Chemical syntheses of caffeoyl and 5-OH coniferyl aldehydes and alcohols and determination of lignin o-methyltransferase activities in dicot and monocot species

Article abstract of DOI:10.1016/S0031-9422(01)00391-0

To investigate the substrate preferences of O-methyltransferases in the monolignol biosynthetic pathways, caffeoyl and 5-hydroxy coniferyl aldehydes were synthesized by a new procedure involving a Wittig reaction with the corresponding hydroxybenzaldehydes. The same procedure can also be used to synthesize caffeoyl and 5-hydroxyconiferyl alcohols. Relative O-methyltransferase activities against these substrates were determined using crude extracts and recombinant caffeic acid O-methyltransferase from alfalfa (Medicago sativa), and crude extracts from the model legume. Medicago truncatula, tobacco, wheat and tall fescue. Extracts from all these species catalyzed methylation of the various monolignol aldehydes and alcohols more effectively than the corresponding hydroxycinnamic acids.

Full text of DOI:10.1016/S0031-9422(01)00391-0

Phytochemistry 58 (2001) 1035–1042
www.elsevier.com/locate/phytochem
Chemical syntheses of caffeoyl and 5-OH coniferyl aldehydes
and alcohols and determination of lignin O-methyltransferase
activities in dicot and monocot species
Fang Chen, Parvathi Kota, Jack W.Blount, Richard A.Dixon*
Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, USA
Received 3 July 2001; accepted 27 July 2001
Abstract
To investigate the substrate preferences of O-methyltransferases in the monolignol biosynthetic pathways, caffeoyl and 5-hydroxy
coniferyl aldehydes were synthesized by a new procedure involving a Wittig reaction with the corresponding hydroxybenzaldehydes.
The same procedure can also be used to synthesize caffeoyl and 5-hydroxyconiferyl alcohols.Relative O-methyltransferase activities
against these substrates were determined using crude extracts and recombinant caffeic acid O-methyltransferase from alfalfa (Medi-
cago sativa), and crude extracts from the model legume Medicago truncatula, tobacco, wheat and tall fescue.Extracts from all these
species catalyzed methylation of the various monolignol aldehydes and alcohols more effectively than the corresponding hydroxy-
cinnamic acids. # 2001 Elsevier Science Ltd.All rights reserved.
Keywords: Medicago sativa; Medicago truncatula; Nicotiana tabacum; Triticum aestivum; Festuca arundinacea; Leguminosae; Solanaceae; Gramineae;
Syntheses; Caffeoyl aldehyde; 5-Hydroxyconiferaldehyde; O-Methyltransferase; Lignin
1. Introduction
alfalfa (Medicago sativa).Alfalfa is the world’s major
forage legume and a species in which lignification has
important implications for forage digestibility and
therefore utilization efficiency (Jung et al., 1997). To
fully understand the opportunities for, and con-
sequences of, genetically modifying lignin O-methyl-
transferases (OMTs), it is important to determine
relative OMT activities against all potential substrates
in the monolignol pathway.Unfortunately, caffeoyl-
and 5-hydroxyconiferyl aldehydes and alcohols are not
commercially available, and present some challenges in
synthesis due to their o-dihydroxy ring substitution.In
the past, 4-hydroxycinnamyl alcohol was synthesized by
reducing 4-hydroxycinnamic acid esters with lithium
aluminum hydride or diisobutylaluminum hydride
(DIBALH) (Quideau and Ralph, 1992; Terashima et al.,
1995).4-Hydroxycinnamaldehyde was obtained by
reduction of cinnamic acid ester or chloride followed by
oxidation of the alcohol by manganese dioxide.These
protocols involve many reaction steps and separations,
leading to poor yields and difficulties with purification.
Daubresse et al.(1994) developed a mild protocol to syn-
thesize coumaryl, coniferyl, and sinapyl aldehydes and
alcohols, performed under phase-transfer conditions.
