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Publicly Available Published by De Gruyter September 22, 2016

Characteristic of β-O-4 structures in different reaction wood lignins of Eusideroxylon zwageri T. et B. and four other woody species

  • Deded Sarip Nawawi , Takuya Akiyama EMAIL logo , Wasrin Syafii and Yuji Matsumoto
From the journal Holzforschung

Abstract

Lignin analyses were performed on the reaction woods of five tropical wood species. The reaction woods of these five species and that of Gnetum gnemon L. (previously reported) were categorized into three types based on eccentric thickening growth and p-hydroxyphenyl/guaiacyl/syringyl (H/G/S) ratios: compression wood (CW) containing GH-lignin observed in gymnosperms (GH-lignin-CW), tension wood (TW) containing GS-lignin observed in angiosperms (GS-lignin-TW), and reaction wood that resembles CW and contains GS-lignin (GS-lignin-CW). GS-lignin-CW is an unusual type that was found in the angiosperm Eusideroxylon zwageri and in the gymnosperm G. gnemon. The erythro/threo ratio of the β-O-4 structures and the S/G ratio were higher on the upper side (usW) of the leaning wood stem or branch, and both ratios decreased along the periphery of the stem toward the lower side (lsW). Except for a difference in thickening growth, these distribution patterns were similar to the GS-lignin-TW patterns for Melia azedarach L. and Avicennia sp. Reaction wood of Paraserianthes falcataria (L.) Nielsen was also classified as a GS-lignin-TW, but this was lacking a clear distribution pattern. In contrast, the GH-lignin-CW of the usW of Pinus merkusii Jungh. et de Vriese had a low erythro/threo ratio, which increased toward the lsW along with increasing lignin contents and H/G ratios.

Introduction

Lignin, a major polymer component of plant cell walls, provides the strength, water resistance, and rigidity of cell walls, thereby providing the mechanical support that allows vascular plants to stand upright. The amount and chemical structure of lignin vary among woody species, with the variations particularly occurring within the reaction wood (RW) of stems or branches (Timell 1983; Timell 1986; Donaldson 2001; Kiyoto et al. 2015). Lignin distribution is thought to play an important role in the adaption of trees to their environment. Regardless of the RW type, the lignin content is relatively higher on the lower side (lsW) of the leaning woody stem than on the upper side (usW) (Bland 1958; Yoshida et al. 2002; Pilate et al. 2004; Yoshinaga et al. 2012).

In response to longitudinal growth stress, coniferous gymnosperms form compression wood (CW) through eccentric radial growth of the lsW. The lsW is more lignified than the usW, and it is enriched with p-hydroxyphenyl units (H-units) (Timell 1986). In contrast, normal and opposite wood lignins (the usW) are primarily composed of guaiacyl units (G-units) and a smaller amount of H-units (Timell 1986; Lapierre et al. 1988; Fukushima and Terashima 1991; Yeh et al. 2006; Nanayakkara et al. 2009). In many cases, angiosperms form tension wood (TW) by eccentric radial growth of the usW, which is less lignified than the lsW (Bland 1958; Timell 1969; Akiyama et al. 2003; Pilate et al. 2004). Similar to conifers that form CW, several angiosperm species show eccentric growth of the lsW in stems or branches. These unusual RWs are found in Gardenia jasminoides Ellis (Aiso et al. 2013), Hebe salicifolia G. Forst. (Pennel) (Kojima et al. 2012), Viburnum odoratissimum var. awabuki (K. Koch) Zabel (Wang et al. 2010), Buxus sempervirens L. (Baillères et al. 1997), Buxus microphylla var. insularis Nakai (Yoshizawa et al. 1993; Yoshizawa et al. 1999), Pseudowintera colorata (Raoul) Dandy (Kučera and Philipson 1978; Meylan 1981), and Phyllocladus alpinus Hook.f. (Kučera and Philipson 1977).

