NMR evidence for benzodioxane structures resulting from incorporation of 5-hydroxyconiferyl alcohol into lignins of O-methyltransferase-deficient poplars

Article abstract of DOI:10.1021/jf001042+

Benzodioxane structures are produced in lignins of transgenic poplar plants deficient in COMT, an O-methyltransferase required to produce lignin syringyl units. They result from incorporation of 5-hydroxyconiferyl alcohol into the monomer supply and confirm that phenols other than the three traditional monolignols can be integrated into plant lignins.

Full text of DOI:10.1021/jf001042+

86
J. Agric. Food Chem. 2001, 49, 86−91
NMR Evid en ce for Ben zod ioxa n e Str u ctu r es Resu ltin g fr om
In cor p or a tion of 5-Hyd r oxycon ifer yl Alcoh ol in to Lign in s of
O-Meth yltr a n sfer a se-Deficien t P op la r s
J ohn Ralph,*,†,‡ Catherine Lapierre,§ Fachuang Lu,†,‡ J ane M. Marita,†,‡ Gilles Pilate,#
J an Van Doorsselaere, Wout Boerjan, and Lise J ouanin|
U.S. Dairy Forage Research Center, Agricultural Research Service, U.S. Department of Agriculture,
Madison, Wisconsin 53706; Department of Forestry, University of Wisconsin, Madison, Wisconsin 53706;
Laboratoire de Chimie Biologique, Institut National Agronomique, 78850 Thiverval-Grignon, France;
Unite´ Ame´lioration, Ge´ne´tique et Physiologie Forestie`res, INRA Orleans, B.P. 20619 Ardon,
45166 Olivet, France; Departement Plantengenetica, Vlaams Interuniversitair Instituut voor
Biotechnologie, Ledeganckstraat 35, 9000 Gent, Belgium; and Biologie Cellulaire, INRA,
78026 Versailles Cedex, France
Benzodioxane structures are produced in lignins of transgenic poplar plants deficient in COMT, an
O-methyltransferase required to produce lignin syringyl units. They result from incorporation of
5-hydroxyconiferyl alcohol into the monomer supply and confirm that phenols other than the three
traditional monolignols can be integrated into plant lignins.
Keyw or d s: Lignin; benzodioxane; 5-hydroxyconiferyl alcohol; lignan; O-methyltransferase; anti-
sense; sense-upregulation; transgenic plants; NMR
INTRODUCTION
downregulated, incorporation of hydroxycinnamyl al-
dehydes into the lignins increased and new products
were found (10).
Lignins are traditionally described as plant polymers
resulting from dehydrogenative polymerization (via
radical coupling reactions) of three primary phenylpro-
panoid monomers, p-coumaryl (4-hydroxycinnamyl), co-
niferyl (4-hydroxy-3-methoxycinnamyl), and sinapyl
(3,5-dimethoxy-4-hydroxycinnamyl) alcohols, analogues
varying in their degree of methoxylation (1). They
represent a class of complex polymeric natural products
present in large quantities in the cell walls of terrestrial
plants.
Recent advances in genetic engineering have allowed
researchers to perturb the monolignol biosynthetic
pathway (2-5), allowing normally minor components to
be substantially enhanced and therefore structurally
analyzed (6-10). This approach provides valuable in-
sights into the control of lignification and into the
remarkable biochemical flexibility of the lignification
system. For example, Arabidopsis plants completely
devoid of syringyl units in their lignins were found in a
chemical-mutagenesis-derived mutant (11, 12) deficient
in ferulate 5-hydroxylase, for which the preferred
substrate is now recognized to be coniferyl aldehyde
rather than ferulate (13, 14). More strikingly, when the
gene was reintroduced into the mutant background,
with a suitable promoter, the guaiacyl level was <3%,
far lower than in any plant reported to date (15).
Similarly, when the final enzyme on the monolignol
pathway [cinnamyl alcohol dehydrogenase (CAD)] was
Benzodioxane lignans resulting from cross-coupling
of monolignols (coniferyl or sinapyl alcohol) with 5-hy-
droxyconiferyl alcohol have been reported. Compound
1, in Figure 1a, the cross-product of sinapyl alcohol and
5-hydroxyconiferyl alcohol, was isolated from the wood
of Xanthoxylum nitidum (Fagara nitida) (16), a plant
used as a folk medicine in the tropics. NMR methods
(selective INEPT) were used to identify the coupling
regiochemistry (4-O-â/5-O-R versus 5-O-â/4-O-R). Glu-
cosides of the cross-products of coniferyl alcohol and
5-hydroxyconiferyl alcohol, isomers 2a and 2b, were
isolated from whole plants of Pedicularis verticillata
(17), a Chinese medicinal plant.
