibitor (Invitrogen), and
total volume of 30 mL. The reaction was incubated at 428C for 3 h and
terminated by addition of 10 mL 0.5 M EDTA, pH 8.0. The RNA was
degraded with alkaline treatment (10 mL 1 N NaOH) during 15 min at 658C
and neutralized with 50 mL 1 M N-(2-hydroxyethyl)piperazine-N9-
PCR cleanup kit according to the manufacturer’s instructions and eluted
with 30 mL of 4 mM KPO4, pH 8.5. Concentrations were determined
spectrophotometrically with a NanoDrop ND100 (NanoDrop Technolo-
gies). Aliquotsof800 ngcDNA weredried in a speedvac (DNA SpeedVac;
Thermosavant). Cy3 and Cy5 monoreactive dried dyes (GE-Healthcare)
were dissolved in 120 mL0.1 M NaHCO3,pH 9.0, and 15 mLofCy3 or Cy5
was added to 800 ng cDNA and incubated in the dark for 2 h at room
temperature. Unincorporated dyes were removed with the Cyscribe GFX
purification kit (GE-Healthcare) according to manufacturer’s instructions,
after adjustment of the volume to 50 mL with water. cDNAs for each
hybridization,labeled withCy3andCy5, wereelutedin thesamevial.Dye
incorporation was evaluated spectrophotometrically with a NanoDrop
ND100. The labeled cDNA was lyophilized to a volume of 82 mL.
The POP2 Populus glass-spotted cDNA microarrays were used for this
study (http://www.populus.db.umu.se) and hybridized in an automated
slide processor (Lucidea ASP Hybridization Station; Ge-Healthcare) as
described by Taylor et al. (2005) according to an all pairwise-comparison
design (see Supplemental Figure 2 online). A dye-swap replication was
done for each hybridization. Arrays were scanned with a ScanarrayLite
4000 microarray analysis system scanner (Packard-Bell). Laser settings
and photomultiplier tube were adjusted to obtain overall similar signal
3686 The Plant Cell
saturated spots. The obtained images were analyzed with Genepix Pro
5.1 (Axon Instruments). The spot diameter resize feature was set to
minimum 80% and maximum 120%. The composite pixel intensity was
set to 100. The median background pixel intensity was subtracted from
the mean spot pixel intensity applying the local method (default option).
All data were manipulated and computed with the SAS system software
package for windows V8. A spot was considered good when the fore-
ground intensity was higher than twice the background SD in both
channels, or the foreground intensity was higher than four times the
background SD in one channel and higher than one background SD in the
other channel. From the 25,728 cDNAs spotted on the array, 24,735 were
11,274 Populus cDNAs, an F-value could be calculated. For the others,
the expression remained below background on most arrays.
Metabolite Profiling by GC-MS
For metabolite profiling by GC-MS, young developing xylem was sam-
pled fromstemsof3-month-old greenhouse-grown poplars asdescribed
for the phenolic analysis (Morreel et al., 2004a, 2004b). Nineteen wild-
type poplars and eight, nine, and 14 poplars of the transgenic lines
FAS13, FS40, and FS3, respectively, were analyzed separately. Extrac-
tion using hot methanol:chloroform:water (3:2:4 [v/v/v]) and derivatization
of the polar metabolites were performed as previously described (Kaplan
et al., 2004; Desbrosses et al., 2005). Ribitol, isoascorbic acid, and
deuterated Ala were added as internal standards, and retention time
indices determined with a C12, C15, C19, C22, C28, C32, and C36n-alkane
mixture. Metabolite profiling was performed according to Desbrosses
et al. (2005) using a Fisons 8000 GC instrument coupled to a Fisons
MD800 mass spectrometer and equipped with an AS800 autosampler
and an electron impact source (ThermoQuest). In general, the GC-MS
profiling protocol yielded standard deviations that are below 6% of the
mean chromatogram peak abundance (Roessner et al., 2000). The quad-
rupole mass spectral and retention time index library (i.e., Q_MSRI) was
used to integrate particular candidate mass-to-charge traces corre-
sponding with known metabolites and with unidentified mass spectra of
metabolites; the latter are referred to as mass spectral metabolite tags
for in the GC-MS chromatograms, of which 159 were known metabolites.
