Holzforschung 2016; 70(7): 593–602
Open Access
Deded Sarip Nawawi, Wasrin Syafii, Takuya Akiyama* and Yuji Matsumoto
Characteristics of guaiacyl-syringyl lignin
in reaction wood in the gymnosperm Gnetum
gnemon L.
DOI 10.1515/hf-2015-0107
Received May 7, 2015; accepted November 15, 2015; previously
­published online January 6, 2016
Abstract: Gnetum gnemon L. is a unique gymnosperm
s­ pecies showing angiosperm-like features in terms of its
morphology and chemical composition of the cell wall.
Xylan is the main hemicellulose component, and its lignin
is primarily composed of syringyl (S) and guaiacyl (G)
units and small amounts of p-hydroxyphenyl (H) units. In
the present study, in addition to branch, root, bark, and
leaf samples, the reaction wood (RW) taken from the leaning stem of G. gnemon, was investigated mainly by alkaline
nitrobenzene oxidation, ozonation and NMR spectroscopy. The leaning stem was wider on the lower side of the
wood stem (lsW) than on the upper side (usW), similar to
the case for compression wood (CW) in gymnosperms. The
usW contained lignin with a higher S/G ratio, and ­β-O-4
structure had a higher erythro/threo ratio, while both
ratios decreased around the periphery of the stem towards
the lsW. The lignin content was higher towards the lsW.
Overall, the lignin composition in the RW of this tree was
*Corresponding author: Takuya Akiyama, Wood Chemistry
Laboratory, Department of Biomaterial Sciences, Graduate School of
Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku,
Tokyo 113-8657, Japan; and Japan Science and Technology Agency
(JST), PRESTO, Kawaguchi, Saitama 332-0012, Japan,
e-mail: [email protected]
Deded Sarip Nawawi: Wood Chemistry Laboratory, Department
of Biomaterial Sciences, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657,
Japan; and Division of Forest Products Chemistry, Faculty of Forestry,
Department of Forest Products, Bogor Agricultural University (IPB),
Kampus IPB Darmaga Bogor 16680, Indonesia
Wasrin Syafii: Division of Forest Products Chemistry, Faculty
of Forestry, Department of Forest Products, Bogor Agricultural
University (IPB), Kampus IPB Darmaga Bogor 16680, Indonesia
Yuji Matsumoto: Wood Chemistry Laboratory, Department of
Biomaterial Sciences, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657,
Japan
similar to that in the tension wood of angiosperms. The
H-units were minor components in the lignin, but the content was higher towards the lsW, which resembles the distribution of the H-units in a gymnosperm CW.
Keywords: compression wood, erythro and threo forms of
β-O-4 structure, Gnetales, guaiacyl-syringyl lignin, hemicelluloses, p-hydroxyphenyl, plant evolution, reaction
wood, tension wood, xylan
Introduction
The amount of lignin in secondary cell walls of plants and
the chemical structure of the lignin vary between species,
and these variations are closely related to plant evolution
(Higuchi et al. 1977; Vanholme et al. 2010). Guaiacyl (G)
lignin, which is primarily composed of the G units together
with a small amount of p-hydroxyphenyl (H) units, is
typical for ferns and conifers of gymnosperms (Sarkanen
and Hergert 1971; Higuchi et al. 1977; Weng et al. 2008).
Angiosperm lignins additionally contain syringyl (S)
units, and this form is called GS lignin (Higuchi et al. 1977;
Boerjan et al. 2003). However, some species, other than
angiosperms, have been found to contain S units in their
lignins. Substantial proportions of S units are present in
lignin in Selaginella (Jin et al. 2005; Weng et al. 2008; Weng
et al. 2010), Tetraclinis articulata Vahl (Creighton et al.
1944; Leopold and Malmström 1952; Gómez Ros et al. 2007)
and Gnetales species (Creighton et al. 1944; Melvin and
Stewart 1969; Sarkanen and Hergert 1971; Jin et al. 2007).
Gnetales is a small group comprising three genera (80–
100 species; Ephedra, Gnetum, and Welwitschia), and is
often classified to gymnosperms as well as Ginkgo, cycads,
and conifers. However, the exact phylogenetic position of
Gnetales in seed plants seems to be still obscure (Christenhusz et al. 2011). Gnetales has been considered as the sister
group of angiosperms because of its angiosperm-like morphology (Doyle 1996). However, based on molecular genetics studies Gnetales species are more closely related to
©2016, Takuya Akiyama et al., published by De Gruyter.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.