In our previous study of RW lignins (Nawawi et al. 2016), it was demonstrated that Gnetum gnemon L., a gymnosperm from the order Gnetales, forms an unusual RW. The lsW of its leaning stem shows eccentric growth, similar to CW in conifers, although Shirai et al. (2015) reported that the usW of this species shows eccentric growth, similar to TW in angiosperms. The G. gnemon CW primarily contains syringyl units (S-units), G-units, and a smaller amount of H-units in its GS-lignin, and the S/G ratio of its usW is higher than that of its lsW (Nawawi et al. 2016). Similarly, the distributions of S- and G-units in the TW of angiosperm stem RWs are usually uneven (Bland 1958; Timell 1969; Aguayo et al. 2010; Al-Haddad et al. 2013). Several studies on angiosperm RW resembling CW have shown that GS-lignin in CW of the usW has a high S/G ratio. For example, GS-lignin-CW of the usW has high S/G ratios in B. microphylla and G. jasminoides (determined through the Mäule and Wiesner color reactions, see Yoshizawa et al. 1993; Aiso et al. 2013), in B. sempervirens (by thioacidolysis; Baillères et al. 1997), and in V. odoratissimum (by means of alkaline nitrobenzene oxidation, NBO, see Wang et al. 2010). However, information regarding the unusual RW that resembles CW in angiosperms is limited to the lignin content and S/G compositions of these species.

In many cases, the S/G composition affects the composition of the different lignin interunit linkages (Ralph et al. 2006; Stewart et al. 2009; Weng et al. 2011) and influences the characteristics of arylglycerol-β-aryl ether (β-O-4) structures, which are the most predominant type of lignin linkages (Akiyama et al. 2003; Akiyama et al. 2005; Akiyama et al. 2015). In our study of CW from G. gnemon lsW, we found through ozonation that the leaning stem is distinguishable from the usW in terms of the β-O-4 stereo structures (Nawawi et al. 2016).

There appears to be a pattern in the structural differences of lignins in the typical angiosperm TW and coniferous CW. That is, the S/G ratio of the usW of TW tends to be high, and the H/G ratio of the lsW of coniferous CW and the lignin content of the lsW of both CW and TW are high.

In the present paper, the RW resembling CW in a tropical angiosperm, Eusideroxylon zwageri T. et B. (ulin wood), will be investigated by comparing its lignin against the typical GS-lignin-TW and GH-lignin-CW, as well as against the GS-lignin-CW in the gymnosperm G. gnemon. These different types of reaction woods will be compared in terms of lignin content, composition of H, G, and S-units, and the β-O-4 stereo chemistry.

Materials and methods

All chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Japan) or Tokyo Chemical Industry, Co., Ltd. (Japan). Woody stems and branches were harvested in Bogor, West Java, or Bengkulu, Sumatra Indonesia (Table 1 and Figure 1). Wood disks were collected from leaning stems and branches of a tropical gymnosperm (Pinus merkusii Jungh. et de Vriese) and four angiosperms (Paraserianthes falcataria (L.) Nielsen, Melia azedarach L., E. zwageri T. et B., and Avicennia sp.). Wood disks were obtained from P. merkusii and P. falcataria trees at three different heights above ground (1.7, 2.3, and 2.8 m for P. merkusii; 0.5, 1.5, and 2.5 m for P. falcataria). The collection locations and the diameter of the wood disks are listed in Table 1.

Table 1:

Woody species and their type of reaction wood (RW) and lignin.

Woody species (common name)ClassSampleDiameterc (cm)Place of harvestdRW typeeLignin typef
Pinus merkusii (Sumatran pine)GymnospermStema30BogorCWG
Branch13BogorCWG
Gnetum gnemonGymnospermStemb22BogorCWGS
Branchb7BogorCWGS
Eusideroxylon zwageri (ulin)AngiospermBranch13BogorCWGS
Melia azedarachAngiospermStem27BogorTWGS
Branch4BogorTWGS
Avicennia sp.AngiospermStem28BengkuluTWGS
Paraserianthes falcatariaAngiospermStema26BogorTWGS
Branch12BogorTWGS

aWood disks from three different heights above ground. bNawawi et al. (2016). cDiameter of the wood disk collected. dBogor and Bengkulu provinces in Indonesia. eEccentric growth was observed in the upper side (tension wood side: TW) or the lower side (compression wood side: CW) of a branch or leaning stem. fG-lignin, guaiacyl lignin; GS-lignin, guaiacyl-syringyl lignin.