The first suggestion that 5-hydroxyconiferyl alcohol
can be incorporated into lignins was the observation of
the benzodioxane 3 (Figure 1b) in hydrogenolysis prod-
ucts from Fraxinus mandshurica var. japonica (18).
Later, analytical thioacidolysis produced 5-hydroxyguai-
acyl monomers from a brown-midrib mutant of maize
(19), specifically the bmr3 mutant deficient in caffeic
acid O-methyltransferase (COMT) (20), an O-methyl-
transferase operating late in the lignin biosynthetic
pathway. The enzyme, which now appears to preferen-
tially methylate 5-hydroxyconiferyl aldehyde (21) but
will also methylate 5-hydroxyconiferyl alcohol (22), is
one of two enzymes required to produce lignin syringyl
(3,5-dimethoxy-4-hydroxyphenyl) units. J acquet (23)
postulated benzodioxane structures in lignins following
extensive work demonstrating the role of 5-hydroxyco-
niferyl alcohol in the lignification of COMT-deficient
plants. 5-Hydroxyguaiacyl compounds were also noted
in pyrolysates of a wider variety of plants, mainly
tropical grasses and tropical angiosperm woody plants
(24). More recently, with one of the COMT-deficient
* Corresponding author [telephone (608) 264-5407; fax (608)
264-5147; e-mail jralph@facstaff.wisc.edu].
† U.S. Dairy Forage Research Center.
‡ Department of Forestry.
§ Laboratoire de Chimie Biologique.
#
Unite´ Ame´lioration.
Departement Plantengenetica.
| Biologie Cellulaire.
10.1021/jf001042+ CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/28/2000

Benzodioxanes in Lignins of COMT-Deficient Poplar
J. Agric. Food Chem., Vol. 49, No. 1, 2001 87
F igu r e 1. (a) Benzodioxane lignans 1 and 2 previously reported. (b) Benzodioxanes 3-5 observed in lignin degradation products.
lignins were then extracted into CHCl3 and washed with
aqueous EDTA (6 mM, pH 8.0) to remove trace metal con-
taminants.
poplar transgenics used in the study reported here, a
new compound was found among the products of thio-
acidolysis followed by Raney nickel desulfurization (9).
The mass spectrum suggested that it was compound 4
(Figure 1b) or an isomer. We also have preliminary
evidence for the release of compound 5 by derivatization
followed by reductive cleavage, the “DFRC” method (25,
26). None of these methods, however, establishes the
presence of benzodioxanes as such in the lignin polymer.
Here we provide NMR evidence for benzodioxane
units in lignins isolated from poplar (Populus tremula
× Populus alba) transgenics deficient in COMT. The
presence of benzodioxane structures strongly suggests
that 5-hydroxyconiferyl alcohol (a hydroxycinnamyl
alcohol not normally associated with lignification) in-
corporates as a monomer into the lignin.
NMR Sp ectr oscop y. NMR spectra were acquired on a
1
Bruker DRX-360 instrument fitted with a 5-mm H/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 (δC 29.80, δH 2.04).
We used the standard Bruker implementation of the gradient-
selected inverse (1H-detected) HMQC experiment (32, 33) for
the spectra in Figure 3.
Syn th esis of Ben zod ioxa n e Mod el Com p ou n d 13 {3,5-
Bis(h yd r oxym et h yl)-2-(4-h yd r oxy-3-m et h oxyp h en yl)-5-
m eth oxyben zo[1,4]d ioxa n e}. Methyl 4,5-dihydroxy-3-meth-
oxybenzoate (methyl 3-O-methylgallate) was prepared from
methyl gallate by methylation of the borate complex (34).
Coniferyl alcohol was prepared from coniferaldehyde by reduc-
tion using sodium triacetoxyborohydride in ethyl acetate (35).
Radical coupling was accomplished using the method of She
et al. (36). Ag2CO3 (2.53 g, 9.20 mmol) was added to a solution
of methyl 3-O-methylgallate (900 mg, 4.55 mmol) and coniferyl
alcohol (954 mg, 5.3 mmol) in acetone/benzene (1:2, 100 mL)
and the mixture stirred for 18 h at room temperature.
