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J. Biol. Chem., Vol. 275, Issue 47, 36899-36909, November 24, 2000
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From the a Vakgroep Moleculaire Genetica & Departement
Plantengenetica, Vlaams Interuniversitair Instituut voor
Biotechnologie, Universiteit Gent, B-9000 Gent, Belgium,
c Laboratoire de Chimie Biologique, Institut National
Agronomique Paris-Grignon, F-78850 Thiverval-Grignon, France,
d Vakgroep Organische Scheikunde, Universiteit Gent, B-9000
Gent, Belgium, e Laboratorium Medicinale Scheikunde, Katholieke
Universiteit Leuven, B-3000 Leuven, Belgium, f Vakgroep
Biochemie, Fysiologie en Microbiologie, Universiteit Gent, B-9000 Gent,
Belgium, and h U.S. Dairy Forage Research Center, U.S.
Department of Agriculture-Agricultural Research Service,
Madison, Wisconsin 53706-1108
Received for publication, August 1, 2000
Caffeoyl-coenzyme A
O-methyltransferase (CCoAOMT) methylates, in
vitro, caffeoyl-CoA and 5-hydroxyferuloyl-CoA, two possible precursors in monolignol biosynthesis in vivo. To clarify
the in vivo role of CCoAOMT in lignin biosynthesis,
transgenic poplars with 10% residual CCoAOMT protein levels in the
stem xylem were generated. Upon analysis of the xylem, the affected
transgenic lines had a 12% reduced Klason lignin content, an 11%
increased syringyl/guaiacyl ratio in the noncondensed lignin fraction,
and an increase in lignin-attached p-hydroxybenzoate but
otherwise a lignin composition similar to that of wild type. Stem xylem of the CCoAOMT-down-regulated lines had a pink-red coloration, which
coincided with an enhanced fluorescence of mature vessel cell walls.
The reduced production of CCoAOMT caused an accumulation of
O3- Lignin is, second to cellulose, the most abundant organic compound
in the terrestrial biosphere. In different tree species, lignin content
varies between 15 and 36% of the dry weight of wood (1). Lignin is a
major constituent of cell walls of fibers and tracheary elements and
provides these cells rigidity for structural support and impermeability
for water transport. For the production of high-quality paper, lignin
is considered as a negative factor because it must be extracted from
the cellulose fraction by energy-requiring and polluting methods. For
this reason, there is considerable interest in modifying lignin by
genetic engineering to improve its extractability from wood (2-5).
Lignin monomer biosynthesis starts with the deamination of
phenylalanine to produce cinnamic acid (Fig.
1). Further enzymatic reactions include
the hydroxylation of the aromatic ring, the methylation of selected
phenolic hydroxyl groups, the activation of the cinnamic acids to
cinnamoyl-CoA esters, and the reduction of these esters to
cinnamaldehydes and cinnamyl alcohols. The precise order in which these
reactions occur is not yet fully resolved. In dicotyledonous plants,
lignin is composed mainly of guaiacyl
(G)1 and syringyl (S) units
that are monomethoxylated (C-3) and dimethoxylated (C-3, C-5) and
derived from coniferyl alcohol and sinapyl alcohol, respectively. The
lignin monomers are transported to the cell wall and are subsequently
polymerized, resulting in the deposition of a cross-linked polymer.
Although most of the lignin biosynthesis enzymes have been
characterized at the molecular level, their precise role in determining
lignin amount and composition still needs to be clarified.
Based on in vitro data, it has been generally accepted that
the methylation reactions in lignin biosynthesis occur exclusively at
the cinnamic acid level and that they are catalyzed by a bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase
(COMT) (1). However, the analysis of transgenic tobacco and poplar with
suppressed COMT activity has shown that COMT is mainly or exclusively
implicated in the biosynthesis of S monomers (6-9). Later, it was
shown that the methylation of the lignin precursors could also occur at
the level of the cinnamoyl-CoA esters by
caffeoyl-CoA-3-O-methyltransferase (10), and it was proposed
that CCoAOMT was predominantly involved in the biosynthesis of G units.
Transgenic tobacco plants down-regulated for CCoAOMT, however, had a
reduced lignin amount, indicating that CCoAOMT regulates both G and S
unit production (11). Subsequently, in vitro activity assays
have shown that COMT is also active at the cinnamoyl-CoA,
cinnamaldehyde, and cinnamyl alcohol levels (12-14) and that
5-hydroxyconiferaldehyde competitively inhibits the methylation of
caffeic acid to ferulic acid and of 5-hydroxyferulic acid to sinapic
acid (15). Additionally, coniferyl aldehyde has been demonstrated to
inhibit the conversion of ferulic acid to 5-hydroxyferulic acid in a
noncompetitive manner (13). Together, these data, obtained from
in vitro enzymatic assays, have suggested that G and S units
are biosynthesized via caffeoyl-CoA and feruloyl-CoA to coniferaldehyde
and then diverted to S units via 5-hydroxyconiferaldehyde and
sinapaldehyde. Furthermore, these data also suggested that the pathway
from caffeic acid to sinapic acid is not used for lignin precursor
biosynthesis. Given our sparse knowledge on the cell-specific
localization of these enzymes in the different cell types of wood (16),
the actual pool sizes of pathway intermediates in the different cell
types, their effects on the kinetics of the monolignol biosynthesis
enzymes (13, 15, 17), and the expression levels of the corresponding
genes (18), the metabolic outcome of altering one particular step of
the pathway in vivo still remains largely unpredictable.
Here, we have analyzed the metabolic consequences of reducing CCoAOMT
production in transgenic poplar. We show that a down-regulation of
CCoAOMT by 90% reduces the Klason lignin amount by 12%, increases the
S/G composition ratio by 11%, and increases the content of p-hydroxybenzoate in the lignin polymer. In addition, we
demonstrate by HPLC (along with MS and 1H NMR) that
O3- Construction of Sense and Antisense Vectors
All DNA recombinant techniques were performed essentially as
described (19). The binary vector pBIBHYG (20) is derived from pBIN19
(21), in which the neomycin phosphotransferase gene is replaced by the
hygromycin phosphotransferase gene. This binary vector was used to
transform poplar with four different constructs harboring DNA sequences
from a Populus trichocarpa "Trichobel" full-length
CCoAOMT-1B cDNA (accession number AJ224894), cloned in
vector pBlueScript SK (Stratagene, La Jolla, CA) (plasmid name, pccoaomt-1b). The CCoAOMT sequences were placed downstream
from the duplicated 250-bp upstream enhancer of the cauliflower mosaic virus (CaMV) 35S RNA promoter (P70) (22).