Figure 1: Cross sections of wood disks obtained from the leaning stem or branch of different woody species: (a) Pinus merkusii, (b) Eusideroxylon zwageri, (c) Melia azedarach, (d) Avicennia sp., and (e) Paraserianthes falcataria. Arrows in the pictures indicate the pith position.
Figure 1:

Cross sections of wood disks obtained from the leaning stem or branch of different woody species: (a) Pinus merkusii, (b) Eusideroxylon zwageri, (c) Melia azedarach, (d) Avicennia sp., and (e) Paraserianthes falcataria. Arrows in the pictures indicate the pith position.

Wood blocks were cut from the wood disks at different positions along the periphery of the disks. The position along the periphery was defined in angles, as shown in Figure 1, with 0 (360°) being on the lsW and 180° being on the usW. Wood blocks were also cut from the usW and lsW of branches of P. merkusii, P. falcataria, and M. azedarach.

Each wood block was ground in a Wiley mill to obtain 40- to 60-mesh wood meals, which were pre-extracted with ethanol/benzene mixture (1:2, v/v) for 8 h in a Soxhlet apparatus and were then subjected to the Klason lignin determination and alkaline NBO. Finer wood meals were prepared from the pre-extracted wood meals for ozonation by means of a vibratory ball mill (Retsch, type MM200, Verder Scientific Co., Ltd., Tokyo, Japan) at a vibration frequency of 30 s−1 for 10 min.

The total lignin content is the sum of the Klason lignin content and the acid-soluble lignin content (Dence 1992). A pre-extracted wood meal (500 mg) was hydrolyzed with 72% H2SO4 solution (5 ml) for 3 h at room temperature (r.t.). The mixture was diluted with deionized water to obtain a 3% H2SO4 solution, which was then autoclaved at 120°C for 30 min. The resulting suspension was filtered through a fine glass filter, and the insoluble residue (Klason lignin) was dried at 105°C overnight and then weighed. The concentration of acid-soluble lignin in the filtrate was determined by measuring the UV absorption of the filtrate at 205 nm with the absorption coefficient 110 l g−1 cm−1 (Swan 1965).

The proportions of S and G units in lignin were evaluated through the alkaline NBO method (Chen 1992) with minor modifications as described by Akiyama et al. (2005). Wood meals (40 mg), 2 M NaOH solution (4 ml), and nitrobenzene (0.25 ml) were sealed in a 10-ml stainless-steel autoclave and heated to 170°C for 2 h. The reaction mixture was cooled with ice water, and the internal standard (IS), ethyl vanillin (3-ethoxy-4- hydroxybenzaldehyde; 6 μmol), was added. The mixture was then washed three times with CH2Cl2 (15 ml) and acidified with 2 M HCl solution. The acidified aqueous layer was extracted three times with CH2Cl2 (20 ml) and once with diethyl ether (15 ml). The organic layers were combined, washed with water (20 ml), and dried over Na2SO4. After filtration, the solution was dried at 30°C at low pressure. The residue was trimethylsilylated with bis(trimethylsilyl)acetamide (100 μl) at 100°C for 10 min and then analyzed by gas chromatography (GC-FID) with a Shimadzu 17A instrument equipped with an IC-1 column. The 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 in terms of the lignin content of the wood meals. The syringyl ratio is defined as (Sald+Sacid)/(Sald+Sacid+Vald+Vacid).