Following filtration through silica gel and solvent evaporation,
the product was purified by flash chromatography using 2:1
petroleum ether (bp range 30-60 °C)/ethyl acetate to yield
the â-O-4/R-O-5-coupled product {methyl 2-(4-hydroxy-3-meth-
oxyphenyl)-3-(hydroxymethyl)-5-methoxybenzo[1,4]dioxane-7-
carboxylate} in 65% yield: δC/δH (assignment) 76.8/4.99 (R),
79.6/4.12 (â), 61.5/3.82,3.52 (γ). Reduction of the ester (150 mg)
with DIBAL (8 equiv) (37) and collection of the white solid
triturated from acetone produced the required compound 13
(115 mg, 85%). trans-13: δH 3.47 (1H, m, γ1), 3.76 (1H, m, γ2),
3.81 (3H, s, 5H-3-OMe), 3.85 (3H, s, G-3-OMe), 3.99 (1H, ddd,
J ) 7.9, 3.9, 2.5 Hz, â), 4.49 (2H, m, 5H-R), 4.95 (1H, d, J )
7.9 Hz, R), 6.53 (1H, ddd, J ) 1.8, 0.75, 0.75 Hz, 5H-6), 6.60
(1H, d, J ) 1.8 Hz, 5H-2), 6.86 (1H, d, J ) 8.1 Hz, G-5), 6.94
(1H, ddd, J ) 8.1, 1.9, 0.5 Hz, G-6), 7.09 (1H, d, J ) 1.9 Hz,
G-2), 7.80 (1H, s, G-4-OH); δC 56.26 (5H-3-OMe), 56.32 (G-3-
OMe), 61.8 (γ), 64.6 (5H-R), 77.0 (R), 79.3 (â), 104.1 (5H-2),
108.6 (5H-6), 111.9 (G-2), 115.7 (G-5), 121.5 (G-6), 129.5 (G-
1), 133.2 (5H-4), 135.5 (5H-1), 145.2 (5H-5), 147.9 (G-4), 148.4
(G-3), 149.8 (5H-3). Acetylation with 1:1 pyridine/acetic an-
hydride produced the triacetate 13-Ac as a colorless oil: δH
1.99 (3H, s, 5H-R-OAc), 2.03 (3H, s, γ-OAc), 2.24 (3H, s, G-4-
OAc), 3.83 (3H, s, 5H-3-OMe), 3.84 (3H, s, G-3-OMe), 4.02 (1H,
dd, J ) 12.4, 4.3 Hz, γ1), 4.28 (1H, dd, J ) 12.4, 3.4 Hz, γ2),
4.40 (1H, ddd, J ) 7.7, 4.3, 3.4 Hz, â), 4.98 (2H, s, 5H-R),
5.04 (1H, d, J ) 7.7 Hz, R), 6.61 (1H, d, J ) 1.9 Hz, 5H-6),
6.66 (1H, d, J ) 1.9 Hz, 5H-2), 7.08 (1H, dd, J ) 8.1, 1.7 Hz,
G-6), 7.11 (1H, d, J ) 8.1 Hz, G-5), 7.26 (1H, d, J ) 1.7 Hz,
G-2); δC 20.45 (G-4-OAc), 20.49 (γ-OAc), 20.8 (5H-R-OAc), 56.36
(G-3-OMe), 56.44 (5H-3-OMe), 63.4 (γ), 66.4 (5H-R), 76.0 (â),
77.0 (R), 106.1 (5H-2), 110.4 (5H-6), 112.8 (G-2), 120.8 (G-6),
123.9 (G-5), 129.9 (5H-1), 133.7 (5H-4), 136.0 (G-1), 141.5
MATERIALS AND METHODS
Gen er a l. Commercially available chemicals were from
Aldrich, Milwaukee, WI, and used without further purification
unless otherwise stated. Ion-trap mass spectra were run on a
Thermoquest (Austin, TX) Trace 2000 GC (Austin, TX) coupled
to a Thermoquest GCQ ion-trap MS.
Tr a n sgen ic P op la r s. The transgenic poplars examined
were (a) an attempted sense-overexpressed transgenic line
with a double 35S promoter in which the COMT activity was
close to zero due to a gene silencing phenomenon, as described
recently (9)ssimilar sense suppression was earlier observed
in quaking aspen (27) and (b) an antisense COMT poplar also
previously described (28, 29).