Based onthecorresponding massspectralidentifiers,furtherinformation
on the metabolites can be found at http://csbdb.mpimp-golm.mpg.de/
csbdb/gmd/msri/gmd_smq.html. GC-MS spectral manipulation and in-
tegration were done with MassLab software version 1.4v (ThermoQuest).
Correlation networks of metabolites were constructed for the wild type,
FAS13, FS40, and FS3 based onthe GC-MS data fromseven, eight, nine,
and 14 biological replicates, respectively. Compounds that could be
quantified in at least 80% of all individuals were selected for correlation
analysis.Afterlogarithmic transformation, Pearson product-momentcor-
relation coefficients were calculated with S-plus version 6.1 (Insightful).
Correlation networks were subsequently generated with the Fruchterman-
Reingold three-dimensional algorithm in Pajek (http://vlado.fmf.uni-lj.si/
Stems of 6-month-old greenhouse-grown wild-type, FS3, FS40, and
FAS13 poplars were analyzed by FTIR spectroscopy. Attenuated total
reflection spectra of reference compounds and of 30-mm-thick xylem
sections were recorded with an FTIR spectrometer Equinox 55 (Bruker
Optics) combined with a DuraSamplIR II ATR unit (SensIR Europe) at a
resolution of 4 cm?1, 32 scans. The reference compounds analyzed were
ferulic acid, methyl ferulate, ethyl ferulate, a ferulate-glucose ester (Me-
6-O-FA-b-D-Glc), a ferulate-galactose ester (Me-6-O-FA-b-D-Gal), a syn-
thetic lignin DHP made of 50:50 coniferyl:sinapyl alcohol, and a synthetic
lignin DHP made of 50:50 coniferyl:sinapyl alcohol and 5% ferulic acid
(Ralph et al., 2007b). Dried sections of four plants per poplar line were
analyzed and, per section, spectra of three spots (1-mm diameter) were
recorded. Among the 36 spots analyzed from the transgenic poplars, 12
were from noncolored, 19 from weakly colored, and five from intensely
Spectral data were evaluated as described by Naumann et al. (2005).
The spectra were vector-normalized, and peak areas were calculated by
integration of the absorbance units from the baseline, over the wave
number range defining the absorption band (Opus 5.5 software, method
A; Bruker Optics). Fifteen absorption bands were defined and integrated:
1778to 1691(1), 1691to 1612(2),1612to 1554(3), 1527to 1486(4),1486
to 1442 (5), 1442 to 1397 (6), 1397 to 1349 (7), 1349 to 1293 (8), 1293 to
118 (9), 1188 to 1145 (10), 1145 to 1096 (11), 1096 to 999 (12), 999 to 917
(13), 917 to 881 (14), and 683 to 649 cm?1(15).
The NMR spectra were acquired on a Bruker Biospin DMX-500 instru-
ment fitted with a cryogenically cooled 5-mm TXI1H/13C/15N gradient
probe with inverse geometry (proton coils closest to the sample). Acet-
ylated lignin preparations (60 to 80 mg) were dissolved in 0.5 mL CDCl3;
the central chloroform solvent peak was used as internal reference
(dC77.0, dH7.26 ppm). The standard Bruker implementations of one-
dimensional and two-dimensional (gradient-selected,1H-detected HSQC,
HSQC-TOCSY, and HMBC) NMR experiments were used for structural
elucidation and assignment authentication. Data for model compounds
can be found in the ‘‘NMR Database of lignin and cell wall model com-
pounds’’ available athttp://www.dfrc.ars.usda.gov/software.html. TOCSY
experiments used a 100-ms mixing time; HMBC spectra used an 80-ms
long-range coupling delay. Normal HSQC experiments at 500 MHz typ-
ically had the following parameters: acquired from 8.6 to 2.4 ppm in
F2 (1H) with 1864 data points (acquisition time 200 ms), 158 to 40 ppm in
F1 (13C) with 256 increments (F1 acquisition time 8.6 ms) of 72 scans with
a 1-s interscan delay, total acquisition time of 6.5 h; the d24delay was set
to 1.72 ms (;1/4 J). Processing used typical matched Gaussian apo-
dization in F2 and squared sine-bell in F1. One level of linear prediction in
F1 (32 coefficients) gave improved F1 resolution but was not required.