Construction of a 5' Antisense Construct
A 5' CCoAOMT PCR fragment was generated using primers
5'-AGAAGGGATCCACAATAATGGCC-3' and 5'-GGCCATTGACAAGCTTCAAAAGC-3'
(priming at positions Construction of an Internal Antisense Construct
A 571-bp HindIII/BamHI CCoAOMT
fragment was generated by digesting pccoaomt-1b. This fragment was
cloned into the HindIII/BamHI-digested vector
OMTmutpBSrev, kindly provided by U. Matern (Institute of Pharmaceutical
Biology, Marburg, Germany), resulting in plasmid pCCmut. pCCmut was
digested with KpnI and BamHI, and the
CCoAOMT fragment was ligated in the
KpnI/BamHI-digested pUC19. This construct was
digested with EcoRI and BamHI and ligated in
EcoRI/BamHI-digested pLBR19, resulting in
pLBR19-571. The 1.9-kilobase pair
KpnI/XbaI P70-CCoAOMT fragment,
derived by partial digestion of pLBR19-571, was ligated in the
KpnI/XbaI-digested pBIBHYG vector, resulting in
vector pIn-asCCoAOMT.
Construction of the Full-length Antisense Construct
The 5' CCoAOMT PCR fragment, obtained as described
above, was cloned in pGEM-T (Promega, Madison, WI). This construct was digested with HindIII/PstI, and the
CCoAOMT fragment was transferred into the
HindIII/PstI site of pccoaomt-1b. The resulting
plasmid was digested with BamHI and the fragment was cloned
in the BamHI site of pLBR19 in the antisense orientation.
This construct was digested with KpnI/XbaI and
the P70-CCoAOMT fragment was ligated into
KpnI/XbaI-digested pBIBHYG, resulting in vector
pFl-asCCoAOMT.
Construction of the Full-length Sense Construct
Plasmid pccoaomt-1b was digested with PstI and
KpnI, and the CCoAOMT fragment was ligated in the
PstI/KpnI site of pUC19. This plasmid was
digested with EcoRI, and the CCoAOMT fragment was
ligated in the EcoRI site of pLBR19 in the sense
orientation. This plasmid was partially digested with KpnI
and XbaI, and the 2.5-kilobase pair
P70-CCoAOMT fragment was ligated into the
KpnI/XbaI site of pBIBHYG, resulting in vector psCCoAOMT.
Poplar Transformation and Regeneration
Wild-type Populus tremula × Populus alba (INRA,
number 717 1-B4) was transformed with all four constructs. The
transformation via Agrobacterium tumefaciens and
regeneration procedures were essentially as described (23). One shoot
was isolated from each stem explant to ensure that the transgenic lines
were derived from independent transformation events.
Production of Antibodies against Poplar CCoAOMT
The PCR product described above was digested with
BamHI/HindIII and ligated together with a
HindIII/XhoI fragment from the CCoAOMT-1B cDNA into the
BamHI/XhoI site of the pGEX-4T-1 expression vector (Amersham Pharmacia Biotech), resulting in vector
pGEX::CCoAOMT. CCoAOMT was expressed as a glutathione
S-transferase-CCoAOMT fusion protein. Fusion protein
expression was induced with 0.1 mM isopropyl Immunoblot Analysis
Samples were taken from the base of the stem of 6-month-old
greenhouse-grown poplars. Xylem tissue was obtained by scraping a 5-cm
debarked stem with a scalpel. This tissue was subsequently homogenized
in liquid N2. Pink-red-colored and whitish xylem from line
asccoaomt-1 were separated with a scalpel. Proteins were extracted with 60 mM Tris (pH 6.8) containing 0.03% (w/v)
dithiothreitol and 0.5% (w/v) polyvinylpolypyrrolidone and the
proteinase inhibitor CompleteTM Mini (Roche Diagnostics,
Brussels, Belgium). SDS-polyacrylamide gel electrophoresis was carried
out according to standard procedures. Proteins were blotted on Hybond C
super membranes (Amersham Pharmacia Biotech). Immunodetection was
performed according to standard laboratory methods, using an
anti-rabbit IgG alkaline phosphatase conjugate (Roche Diagnostics) and
the color development reagents 5-bromo-4-chloro-3-indolyl phosphate
p-toluidine salt (Duchefa, Haarlem, The Netherlands) and
p-nitro blue tetrazolium chloride (Duchefa). The titer of
the CCoAOMT antibodies and anti-rabbit IgG secondary antibodies in the
immunodetection procedure was 1:1500 and 1:2500, respectively.
Alternatively, CCoAOMT protein amounts were quantified by measuring the
hybridization intensities with a PhosphorImager 445SI (Amersham
Pharmacia Biotech), after using 35S-labeled anti-rabbit IgG
(Amersham Pharmacia Biotech) (dilution 1:2500) as secondary antibodies.
Histology of Stem Sections
Stem cross-sections (60 µm thick) were made from the base of
6-month-old greenhouse-grown poplars with a sledge microtome (Jung,
Heidelberg, Germany). Mäule and Wiesner staining were performed
essentially as described (6). Autofluorescence of stem cross-sections
was observed with an Axioskop microscope (Zeiss, Jena, Germany)
equipped with a fluorescence device using a filter combination of
450-490 nm excitation and 515-565 nm emission in conjunction with a
100-watt mercury burner.
Lignin Analyses
Klason lignin analysis, thioacidolysis, and analysis of
low-molecular-weight phenolics released by mild alkaline hydrolysis were essentially as described (9). For lignin isolation and subsequent
NMR analysis, three to four stems from each line of 7-month-old poplars
were pooled. Before the poplar stem cell walls were isolated, the stems
were cut into 1-2-cm pieces and ground to pass a 1.0-mm screen of a
cyclone mill (Udy Corp., Fort Collins, CO). Lignin isolations were
essentially as described previously (24, 25). Soluble phenolics,
carbohydrates, and other components were removed by successive
extractions with water, methanol, acetone, and chloroform. Most of the
colored material was removed by the water and methanol extraction
cycles. The isolated cell wall material was ball-milled, suspended in
50 mM acetate buffer (pH 5.0), and treated with 30 mg of
cellulase (Cellulysin; Calbiochem-Novabiochem, Bad Soden, Germany) per
1 g of ball-milled material. Cell wall digestions ran for 8 days
with fresh enzyme and buffer being added after 2.5 and 5 days of
incubation at 30 °C. The resulting lignin polysaccharide complex was
subjected to fractionation in 96:4 (v/v) dioxane/water, reflective of
standard "milled wood lignin" conditions (26). The final yields of
the dioxane-soluble lignin fractions were 13.0, 11.2, and 11.6% of the
total cell wall material, and 73, 72, and 69% of the total lignins for
wild-type, sccoaomt-16, and sccoaomt-29, respectively. Isolated lignins
(200 mg of each) were acetylated overnight with acetic
anhydride/pyridine. The solvents were removed by coevaporation with
95% ethanol and traces of ethanol by coevaporation with acetone. The
acetylated lignins were then extracted into CHCl3 and
washed with aqueous EDTA (6 mM, pH 8.0) to remove trace
metal contaminants. Proton-decoupled 13C NMR spectra were
taken on a Bruker DRX-360 instrument (Bruker, Karlsruhe, Germany)
fitted with a 5-mm 1H/broadband gradient probe with inverse
geometry (proton coils closest to the sample). Acetylated lignins
(EDTA-washed, 100 mg) were dissolved in 0.4 ml of
acetone-d6; unacetylated lignins (not EDTA-washed, 60 mg) were dissolved in acetone-d6
(0.35 ml) and deuterium oxide (D2O) (0.05 ml). The central
acetone solvent peak was used as internal reference ( Analysis of Soluble Phenolics
Xylem tissue (approximately 100 mg) was obtained by scraping the
debarked stem of 4-month-old greenhouse-grown poplars with a scalpel,
homogenized in liquid N2, and extracted with 5 ml of methanol. The supernatant was dried, and samples were extracted with
cyclohexane/water containing 0.1% trifluoroacetic acid (1:1, v/v). The
phenolic compounds in the aqueous phase were separated and quantified
by reversed-phase HPLC using a Luna C18 column (Phenomenex, Torrance,
CA) (5 µm, 250 × 4.6 mm) with gradient elution by an HPLC
Waters 625LC system (Waters, Milford, MA), employing an increasing
gradient of methanol-acetonitrile (25:75, v/v) acidified with 0.1%
trifluoroacetic acid (solvent B) in 0.1% aqueous trifluoroacetic acid
(solvent A). The following gradient elution conditions were used:
time = 0 min/0% B; time = 25 min/60% B; time = 27 min/100% B; flow rate = 1.5 ml/min; temperature 40 °C;
injection loop = 20 µl. All solvents were of HPLC grade purity. Absorbance spectra were recorded with a Waters 996 diode array detector
by scanning from 200 to 450 nm. The peak height was quantified at the
maximum absorbance value between 230 and 450 nm. Data collection and
integration was done with the Millennium software (Waters).