Lign in Isola tion . Three to four stems from each line of
7-month-old poplars (control and the two transgenics) were
pooled in their respective groups. Before isolation of the poplar
stem cell walls, 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 de-
scribed previously (6, 30). 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 (three cycles of 0.5 h on, 0.5 h off; stainless steel ball
bearings, stainless steel vessels, under ambient atmosphere),
suspended in 50 mM acetate buffer (pH 5.0), and treated with
30 mg of cellulase (Cellulysin Calbiochem-Novabiochem) 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
(31). The final yields of the dioxane soluble lignin fractions
were 65% of the total (Klason) lignins in all three samples.
Isolated lignins (200 mg of each) were acetylated overnight
using acetic anhydride/pyridine, and the solvents were re-
moved by coevaporation with 95% ethanol. Traces of ethanol
were removed by coevaporation with acetone. The acetylated

88 J. Agric. Food Chem., Vol. 49, No. 1, 2001
Ralph et al.
F igu r e 2. Mechanistic scheme for the production of benzodioxanes 12 in lignins via incorporation of 5-hydroxyconiferyl alcohol
6 into a guaiacyl lignin. Only the pathways producing â-ether units are shown, but â-5- (phenylcoumaran) units are also possible.
Cross-coupling with syringyl units is also possible but must be less pronounced in lignins which have a low syringyl content due
to strong COMT downregulation. G ) guaiacyl unit; 5H ) 5-hydroxyguaiacyl unit.
(G-4), 144.9 (5H-5), 145.0 (5H-3), 152.6 (G-3), 168.9 (G-4-OAc),
170.6 (γ-OAc), 170.8 (5H-R-OAc); ms, m/z (rel abundance) 474
(100, M+), 432 (25), 415 (11), 372 (26), 313 (25), 271 (45), 222
(19), 179 (10). Authentication of the 4-O-â/5-O-R-regiochem-
istry was from long-range 13C-1H (HMBC) correlations be-
tween proton-R (4.98 ppm) and the 5H-5-carbon (144.9 ppm);
deficient poplar lignins. As with the recently identified
dibenzodioxocins (38-40), the benzodioxanes are readily
apparent in short-range 13C-1H correlation spectra
(HMQC or HSQC) of acetylated isolated lignins. Well-
resolved contours centered at δC/δH of 76.8/4.98 (R) and
75.9/4.39 (â) are diagnostic; the γ-correlations overlap
with those in other lignin units. Anticipated shifts occur
following acetylation; in the unacetylated lignins (not
shown), the correlations are centered at δC/δH of 76.5/
4.87 (R) and 78.9/4.06 (â). As seen in Figure 3d, the side-
chain correlations agree well with those in a model
compound for the acetylated trans-benzodioxane, 13-Ac,
synthesized by biomimetic cross-coupling reactions be-
tween coniferyl alcohol and a 5-hydroxyguaiacyl unit.
The R-proton shift deviated the most, presumably
because this is an acetylated (and therefore originally
free-phenolic) model rather than a phenol-etherified
structure, which would correspond to most of the units
in the lignin polymer. The presumed cis-benzodioxane
model cis-13-Ac, had its R-proton at 5.38 ppm, signifi-
cantly downfield of that from the trans isomer (at 4.98
ppm); its location beyond the CR/HR contour in either
of the HMQC spectra (Figure 3b,c) suggests that the
trans isomer is the major benzodioxane isomer in the
lignins as well. The 4-O-â/5-O-R- versus 5-O-â/4-O-R-
regiochemistry of the benzodioxane model 13-Ac was
established from long-range 13C-1H (HMBC) correla-
tions between proton-R (4.98 ppm) and the 5H-5-carbon
(144.9 ppm). Strictly, this regiochemical assignment
could not be authenticated in the lignins as the required
long-range coupling delay of >200 ms was well beyond
their proton relaxation times. The assignment here is
therefore by analogy with the coupling reactions used
to produce model 13 and lignan 1 (16) and those
required to produce the structure in lignins. Indepen-
dent authentication must be sought. The possibility that
the correlations might be due to catechol structures
(without the methoxylated aromatic) known to be in the
extractives of poplars is unlikely to be due to the absence
of such structures and the presence of the 5-hydroxy-
guaiacyl monomer and putative benzodioxane 4 in
thioacidolysis products (9).