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594 D.S. Nawawi et al.: Lignin in the reaction wood of Gnetum gnemon
gymnosperms than angiosperms (Winter et al. 1999; Bowe
et al. 2000; Chaw et al. 2000; Soltis et al. 2002; Wang 2004;
Zhong et al. 2010). Interestingly, some molecular phylogenetic studies placed Gnetales within conifers as the sister
group of Pinaceae based on the analyses of structural
alteration of the plastid genome (“gnepine” hypothesis)
(Braukmann et al. 2009), whereas some studies using the
large set of nuclear genes supports a “gnetifer” hypothesis,
in which Gnetales are categorized as the sister group of all
conifers (Chaw et al. 1997, 2000; Wickett et al. 2014).
Gnetum gnemon (Gnetales) contains also vessels that
are characteristic for angiosperm wood tissues in addition
to tracheids and fiber tracheids (Carlquist 1994; T
­ omlinson
2001). The leaf cells in G. gnemon also have angiosperm-like
characteristics (Tomlinson and Fisher 2005). Its secondary
cell wall has angiosperm-like features, i.e. the wood contains GS lignin, and a high proportion of 4-O-methylglucuronoxylan in its hemicelluloses (Melvin and Stewart 1969).
G. gnemon also contains glucomannan with a low galactose
content (galactose:glucose:mannose = 0.09:1:1.4), which is
typical for angiosperm hardwoods.
While most Gnetum species are lianas, G. gnemon
is a woody tree and the species forms a reaction wood
(RW). It may be possible to obtain additional information about the classification of G. gnemon based on the
chemical structural features of its RW. Both gymnosperm
and angiosperm trees develop RW with eccentric thickening growth in leaning stems in response to longitudinal
growth stress, but the RW tissues are different in these
plant groups, and their chemical structures are also different in terms of lignin and hemicelluloses composition.
A coniferous gymnosperm generally forms compression wood (CW) on the lower side of the leaning wood
stem or branch (lsW). The lsW of CW is more lignified
than the upper side (usW), and the content of H-units in
the lsW lignin is higher than in the usW lignin (Timell
1986; ­Fukushima and Terashima 1991; Yeh et al. 2006;
­Nanayakkara et al. 2009). In contrast, a general angiosperm wood forms tension wood (TW) on the usW. The
usW of TW is less lignified than the lsW, and the usW
lignin tends to show a higher S/G ratio (Bland 1958;
Akiyama et al. 2003). However, some exceptions have
been found in angiosperm RW, which showed eccentric
radial growth on the lsW similarly to the case for CW.
These include Phyllocladus alpinus Hook.f. (Kučera and
Philipson 1977), Pseudowintera colorata (Raoul) Dandy
(Kučera and Philipson 1978; Meylan 1981), Buxus microphylla var. insularis NaKai (­Yoshizawa et al. 1993, 1999),
B. sempervirens L. (Baillères et al. 1997), Viburnum odoratissimum var. awabuki (Wang et al. 2010), and Hebe salicifolia G. Forst. (Pennel) (Kojima et al. 2012).
The neutral sugar composition of hemicelluloses are
also different in TW and CW as the latter often yields a large
amount of galactose, indicating that the lower cellulose
content is accompanied by higher galactose and lignin contents (Timell 1969; Timell 1986; Nanayakkara et al. 2009;
Kibblewhite et al. 2010). In TW, the lsW of a trunk will generally give rise to a relatively larger amount of xylose and
thus the lower yield of glucose originating from cellulose
is compensated (Timell 1969). Interestingly, in the study of
Altaner et al. (2010) concerning the cell wall structure of
an inclined stem of Cycas micronesica K.D. Hill (cycad), the
tracheid tissue gave rise to more xylose than mannose by
neutral sugar analysis, which is unusual for gymnosperms.
This study focuses on the chemical structures of
G. gnemon RW, and the characteristics of the hemicelluloses and lignin were investigated by neutral sugar analysis, alkaline nitrobenzene oxidation, ozonation methods
and NMR spectroscopy, aiming at understanding whether
the RW of G. gnemon is gymnosperm- or angiosperm-like.