Statistical Analyses
Pearson product-moment correlation analyses were performed using
GraphPad Prism version 2.01. Calculation of the correlation coefficient
(r2) was based on peak concentration values
obtained from two harvests (July 1998 and May 1999), containing several
ramets for lines sccoaomt-16 and sccoaomt-29 (12 plants in total).
Mass Spectrometry
The molecular weight and the daughter ion spectra of the
accumulating glycosides were determined using a nanoelectrospray ionization source operated in the negative mode on a hybrid quadrupole time-of-flight mass spectrometer (Micromass, Manchester, United Kingdom) using borosilicate capillaries (Protana, Odense, Denmark). The
conditions were as follows: solvent = acetonitrile/water/formic acid (1:0.99:0.01 (v/v/v)); concentration loaded = 5 pmol/µl; electrospray ionization probe voltage = 1H NMR Spectroscopy
Approximately 250 µg of the glycosides were dissolved in 0.7 ml of D2O (Aldrich). The spectra were recorded with a
UNITY-500 spectrometer (Varian Analytical Instruments, Palo Alto, CA).
The experiments were performed with a 5-mm inverse detection probe equipped with pulsed magnetic field gradient coils. The standard Varian
software Vnmr version 5.3b was used throughout. The spectra were run at
27 °C and referenced to the residual water peak at Structural Assignment of Compounds X, D, and E
In MS/MS, compound X had a [M
Modifications in Lignin and Accumulation of Phenolic Glucosides
in Poplar Xylem upon Down-regulation of Caffeoyl-Coenzyme A
O-Methyltransferase, an Enzyme Involved in Lignin
Biosynthesis*
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-D-glucopyranosyl-caffeic
acid,
O4--D-glucopyranosyl-vanillic
acid, and
O4--D-glucopyranosyl-sinapic
acid (GSA), as authenticated by 1H NMR. Feeding experiments
showed that
O3--D-glucopyranosyl-caffeic
acid and GSA are storage or detoxification products of caffeic and
sinapic acid, respectively. The observation that down-regulation of
CCoAOMT decreases lignin amount whereas GSA accumulates to 10% of
soluble phenolics indicates that endogenously produced sinapic acid is
not a major precursor in syringyl lignin biosynthesis. Our in
vivo results support the recently obtained in vitro
enzymatic data that suggest that the route from caffeic acid to sinapic
acid is not used for lignin biosynthesis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Biosynthetic pathway for cinnamyl alcohols
and production of side product phenolics. Enzymatic reactions
involved in the cinnamyl alcohol biosynthesis in poplar are indicated
in black. The formation of sinapoyl-CoA and its conversion
to sinapaldehyde are indicated in blue; the relevance
of these conversions in monolignol biosynthesis is uncertain, as
mentioned above. Conversions leading to the synthesis of side
product phenolics are indicated in red and are markedly
up-regulated when CCoAOMT expression is reduced. The reduced
flux from caffeoyl-CoA to feruloyl-CoA, caused by the down-regulated
CCoAOMT expression causes an elevated production of caffeic
acid that is detoxified to GCA. GVA synthesis is probably up-regulated
because of an increased availability of caffeic acid (48). Caffeic acid
can be converted to sinapic acid by the sequential activity of
COMT/AldOMT, ferulic acid 5-hydroxylase (F5H)/coniferyl
aldehyde 5-hydroxylase (CAld5H), and COMT/AldOMT. Because of
the lack of 4CL activity toward sinapic acid, it cannot take part in
syringyl biosynthesis, and it is detoxified and stored as GSA.
p-Hydroxybenzoic acid formation can be mediated by a
-oxidation mechanism (conversion 1) (47) or, more probably, by an
NAD-dependent p-hydroxybenzaldehyde
dehydrogenase (conversion 2) (52) acting on p-coumaric acid.
The formation of sinapyl alcohol from coniferyl alcohol remains
ambiguous (5, 13, 14, 17, 27). PAL, phenylalanine
ammonia-lyase; C4H, cinnamic acid 4-hydroxylase;
C3H, coumaric acid 3-hydroxylase; CCoA3H,
coumaroyl-CoA 3-hydroxylase; CCR, cinnamoyl-CoA reductase;
CAD, cinnamyl alcohol dehydrogenase.
-D-glucopyranosyl-caffeic acid
(GCA),
O4--D-glucopyranosyl-vanillic
acid (GVA), and
O4--D-glucopyranosyl-sinapic acid
(GSA) accumulate in the methanol-soluble phenolics fraction of the
transgenic wood. These data indicate that when the flux toward
monolignol biosynthesis is reduced in vivo by
down-regulating CCoAOMT, the pathway from caffeic acid to sinapic acid
is followed. Furthermore, because the lignin amount is reduced and GSA
accumulates up to 10% of the total soluble phenolics, our data
indicate that sinapic acid is not used as a primary lignin precursor.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
17 and +240 relative to the ATG start codon of
CCoAOMT-1B, respectively), hereby introducing a
BamHI site at the 5'-end of the coding region. After
BamHI and HindIII digestion, the fragment was
cloned in the BamHI/HindIII sites of vector
pLBR19. pLBR19 is a pUC19-derived vector containing the P70 and the
CaMV terminator sequence, kindly provided by L. Jouanin (INRA
Versailles, France). Subsequently, the KpnI/XbaI
P70-CCoAOMT fragment was cloned into the
KpnI/XbaI sites of pBIBHYG, resulting in vector
p5'-asCCoAOMT.