The current concept of monolignol biosynthesis is a
metabolic grid, through which side chain reduction and
ring hydroxylation/methylation can occur at several
different levels (reviewed in Lewis and Yamamoto 1990,
Whetten and Sederoff 1995, Dixon et al., 2001). This
concept was first proposed following the discovery of
apparently independent pathways for monolignol O-
methylation at the levels of hydroxycinnamic acids and
their coenzyme A esters, catalyzed by caffeic acid O-
methyltransferase (COMT) and caffeoyl CoA O-methyl-
transferase (CCoAOMT), respectively (Ye et al., 1994;
Higuchi 1996).However, in recent years, increasing evi-
dence has pointed to the hydroxylation/methylation of
cinnamyl aldehydes or cinnamyl alcohols in lignin bio-
synthesis (Chen et al., 1999; Osakabe et al., 1999; Li et
al., 2000).
Our recent studies (Guo et al., 2001) have centered on
genetic manipulation of the O-methylation reactions of
guaiacyl (G) and syringyl (S) lignin biosynthesis in
* Corresponding author.Fax: +1-580-221-7380.
E-mail address: radixon@noble.org (R.A. Dixon).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd.All rights reserved.
PII: S0031-9422(01)00391-0

1036
F. Chen et al. / Phytochemistry 58 (2001) 1035–1042
We tried to use this procedure for synthesis of caffeoyl
aldehyde and 5-hydroxyconiferaldehyde but without suc-
cess.Nevertheless, the idea of using a Wittig reaction
(McMurry, 2000) with (1,3 - dioxolan - 2 - yl) - methyltri-
phenylphosphonium bromide inspired us to develop a
new procedure for synthesis of these compounds.We
have found that 18-crown-6 can effectively catalyze the
Wittig reaction and give good yields for synthesis of
monolignol precursors.We here describe the synthetic
procedures, and the use of caffeoyl and 5-hydroxyconiferyl
aldehydes and alcohols to determine substrate preferences
for OMTs in extracts from several plant species.
prepared from 5-hydroxyvanillin (Osakabe et al., 1999).
This protocol demands several steps of reaction and
purification (Fig.1), and gives only moderate overall
yield (reported to be around 25%).Daubresse et al.
(1994) have described mild syntheses of cinnamyl alde-
hydes and alcohols from 4-acetoxybenzaldehyde.The
key step for this procedure is a Wittig reaction with (1,3-
dioxolan-2-yl-methyl)-triphenylphosphonium
bromide
catalyzed by tris-[2-(2-methoxyethoxy)-ethyl]amine (TDA-
1).We therefore attempted to prepare caffeoyl aldehyde
and 5-hydroxyconiferaldehyde using this procedure.
However, in our hands, the yields of dioxolanes
obtained from 3,4-diacetoxybenzaldehyde and 3,4-di-
acetoxy-5 methoxybenzaldehyde were too low and the
predominant products of the reaction were the corre-
sponding piperonals even if the phenolic groups were
already acetylated.To exploit the convenience of
synthesis of cinnamyl aldehydes and alcohols with (1,3-
dioxolan-2-yl-methyl)-triphenylphosphonium bromide,
we tried two alternative catalysts for Wittig reactions, 18-
crown-6 (1,4,7,10,13,16-hexaoxacyclooctadecane) and
tetrabutylammonium bromide, and found that 18-
crown-6 most efficiently catalyzed the reactions with
3,4-diacetoxy-5-methoxybenzaldehyde and 3,4-diacet-
oxybenzaldehyde.The new procedure (Fig.2) described
here requires fewer steps than previous methods, can be
2. Results and discussion
2.1. New synthetic approaches for hydroxycinnamyl
aldehydes and alcohols
To investigate fully the monolignol biosynthetic path-
way, it is a prerequisite to obtain all potential substrates in
the pathway.Caffeoyl aldehyde, caffeoyl alcohol, 5-
hydroxyconiferaldehyde and 5-hydroxyconiferyl alcohol
are all potential substrates for monolignol O-methyl-
transferase but are not commercially available at this
time.In recent reports, 5-hydroxyconiferaldehyde was
Fig.1. Synthesis of 5-hydroxyconiferaldehyde from 5-hydroxyvanillin and monoethyl malonate.