3
the small J C5H-5-HR coupling constant (apparently ∼1 Hz)
required an HMBC coupling delay of at least 200 ms to readily
observe this correlation. This regiochemistry in the rapidly
relaxing lignins could not be authenticated as the required
long-range coupling delay was beyond their proton relaxation
times. A trans configuration was assigned from coupling
constants (J Râ ) 7.7 Hz); traces of the presumed cis isomer (δ
5.38, J Râ ) 2.7 Hz, R) were also found.
RESULTS AND DISCUSSION
Radical cross-coupling of monolignol radicals 10•
(Figure 2) with the growing lignin oligomer/polymer
(radicals) 7• is the major reaction occurring during
lignification (1). Consequently, it is anticipated that if
5-hydroxyconiferyl alcohol 6 incorporates into lignins,
it will likely do so via radical 6• reacting at its favored
â-position with lignin radicals 7•, at their 4-O- or
5-positions for guaiacyl radicals (only the 4-O-coupling
is shown in Figure 2); syringyl lignin units can react
only at their 4-O-positions. Following trapping by water
of the quinone methide intermediate 8 in the conven-
tional way, a new 5-hydroxyguaiacyl unit 9 is produced.
These bis(phenol)s can also generate radicals via oxida-
tive processes, typically peroxidase and hydrogen per-
oxide, or laccase and oxygen in vivo. Radicals 9• react
readily at their 4-O- positions with monolignol radicals
10• (at their favored â-positions) as seen in the synthesis
of 13. What follows is a logical trapping of the quinone
methide internally by the phenolic 5-OH, effecting
rearomatization and generating a benzodioxane struc-
ture 12. These units may then incorporate more fully
into the lignin by further radical coupling reactions with
normal lignin monomers 10• or radicals from the novel
5-hydroxyconiferyl alcohol monomer 6.
NMR spectra (Figure 3) provide elegant proof that
benzodioxanes are major structures in the two COMT-

Benzodioxanes in Lignins of COMT-Deficient Poplar
J. Agric. Food Chem., Vol. 49, No. 1, 2001 89
F igu r e 3. Presence of benzodioxane structures in COMT-deficient transgenic plants diagnostically revealed by NMR. Partial
spectra from gradient HMQC NMR experiments highlight new peaks (not in the control) for benzodioxane units H in acetylated
lignins from COMT-downregulated transgenic poplars and similar correlations from a benzodioxane model 13-Ac. The major unit
coding is the same as that used in a recent review on NMR of lignins (40). It is suggested that H be reserved for these
5-hydroxyguaiacyl benzodioxane units in lignin NMR spectra.
Recent studies have begun to elucidate the propensity
for the monolignols to cross-couple with (free-phenolic)
guaiacyl and syringyl units in lignins (41, 42). Cross-
coupling of hydroxycinnamaldehydes into lignins has
been recently demonstrated (10). The cross-coupling of
5-hydroxyconiferyl alcohol into the lignins of the COMT-
deficient poplar plants, by the logical mechanism in
Figure 2, is further evidence for our contention that
lignification is metabolically plastic (43). Degradation
experiments are planned to complement these NMR
findings and to establish how intimately 5-hydroxyco-
niferyl alcohol is incorporated into the lignin polymer.
COMT-deficient poplars incorporate 5-hydroxyconifer-
yl alcohol into the lignin polymer whereas the wild-type
controls do not. The statement on lignification by Lewis
(44) that “There is, however, no known precedent for
the free interchange of monomeric units in any biopoly-
mer assembly, then or now, and no biochemical evi-
dence...” must therefore be revisited. When the flux to
the final syringyl monolignol is restricted, a phenolic
intermediate other than the traditional monolignols can
be utilized to make modified lignin polymers with
properties apparently sufficient to accommodate the
water transport and mechanical strengthening roles of
lignin and to allow the plant to be viable, as we have
noted previously (6, 10, 43). Whether such plants will
be able to confront the rigors of a natural environment
replete with a variety of pathogens remains to be
determined. However, having the metabolic flexibility
to allow polymerization of products of incomplete mono-
lignol synthesis along with the traditional monolignols
is a significant advantage to plants; in a single genera-
tion these plants have circumvented genetic obstacles
to remain viable.
ACKNOWLEDGMENT
We are grateful to Paul Schatz (University of Wiscon-
sinsMadison) for discussion on the related lignans and
to anonymous reviewers who provided useful comments.

90 J. Agric. Food Chem., Vol. 49, No. 1, 2001
Ralph et al.