-D-thiogalactoside after 4 h of bacterial growth,
according to the Bulk and RediPack glutathione S-transferase
purification protocol (Amersham Pharmacia Biotech). The fusion protein
was purified using the glutathione-Sepharose 4B RediPack columns
(Amersham Pharmacia Biotech). The fusion protein was cleaved with
thrombin. The glutathione S-transferase and CCoAOMT protein
fragments were then separated by SDS-polyacrylamide gel
electrophoresis. Approximately 200 mg of CCoAOMT protein, cut out from
the polyacrylamide gel, was used for immunization of a rabbit according
to standard procedures at the Laboratoire d'Hormonologie Animale
(Marloie, Belgium).
C
29.80). Peaks for unacetylated and acetylated
p-hydroxybenzoic acid esters were assigned by comparison with data from methyl or ethyl p-hydroxybenzoates in the NMR
Database of Lignin and Cell Wall Model
Compounds,2 entries 17, 18, and 94.
1.4 kV; cone
voltage = 40 V; source temperature = 40 °C; collision
energy = 30 eV; collision gas = argon; collision
pressure = 1 × 10
3 torr. The
collision-induced dissociation was performed on the [M H] ions. MS data were acquired in the m/z
range from 100 to 600 Da, and MS/MS data were acquired in the range
from 50 to 450 Da.
4.76. The
1H-90° pulses were 5.5 µs. Presaturation was chosen to
suppress the water peak by using a saturation delay of 2.5 s at a
power of 1 dB. Data were analyzed with the software package from
Advanced Chemistry Development Inc. (Toronto, Canada). Nuclear
Overhauser effect (NOE) difference spectroscopy was performed
essentially as described (28).
H]
molecular ion at m/z 329.3 and fragment ions at
m/z 108.1, 123.2, 152.2, and 167.2; compound D
had a [M H] ion at m/z
341.3 and fragment ions at m/z 135.2 and 179.2;
and compound E had a [M H] ion at
m/z 385.3 and fragment ions at m/z
149.2, 164.2, 208.2, and 223.2. All three compounds showed a loss of
162 Da, suggesting an O-glycosidic bond. Molecular masses of
167.2, 179.2, and 223.2 Da gave evidence that the respective aglycones
of compounds X, D, and E were isovanillic or vanillic, caffeic, and
sinapic acid analogues, respectively. The assignment of the isomeric
form was derived from 1H NMR (Table
I).
1H NMR data for products X, D, and E
Structure Assignment of the Glycosylated Vanillic Acid
Analogue (Compound X)--
The resonances for H-6A' and H-6B' were
typical for the resonances of an exocyclic CH2OH group in
glucopyranose. These data and the data in Table I demonstrated that the
sugar residue was a -D-glucopyranosyl residue (29). The
1H NMR data showed three resonances in the region of the
aromatic protons (two doublets: 7.194 with J = 8.4 Hz and 7.577 with J = 1.7 Hz; one doublet of
doublets: 7.510 with J = 8.4 and 1.7 Hz), which is
typical for the substitution pattern of a phenyl ring with two vicinal
protons and one isolated proton meta-positioned to one of
these. Although two possibilities could be considered, GVA or
O3--D-glucopyranosyl-isovanillic
acid, extensive NOE difference spectroscopy experiments clearly pointed
to the former.
Structure Assignment of the Glycosylated Caffeic Acid Analogue
(Compound D)--
The nature of the sugar was derived from the
1H NMR data and led to the identification of a
-D-glucopyranosyl residue. The substitution pattern of
the phenyl group was derived from the splitting of the three
resonances. Two possibilities for compound D could be considered:
O4--D-glucopyranosyl-caffeic acid
or GCA. The latter structure was confirmed by the NOE difference
spectroscopy experiments. The 1H NMR spectrum of compound D
was a mixture of the trans and cis isomers of
which the parameters were assigned from the magnitude of the vicinal
coupling constant of the double bond (16.1 Hz for trans,
12.5 Hz for cis) as well as from the chemical shifts as calculated from Pascual's tables (30).
Structure Assignment of the Glycosylated Sinapic Acid Analogue
(Compound E)--
At 5.036, a disturbed doublet for the anomeric
proton was found. The magnitude of these coupling constants pointed to
a -D form of the gluco-configuration (29). Integration
of the 1H NMR showed two O-methyl substituents
on the aromatic system, collapsing at 3.867. A singlet at 6.983 that integrated for two protons was found in the aromatic region. NOE
difference spectroscopy and an H-predictor consideration (Advanced
Chemistry Development) confirmed that compound E was GSA, whereas
O4--D-glucopyranosyl-2,6-dimethoxy-4-hydroxycinnamic
acid as an alternative possibility was rejected. GSA was a mixture of
cis and trans isomers (30).
Feeding Experiments
Internodes of approximately 15-cm length with one leaf were cut
from 3-month-old, greenhouse-grown wild-type poplars. These internodes
were put for 16 h overnight into Falcon tubes containing 10 ml of
either distilled water or a 1 mM concentration of the following cinnamic acids: caffeic acid, ferulic acid, 5-hydroxyferulic acid, or sinapic acid. Xylem extraction and HPLC separation of the
metabolites were performed as described above. Quantification was based
on the maximum absorbance value between 230 and 450 nm and expressed as
percentage peak height, i.e. the height of the peak of
interest relative to the sum of all peak heights in the chromatogram.
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Identification of Transgenic Poplars with Reduced CCoAOMT Production-- We have previously shown that CCoAOMT is encoded by two genes in poplar (31). Both genes are expressed at similar levels in poplar stems.3 Because the cDNAs that correspond to both genes share 92% nucleotide identity in the coding region, one of the cDNAs (CCoAOMT-1B) was chosen to make sense and antisense constructs to down-regulate the expression of both CCoAOMT genes. Three CCoAOMT cDNA fragments containing a 235-bp 5'-coding region, 571-bp internal coding sequence, or the complete 744-bp coding sequence of CCoAOMT-1B were cloned in the antisense orientation downstream from the constitutive, double CaMV 35S promoter (P70) in a binary vector. In addition, the entire CCoAOMT-1B sequence was placed behind the P70 promoter in sense orientation. Stem explants from poplar (P. tremula × P. alba clone 717 1-B4) were transformed with A. tumefaciens harboring the binary vectors, and 14-20 independent transformants were obtained for each construct.
The transgenic poplars were screened by protein gel blot analysis for a reduced or increased CCoAOMT protein level in the stem xylem. This analysis was chosen to screen the transgenic lines because bispecific COMT has been shown to O-methylate cinnamoyl-CoA esters too (12, 32) and, consequently, to interfere with the classical CCoAOMT activity assay (33). The antibodies used were generated against a recombinant poplar CCoAOMT protein and reacted with the two poplar CCoAOMT isoforms (34).
Of all transgenic plants analyzed, only those transformed with a sense
construct had a strongly reduced protein amount. None of the transgenic
lines harboring the sense construct showed an increased CCoAOMT level
in the stem xylem. Out of 14 transgenic lines transformed with the
sense construct, the first screening yielded four lines with reduced
CCoAOMT protein amount. In subsequent harvests, two lines, sccoaomt-16
and sccoaomt-29, were shown to be stably down-regulated (Fig.