Materials and methods
Dioxane was distilled over Na. All other chemicals were purchased
from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) or Tokyo
Chemical Industry, Co., Ltd. (Tokyo, Japan). NMR spectra (proton and
gradient HSQC) were acquired by a Bruker Avance 600 MHz spectrometer fitted with a 5 mm TCI gradient cryoprobe (Bruker, Fällanden,
Switzerland). Acetylated milled wood lignin ­(Acetylated MWL, 50 mg)
was dissolved in 0.5 ml CDCl3. The central chloroform solvent peak
was used as an internal reference (δH 7.26, δC 77.0 ppm). The traditional numbering system for lignins (Sarkanen and Ludwig 1971) was
followed rather than the systematic IUPAC numbering scheme. In
the HSQC experiments, 1846 data points were acquired from 10.5 to
-0.5 ppm in F2 (1H), with an a
­ cquisition time of 140 ms, and from 200
to 0 ppm in F1 (13C) with 512 increments, 16 scans, and a 1.0 s interscan
delay, with a total acquisition time of 2 h 39 min. Processing the final
matrix of 2 k by 1 k data points was performed by means of a squared
sine-bell in both F2 and F1. The NMR signals were assigned according
to the literature based on acetylated lignin model compounds and the
NMR database (Ralph et al. 2009). The # refers to the library number:
in case of β-O-4 (#3, #74, #29, #97, #98, #214, Hauteville et al. 1986;
Sipilä and Syrjänen 1995), β-β (#109 and #123), β-5 (#2005 and Li et al.
1997), and dibenzodioxocin (#278 and Karhunen et al. 1995).
Samples were collected from a G. gnemon tree with a ­leaning stem
in Bogor, Indonesia. A wood disk with 22 cm Ø, a branch (8 cm Ø), a
lignified root (4 cm Ø), and bark and leaf samples were collected (Figure 1). Blocks of wood were cut from the xylem at six positions along
the periphery of the disk (Figure 2). The angle around the periphery
of the wood disk from the leaning stem was defined as shown in Figure 2, with 0° (360°) being on the lower side of the wood stem (lsW)
and 180° being on the upper side (usW). Eccentric thickening growth
had occurred on the lsW of the disk, indicating that this side contains
reaction wood (RW), more precisely compression wood (CW).
Each wood block, branch, root, bark, and leaf sample was
ground in a Wiley mill to give a 40–60-mesh powder. Each cell wall
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D.S. Nawawi et al.: Lignin in the reaction wood of Gnetum gnemon 595
Leaning tree
(G. gnemon)
Branch, root
bark, leaf
Stem wood disc
usW
180°
120°
240°
60°
300°
0°
lsW
Cell wall meals
Klason lignin
NBO
Ozonation
Wood meals
at 0°(lsW), 60°
120°, 180°(usW)
240°, 300°
MWL
(lsW, usW)
Neutral sugar analysis
NMR
Figure 1: Gnetum gnemon samples and their analysis methods
examined in this study. usW: upper side of the wood stem, lsW: lower
side of the wood stem, MWL: milled wood lignin, NBO: nitrobenzene
oxidation. Figure 2 shows the sampling from wood discs.
Upper side
180°
120°
240°
22 cm
Pith
Heartwood
60°
Sampling position
Sapwood
300°
0°
Lower side
Figure 2: Cross-section of the Gnetum gnemon leaning stem, with
the positions marked of sampling. The peripheral position at 0°
( = 360°) is the lsW (compression wood side) and the position at
180° is the usW (the opposite wood side).
sample was pre-extracted with ethanol/benzene (1:2, v/v) for 8 h in a
Soxhlet apparatus. Then, the Klason lignin content was determined
and the wood was submitted to alkaline nitrobenzene oxidation
(NBO). A fine pre-extracted powder was prepared, in a vibratory ball
mill (Retsch type MM200, Verder Scientific Co., Ltd., Tokyo, Japan,
vibration rate of 30 s-1 for 10 min) before ozonation analysis.