F. Chen et al. / Phytochemistry 58 (2001) 1035–1042
1037
Fig.2. Synthesis of caffeoyl and 5-hydroxyconiferyl aldehydes by a Wittig reaction with the corresponding hydroxybenzaldehydes and (1,3-dioxo-
lan-2-yl-methyl)- triphenylphosphonium bromide.
performed in ambient atmosphere, and gives higher
yields than previous methods (50–60% being routine in
our hands).
3,4-diacetoxyconiferaldehyde with a yield of approxi-
mately 70%.Fig.5 shows the mass spectra of the
obtained products.The results reported here show that
a Wittig reaction with (1,3-dioxolan-2-yl-methyl)-tri-
phenylphosphonium bromide is a relatively straightfor-
ward method for synthesis of caffeoyl and 5-hydroxy-
coniferyl aldehydes and alcohols in good yield.The
method can be performed with limited demands on
synthetic expertise.
3,4 - Dihydroxybenzaldehyde and 3,4 - dihydroxy - 5 -
methoxybenzaldehyde can be acetylated at both phen-
olic hydroxyls using acetic anhydride under alkaline
conditions.The acetylated products were then mixed
with (1,3-dioxolan-2-yl-methyl)- triphenylphosphonium
bromide, 18-crown-6 and solid K2CO3 in dry dichloro-
methane to produce the corresponding dioxolanes.The
process can easily be monitored by GC/MS in one-hour
intervals to ensure the maximum yield.Dioxolanes are
obtained as isomers with a Z/E ratio of about 60/40, as
described by Daubresse et al.(1994).After removal of
the solid phase by filtration, the mixture can be hydro-
lyzed without further purification.Acid hydrolysis of
the dioxolanes leads to nearly pure (E )-acetylated alde-
hydes as revealed by GC/MS.After purification of the
3,4-diacetoxyconiferyl aldehyde by column chromato-
graphy, the acetyl groups can be removed by trans-
esterification in absolute EtOH.Fig.3 shows the mass
spectra of the resulting caffeoyl aldehyde and 5-hydroxy-
coniferaldehyde after trimethylsilylation.Methoxime
derivatization of the products was also conducted to con-
firm the presence of the aldehyde groups.Fig.4 shows the
mass spectra of caffeoyl aldehyde and 5-hydroxycon-
iferaldehyde after methoximation and trimethylsilylation.
Caffeoyl alcohol and 5-hydroxyconiferyl alcohol can
be obtained by condensation of 3,4-dihydroxybenz-
aldehyde or 5-hydroxyvanillin, respectively, with mono-
ethyl malonate according to the method of Umezawa et
al.(1991).However, the yield for these compounds
(around 20%) is much lower than that reported for the
synthesis of coniferyl alcohol (around 50%).With the
procedure described by Daubresse et al.(1994), we were
able to synthesize caffeoyl alcohol and 5-hydroxyconi-
feryl alcohol from 3,4-diacetoxycaffeoyl aldehyde and
2.2. Substrate preferences of O-methyltransferases in
dicots and monocots
To determine if the various hydroxycinnamyl alde-
hydes and alcohols were effective substrates for alfalfa
COMT, the alfalfa COMT cDNA (Inoue et al., 1998)
was cloned into the pET15b vector and expressed in E.
coli.The His-tagged recombinant COMT enzyme was
purified to homogeneity from the soluble protein using
nickel affinity chromatography.Enzyme assays were
conducted with the usual substrates, caffeic acid, 5-
hydroxyferulic acid, and caffeoyl and 5-hydroxy-coniferyl
aldehydes and alcohols.HPLC/diode-array analyses with
parallel radio-detection showed that all these com-
pounds acted as substrates for recombinant alfalfa
COMT and gave the predicted products (Parvathi et al.,
2001).The relative activites are summarized in Table 1.