2). The amount of residual CCoAOMT protein present in xylem extracts was measured by quantifying the
hybridization intensity of the 29-kDa band, corresponding to CCoAOMT,
in immunoblot experiments using 35S-labeled secondary
antibodies. When the CCoAOMT protein level of wild type was set at
100 ± 35.8 (n = 4), the CCoAOMT protein levels of
lines sccoaomt-16 and sccoaomt-29 were 10.2 ± 4.0 (n = 3) and 8.4 ± 1.9 (n = 3),
respectively.
|
CCoAOMT Down-regulation Coincides with Altered Xylem Coloration and
Enhanced Vessel Cell Wall Fluorescence--
After the bark was peeled
off, the stem xylem of the transgenic lines with a reduced CCoAOMT
protein level displayed a pink-red coloration (Fig.
3D). Line asccoaomt-1,
harboring the full-length antisense construct, had a mottled pink-red
coloration (data not shown). A strong reduction of the CCoAOMT amount
was observed in the pink-red-colored xylem region, whereas a normal
CCoAOMT amount was detected in neighboring whitish xylem, showing the direct relation between color change and reduced CCoAOMT protein level
(Fig. 2). A similar variable pattern of gene silencing has been
observed in transgenic poplars transformed with a sense COMT construct (8). The molecular mechanisms responsible for these variable
gene silencing effects are not fully understood yet (35, 36).
|
Microtome sections of the pink-red-colored stem from lines sccoaomt-16 and sccoaomt-29 showed an intense fluorescence upon UV microscopy, which was not observed in sections of wild-type poplar (Fig. 3, A-C). This intense fluorescence was also seen in the pink-red-colored xylem from line asccoaomt-1 and was absent from the neighboring whitish xylem. The intense fluorescence was predominantly found in the cell walls of mature vessel cells, whereas the cell walls of the vessel cells close to the cambium did not show this fluorescence.
The overall plant morphology of the transgenic lines with a reduced CCoAOMT activity was indistinguishable from wild type. By genomic DNA gel blot, lines asccoaomt-1, sccoaomt-16, and sccoaomt-29 were found to be independent transformants (data not shown).
Analysis of Lignin and Associated Low Molecular Weight
Phenolics--
To determine the effect of CCoAOMT down-regulation on
the quantity and quality of lignin, the Klason method and
thioacidolysis were used along with NMR of isolated lignin fractions.
The Klason method quantifies lignin by weighting the residue left after
hydrolysis of the cell wall polysaccharides with sulfuric acid.
Thioacidolysis and subsequent gas chromatography analysis identify
monomers that are released by selectively breaking the intermonomeric
-O-4 bonds, the major interunit linkages in lignin (37).
An additional analysis of the released dimeric structures allows the
determination of different types of carbon-carbon and diphenyl ether
bonds between monomers (9). 13C NMR of isolated lignin
fractions provides compositional and structural fingerprints for
comparison; detailed studies can identify the nature of individual
components (38).
Klason lignin analysis and thioacidolysis were performed on 6-month-old
greenhouse-grown wild-type poplars and on lines sccoaomt-16 and
sccoaomt-29, harvested in November 1997 and August 1998 (Table II). The lignin analyses showed that the
amount of lignin in the stem varied according to the period at which
the plants were harvested, as already observed for poplar (7). Overall,
a 12% reduction in Klason lignin content was observed in the
down-regulated lines. In addition, an increase of 11% in the S/G ratio
of -O-4-linked monomers was found. The type of chemical
bonds between monomers was not altered; no difference was observed in
the relative frequency of monomers involved in -O-4
bonds, as given by the S + G/Klason values, nor was there any change in
the relative frequency of different types of condensed bonds. The
thioacidolysis procedure applied to permethylated samples allows the
determination of the relative amount of monomers that have free
phenolic end groups (39). Compared with wild-type lignin, the relative
amount of free phenolic end groups on G or S monomers did not differ
significantly in lignin of poplars from lines sccoaomt-16 and
sccoaomt-29 (data not shown).
|
13C NMR analysis of lignin fractions obtained from
7-month-old poplars indicated that lignin from lines sccoaomt-16 and
sccoaomt-29 had only minor structural variations compared with lignin
from wild type (Fig. 4). The core lignin
was almost indistinguishable, but the lignin-attached
p-hydroxybenzoate component was more prominent in the
CCoAOMT-reduced lines. Because these lignins are essentially free of
carbohydrates, the p-hydroxybenzoate groups are attached to
lignin, assumedly at the 
-side chain positions. Like
p-coumarates on grass lignin (24), the
p-hydroxybenzoates have free phenolic end groups as shown by
the chemical shifts of all of the structures' carbons that move in a
predictable way when spectra of acetylated lignins (Fig. 4,
A and B) are compared with those from
unacetylated lignins (Fig. 4, C and D); see
compounds 17 (methyl 4-acetoxybenzoate), 18 (methyl 4-hydroxybenzoate),
and 94 (ethyl 4-hydroxybenzoate) in the NMR Database of Lignin and Cell
Wall Model Compounds.2
|
Alkaline hydrolysis of extract-free wood followed by gas chromatography-MS analysis was performed to recover hydroxybenzaldehydes and hydroxybenzoic acids, associated with lignified cell walls (9, 40). The yield of vanillin or syringaldehyde in the transgenic poplars did not differ compared with wild type. However, in agreement with the lignin characterization by 13C NMR, the yield of p-hydroxybenzoic acid, calculated on the basis of lignin content, increased 2-fold in both transgenic lines as compared with wild type (Table II).
Cross-sections of the stem were taken, but neither the Mäule reaction, which stains S units red, nor the Wiesner reaction, which is indicative for cinnamaldehydes (41), revealed any differences between the wild-type poplars and the lines with reduced CCoAOMT expression (data not shown).
Taken together, these data suggest that a reduced CCoAOMT production leads to a reduced synthesis of both G and S units. Whereas the type of interunit bonds and the amount of free phenolic groups on monomers were not affected, a slight increase in the S/G ratio in the noncondensed lignin fraction was detected. Except for an increase in associated p-hydroxybenzoate, no differences were observed in lignin-associated phenolics in the CCoAOMT-reduced lines compared with wild type.
Changes in the Accumulation of Soluble Phenolics in Wood of
Transgenic Poplars--
The lignin analyses described above suggest
that a significant fraction of the phenylpropanoids that can
potentially be produced are not incorporated into the lignin polymer.