Milled wood lignin (MWL) was isolated according to Björkman
(1956) with minor modifications. The pre-extracted wood meals prepared from the usW (180°) and the lsW (0°) of the G. gnemon wood
disk were ground further in a ball mill (planetary mill; Pulverisette
5, Fritsch Japan Co., Ltd. Yokohama, Japan; 300 rpm) in an environment at 4°C for 11 h, while the milling was interrupted after every
15 min for 15 min to avoid excessive heat development. Zirconium
dioxide jars (500 ml) containing ZrO2 balls (80 pieces, each with a
10 mm Ø) were used. Each ball-milled wood meals (20.3 g for the usW
(180°) sample and 14.0 g for the lsW (0°) sample) was extracted twice
with dioxane/water (96:4, v/v, 10 ml g-1 sample) overnight at r.t. Each
extract, called crude MWL, was freeze-dried, suspended in water,
and then filtered through a hydrophilic PTFE membrane filter (0.2
μm pore size; Advantec Toyo Kaisha, Ltd., Tokyo, Japan). The residue
was dissolved in a small amount of dioxane/water (96:4, v/v, 1 ml),
then poured into diethyl ether (100 ml) to precipitate the MWL. The
MWL yields were 0.99 g (180° sample) and 1.30 g (0° sample), which
are equivalent to 17.6% and 32.1% yields, respectively. Each MWL
sample (100 mg) was acetylated overnight at r.t. with Ac2O (0.5 ml)
and pyridine (1.5 ml).
The lignin content (L) of a pre-extracted wood meal or cell wall
meal sample was determined as the sum of the Klason lignin (KL)
and the acid-soluble lignin (ASL) contents (Swan 1965; Dence 1992)
according to Akiyama et al. (2005).
Neutral sugar analysis was carried out based on the alditol
acetate method (Borchardt and Piper 1970; Blakeney et al. 1983) with
minor modifications. A pre-extracted wood meal sample (100 mg) was
treated with 72% H2SO4 (1 ml) for 4 h at r.t. The mixture was diluted
with deionized water to give to a H2SO4 concentration of 4%; then the
mixture was heated to 120°C for 1 h in an autoclave. The resulting suspension was cooled, filtered, and washed with water until the filtrate
solution had a total volume of 100 ml. A 1 mg ml-1 myo-inositol solution (1 ml) was added to an aliquot of the filtrate (5 ml) as an internal
standard. The pH of the hydrolysate was adjusted to 5.5 with Ba(OH)2
solution. After centrifugation, the monosaccharides in the supernatant were reduced with NaBH4 (20 mg) at r.t. for 2 h; then the excess
NaBH4 was decomposed with acetic acid. The solution was dried
under reduced pressure, and the boric acid was removed by repeated
evaporation with a small amount of MeOH (3 × 2 ml). The residue was
acetylated by adding 2 ml of Ac2O and heating the mixture at 120°C
for 3 h. The resulting alditol acetates were analyzed by GC-FID (Shimadzu 14B, Kyoto, Japan, using a TC-17 column, GL science, Tokyo,
Japan). The monosaccharide yields (i.e. glucose, mannose, xylose,
galactose, and arabinose) are recalculated as a polysaccharide yield
by a factor 0.88 (132/150) for pentose and 0.90 (162/180) for hexose.
The proportions of S and G units were evaluated by the alkaline nitrobenzene oxidation (NBO) (Chen 1992) according to the
procedure of Akiyama et al. (2005). The NBO products of wood meal
samples were analyzed as trimethylsilyl derivatives by GC-FID (Shimadzu 17A, Kyoto, Japan, IC-1 column, GL science, Tokyo, Japan).
The product yields of syringaldehyde (Sald) and syringic acid (Sacid),
vanillin (Vald) and vanillic acid (Vacid), and p-hydroxybenzaldehyde
(Hald) and p-hydroxybenzoic acid (Hacid) are expressed based on the
sample’s lignin content. The syringyl ratio was defined as (Sald+Sacid)/
( Sald+Sacid+Vald+Vacid).
The erythro/threo ratios of the β-O-4 structures in the lignin of
wood meals and cell wall samples were determined by the ozonation
method (Matsumoto et al. 1986) according to Akiyama et al. (2002).
The sample weight was 50 mg and 10 μmol of erythritrol was applied
as internal standard, and an IC-1 column was applied for the GC separation of the ozonation products. The yields of erythronic acid (E)
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596 D.S. Nawawi et al.: Lignin in the reaction wood of Gnetum gnemon
and threonic acid (T) in the ozonation products were determined. The
erythro ratio was defined as the E/(E+T) ratio.