The erythro/threo ratio of the β-O-4 structures in lignin was determined through ozonation (Akiyama et al. 2002). Fine wood meals (50 mg) were suspended in AcOH/H2O/MeOH mixture (16:3:1, v/v, 30 ml) in a round-bottom flask, which was then placed in an ice bath. Oxygen containing 3% ozone was bubbled into the suspension at a rate of 0.5 l min−1 for 2 h, and the suspension was stirred continuously. The residual ozone was removed by bubbling oxygen through the mixture, and then the solvent was removed at 40°C at low pressure. Traces of AcOH were removed by repeatedly adding a small amount of water (0.5 ml) and by evaporating the mixture between each addition. The ozonation products were saponified with 0.1 M NaOH solution (20 ml) at r.t. overnight. The IS erythritol (10 μmol) was then added. The reaction mixture was filtered, and the soluble fraction was passed through a column filled with cation exchange resin (10–15 ml; Dowex-50 W-X4 in the NH4+ form). The column was eluted with water until an eluent pH of approximately 7–8 was reached, and the total volume eluted was adjusted to 100 ml. An aliquot of the eluent (2 ml) was dried at low pressure at 40°C. The residue was trimethylsilylated with dimethyl sulfoxide (DMSO) (300 μl), hexamethyldisilazane (200 μl), and trimethylchlorosilane (100 μl) at 60°C for 30 min. The upper layer, which contains the product, was then subjected to GC-FID using a Shimadzu 17A with an IC-1 column to determine the yields of erythronic acid (E) and threonic acid (T) in the ozonation product. The erythro ratio is defined as the E/(E+T) ratio.

Results and discussion

GH-lignin-CW

Eccentric growth was observed in the lsW of the P. merkusii leaning stem (Figure 1), similar to CW of pine and other coniferous species (Timell 1986; Donaldson and Knox 2012). Uncondensed-type NBO products of the P. merkusii CW were mostly G-type products (V=Vald+Vacid) and a smaller amount of H-type products (H=Hald+Hacid; Table 2). The amounts of both lignin and H-type products were higher in the lsW than in the usW, typical of CW (Timell 1986; Lapierre et al. 1988; Fukushima and Terashima 1991; Yeh et al. 2006; Nanayakkara et al. 2009). Figure 2 shows the distributions of lignin and NBO products around the periphery of the stem. The relative yield of H-type products relative to the total NBO products [defined as H/(H+V) ratio] was <2% in the usW, reaching up to 14% toward the lsW. It is noteworthy that all data from P. merkusii show a high correlation between lignin content and p-hydroxyphenyl ratios (Figure 3a, R2=0.98).

Table 2:

Lignin content, yields of nitrobenzene oxidation (NBO) products, and ozonation products obtained from the reaction wood (RW) of Pinus merkusii (GH-lignin-CW).

RW sampleSampling positionLignin contentc (%)NBO yieldd (μmol g−1)H/(H+V)e (%)Ozonationf (μmol g−1)E/(E+T)g (%)
HVE+T
Stem (disk 1)ausWb28.13719481.9103349.3
lsWb33.9231162510.793051.9
Stem (disk 2)ausWb27.53819311.995549.5
lsWb34.9215151912.480253.7
Stem (disk 3)ausWb28.23418651.891249.7
lsWb35.7236144014.171153.5
BranchusWb27.73619591.895549.9
lsWb31.413017207.192851.1

aWood disk samples 1, 2, and 3 from the stem at 2.8, 2.3, and 1.7 m above ground, respectively. bThe upper side (usW) and lower side (lsW) are the peripheral positions on the wood disk at 180° and 0°, respectively (Figure 1). cLignin content, total of Klason lignin and acid soluble lignin. dH, total of p-hydroxybenzaldehyde and p-hydroxybenzoic acid; V, total of vanillin and vanillic acid. Yield is based on lignin. eH/(H+V), p-hydroxyphenyl ratio. fE, erythronic acid; T, threonic acid. gE/(E+T), erythro ratio. Yield is based on lignin.

Figure 2: Structural differences in Pinus merkusii lignins within the reaction wood (GH-lignin-CW): (a) lignin content, (b) p-hydroxyphenyl ratio determined from the yields of NBO products, and (c) erythro ratio of β-O-4 structures determined by the ozonation method. The peripheral positions at 0° and 180° are the lsW and usW, respectively, of the leaning stem (Figure 1 shows the sampling positions). Lignin content is Klason lignin and acid-soluble lignin; p-hydroxyphenyl ratio, H/(H+V); erythro ratio, E/(E+T); H, total of p-hydroxybenzaldehyde and p-hydroxybenzoic acid; V, total of vanillin and vanillic acid; E, erythronic acid; T, threonic acid.
Figure 2:

Structural differences in Pinus merkusii lignins within the reaction wood (GH-lignin-CW): (a) lignin content, (b) p-hydroxyphenyl ratio determined from the yields of NBO products, and (c) erythro ratio of β-O-4 structures determined by the ozonation method. The peripheral positions at 0° and 180° are the lsW and usW, respectively, of the leaning stem (Figure 1 shows the sampling positions). Lignin content is Klason lignin and acid-soluble lignin; p-hydroxyphenyl ratio, H/(H+V); erythro ratio, E/(E+T); H, total of p-hydroxybenzaldehyde and p-hydroxybenzoic acid; V, total of vanillin and vanillic acid; E, erythronic acid; T, threonic acid.

Figure 3: Relationships between p-hydroxyphenyl (H)/guaiacyl (G) composition and lignin content, and the erythro and threo isomeric forms of β-O-4 structures in the reaction wood in a Pinus merkusii stem: (a) lignin content vs. H-ratio, (b) erythro ratio vs. H-ratio, (c) yields of ozonation products vs. H. H/(H+V), H-ratio; E/(E+T), erythro ratio; H, total of p-hydroxybenzaldehyde and p-hydroxybenzoic acid; V, total of vanillin and vanillic acid; E, erythronic acid; T, threonic acid.
Figure 3:

Relationships between p-hydroxyphenyl (H)/guaiacyl (G) composition and lignin content, and the erythro and threo isomeric forms of β-O-4 structures in the reaction wood in a Pinus merkusii stem: (a) lignin content vs. H-ratio, (b) erythro ratio vs. H-ratio, (c) yields of ozonation products vs. H. H/(H+V), H-ratio; E/(E+T), erythro ratio; H, total of p-hydroxybenzaldehyde and p-hydroxybenzoic acid; V, total of vanillin and vanillic acid; E, erythronic acid; T, threonic acid.

The pine CW lignin was further characterized by ozonation according to Akiyama et al. (2002). The content of β-O-4 structures and their erythro/threo ratios were evaluated from the yields of E and T, which were released from the side-chain of the β-O-4 structures by ozonation. In all sampled wood disks, the total yield of E and T of the lsW was lower than that of the yield of the usW (e.g. 1033 and 930 μmol g−1 lignin, respectively, for usW and lsW of stem 1, see Table 2). The interpretation is that β-O-4 structures are less abundant in the lsW lignin (on the CW side) than those in the usW lignin. The E and T yields from all pine samples are negatively correlated with the corresponding H/(H+V) ratios (Figure 3c). This relationship implies that the frequency of the β-O-4 coupling reaction during lignin polymerization is low when the proportion of H-units is high.

The proportion of erythro and threo forms of β-O-4 structures was nearly 50:50 in the usW, but the proportion of the erythro form was slightly higher toward the lsW (Figure 2): the E/(E+T) ratio was 49.5%±0.2% in the usW and 52.8%±0.9% in the lsW for the three CW stem samples (Table 2). In our previous study, the E/(E+T) ratio was within the 50.0%±0.6% range for five gymnosperm species that are mostly composed of G-units, which was determined by ozonation (Akiyama et al. 2005). The E/(E+T) ratio of the lsW of P. merkusii stem is outside this range, thus indicating that its CW lignin contains more of the erythro form of β-O-4 structures than the threo form.

It is worth noting that the distribution of erythro and threo forms is correlated with the distribution of H- and G-units in the CW of the pine stem (Figure 3b). A similar relationship for the CW of loblolly pine (Pinus taeda L.) was detected by Yeh et al. (2006). The coefficients of correlation (R2) between the erythro ratio [E/(E+T)] and the p-hydroxyphenyl ratio for the P. merkusii tree stem sampled at three different heights above ground are high (0.99, 0.94, and 1.00 m for disks 1, 2, and 3, respectively; Figure 2). The higher erythro ratio on the lsW (the CW side) is the consequence of lower abundance of the threo form (Figure 3b and c). The amounts of both erythro and threo forms decreased with increasing H/G ratios; however, the decrease in the erythro form was less than that of the threo form.