Complementary to the analysis of lignin composition, we compared the
phenolic metabolites present in the methanol-extractable fraction of
xylem cells of wild-type and lines sccoaomt-16 and
sccoaomt-29 by HPLC (Fig. 5). Three peaks
(X, D, and E) were found to be more abundant in extracts of the
CCoAOMT-down-regulated lines. Remarkably, the HPLC fractions containing
compounds D and E were fluorescent, whereas no other HPLC fraction,
derived either from wild type or from the transgenics, showed this
fluorescence.
|
The absorbance spectra (Fig. 6) of
compound X (
max = 253.5 and 291.3 nm) and compound D
(max = 314.9 nm) indicated X to be a dihydroxybenzoic
acid analogue and D a cinnamic acid analogue, respectively. No
structural assumptions could be made for compound E (max = 299.5 nm). Mild acid hydrolysis of the isolated compounds resulted in
more hydrophobic compounds: x, d, and e (Fig. 6). The retention time
and spectra of x, d, and e were consistent with those of isovanillic or
vanillic acid (max = 260.6 and 292.4 nm), caffeic acid
(max = 241.7 and 324.4 nm), and sinapic acid (max = 324.4 nm) analogues, respectively. To elucidate
the structure of these compounds, they were analyzed by electrospray
ionization mass spectrometry (Fig. 7) and
1H NMR and NOE difference spectroscopy (see "Experimental
Procedures"). These analyses revealed unambiguously that compound X
was GVA, compound D was GCA, and compound E was GSA.
|
|
In wild-type poplars, GCA and GSA were only detected in trace amounts,
at concentrations below 0.05 µg/g of wood. In contrast, the
concentration of GCA and GSA in the wood of CCoAOMT-down-regulated poplars was 1-2.5 µg/g and 4-20 µg/g, respectively. The
concentration of GVA was 0.2-0.4 µg/g in wild-type wood and
increased to 1-2.5 µg/g in the transgenic wood. Fig.
8 shows the relative concentrations of
GCA, GVA, and GSA observed in ramets of the transgenic lines. Pearson
product-moment correlation analysis showed a correlation coefficient
(r2) of 0.71 (p = 0.0006, significance level 
= 0.05) between the concentrations of GCA
and GVA in the transgenic lines. In contrast, the
r2 value for the relationship between GCA and
GSA was 0.36 (p = 0.0398), and a nonsignificant
r2 value of 0.20 (p = 0.1460)
was obtained between GVA and GSA. The concentrations for GCA and GVA
seemed to depend to a large extent on environmental factors; poplars
harvested in July 1998 had a relatively low amount of these compounds,
whereas higher concentrations were observed when the same transgenic
lines were harvested in May 1999. The accumulation of GSA was
especially characteristic of the transgenic trees; it always accounted
for more than 10% of the total phenolics in the CCoAOMT-suppressed poplars (Fig. 8).
|
To investigate whether GCA, GVA, and GSA were derived from free cinnamic acids, caffeic acid, ferulic acid, 5-hydroxyferulic acid, and sinapic acid were fed to young stems of poplar. As shown in Table III, feeding caffeic acid and sinapic acid resulted in the accumulation of GCA and GSA, respectively. Glucosides of ferulic acid or 5-hydroxyferulic acid were not detected. Due to the variable concentrations of GVA in wild-type plants, it could not be conclusively determined from the feeding experiments whether the precursor of GVA was any of the free cinnamic acids.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
CCoAOMT Is Involved in the Biosynthesis of G and S Monomers-- A down-regulation of CCoAOMT by 90% in poplar xylem suppressed the amount of lignin by 12%. This lignin depletion is caused by a reduced synthesis of both G and S monomers. As determined by thioacidolysis, the noncondensed fraction of the lignin had an 11% increased S/G ratio. Recently, Zhong et al. (11) have shown that a down-regulation of CCoAOMT activity by 70% in tobacco resulted in a decrease of 50% in lignin amount. Also in tobacco, a slight increase in the S/G ratio has been observed. The fact that the lignin in the two transgenic species is depleted in both G and S monomers argues against the idea that the methylation step accomplished by CCoAOMT is specific for G monomer synthesis as suggested earlier (7, 10). Indeed, recent in vitro enzymatic assays and feeding experiments have shown that monomethoxylated cinnamyl alcohols and cinnamaldehydes can be converted to S units (13, 17, 31, 42), although the poplar COMT/AldOMT did not methylate 5-hydroxyconiferyl alcohol in vitro (15). These observations imply that methylation of caffeoyl-CoA by CCoAOMT can contribute equally to the synthesis of both G and S units (Fig. 1). Consequently, the reduced synthesis of both G and S units in the transgenic plants can be explained by the reduced flux from caffeoyl-CoA to feruloyl-CoA and does not necessarily involve the methylation of 5-hydroxyferuloyl-CoA, a reaction that is carried out by CCoAOMT in vitro.
Accumulation of Phenolic Acid Glucosides-- To obtain insight into the flux of metabolites through the monolignol biosynthesis pathway, we have compared the methanol-soluble phenolic fraction from xylem of wild-type plants and transgenic plants down-regulated for CCoAOMT. Reduced CCoAOMT caused a strong accumulation of GVA, GCA, and GSA, of which the latter two were barely detectable in wild-type wood. Their identities were unambiguously established by UV, MS, and 1H NMR analysis. Glucosylated forms of hydroxycinnamic acids may occur as esters (where the glucose is attached to the carboxyl group) or as glucosides (where the glucose is attached to the phenolic hydroxyl group). The hydroxycinnamic acid glucose esters have a high group transfer potential and are often found to be intermediates in phenylpropanoid metabolism (43). Hydroxycinnamic acid glucosides have been suggested to arise from a common detoxification mechanism employed in plants against reactive phenylpropanoids (44). GCA has been previously detected after infiltration of poplar leaves with caffeic acid. Also in these studies, it was shown that GCA was specifically glucosylated at the O3 position (45). Experiments with poplar stems indeed showed that GCA accumulates upon feeding with caffeic acid. Similarly, feeding sinapic acid resulted in the accumulation of GSA (Table III). Nevertheless, the corresponding glucosides of ferulic acid and 5-hydroxyferulic acid were not detected upon feeding with ferulic acid and 5-hydroxyferulic acid, respectively. Taken together, our data suggest that down-regulation of CCoAOMT results in a decreased flux of caffeic acid to lignin, leaving a larger pool of caffeic acid to be shunted to GCA and, via the intermediate formation of sinapic acid, to GSA. The variable levels of GVA in the wild-type plants did not allow us to determine whether GVA was derived from any of the free cinnamic acids.
For the biosynthesis of vanillic acid, two biosynthetic pathways have
been proposed. Because vanillic acid and ferulic acid have identical
substitutions on the aromatic ring, vanillic acid could be derived from
feruloyl-CoA by a -oxidation mechanism, in analogy with the
synthesis of p-hydroxybenzoic acid from
p-coumaroyl-CoA (Fig. 1) (46). This pathway is unlikely
because feruloyl-CoA is the product of CCoAOMT and is expected to
accumulate only when it is made through the subsequent action of COMT
and 4-coumaric acid:CoA ligase (4CL). If this were the case, the
putative transient excess of feruloyl-CoA would probably be shunted
into the monolignol rather than into the vanillic acid biosynthesis
pathway. In addition, in vitro experiments have shown that
in the presence of caffeic acid, ferulic acid is not a good substrate
for the poplar Pt4CL1.4 Our
data are in favor of another pathway that is active in Vanilla planifolia and has been proposed by Funk and Brodelius (47); caffeic acid is shunted into the vanillic acid biosynthesis pathway by
O4-methylation to produce isoferulic acid.