Results and discussion
Reaction wood lignin
Wood samples were taken from different parts of a wood
disk from the leaning stem of G. gnemon tree (Figure 1).
Eccentric thickening growth was observed on the lsW of
the disk, and this is typical for compression wood (CW) in
gymnosperms (Figure 2). The CW character is also visible
based on the lignin contents and the neutral sugar composition in the disks (Figure 3a), i.e. the lignin content was
higher in the CW and, as a consequence, the glucose yield
(representative for the relative amount of cellulose) was
lower in the CW.
a
b
Figure 3: Chemical compositions around the periphery of the
Gnetum gnemon leaning stem. (a) Sugar yields obtained through
neutral sugar analysis, (b) lignin contents. The yields of monomeric
sugar released by acid hydrolysis are expressed as polysaccharide yields calculated by the conversion factors 0.88 and 0.90
“monosaccharides to polysaccharides” for pentoses and hexoses,
respectively. The peripheral positions 0° ( = 360°) and 180° are the
lsW and usW of the leaning stem, respectively (Figure 2 shows the
sampling). Glc, Glucose; Xyl, xylose; Man, mannose; Ara, ­arabinose;
Gal, galactose; KL, Klason lignin; ASL, acid-soluble lignin;
Total = KL+ASL.
The main products of the neutral sugar analysis of the
G. gnemon wood were glucose and xylose accompanied
by smaller amounts of mannose, arabinose, and galactose (Figure 3a), which resembled the sugar composition
of angiosperm woods (Timell 1967; Timell 1969). These
data confirmed the findings of Melvin and Stewart (1969),
who isolated 4-O-methylglucuronoxylan from G. gnemon
as the main hemicellulose fraction, while the moiety of
­glucomannan was low.
Nevertheless, the RW of G. gnemon was different from
both a typical tension wood (TW) and CW because of the
lower xylose and higher mannose yields in the lsW. Opposite to the xylose distribution in a common TW, the xylose
yield in the G. gnemon RW was lower at the lsW than from the
usW (Figure 3a). The mannose yield was 3.3 times higher at
the lsW. A large part of the glucose and xylose decrements
were offset by higher mannose yield and lignin content,
thus the total of neutral sugars and lignin contents were
similar at different peripheral positions (81.2±0.71%). The
galactose yield remained low in this context (Figure 3a),
which implied the presence of glucomannan in the lsW
(Melvin and Stewart 1969). The presence of galactan was
not indicated in the RW in the present study (Meier 1962;
Kuo and Timell 1969; Altaner et al. 2007).
HSQC spectra revealed the presence GS lignin in
G. gnemon (Figure 4a). The alkaline NBO yielded substantial amounts of Sald and Sacid as well Vald and Vacid (Table 1).
The S yield was comparable to that of angiosperm species
(0.30–2.44 mmol g -1 lignin; Akiyama et al. 2005), although
the S/V ratio of 1.2 is lower than the corresponding finding
(1.9) of Melvin and Stewart (1969). It was confirmed that
G. gnemon wood contains GS lignin, as was the case for
four Gnetales species: Ephedra trifurca Torr. ex S.Watson,
Gnetum indicum (Lour.) Merr., Welwitschia mirabilis
Hook.f., and Ephedra sinica Stapf (Creighton et al. 1944;
Jin et al. 2007).
The side-chain region in the HSQC spectra of the
G. gnemon MWL samples from both the usW and lsW of
the leaning stem reflected the presence of a GS lignin
(Figure 4b). The spectra were also indicative of the dominance of β-O-4 structures and resinols accompanied by
relatively small amounts of 5-linked structures, such
as phenylcoumarans. These features are typical for GS
lignins (Stewart et al. 2009). Dibenzodioxocin structures
(Karhunen et al. 1995; Wagner et al. 2007; Terashima et al.
2009), which also belong to 5-linked structures, typical for
gymnosperm lignins, were not found in the HSQC spectra.
Although the HSQC peak patterns of the both MWLs
were similar to each other (Figure 4a and 4b), the 1H-NMR
spectra (Figure 4c) showed differences. A positive peak for
S units was found at 6.6 ppm and a broad negative peak
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D.S. Nawawi et al.: Lignin in the reaction wood of Gnetum gnemon 597
a
100
a1) Upper side
b
50
Bβ
b1) Upper side
Cβ
S2/6
Methoxyl
Aγ + ...