LITERATURE CITED
(19) Lapierre, C.; Tollier, M. T.; Monties, B. A new type of
constitutive unit in lignins from the corn bm3 mutant.
C. R. Acad. Sci., Ser. 3 1988, 307, 723-728.
(1) Freudenberg, K.; Neish, A. C. Constitution and Biosyn-
thesis of Lignin; Springer-Verlag: Berlin, 1968.
(2) Dixon, R. A.; Ni, W. Genetic manipulation of the
phenylpropanoid pathway in transgenic tobacco: New
fundamental insights and prospects for crop improve-
ment. Biotechnol. Biotechnol. Equip. 1996, 10, 45-51.
(3) Baucher, M.; Monties, B.; Van Montagu, M.; Boerjan,
W. Biosynthesis and genetic engineering of lignin. Crit.
Rev. Plant Sci. 1998, 17, 125-197.
(20) Vignols, F.; Rigau, J .; Torres, M. A.; Capellades, M.;
Puigdomenech, P. The brown midrib 3 (bm3) mutation
in maize occurs in the gene encoding caffeic acid
O-methyltransferase. Plant Cell 1995, 7, 407-416.
(21) Li, L.; Popko, J . L.; Umezawa, T.; Chiang, V. L. 5-Hy-
droxyconiferyl aldehyde modulates enzymatic methyla-
tion for syringyl monolignol formation, a new view of
monolignol biosynthesis in angiosperms. J . Biol. Chem.
2000, 275, 6537-6545.
(4) Boudet, A.-M. A new view of lignification. Trends Plant
Sci. 1998, 3, 67-71.
(22) Maury, S.; Geoffroy, P.; Legrand, M. Tobacco O-meth-
yltransferases involved in phenylpropanoid metab-
olism. The different caffeoyl-coenzyme A/5-hydroxy-
(5) Whetten, R. W.; MacKay, J . J .; Sederoff, R. R. Recent
advances in understanding lignin biosynthesis. Annu.
Rev. Plant Physiol. Plant Mol. Biol. 1998, 49, 585-609.
(6) Ralph, J .; MacKay, J . J .; Hatfield, R. D.; O’Malley, D.
M.; Whetten, R. W.; Sederoff, R. R. Abnormal lignin in
a loblolly pine mutant. Science 1997, 277, 235-239.
(7) Ralph, J .; Hatfield, R. D.; Piquemal, J .; Yahiaoui, N.;
Pean, M.; Lapierre, C.; Boudet, A.-M. NMR character-
ization of altered lignins extracted from tobacco plants
down-regulated for lignification enzymes cinnamyl-
alcohol dehydrogenase and cinnamoyl-CoA reductase.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12803-12808.
(8) Ralph, J .; Kim, H.; Peng, J .; Lu, F. Arylpropane-1,3-diols
in lignins from normal and CAD-deficient pines. Org.
Lett. 1999, 1, 323-326.
feruloyl-coenzyme
A
3/5-O-methyltransferase and
caffeic acid/5-hydroxyferulic acid 3/5-O-methyltrans-
ferase classes have distinct substrate specificities and
expression patterns. Plant Physiol. 1999, 121, 215-
223.
(23) J acquet, G. Structure et re´activite´ des lignines de
gramine´es et des acides phe´noliques associe´s: de´vel-
oppment des me´thodologies d’investigation. Ph.D., In-
stitut National Agronomique, Paris-Grignon, France,
1997.
(24) Suzuki, S.; Lam, T. B. T.; Iiyama, K. 5-Hydroxyguaiacyl
nuclei as aromatic constituents of native lignin. Phy-
tochemistry 1997, 46, 695-700.
(25) Lu, F.; Ralph, J . The DFRC method for lignin analy-
sis. 1. A new method for â-aryl ether cleavage: lignin
model studies. J . Agric. Food Chem. 1997, 45, 4655-
4660.
(26) Lu, F.; Ralph, J . Derivatization followed by reductive
cleavage (DFRC method), a new method for lignin
analysis: protocol for analysis of DFRC monomers. J .
Agric. Food Chem. 1997, 45, 2590-2592.
(27) Tsai, C.-J .; Popko, J . L.; Mielke, M. R.; Hu, W.-J .; Podila,
G. K.; Chiang, V. L. Suppression of O-methyltransferase
gene by homologous sense transgene in quaking aspen
causes red-brown wood phenotypes. Plant Physiol. 1998,
117, 101-112.