Volume integration of contours in HSQC plots was done using Bruker’s
TopSpin 1.3 software as described recently (Ralph et al., 2006). For
quantification of S/G distributions, only the carbon-2 correlations from G
units, and the carbon-2/6 correlations from S units were used, and the G
integrals were logically doubled. No correction factors were necessary,
determined from two-model compounds, coniferyl p-hydroxybenzoate,
and the g-p-hydroxybenzoate ester of a syringyl b-ether dimer. For
quantification of the various interunit linkage types, the following well-
resolved contours (see Figure 4) were integrated: Aa, Ba, Ca, Sa, and
X1g.Integral correctionfactors determinedpreviously(Ralphet al., 2006)
were used: Aa 1.00, Ba 0.71, and Ca 1.06; Sa and X1g were either not or
not reliably determined and were assumed as 1.00. These values were
usedto correct thevolumeintegrals toprovide the estimatesofunit ratios
in Supplemental Table 2 online.
for chemical pulping analysis. For each line (FS3, FS30, FAS13, FAS18,
and wild type), five stems were harvested, each derived from a distinct
block from the field trial. Trees were debarked and cut into chips with a
wood log chipper and screened to remove the coarse and fine elements
with a chip size classifier (Lecourt et al., 2006). The kraft pulping process
Downregulation of CCR in Poplar3687
(Gullichsen and Fogelholm, 2000) was simulated at laboratory scale on
200 g of oven-dried wood chips in small pressurized reactorsin a rotating
oil-thermostatic bath under the following conditions: active alkali ¼ 16
to 20%, sulfidity ¼ 25%, liquor/wood ratio ¼ 4, temperature rise to 1708C
over 90 min, and maintained for 1 h for cooking. Pulps were washed and
screened on a 150-mm slotted screen to determine uncooked particles
and pulp yield. Kappa number and pulp viscosity were determined
according to international standards (NF ISO 302 and ISO 5351-1).
Statistical Analyses of the Data
Microarray Data Deposition
Data have been deposited in the UPSC-BASE Populus transcriptomics
online (http://www.upscbase.db.umu.se/) (Sjo ¨din et al., 2006) under
experiment number 0015.
Sequence data from this article can be found in the GenBank/EMBL
full-length CCR cDNA).
The following materials are available in the online version of this article.
Supplemental Figure 1. Long-Wavelength UV-Excited Autofluores-
Supplemental Figure 2. Aromatic Regions of HSQC Spectra from
MWEL Illustrating Compositional Changes.
Supplemental Figure 3. Partial HSQC NMR Spectra.
Supplemental Figure 4. Microarray Design.
Supplemental Figure 5. Ascorbate, Hemicellulose, and Pectin Me-
Supplemental Figure 6. Metabolite Correlation Networks.
Supplemental Figure 7. FTIR Spectra Overlay.
Supplemental Figure 8. Chemical Pulping Characteristics.
Supplemental Table 1. CCR Transcript Abundances.
Supplemental Table 2. Lignin Profile in Young Developing Xylem.
Supplemental Table 3. Statistical Analysis of Lignin Characteristics.
Supplemental Table 4. Concentration of Phenylpropanoids.
Supplemental Table 5. Identity, Expression, and Functional Classi-
fication of the Genes.
Supplemental Table 6. Number of Vertices and Edges in Correlation
Supplemental Table 7. Carbohydrate Analyses Raw Data.
Supplemental Table 8. FTIR Analysis.
We thank Mark Van Montagu and Dirk Inze ´ for support, the technical
staff that managed the field trial, Fre ´de ´ric Le ´ge ´e for Klason lignin
determination, Nade `ge Millet and Bart Ivens for in vitro culture work and
plant care, Franc ¸oise Laurans and Alain Moreau for histochemical work,
Marie-Claude Lesage-Descauses for quantitative PCR analysis, Isabelle
Paintrand for skillful assistance with the light microscopy, Jørgen Holst
Christensen for helpful discussions, Lise Jouanin for providing the
pLBR19 vector, and Martine De Cock for help with the manuscript.
This work was conducted in the framework of the European Union
Research Program TIMBER (FAIR-CT-95-0424), EDEN (QLK5-CT-2001-
00443), and COPOL (QLK5-CT-2000-01493); partial funding to H.K. and
J.R. was from the U.S. Department of Energy, Energy Biosciences
Received July 11, 2007; revised October 12, 2007; accepted October 19,
2007; published November 16, 2007.
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