110
G2
60
Bγ
Aα
G5
G6
13
a2)
6.0
80
Cα
Bα
8.0 7.5 7.0 6.5 1H
100
C
13
Aβ
C
130
ppm
Lower side
70
Cγ
120
5.5
90
4.5 1H 4.0
5.0
3.0 ppm
3.5
50
Bβ
b2) Lower side (compression side)
Cβ
S2/6
Methoxyl
Aγ + ...
110
G2
60
Bγ
Aα
G5
G6
13
70
Cγ
120
C
13
Aβ
C
130
ppm
Cα
Bα
8.0 7.5 7.0 6.5 1H
6.0
80
5.5
90
4.5 1H 4.0
5.0
3.0 ppm
3.5
OCH3
6
S
MeO
HO
OMe
O
6
5
G
β
α
γ
HO
O 4
5
β
α
OCH3
O
β
Methoxyl
α O
O
O
A
β-aryl ether
(β-O-4)
OMe
γ O
OCH3
OCH3
2
O
c
O
γ
HO
2
OCH3
O
B
phenylcoumaran
(β-5)
OCH3
C
resinol
(β-β)
Unresolved,
Unassigned,
saccharides etc.
c) Gnetum gnemon (MWL-Ac)
G2
G5 S2/6
G6
CHCl3
β-O-4
(α position)
c1) Upper side
c2) Lower side
Methoxyl
c3) Diff. spectrum
(c1 – c2)
8.0
7.0
6.0
5.0
4.0
H
1
ppm 3.0
Figure 4: NMR spectra of the acetylated MWLs isolated from the usW (a1, b1 and c1) and the lsW (a2, b2 and c2) of the leaning Gnetum
gnemon trunk (CDCl3 NMR solvent). (a) aromatic regions, (b) side chain regions of short-range 13C-1H correlation (HSQC) spectra. (c1 and c2)
1
H-NMR spectra. (c3) Difference spectrum obtained by subtracting the 1H-NMR spectrum c2 from the spectrum c1. The 1H-NMR spectra were
normalized prior to the subtraction procedure being performed so that the total signal intensities from 2.5 to 8 ppm remained the same.
S, Syringyl; G, guaiacyl; MWL, milled wood lignin; Ac, acetylated. Figure 2 shows the sampling from the trunk.
for G units at 6.9–7.0 ppm when the lower-side 1H-NMR
spectrum was subtracted from the upper-side spectrum,
indicating that the S/G ratio was higher in the MWL of
usW. However, these results cannot be generalized for the
whole lignin, as the MWL yields were low, more precisely
MWL yields in the present study was 17.6% for the usW,
which was much lower than the 32.1% yield for the lsW.
The difference in the yields may influence the comparison
of S/G ratios of usW and lsW because the partitioning of
lignin often occurs during isolation (Fujimoto et al. 2005;
Capanema et al. 2015). In this context, the results of NBO
of the wood meals are more representative of the whole
lignin, although the condensed units in lignin do not contribute for the S/G ratio determination by this method.
The proportion of S-type products in total of S- and
G-type products (syringyl ratio) obtained by NBO on
the leaning G. gnemon stem is presented in Figure 5a1.
The highest ratio was found at the 180° position (usW),
and the ratio decreased towards the lsW (i.e. in the CW
moiety), this tendency is typical for TW stems (Bland 1958;
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598 D.S. Nawawi et al.: Lignin in the reaction wood of Gnetum gnemon
Table 1: Lignin content (L) and yields of nitrobenzene oxidation (NBO) and ozonation (OZ) for the Gnetum gnemon stem, branch, root, bark,
and leaf samples.