(28) Van Doorsselaere, J .; Baucher, M.; Chognot, E.; Chab-
bert, B.; Tollier, M.-T.; Petit-Conil, M.; Leple´, J .-C.;
Pilate, G.; Cornu, D.; Monties, B.; Van Montagu, M.;
Inze´, D.; Boerjan, W.; J ouanin, L. A novel lignin in
poplar trees with a reduced caffeic acid/5-hydroxyferulic
acid O-methyltransferase activity. Plant J . 1995, 8,
855-864.
(29) Lapierre, C.; Pollet, B.; Petit-Conil, M.; Toval, G.;
Romero, J .; Pilate, G.; Leple, J . C.; Boerjan, W.; Ferret,
V.; De Nadai, V.; J ouanin, L. Structural alterations of
lignins in transgenic poplars with depressed cinnamyl
alcohol dehydrogenase or caffeic acid O-methyltrans-
ferase activity have an opposite impact on the efficiency
of industrial kraft pulping. Plant Physiol. 1999, 119,
153-163.
(30) Ralph, J .; Hatfield, R. D.; Quideau, S.; Helm, R. F.;
Grabber, J . H.; J ung, H.-J . G. Pathway of p-coumaric
acid incorporation into maize lignin as revealed by
NMR. J . Am. Chem. Soc. 1994, 116, 9448-9456.
(31) Bjo¨rkman, A. Studies on finely divided wood. Part I.
Extraction of lignin with neutral solvents. Sven. Pap-
perstidn. 1956, 59, 477-485.
(9) J ouanin, L.; Goujon, T.; de Nada¨ı, V.; Martin, M.-T.;
Mila, I.; Vallet, C.; Pollet, B.; Yoshinaga, A.; Chabbert,
B.; Petit-Conil, M.; Lapierre, C. Lignification in trans-
genic poplars with extremely reduced caffeic acid O-
methyltransferase activity. Plant Physiol. 2000, 123,
1363-1373.
(10) Kim, H.; Ralph, J .; Yahiaoui, N.; Pean, M.; Boudet, A.-
M. Cross-coupling of hydroxycinnamyl aldehydes into
lignins. Org. Lett. 2000, 2, 2197-2200.
(11) Meyer, K.; Cusumano, J . C.; Somerville, C.; Chapple,
C. C. S. Ferulate-5-hyrdoxylase from Arabidopsis thaliana
defines a new family of cytochrome P450-dependent
monooxygenases. Proc. Natl. Acad. Sci. U.S.A. 1996, 93,
6869-6874.
(12) Meyer, K.; Shirley, A. M.; Cusumano, J . C.; Bell-Lelong,
D. A.; Chapple, C. Lignin monomer composition is
determined by the expression of a cytochrome P450-
dependent monooxygenase in Arabidopsis. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 6619-6623.
(13) Humphreys, J . M.; Hemm, M. R.; Chapple, C. Ferulate
5-hydroxylase from Arabidopsis is a multifunctional
cytochrome P450-dependent monooxygenase cata-
lyzing parallel hydroxylations in phenylpropanoid me-
tabolism. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10045-
10050.
(14) Osakabe, K.; Tsao, C. C.; Li, L.; Popko, J . L.; Umezawa,
T.; Carraway, D. T.; Smeltzer, R. H.; J oshi, C. P.;
Chiang, V. L. Coniferyl aldehyde 5-hydroxylation and
methylation direct syringyl lignin biosynthesis in an-
giosperms. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8955-
8960.
(15) Marita, J .; Ralph, J .; Hatfield, R. D.; Chapple, C. NMR
characterization of lignins in Arabidopsis altered in the
activity of ferulate-5-hydroxylase. Proc. Natl. Acad. Sci.
U.S.A. 1999, 96, 12328-12332.
(16) Ishikawa, T.; Seki, M.; Nishigaya, K.; Miura, Y.; Seki,
H.; Chen, I.-S.; Ishii, H. Studies on the chemical
constituents of Xanthoxylum nitidum (Roxb.) D. C.
(Fagara nitida Roxb.). III. The chemical constituents of
the wood. Chem. Pharm. Bull. 1995, 43, 2014-2018.
(17) Su, B.-N.; Li, Y.; J ia, Z.-J . Neolignan, phenylpropanoid
and iridoid glycosides from Pedicularis verticillata.
Phytochemistry 1997, 45, 1271-1273.