Subsequently, O3-methylation, oxidative
decarboxylation, and demethylation at the p position
ultimately result in vanillic acid (47). This pivotal role of caffeic
acid as both a lignin and vanillic acid intermediate has been suggested
by experiments, in which the addition of a 4CL inhibitor,
3,4-(methylenedioxy)-cinnamic acid, to V. planifolia cell
suspension cultures resulted in an increase of vanillic acid formation
with a simultaneous decrease in the biosynthesis of ligneous material
(48).
The accumulation of GCA, GVA, and GSA in the CCoAOMT-down-regulated plants is in agreement with recent data obtained from in vitro enzyme studies. It has been shown that coniferaldehyde inhibits the hydroxylation of ferulic acid to 5-hydroxyferulic acid in a noncompetitive manner and that 5-hydroxyconiferaldehyde competitively inhibits the methylation of caffeic acid and 5-hydroxyferulic acid (13, 15). Thus, when the concentrations of coniferaldehyde and 5-hydroxyconiferaldehyde are sufficiently high, the pathway from caffeic acid to sinapic acid is not active. Our data suggest that because of the reduced expression of CCoAOMT, the concentrations of coniferaldehyde and 5-hydroxyconiferaldehyde are reduced and, as a consequence, the path from caffeic acid to sinapic acid is used, leading to the accumulation of GCA, GVA, and GSA, which are storage and/or detoxification products of the acids formed.
Sinapic Acid May Not Participate in S Monomer Biosynthesis in
Vivo--
Whereas the concentrations of GCA and GVA sometimes
approached the detection limit in the transgenic plants, GSA
concentrations always largely exceeded those found in wild-type
poplars. In Brassicaceae, sinapic acid is used for the
synthesis of sinapic acid esters (43). However, the produced sinapic
acid in the CCoAOMT-down-regulated poplars is converted into its
O4--D-glucoside. There is some
dispute about the ability to convert sinapic acid to sinapyl alcohol,
because 4CL is inefficient in the activation of sinapic acid to its CoA
ester. Although Grand et al. (49) showed that the poplar 4CL
can convert sinapic acid to its thioester, the three distinct poplar
4CL isoforms have since been shown to share similar hydroxycinnamic
acid substrate utilization profiles but to be inactive against sinapic
acid (50). A similar situation regarding the inactivity of 4CL toward
sinapic acid has been observed in other species too (51). On the other hand, when sinapic acid, labeled with six deuterium atoms at both methoxyl groups, is fed to Robinia pseudoacacia and
Nerium indicum, labeled syringyl units with six deuterium
atoms can be found back in the "derivatization followed by reductive
cleavage" products, strongly indicating that a route from sinapic
acid to sinapyl alcohol does occur in
plants.5 Our data show that
GSA is derived from sinapic acid and accumulates up to 10% of the
soluble phenolics; this result, together with the observation of a
reduced lignin amount in the CCoAOMT-down-regulated plants, however,
indicate that endogenously produced sinapic acid does not play a major
role in the conversion of sinapic acid toward S unit synthesis in
vivo.
Down-regulation of CCoAOMT Results in an Increased Incorporation of
p-Hydroxybenzoate--
Our 13C NMR and alkaline hydrolysis
experiments have shown the increased incorporation of
p-hydroxybenzoate in lignin of CCoAOMT-down-regulated poplars. p-Hydroxybenzoic acid formation can be mediated by
a -oxidation mechanism acting on p-coumaroyl-CoA (Fig. 1,
conversion 1) (46) or, more probably, by an NAD-dependent
p-hydroxybenzaldehyde dehydrogenase (conversion 2) (52)
acting on p-coumaric acid. The p-hydroxybenzoate
units are attached to lignin units and are assumed to arise from the
incorporation of p-hydroxybenzoylated monolignols during the
lignification process, giving rise to -acylated units in the lignin
(53). Analogous p-coumarate units are known to be
exclusively attached to lignin side chain -positions (24), predominantly on syringyl units (38, 54).
Down-regulation of CCoAOMT Results in Increased Vessel Cell Wall Fluorescence-- It remains to be investigated whether the quantitative changes, i.e. the accumulation of the phenolic glucosides, the increased incorporation of p-hydroxybenzoate, and the reduced lignin amount, occur in all cell types or only in the vessels. Promoter-GUS analyses and immunolocalizations have shown that in poplar xylem, CCoAOMT is most strongly expressed in developing vessels and in contact ray cells, whereas it is barely detectable in isolation rays and fibers (16). Consequently, the reduction in CCoAOMT is expected to affect lignin biosynthesis in a cell-specific manner. This cell-specific effect is exemplified by UV microscopy that showed an increased fluorescence specifically in the vessel cell walls. At present, it is still unclear whether the altered vessel cell wall fluorescence is due to the glucosides or to the increased incorporation of p-hydroxybenzoate. Because O4-glucosylated cinnamyl alcohols are the transport forms of the monolignols and because UDP-glucose:coniferyl glucosyl transferase, the enzyme responsible for the glucosylation of cinnamyl alcohols also acts upon sinapic acid (55), it is tempting to speculate that at least a fraction of GSA, GVA, or GCA can also be transported to the cell wall. Such an association may account for the altered cell wall fluorescence, because both GCA and GSA were shown to be responsible for a new fluorescence only present in the transgenic plant extracts. However, except for an increase in p-hydroxybenzoic acid, no differences in lignin-associated low molecular weight phenolics were observed in the transgenic lines. Possibly, the quantity of incorporated GVA, GCA, and/or GSA is insufficient to be revealed by either alkaline hydrolysis, thioacidolysis, or NMR, because these compounds may be present at elevated levels only in the vessel cell walls, which account for 10% of the total cell wall volume of wood (56). Although not conclusive, the polymerization of p-hydroxybenzoic acid to sections from wild-type stems using horseradish peroxidase and H2O2 intensified the fluorescence of xylem cell walls (data not shown). Further lignin structural analyses are necessary to clarify unambiguously the nature of the fluorescence.
Similarly, the presumed cell-specific effect of down-regulating CCoAOMT could also explain why only a 12% reduction in total lignin content was observed in the transgenic lines with 90% reduced CCoAOMT protein amount. Because vessel cell walls are richer in G units than fiber cell walls, the preferential effect in vessel cell walls may also be the reason why the S/G lignin composition is slightly higher. If the lower lignin content could be ascribed mainly to the vessels, it remains curious that the overall structure of the cell walls is not affected. In tobacco, a reduction of 50% in Klason lignin amount by down-regulating cinnamoyl-CoA reductase results in a collapse of cell wall structure (57). In contrast, down-regulation of 4CL in poplar yielded a 45% reduced lignin amount, but without detrimental effect on cell wall structure. In the latter plants, the reduced lignin content is compensated for by an increased cellulose deposition (58). Further experiments are needed to investigate whether an increase in cellulose content also complements the reduced lignin content in the CCoAOMT-down-regulated poplars.