KLb (%) ASLb
(%) Stem, usWa Stem, lsWa Branch, usWa Branch, lsWa Root
Bark
Leaf
25.56 27.06 24.50 25.33 26.57 28.50 18.07 2.19 1.83 2.56 2.17 2.18 4.39 5.94 Sample
Total Lb
(%) 27.74 28.89 27.06 27.50 28.75 32.89 24.01 Yield (mmol g-1 l)
NBO S:V:Hc OZ
E:Td (S+G+H) (E+T)
1.23:1:0.02 0.85:1:0.02 1.82:1:0.03 1.23:1:0.03 0.82:1:0.03 0.52:1:0.10 0.18:1:0.87 2.14:1 1.86:1 2.33:1 2.14:1 1.85:1 1.67:1 1.20:1 2.17 2.15 2.23 2.21 1.87 1.30 0.24 1.33
1.20
1.28
1.25
0.93
0.49
0.10
The upper side (usW) and lower side (lsW) are the peripheral positions 180° and 0°, respectively, on the wood disk shown in Figure 2. bKL,
Klason lignin; ASL, acid soluble lignin; total L=KL+ASL. cS, Total syringaldehyde+syringic acid; V, total vanillin+vanillic acid; and H, total
p-hydroxybenzaldehyde+p-hydroxybenzoic acid. dE: erythronic acid, T: threonic acid.
a
Akiyama et al. 2003). As shown in Figure 5a2, the higher
syringyl ratio on the usW can be attributed not only to
a higher yield of S-type degradation producs but also to
a lower yield of G-type products. It is noteworthy that a
similar distribution of S and G units were found in CWs
a
b
a1
erythro ratio [E/(E+T)]
b1
a2
b2
Figure 5: Structural differences in the Gnetum gnemon lignins
within the reaction wood stem. (a) The syringyl ratio (a1) determined
from the yields of NBO products (a2). (b) The erythro ratio of the
β-O-4 structures (b1) determined from the yields of the ozonation
products (b2). The peripheral positions 0° ( = 360°) and 180° are the
lsW and usW of the leaning stem, respectively (Figure 2 shows the
sampling). Syringyl ratio S/(S+V), erythro ratio E/(E+T). S, total of
syringaldehyde and syringic acid; V, total of vanillin+vanillic acid; H,
p-hydroxybenzaldehyde+p-hydroxybenzoic acid; E, erythronic acid
erythronic acid; T, threonic acid.
of angiosperm woods although the information about the
S/G ratio of the CW lignin is limited to a few species, in
which a higher S/G ratio in the usW was indicated in the
RW wood of B. microphylla by the Mäule and Wiesner color
reactions, of B. sempervirens by thioacidolysis, and of
V. odoratissimum by NBO (Yoshizawa et al. 1993; Baillères
et al. 1997; Wang et al. 2010).
On the basis of the total yields of erythronic (E) and
threonic (T) acids obtained by ozonation (Matsumoto
et al. 1986; Akiyama et al. 2002), the β-O-4 structures in
the G. gnemon lignin were further characterized. The total
yield of the ozonation products (E+T) was 1.33 mmol g -1
lignin (Table 1 and Figure 5b2), which corresponds to 0.27
per C9 phenylpropanoid unit, assuming that the C9 units
have a molecular weight of 200. This value is comparable
to that found for various wood species (1.07–1.85 mmol g -1
lignin) by Akiyama et al. (2005).
The G. gnemon lignin contained β-O-4 structures with
more erythro than threo forms, as typical for GS lignins
(Figure 5b). In gymnosperm lignins the erythro and threo
forms are in a similar range, but in angiosperm lignins
the erythro form dominates as demonstrated by NMR
­(Lundquist 1979; Nimz et al. 1984; Bardet et al. 1998) and
ozonation studies (Matsumoto et al. 1986; Akiyama et al.
2005; Akiyama et al. 2015). The predominance of the
erythro form is the result of stereo-selective water addition
to a quinone methide intermediate during biosynthesis,
and the selectivity is different for a S- or a G-type aromatic
ring (Figure 6) (Brunow et al. 1993). The proportion of the
erythro form (erythro ratio) was highest at the usW (180°)
of the leaning G. gnemon stem and lowest at the lsW (0°),
as was the case for the syringyl ratio (Figure 5a1). The
higher erythro ratio on the usW was the consequence of
the higher erythronic acid yield but not of the lower threonic acid yield (Figure 5b2). Accordingly, the G. gnemon
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D.S. Nawawi et al.: Lignin in the reaction wood of Gnetum gnemon 599
CH2OH
R′
H
C
O
H
C
OH
R′
R
R
O erythro form
CH2OH
R′
H
C
O
HO
C
H
R′
R
O threo form
R
R′
H
OCH3
OCH3
R″
H
: p-hydroxyphenyl
H
: guaiacyl
OCH3: syringyl
β-O-4 Structures
Figure 6: Structural variation of β-O-4 structures.
lignin on the usW contains more β-O-4 structures than did
the lignin on the lsW. The erythro ratio and syringyl ratio
distributions in the G. gnemon RW were similar to those
found in TW of the angiosperm Liriodendron tulipifera L.