(32) Ruiz-Cabello, J .; Vuister, G. W.; Moonen, C. T. W.; Van
Gelderen, P.; Cohen, J . S.; Van Zijl, P. C. M. Gradient-
enhanced heteronuclear correlation spectroscopy. Theory
and experimental aspects. J . Magn. Reson. 1992, 100,
282-302.
(33) Vuister, G. W.; Ruiz-Cabello, J .; Van Zijl, P. C. M.
Gradient-enhanced multiple-quantum filter (ge-MQF).
A simple way to obtain single-scan phase-sensitive
HMQC spectra. J . Magn. Reson. 1992, 100, 215-220.
(34) Scheline, R. R. A rapid synthesis of 3-O-methylgallic
acid. Acta Chem. Scand. 1966, 20, 1182.
(18) Hwang, B. H.; Sakakibara, A. Hydrogenolysis of proto-
lignin. XVIII. Isolation of a new dimeric compound with
a heterocycle involving R,â-diether. Holzforschung 1981,
35, 297-300.

Benzodioxanes in Lignins of COMT-Deficient Poplar
J. Agric. Food Chem., Vol. 49, No. 1, 2001 91
(35) Lu, F.; Ralph, J . Highly selective syntheses of coniferyl
and sinapyl alcohols. J . Agric. Food Chem. 1998, 46,
1794-1796.
(36) She, X.; Gu, W.; Wu, T.; Pan, X. A convenient synthesis
of (()-3-methoxybenzodioxane-4,9,9′-triol neolignan, and
methyl ethers of isoamericanol A and isoamericanin A.
J . Chem. Res. (S) 1999, 100-101.
(37) Quideau, S.; Ralph, J . Facile large-scale synthesis of
coniferyl, sinapyl, and p-coumaryl alcohol. J . Agric. Food
Chem. 1992, 40, 1108-1110.
(38) Karhunen, P.; Rummakko, P.; Sipila¨, J .; Brunow, G.;
Kilpela¨inen, I. Dibenzodioxocins; a novel type of linkage
in softwood lignins. Tetrahedron Lett. 1995, 36, 169-
170.
(41) Syrjanen, K.; Brunow, G. Oxidative cross coupling of
p-hydroxycinnamic alcohols with dimeric arylglycerol
â-aryl ether lignin model compounds. The effect of
oxidation potentials. J . Chem. Soc., Perkin Trans. 1
1998, 3425-3429.
(42) Syrjanen, K.; Brunow, G. Regioselectivity in lignin
biosynthesis. The influence of dimerization and cross-
coupling. J . Chem. Soc., Perkin Trans. 1 2000, 183-187.
(43) Sederoff, R. R.; MacKay, J . J .; Ralph, J .; Hatfield, R. D.
Unexpected variation in lignin. Curr. Opin. Plant Biol.
1999, 2, 145-152.
(44) Lewis, N. G. A 20th century roller coaster ride: a short
account of lignification. Curr. Opin. Plant Biol. 1999,
2, 153-162.
(39) Karhunen, P.; Rummakko, P.; Sipila¨, J .; Brunow, G.;
Kilpela¨inen, I. The formation of dibenzodioxocin struc-
tures by oxidative coupling. A model reaction for lignin
biosynthesis. Tetrahedron Lett. 1995, 36, 4501-4504.
(40) Ralph, J .; Marita, J . M.; Ralph, S. A.; Hatfield, R. D.;
Lu, F.; Ede, R. M.; Peng, J .; Quideau, S.; Helm, R. F.;
Grabber, J . H.; Kim, H.; J imenez-Monteon, G.; Zhang,
Y.; J ung, H.-J . G.; Landucci, L. L.; MacKay, J . J .;
Sederoff, R. R.; Chapple, C.; Boudet, A. M. Solution-state
NMR of lignins. In Progress in Lignocellulosics Char-
acterization; Argyropoulos, D. S., Rials, T., Eds.; TAPPI
Press: Atlanta, GA, 1999; pp 55-108.
Received for review August 24, 2000. Revised manuscript
received October 20, 2000. Accepted October 24, 2000. We are
grateful for partial funding through USDA-NRI (Grant 99-
02351 in Improved Utilization of Wood and Wood Fiber) and
to the European Community under the OPLIGE (Optimization
of Lignin in Crop Industrial Plants through Genetic Engineer-
ing) and TIMBER (Tree Improvement Based on Lignin Engi-
neering) projects.
J F001042+