These results provide in vitro evidence for a potential
alternative pathway to monolignols involving methyla-
tion of hydroxycinnamyl aldehydes and alcohols.
To determine whether OMT activity against all the
hydroxycinnamyl aldehydes and alcohols was also
found in other plant species, we assayed crude extracts
from alfalfa, Medicago truncatula (a close relative of
alfalfa), tobacco, wheat and tall fescue.The results
(Table 1) indicate that, in each case, all substrates were
effectively methylated, and, in each case, caffeic acid was

1038
F. Chen et al. / Phytochemistry 58 (2001) 1035–1042
Fig.3. Mass spectra of caffeoyl aldehyde ( A) and 5-hydroxyconiferaldehyde (B) after TMSi derivatization.
the poorest substrate.In alfalfa, M. truncatula, tobacco
and fescue, 5-hydroxyferulic acid, caffeoyl aldehyde,
caffeoyl alcohol and 5-hydroxyconiferyl alcohol were
methylated much more effectively than caffeic acid.In
contrast, 5-hydroxyconiferaldehyde was methylated
with about the same efficiency as caffeic acid in extracts
from alfalfa and M. truncatula, whereas this compound
was as good a substrate as the other aldehydes and
alcohols in extracts from wheat.There were no obvious
differences in substrate preferences between monocots
and dicots in this experiment (compare alfalfa and tall
fescue).
alkaloid norcoclaurine.It is not clear whether these
enzymes are also active with caffeoyl aldehyde or 5-
hydroxyconiferaldehyde, although this would not
appear unlikely.To fully address OMT substrate pre-
ferences in vivo, or the potential activities of the large
number of OMTs now appearing in plant gene EST
databases or being identified from proteomic analyses
(Van der Mijnsbrugge et al., 2000; Bell et al., 2001),
even broader ranges of substrates will be needed.The
relatively facile synthetic procedure reported here will
make such substrates more widely available.
Recently, some OMTs have been found to have an
unexpectedly broad range of substrates and even to
exhibit functional overlap between different biosynthetic
pathways.For example, a series of 38 potential sub-
strates have been tested with four recombinant OMTs
of Thalictrum tuberosum (Frick and Kutchan, 1999;
Frick et al., 2001). Some of the enzymes showed similar
catalytic activity with catechol, caffeic acid and the
3. Experimental
3.1. Plant material, enzymes and chemicals
All plants used in this study were grown under stan-
dard greenhouse conditions.Stem tissues were collected
and ground under liquid N2.The powdered tissues were

F. Chen et al. / Phytochemistry 58 (2001) 1035–1042
1039
Fig.4. Mass spectra of caffeoyl aldehyde ( A) and 5-hydroxyconiferaldehyde (B) after methoxime derivatization and TMSi derivatization.
extracted and the soluble proteins were used for enzyme
assay (Parvathi et al., 2001). Expression of alfalfa
COMT in E. coli was performed as described previously
(Parvathi et al., 2001). The enzyme was extracted from
the E. coli cells by sonication and purified from the
soluble fraction using His.bind resin (Novagen, Madi-
son, WI, USA) according to the manufacturer’s proto-
col.3,4-Dihydroxybenzaldehyde, 5-hydroxy vanillin,
(1,3-dixoxolan-2-yl-methyl)-triphenylphosphosphonium
bromide, 18-crown-6, acetic anhydride, and ethyl ace-
tate were purchased from Aldrich-Sigma Co (St.Louis,
MO, USA) and used without further purification.
crown-6 (0.05 mmol) were added. The reaction mixture
was kept at room temperature for 8 h.The organic
phase was separated from the solid phase by filtration.