(Akiyama et al. 2003).
H-units were not observed in the HSQC spectra of
the G. gnemon lignin, probably because of their low concentrations, but NBO of the G. gnemon RW yielded small
amounts of H-type products (Table 1 and Figure 5a2).
The low yields 0.018–0.027 mmol g -1 lignin tended to be
slightly higher on the lsW than on the usW. The distribution of H-type products in the trunk was similar to that in
a typical CW, whereas overall the RW lignin of G. gnemon
had the general characteristics of a TW lignin.
Branch, root, bark, and leaf lignin
The lignin contents in the G. gnemon stem, branch, root,
bark, and leaf samples, determined as the sum of the
klason lignin (KL) and acid-soluble lignin (ASL) contents,
varied over a wide range, from 24.0 to 32.9% (Table 1). The
ASL contents of the stem and branch were 2.2–2.6% on a
dry wood weight basis. In general, the ASL contents of
gymnosperm stems are less than 0.5% (Musha and Goring
1974). The ASL contents found in the G. gnemon samples
were within a range found for angiosperms GS lignins
(Akiyama et al. 2005).
The branch, root, bark, and leaf samples contained
GS lignin, as did the RW trunk (Table 1). NBO gives rise to
higher S yields and simultaneously to lower G yields. Consequently, a wide range of syringyl ratios was found (0.55,
0.65, and 0.45 for stem, branch, and root, respectively),
revealing the structural variation of lignins in these parts.
The proportion of H-type product to the total oxidation
products, H/(S+G+H), was much higher for the bark and
leaf samples than for the other. The ratio was 6.2% for
the bark and 42.4% for the leaves, while the ratios for the
stem, branch, and root xylem samples were all less than
2%, similar to the ratios found for wood stems of different species (Akiyama et al. 2005). The total yields (S+G+H)
were significantly lower for the bark and leaf samples
than for the other samples, as were the ozonation product
yields (E+T). The low yields found for the bark and leaf
samples could have been partially caused by the uncertainty in the results of the KL determination for leaf and
bark because of the presence of other condensable products than lignin (Chang and Mitchell 1955; Solar et al.
1992; Jin et al. 2003; Chow et al. 2008). The results for the
branch were similar to the results for the RW in stem, the
usW of the branch having a slightly lower lignin content,
a higher syringyl ratio, and a higher erythro ratio than the
lsW of the branch (Table 1). Furthermore, the erythro ratios
of the β-O-4 structures in the leaf, bark, stem, branch, and
root samples varied widely, and closely matched to the
distribution of the syringyl ratios (R2 = 0.976).
Conclusions
Eccentric thickening growth was found on the lsW of the
leaning trunk of a G. gnemon tree, similar to that expected in
gymnosperm compression wood (CW). However, G. gnemon
contained GS lignin and its hemicellulose composition was
similar to that woody angiosperms. Overall, the lignin in
the G. gnemon reaction wood (RW) stem had similar characteristics to tension wood (TW). Specifically, the usW of the
leaning stem contained a lower lignin content, lignin with
a higher S/G ratio, and β-O-4 units with a higher erythro/
threo ratio than did the lsW. Unlike lignin in most CW, the G.
gnemon lignin from the leaning stem contained only trace
amounts of p-hydroxyphenyl (H) units. However, slightly
more H units were found in the lignin from the lsW than
from the usW of the stem, implicating that the RW lignin
in G. gnemon conserves some characteristics of CW lignin.
Acknowledgments: This work was supported by a
­Grant-in-Aid for Scientific Research (17208015) from the
Ministry of Education, Culture, Sports, Science, and
­Technology of Japan (MEXT) and the Japan Science and
Technology Agency, PRESTO. NMR experiments were carried out at the Yokohama City University in Japan, with
support from the Project for Creation of Research Platforms
and Sharing of Advanced Research Infrastructure (MEXT).
The authors also gratefully acknowledge two anonymous
reviewers provided helpful comments on this paper.
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