For the erythro form to be produced in excess during lignin biosynthesis, stereo-preferential water addition must occur to the β-O-4-bonded quinone methide (QM) intermediate (Figure 4). The QM intermediate is formed as a result of the β-O-4 coupling reaction between a monolignol radical and a radical on a growing lignin polymer. Water can be added to either face of the planar QM. This step produces two isomeric forms of the β-O-4 structures, i.e. the erythro and threo forms (e.g. water addition to the Si face leads to the erythro configuration as shown in Figure 4). In a model experiment, however, water addition to a G-type QM did not promote the erythro form, although in case of S-type QMs, the formation of the erythro configuration was dominating (Brunow et al. 1993). In addition, the erythro/threo ratio was 50:50 in gymnosperm lignins (containing mainly G-lignin). As indicated by the experimental data, the erythro configuration is preferentially formed in the CW of P. merkusii, depending on the presence of H-units. However, the mechanism of this reaction is still unknown.

Figure 4: Formation of the erythro and threo forms of β-O-4 structures by addition of water to quinone methide intermediates.
Figure 4:

Formation of the erythro and threo forms of β-O-4 structures by addition of water to quinone methide intermediates.

GS-lignin-CW

Eusideroxylon zwageri differed from the other three angiosperms as its branch (13 cm Ø) showed eccentric growth of the lsW (Figure 1). The lignin content of the lsW is higher than that of the usW. The RW of this species is atypical, similar to that found in some angiosperms (Kučera and Philipson 1977; Kučera and Philipson 1978; Meylan 1981; Yoshizawa et al. 1993; Baillères et al. 1997; Yoshizawa et al. 1999; Wang et al. 2010; Kojima et al. 2012; Aiso et al. 2013).

The NBO products from the E. zwageri wood meal primarily consisted of S-type products (S=Sald+Sacid) and G-type products and included only <1% of H-type products (Table 3). The syringyl ratio [S/(S+V)] from the branch lsW was 11% (0.12 as S/V ratio), which is lower than the ratios of 16 other woody angiosperm species (15% or more) reported by Akiyama et al. (2005). Therefore, E. zwageri lignin may be regarded as a G-rich GS-lignin. The syringyl ratio of E. zwageri CW increased around the periphery of the stem, reaching 20% toward the usW (Figure 5a). The erythro ratio of the β-O-4 structure [erythro/(erythro+threo)] of the usW was also higher than that of the lsW. The erythro ratio distribution within the wood disk is correlated with the S-ratio distribution with 0.92 R2 between E/(E+T) and S/(S+V). These E. zwageri CW data resemble to those of the CW of the gymnosperm G. gnemon (Nawawi et al. 2016). The GS-lignin-CW of the usW of both species showed higher syringyl and erythro ratios than those of the lsW, although the S-units were more abundant in G. gnemon lignin [S/(S+V)≈46–55%] than in E. zwageri lignin.

Table 3:

Lignin content, yields of nitrobenzene oxidation (NBO) products, and ozonation products obtained from the reaction woods of Eusideroxylonzwageri, Melia azedarach, Avicennia sp., and Paraserianthes falcataria (GS-lignin-CW and GS-lignin-TW).

Wood speciesRW sampleSampling positionLignin contentd (%)NBO yielde (μmol g−1)S/(S+V)f (%)Ozonation yieldg (μmol g−1)E/(E+T)h (%)
HVSE+T
Eusideroxylon zwageriBranchusWb40.614129932520.096554.3
lsWb40.812140117511.186153.0
Melia azedarachStemusWb30.79112578441.1130061.7
lsWb31.910129763532.9106659.3
BranchusWc29.981066103149.2119664.4
lsWc30.710117177539.8104761.8
Avicennia sp.StemusWb27.912472199280.9127172.7
lsWb28.411528190578.3130671.9
Paraserianthes falcatariaStem (disk 1)ausWb29.822984137058.0132967.1
lsWb29.222976146560.0132568.1
Stem (disk 2)ausWb28.1281024125655.0135766.2
lsWb28.819978145459.7142467.6
Stem (disk 3)ausWb28.623995121955.0125366.6
lsWb29.222950133058.2142567.2
BranchusWc18.214778136563.7117367.9
lsWc28.711975133957.9135166.9