Aqueous HCl (10%, 50 ml) was added to the organic
portion and the mixture stirred at room temperature for
a further 6 h.During this period, a small amount of
organic phase was taken and subjected to GC/MS ana-
lysis.After 2 h, 3,4-diacetoxyconiferaldehyde could be
detected (MS m/z: 278, 236, 194, 177, 166, 151).At the
end of the reaction, the mixture was diluted with 50 ml
H2O and extracted three times with 100 ml CH2Cl2.The
combined organic layers were washed with saturated
NaHCO3 and saturated aqueous NaCl solution succes-
sively, dried over MgSO4 and evaporated under
vacuum.The residue was dissolved in a small amount of
CH2Cl2 and passed through a silica gel column (eluted
with CH2Cl2: EtOAc, 90:10, v/v).The eluate was mon-
itored by TLC and the portions containing 3,4-diace-
toxyconiferaldehyde were pooled.After evaporation of
3.2. Synthesis procedures
3,4-Diacetoxy-3-methoxybenzaldehyde (5 mmol) and
(1,3-dioxolan-2-yl-methyl)-triphenylphosphosphonium
bromide (5 mmol) were dissolved in CH2Cl2 (80 ml)
with vigorous stirring.Solid K CO3 (5 mmol) and 18-
2

1040
F. Chen et al. / Phytochemistry 58 (2001) 1035–1042
Fig.5. Mass spectra of caffeoyl alcohol ( A) and 5-hydroxyconiferyl alcohol (B) after TMSi derivatization.
Table 1
Methylation of a range of potential monolignol precursors by purified recombinant alfalfa COMT and crude preparations from alfalfa and four
other speciesa
Substrate (50 mM)
OMT activity (pkat mgÀ1 protein)
Recombinant alfalfa COMT
Alfalfa
M. truncatula
Tobacco
Wheat
Tall fescue
Caffeic acid
2989Æ105
9235Æ110
11408Æ725
17286Æ364
1655Æ138
5965Æ624
4.9Æ0.3
15.0Æ0.9
14.4Æ0.9
24.1Æ0.4
5.2Æ0.3
6.4Æ0.1
14.7 Æ1.5
13.3Æ0.7
19.1Æ0.6
6.9Æ0.5
1.6Æ0.1
20.3Æ1.3
20.1Æ0.4
23.8Æ1.7
5.9Æ0.1
7.6Æ0.2
0.7Æ0.04
5.8Æ0.3
5.9Æ0.1
4.6Æ0.3
5.7Æ0.1
4.2Æ0.1
2.6Æ0.3
31.3Æ1.3
24.0Æ0.5
28.4Æ1.1
9.4Æ0.9
5-OH Ferulic acid
Caffeoyl aldehyde
Caffeoyl alcohol
5-OH Coniferaldehyde
5-OH Coniferyl alcohol
13.0Æ0.8
16.0Æ0.7
26.3Æ2.0
Data represent the mean and spread of values for two replicate assays.
a
Crude enzyme extracts from alfalfa, M. truncatula and tobacco were prepared from the 6–9th internodes, the extract from wheat was from the
pseudo stem and leaf sheath, and the extract from tall fescue was from the 1st–4th stem internodes.
the solvent, 3,4-diacetoxyconiferaldehyde was obtained
as a white solid (1.05 g, 72% yield). The 3,4-diacetox-
yconiferaldehyde (0.56 g) was dissolved in 100 ml of 0.2
M KOH in 95% EtOH, and stirred for 8 h under N2.
The solvent was removed under vacuum, and the mix-
ture diluted with 50 ml H2O and extracted with ethyl

F. Chen et al. / Phytochemistry 58 (2001) 1035–1042
1041
acetate (50 ml Â3).The combined organic layers were
References
dried over Na2SO4 and evaporated to give 5-OH con-
iferaldehyde (0.29 g).
Bell, C.J., Dixon, R.A., Farmer, A.D., Flores, R., Inman, J., Gon-
zales, R.A., Harrison, M.J., Paiva, N.L., Scott, A.D., Weller, J.W.,
May, G.D., 2001. The Medicago Genome initiative: a model legume
database.Nucleic Acids Res.29, 114–117.