In conclusion, our data show that when CCoAOMT is down-regulated
in vivo, the flux through the path toward monolignol
biosynthesis is reduced, resulting in a lower lignin amount.
Concomitantly, the route from caffeic acid to sinapic acid is followed,
leading to the accumulation of cinnamic acid glucosides. The
observation that the lignin amount is reduced while GSA accumulates to
high levels strongly indicates that sinapic acid is not a main
precursor for S unit biosynthesis. Our in vivo data
complement the recently obtained data from in vitro
enzymatic assays that suggested that the route from caffeic acid to
sinapic acid is not involved in lignin biosynthesis (13, 15).
| |
ACKNOWLEDGEMENTS |
|---|
We thank Frédéric Legée for the determination of Klason lignin, Ulrich Matern for providing the OMTmutpBSrev vector, Lise Jouanin for providing the pLBR19 vector, Geert Goeminne for technical assistance, Jan Van Doorsselaere and Marie Baucher for critical reading of the manuscript, Martine De Cock for help in preparing it, and Rebecca Verbanck for artwork.
| |
FOOTNOTES |
|---|
* This work was supported by Flemish government Geconcerteerde Onderzoeksacties Grant 12052293, Fund for Scientific Research Flanders Grants FWO 1.5.515.98N and G.0040.00, and European Union Grant FAIR-PL95424.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b Recipient of a predoctoral fellowship from Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie.
g Postdoctoral fellow of the Fund for Scientific Research (Flanders).
i Supported by the U.S. Department of Agriculture-Agricultural Research Service and in part through U.S. Department of Agriculture-National Resources Inventory Grant 99-2351 (Improved Utilization of Wood and Wood Fiber Section).
j To whom correspondence should be addressed: Dept. Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium. Tel.: 32-9-2645202; Fax: 32-9-2645349; woboe@gengenp.rug.ac.be.
Published, JBC Papers in Press, August 8, 2000, DOI 10.1074/jbc.M006415200
2 The NMR Database of Lignin and Cell Wall Model Compounds (S. A. Ralph, J. Ralph, W. L. Landucci, and L. L. Landucci) is available on the World Wide Web.
3 H. Meyermans, unpublished data.
4 V. Chiang and C. Tsai, personal communication.
5 K. Fukushima and K. Yamauchi, personal communication.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
G, guaiacyl;
4CL, 4-coumaric acid:CoA ligase;
CaMV, cauliflower mosaic virus;
CCoAOMT, caffeoyl-CoA O-methyltransferase;
COMT, caffeic
acid/5-hydroxyferulic acid O-methyltransferase;
AldOMT, 5-hydroxyconiferaldehyde O-methyltransferase;
GCA, O3--D-glucopyranosyl-caffeic
acid;
GSA, O4--D-glucopyranosyl-sinapic
acid;
GVA, O4--D-glucopyranosyl-vanillic
acid;
HPLC, high pressure liquid chromatography;
MS, mass spectrometry;
MS/MS, tandem mass spectrometry;
NOE, nuclear Overhauser effect;
S, syringyl;
bp, base pair.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Higuchi, T. (1985) in Biosynthesis and Biodegradation of Wood Components (Higuchi, T., ed) , pp. 141-160, Academic Press, Inc., Orlando, FL |
| 2. | Baucher, M., Monties, B., Van Montagu, M., and Boerjan, W. (1998) Crit. Rev. Plant Sci. 17, 125-197 |
| 3. | Christensen, J. C., Baucher, M., O'Connell, A. P., Van Montagu, M., and Boerjan, W. (2000) in Molecular Biology of Woody Plants (Jain, S. M. , and Minocha, S. C., eds), Vol. 1 , pp. 227-267, Kluwer Academic Publishers, Dordrecht, The Netherlands |
| 4. | Boudet, A.-M. (1998) Trends Plant Sci. 3, 67-71 |
| 5. | Whetten, R. W., MacKay, J. J., and Sederoff, R. R. (1998) Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 585-609 |
| 6. | Atanassova, R., Favet, N., Martz, F., Chabbert, B., Tollier, M. T., Monties, B., Fritig, B., and Legrand, M. (1995) Plant J. 8, 465-477 |
| 7. | Van Doorsselaere, J., Baucher, M., Chognot, E., Chabbert, B., Tollier, M.-T., Petit-Conil, M., Leplé, J.-C., Pilate, G., Cornu, D., Monties, B., Van Montagu, M., Inzé, D., Boerjan, W., and Jouanin, L. (1995) Plant J. 8, 855-864 |
| 8. | Tsai, C.-J., Popko, J. L., Mielke, M. R., Hu, W.-J., Podila, G. K., and Chiang, V. L. (1998) Plant Physiol. 117, 101-112 |
| 9. | Lapierre, C., Pollet, B., Petit-Conil, M., Toval, G., Romero, J., Pilate, G., Leplé, J.-C., Boerjan, W., Ferret, V., De Nadai, V., and Jouanin, L. (1999) Plant Physiol. 119, 153-163 |
| 10. | Ye, Z.-H., Kneusel, R. E., Matern, U., and Varner, J. E. (1994) Plant Cell 6, 1427-1439 |
| 11. | Zhong, R., Morrison, W. H., III, Negrel, J., and Ye, Z.-H. (1998) Plant Cell 10, 2033-2046 |
| 12. | Meng, H., and Campbell, W. H. (1998) Plant Mol. Biol. 38, 513-520 |
| 13. | Osakabe, K., Tsao, C. C., Li, L., Popko, J. L., Umezawa, T., Carraway, D. T., Smeltzer, R. H., Joshi, C. P., and Chiang, V. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8955-8960 |
| 14. | Maury, S., Geoffroy, P., and Legrand, M. (1999) Plant Physiol. 121, 215-223 |
| 15. | Li, L., Popko, J. L., Umezawa, T., and Chiang, V. L. (2000) J. Biol. Chem. 275, 6537-6545 |
| 16. | Chen, C., Meyermans, H., Burggraeve, B., De Rycke, R., Inoue, K., De Vleesschauwer, V., Steenackers, M., Van Montagu, M., Engler, G., and Boerjan, W. (2000) Plant Physiol. 123, 853-867 |
| 17. | Humphreys, J. M., Hemm, M. R., and Chapple, C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 10045-10050 |
| 18. | Mavandad, M., Edwards, R., Liang, X., Lamb, C. J., and Dixon, R. A. (1990) Plant Physiol. 94, 671-680 |
| 19. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
| 20. | Becker, D. (1990) Nucleic Acids Res. 18, 203 |
| 21. | Bevan, M. (1984) Nucleic Acids Res. 12, 8711-8721 |
| 22. | Kay, R., Chan, A., Daly, M., and McPherson, J. (1987) Science 236, 1299-1302 |
| 23. | Leplé, J. C., Brasileiro, A. C. M., Michel, M. F., Delmotte, F., and Jouanin, L. (1992) Plant Cell Rep. 11, 137-141 |