aWood disk samples 1, 2, and 3 from the stem at 2.5, 1.5, and 0.5 m above ground, respectively. bThe upper side (usW) and lower side (lsW) are the peripheral positions on the wood disk at 180° and 0°, respectively (Figure 1). cThe upper half (usW) and lower half (lsW) of the branch were examined. dLignin content, total of Klason lignin and acid soluble lignin. eH, total of p-hydroxybenzaldehyde and p-hydroxybenzoic acid; V, total of vanillin and vanillic acid. Yield is based on lignin; S, total of syringaldehyde and syringic acid. fS/(S+V), syringyl ratio. gE, erythronic acid; T, threonic acid. hE/(E+T), erythro ratio. Yield is based on lignin.

Figure 5: Structural differences in the lignins of the GS-lignin-CW and GS-lignin-TW in the stems or branches of (a) Eusideroxylon zwageri, (b) Gnetum gnemon*1, (c) Melia azedarach, (d) Avinennia sp., and (e) Paraserianthes falcataria. The peripheral positions 0° and 180° are the lsW and usW of the leaning stem, respectively (Figure 1 shows the sampling positions). Lignin content, syringyl ratios, and erythro ratios of β-O-4 structures were determined through the Klason method, from the yields of NBO products and from ozonation products, respectively. Lignin content is total of Klason lignin and acid-soluble lignin; syringyl ratio, S/(S+V); erythro ratio, E/(E+T); S, total of syringaldehyde and syringic acid; V, total of vanillin and vanillic acid; E, erythronic acid; T, threonic acid. *1 Experimental data for G. gnemon are from Nawawi et al. (2016).
Figure 5:

Structural differences in the lignins of the GS-lignin-CW and GS-lignin-TW in the stems or branches of (a) Eusideroxylon zwageri, (b) Gnetum gnemon*1, (c) Melia azedarach, (d) Avinennia sp., and (e) Paraserianthes falcataria. The peripheral positions 0° and 180° are the lsW and usW of the leaning stem, respectively (Figure 1 shows the sampling positions). Lignin content, syringyl ratios, and erythro ratios of β-O-4 structures were determined through the Klason method, from the yields of NBO products and from ozonation products, respectively. Lignin content is total of Klason lignin and acid-soluble lignin; syringyl ratio, S/(S+V); erythro ratio, E/(E+T); S, total of syringaldehyde and syringic acid; V, total of vanillin and vanillic acid; E, erythronic acid; T, threonic acid. *1 Experimental data for G. gnemon are from Nawawi et al. (2016).

GS-lignin-TW

Similar to the TW of angiosperms (Pilate et al. 2004; Nakagawa et al. 2012), P. falcataria, M. azedarach, and Avicennia sp. showed eccentric growth of the usW of their leaning stems. With the exception of P. falcataria, the lignin content of their usW tended to be lower than that of their lsW. The S/G distributions in M. azedarach and Avicennia sp. were also similar to those seen in most stems with TW (Timell 1969; Akiyama et al. 2003): the syringyl ratio of the usW (the TW side) was higher than that of the lsW (Figure 5c and d). Examination of the three wood disks taken from the P. falcataria tree at three different heights above ground did not show a distribution pattern of syringyl ratios (Figure 5e).

The GS-lignin-TW species (P. falcataria, M. azedarach, and Avicennia sp.) contained β-O-4 structures with more erythro form than threo form (Table 3; Figure 5c and d). The TW of the usW of M. azedarach and Avicennia sp. showed a proportion of the erythro form higher than that of the lsW, similar to the patterns of the GS-lignin-CW in E. zwageri and G. gnemon and the patterns of the TW of Liriodendron tulipifera L. (Akiyama et al. 2003). The distribution of the erythro ratio is positively correlated with the syringyl ratio distribution for the disks with TW (R2=0.89 for M. azedarach, R2=0.47 for Avicennia sp.; Figure 5c and d). Even though P. falcataria TW did not show clear distribution patterns of erythro and syringyl ratios, these ratios are positively correlated (R2=0.50, 0.82, 0.66 for disks taken from 0.5, 1.5, and 2.5 m above ground, respectively).