5-Hydroxyconiferyl alcohol was synthesized from 3,4-
diacetoxyconiferaldehyde (70% yield) according to the
procedure described by Daubresse et al.(1994).Caffeoyl
aldehyde and alcohol were synthesized using the same
procedure starting from 3,4-diacetoxybenzaldehyde
with overall yield of 49–53%.
Bradford, M.M., 1976. A rapid and sensitive method for the quatita-
tion of microgram quatities of protein utilizing the principle of
protein-dye binding.Anal.Biochem.72, 248–254.
Chen, F., Yasuda, S., Fukushima, K., 1999. Evidence for a novel bio-
synthetic pathway that regulates the ratio of syringyl to guaiacyl
residues in lignin in the differentiating xylem of Magnolia kobus DC.
Planta 207, 597–603.
3.3. Derivatization for GC/MS analysis
Dixon, R.A., Chen, F., Guo, D., Parvathi, K., 2001. The biosynthesis
of monolignols: a ‘‘metabolic grid,’’ or independent pathways to
guaiacyl and syringyl units? Phytochemistry 57, 1069–1084.
Daubresse, N., Francesch, C., Mhamdi F., Rolando, C., 1994. A mild
synthesis of coumaryl, coniferyl, sinapyl aldehydes and alcohol.
Synthesis, 369–371.
Methoxime derivatization of the carbonyl groups of
caffeoyl aldehyde and 5-hydroxyconiferaldehyde was
performed usingꢀ0.5 ml MOX reagent (Pierce, Rockford,
IL, USA) at 60 C for 3 h, followed by evaporation to
dryness with N2.TMSi derivatization of the synthesized
products and methoxime derivatives was performed
using 250 ml MSTFA reagent (Pierce, Rockford, IL,
Frick, S., Kutchan, T.M., 1999. Molecular cloning and functional
expression of O-methyltransferase common to isoquinoline alkaloid
and phenylpropanoid biosynthesis.Plant J.17, 329–339.
Frick, S., Ounaroon, A., Kutchan, T.M., 2001. Combinatorial bio-
chemistry in plants: the case of O-methyltransferases.Phytochem-
istry 56, 1–4.
ꢀ
USA) at 30 C for 2 h.
3.4. Gas chromatography/ mass spectrometry
Gowri, G., Bugos, R.C., Campbell, W.H., Maxwell, C.A., Dixon,
R.A., 1991. Stress responses in alfalfa (Medicago sativa L.) X.
Molecular cloning and expression of S-adenosyl-l-methionine:-
caffeic acid 3-O-methyltransferase, a key enzyme of lignin bio-
synthesis.Plant Physiol.97, 7–14.
The synthesized products were identified by GC/MS.
The GC/MS was performed on a Hewlett Packard 5890
series II gas chromatograph with a 5971 series mass
selective detector (column: HP-1, 60 mÂ0.25 mmÂ0.25
mm film thickness).The mass spectra were obtained
using electron ionization (70 eV) with a 60 to 650 m/z
scanning range.
Guo, D., Chen, F., Inoue, K., Blount, J.W., Dixon, R.A., 2001.
Downregulation of caffeic acid 3-O-methyltransferase and caffeoyl
CoA 3-O-methyltransferase in transgenic alfalfa: impacts on lignin
structure and implications for the biosynthesis of G and S lignin.
Plant Cell 13, 73–88.
Higuchi, T., 1996. Biosynthesis of wood components. In: Timell, T.E.
(Ed.), Biochemistry and Molecular Biology of Wood. Heidelberg,
Springer, Berlin, New York, pp.156–168.
3.5. Enzyme extraction and assay
Inoue, K., Sewalt, V.J.H., Ballance, G.M., Ni, W., Sturzer, C., Dixon,
R.A., 1998. Developmental expression and substrate specificities of
alfalfa caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-
methyltranserase in relation to lignification.Plant Physiol 117, 761–
770.