S and erythro ratios in GS-lignin-CW and TW species

Data from the four GS-lignin species examined in this study and from G. gnemon cover a wide range of S ratios and lignin contents; however, the range within each woody species is narrow (Figure 6a). The lignin content tended to decrease with increasing S ratios, although the relationship is not strong (Figure 6a, R2=0.66). In contrast, the erythro ratio increased from the lsW toward the usW, together with an elevated syringyl ratio, regardless of the type of reaction wood (TW or CW). The S and erythro ratios for all the RW samples containing GS-lignin (GS-lignin-TW and -CW) are highly correlated with R2=0.96 (Figure 6b). The higher erythro ratio of the usW is the consequence of higher abundance of the erythro form of β-O-4 structures instead of lower abundance of the threo form (Figure 6c).

Figure 6: Relationships between syringyl/guaiacyl composition, lignin content, and the erythro and threo forms of β-O-4 structures for the GS-lignin-CW and GS-lignin-TW in stems and branches: (a) lignin content vs. syringyl ratio, (b) erythro ratio vs. syringyl ratio, and (c) yields of ozonation products vs. syringyl ratio. Syringyl ratio, S/(S+V); erythro ratio, E/(E+T); S, total of syringaldehyde and syringic acid; V, total of vanillin and vanillic acid; E, erythronic acid; T, threonic acid. Experimental data for G. gnemon are from Nawawi et al. (2016).
Figure 6:

Relationships between syringyl/guaiacyl composition, lignin content, and the erythro and threo forms of β-O-4 structures for the GS-lignin-CW and GS-lignin-TW in stems and branches: (a) lignin content vs. syringyl ratio, (b) erythro ratio vs. syringyl ratio, and (c) yields of ozonation products vs. syringyl ratio. Syringyl ratio, S/(S+V); erythro ratio, E/(E+T); S, total of syringaldehyde and syringic acid; V, total of vanillin and vanillic acid; E, erythronic acid; T, threonic acid. Experimental data for G. gnemon are from Nawawi et al. (2016).

In G-lignins, the β-O-4 structures consist of approximately equal amounts of the erythro and threo forms, as described above. However, the erythro form predominates in the β-O-4 structures of GS-lignins (Lundquist 1979; Nimz et al. 1984; Matsumoto et al. 1986; Bardet et al. 1998; Akiyama et al. 2005; Akiyama et al. 2015). The relationship between the erythro/threo and S/G ratios of various woody species has already been demonstrated (Akiyama et al. 2005). A similar relationship between these ratios for different parts of woody plants (xylem, bark, leaf, and root; Nawawi et al. 2016) and for different positions within the TW of the angiosperm L. tulipifera (Akiyama et al. 2003) exists. In a model experiment on the addition of water to different QMs (Brunow et al. 1993) as described above, β-syringyl ether-type QM forms more erythro-type β-O-4 structures than the threo type. In the present study, similar relationships were found within stems or branches regardless of the plant group (gymnosperm or angiosperm) and the type of RW (CW or TW). These results support the hypothesis that the S ratio is a crucial factor for the erythro ratio during GS-lignin biosynthesis.

Conclusions

Various types of reaction wood (CW or TW) containing GS-lignin, regardless of the plant group (gymnosperm or angiosperm) from which the wood is derived, showed similar lignin characteristics: the usW had lower lignin content, higher syringyl/guaiacyl ratio, and higher erythro/threo ratio of the β-O-4 structure than those of the lsW. These patterns were observed within the RW containing GS-lignin of a single trunk for five species. The above evidence suggests that the lignin structures of both GS-lignin RWs (GS-lignin-CW and GS-lignin-TW) change in similar ways in response to longitudinal growth stress during lignification. In contrast, the proportion of the erythro form in the lsW of P. merkusii CW (GH-lignin-CW) was higher than that in the usW and was correlated with proportion of H-units in the lignin.

Acknowledgments

This work was supported by the Grant-in-Aid for Scientific Research (17208015 and 15K14767) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT).

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Received: 2016-6-21
Accepted: 2016-8-12
Published Online: 2016-9-22
Published in Print: 2017-1-1

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