Stem internodes from alfalfa were collected and
ground under liquid N2.The powdered tissue was
extracted for 1 h at 4 C in extraction buffer (100 mM
Tris–HCl, pH 7.5, 0.2 mM MgCl2, 2mM DTT and 10%
ꢀ
Jung, H.G., Sheaffer, C.C., Barnes, D.K., Halgerson, J.L., 1997. For-
age quality variation in the US alfalfa core collection.Crop Sci.37,
1361–1366.
glycerol).The samples were then centrifuged at 12,000 g
ꢀ
for 10 min at 4 C and the extracts desalted on PD-10
columns (Pharmacia).The soluble protein fractions
were used for COMT enzyme assay.The protein con-
centration was measured using Bradford’s reagent (Bio-
Rad) with BSA as standard (Bradford, 1976).COMT
activity was assayed as described previously (Gowri et
al., 1991, Inoue et al., 1998).
Lewis, N.G., Yamamoto, E., 1990. Lignin: occurrence, biogenesis and
biodegradation.Annu.Rev.Plant Physiol.Plant Mol.Biol.41, 455–
496.
Li, L., Popko, J., Umezawa, T., Chiang, L.V., 2000. 5-Hydroxy-
coniferyl aldehyde modulates enzymatic methylation for syringyl
monolignol formation, a new view of monolignol biosynthesis in
angiosperms.J.Biol.Chem.275, 6537–6545.
McMurry, J., 2000. Organic Chemistry. Fifth ed. Brooks/Cole, Pacific
Grove, CA, USA.
Osakabe, K., Tsao, C.C., Li, L., Popko, J., Umezawa, T., Carraway,
D.T., Smeltzer, R.H., Joshi, C.T., Chiang, V.L., 1999. Coniferyl
aldehyde 5-hydroxylation and methylation direct syringyl lignin
biosynthesis in angiosperms.Proc.Natl.Acad.Sci.USA 96, 8955–
8960.
Acknowledgements
We thank Drs.Kazuhiko Fukushima and Seiichi
Yasuda (Nagoya University, Nagoya, Japan) for valu-
able discussions and suggestions, David Huhman and
Dr.Lloyd Sumner for assistance with mass spectro-
metry, and Drs.Lloyd Sumner and Hideyuki Suzuki for
critical reading of the manuscript.This work was sup-
ported by The Samuel Roberts Noble Foundation.
Parvathi, K., Chen, F., Guo, D., Blount, J.W., Dixon, R.A., 2001.
Substrate preferences of O-methyltransferases in alfalfa suggest new
pathway for 3-O-methylation of monolignols.Plant J.25, 193–202.
Quideau, S., Ralph, J., 1992. Facile large-scale synthesis of coniferyl,
sinapyl, and p-coumaryl alcohol.J.Agric.Food Chem.40, 1108–
1110.

1042
F. Chen et al. / Phytochemistry 58 (2001) 1035–1042
Terashima, N., Ralph, S.A., Landucci, L.L., 1995. New facile synth-
esis of monolignol glucosides; p-glucocoumaryl alcohol, coniferin
and syringin.Holzforschung 50, 151–155.
characterization, and seasonal variation of xylem proteins.Planta
210, 589–598.
Whetten, R., Sederoff, R., 1995. Lignin biosynthesis. Plant Cell 7,
1001–1013.
Umezawa, T., Davin, L.B., Lewis, N.G., 1991. Formation of lignans
(À)-secoisolariciresinol and (À)-matairesinol with Forsythia inter-
media cell-free extracts.J.Biol.Chem.266, 10210–10217.
Ye, Z., Kneusel, R.E., Matern, U., Varner, J.E., 1994. An alternative
methylation pathway in lignin biosynthesis in Zinnia.Plant Cell 6,
1427–1439.
Van der Mijnsbrugge, K., Meyermans, H., Van Montagu, M., Bauw,
G., Boerjan, W., 2000. Wood formation in poplar: identification,