Holzforschung 2017; 71(1): 11–20

Deded Sarip Nawawi, Takuya Akiyama*, Wasrin Syafii and Yuji Matsumoto Characteristic of β-O-4 structures in different reaction lignins of zwageri T. et B. and four other woody species

DOI 10.1515/hf-2016-0100 erythro/threo ratio, which increased toward the lsW along Received June 21, 2016; accepted August 12, 2016; previously with increasing lignin contents and H/G ratios. ­published online September 22, 2016 Keywords: β-O-4 structure, compression wood, erythro/ Abstract: Lignin analyses were performed on the reaction threo, nitrobenzene oxidation (NBO), ozonation, tension of five tropical wood species. The reaction woods wood of these five species and that of Gnetum gnemon L. (pre- viously reported) were categorized into three types based on eccentric thickening growth and p-hydroxyphenyl/ guaiacyl/syringyl (H/G/S) ratios: compression wood Introduction (CW) containing GH-lignin observed in gymnosperms Lignin, a major polymer component of cell walls, (GH-lignin-CW), tension wood (TW) containing GS-lignin provides the strength, water resistance, and rigidity of observed in angiosperms (GS-lignin-TW), and reaction cell walls, thereby providing the mechanical support that wood that resembles CW and contains GS-lignin (GS- allows vascular to stand upright. The amount and lignin-CW). GS-lignin-CW is an unusual type that was chemical structure of lignin vary among woody species, found in the angiosperm Eusideroxylon zwageri and in the with the variations particularly occurring within the reac- gymnosperm G. gnemon. The erythro/threo ratio of the β- tion wood (RW) of stems or branches (Timell 1983; Timell O-4 structures and the S/G ratio were higher on the upper 1986; Donaldson 2001; Kiyoto et al. 2015). Lignin distribu- side (usW) of the leaning wood stem or branch, and both tion is thought to play an important role in the adaption ratios decreased along the periphery of the stem toward of trees to their environment. Regardless of the RW type, the lower side (lsW). Except for a difference in thickening the lignin content is relatively higher on the lower side growth, these distribution patterns were similar to the GS- (lsW) of the leaning woody stem than on the upper side lignin-TW patterns for Melia azedarach L. and Avicennia (usW) (Bland 1958; Yoshida et al. 2002; Pilate et al. 2004; sp. Reaction wood of Paraserianthes falcataria (L.) Nielsen ­Yoshinaga et al. 2012). was also classified as a GS-lignin-TW, but this was lacking In response to longitudinal growth stress, conifer- a clear distribution pattern. In contrast, the GH-lignin-CW ous gymnosperms form compression wood (CW) through of the usW of Pinus merkusii Jungh. et de Vriese had a low eccentric radial growth of the lsW. The lsW is more lig- nified than the usW, and it is enriched with p-hydroxy- phenyl units (H-units) (Timell 1986). In contrast, normal *Corresponding author: Takuya Akiyama, Department of Biomaterial Sciences, Wood Chemistry Laboratory, Graduate School of and opposite wood lignins (the usW) are primarily com- Agricultural and Life Sciences, The University of Tokyo, posed of guaiacyl units (G-units) and a smaller amount of Bunkyo-ku, Tokyo 113-8657, Japan, H-units (Timell 1986; Lapierre et al. 1988; Fukushima and e-mail: [email protected] Terashima 1991; Yeh et al. 2006; Nanayakkara et al. 2009). Deded Sarip Nawawi: Department of Biomaterial Sciences, Wood In many cases, angiosperms form tension wood (TW) by Chemistry Laboratory, Graduate School of Agricultural and Life eccentric radial growth of the usW, which is less ligni- Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; and Division of Forest Products Chemistry, Department of fied than the lsW (Bland 1958; Timell 1969; Akiyama et al. Forest Products, Faculty of Forestry, Bogor Agricultural University 2003; Pilate et al. 2004). Similar to conifers that form CW, (IPB), Kampus IPB Darmaga Bogor 16680, several angiosperm species show eccentric growth of the Wasrin Syafii: Division of Forest Products Chemistry, Department lsW in stems or branches. These unusual RWs are found of Forest Products, Faculty of Forestry, Bogor Agricultural University in Gardenia jasminoides Ellis (Aiso et al. 2013), Hebe sali- (IPB), Kampus IPB Darmaga Bogor 16680, Indonesia Yuji Matsumoto: Department of Biomaterial Sciences, Wood cifolia G. Forst. (Pennel) (Kojima et al. 2012), Viburnum Chemistry Laboratory, Graduate School of Agricultural and Life odoratissimum var. awabuki (K. Koch) Zabel (Wang et al. Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan 2010), Buxus sempervirens L. (Baillères et al. 1997), Buxus 12 D. S. Nawawi et al.: Reaction wood lignins of E. zwageri and four other species microphylla var. insularis Nakai (Yoshizawa et al. 1993; Akiyama et al. 2015). In our study of CW from G. gnemon ­Yoshizawa et al. 1999), Pseudowintera colorata (Raoul) lsW, we found through ozonation that the leaning stem is Dandy (Kučera and Philipson 1978; Meylan 1981), and distinguishable from the usW in terms of the β-O-4 stereo Phyllocladus alpinus Hook.f. (Kučera and Philipson 1977). structures (Nawawi et al. 2016). In our previous study of RW lignins (Nawawi et al. There appears to be a pattern in the structural differ- 2016), it was demonstrated that Gnetum gnemon L., a ences of lignins in the typical angiosperm TW and conifer- gymnosperm from the order Gnetales, forms an unusual ous CW. That is, the S/G ratio of the usW of TW tends to be RW. The lsW of its leaning stem shows eccentric growth, high, and the H/G ratio of the lsW of coniferous CW and similar to CW in conifers, although Shirai et al. (2015) the lignin content of the lsW of both CW and TW are high. reported that the usW of this species shows eccentric In the present paper, the RW resembling CW in a tropi- growth, similar to TW in angiosperms. The G. gnemon CW cal angiosperm, Eusideroxylon zwageri T. et B. (ulin wood), primarily contains syringyl units (S-units), G-units, and will be investigated by comparing its lignin against the a smaller amount of H-units in its GS-lignin, and the S/G typical GS-lignin-TW and GH-lignin-CW, as well as against ratio of its usW is higher than that of its lsW (Nawawi et al. the GS-lignin-CW in the gymnosperm G. gnemon. These 2016). Similarly, the distributions of S- and G-units in the different types of reaction woods will be compared in TW of angiosperm stem RWs are usually uneven (Bland terms of lignin content, composition of H, G, and S-units, 1958; Timell 1969; Aguayo et al. 2010; Al-Haddad et al. and the β-O-4 stereo chemistry. 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 Materials and methods S/G ratios in B. microphylla and G. jasminoides (deter- mined through the Mäule and Wiesner color reactions, see All chemicals were purchased from Wako Pure Chemical Industries, Yoshizawa et al. 1993; Aiso et al. 2013), in B. sempervirens Ltd. (Japan) or Tokyo Chemical Industry, Co., Ltd. (Japan). Woody (by thioacidolysis; Baillères et al. 1997), and in V. odorat- stems and branches were harvested in Bogor, West Java, or Bengkulu, issimum (by means of alkaline nitrobenzene oxidation, Indonesia (Table 1 and Figure 1). Wood disks were collected NBO, see Wang et al. 2010). However, information regard- from leaning stems and branches of a tropical gymnosperm (Pinus merkusii Jungh. et de Vriese) and four angiosperms (Paraserianthes ing the unusual RW that resembles CW in angiosperms falcataria (L.) Nielsen, Melia azedarach L., E. zwageri T. et B., and is limited to the lignin content and S/G compositions of Avicennia sp.). Wood disks were obtained from P. merkusii and P. these species. falcataria trees at three different heights above ground (1.7, 2.3, and In many cases, the S/G composition affects the com- 2.8 m for P. merkusii; 0.5, 1.5, and 2.5 m for P. falcataria). The collec- position of the different lignin interunit linkages (Ralph tion locations and the diameter of the wood disks are listed in Table 1. Wood blocks were cut from the wood disks at different positions et al. 2006; Stewart et al. 2009; Weng et al. 2011) and influ- along the periphery of the disks. The position along the periphery was ences the characteristics of arylglycerol-β-aryl ether (β- defined in angles, as shown in Figure 1, with 0 (360°) being on the lsW O-4) structures, which are the most predominant type of and 180° being on the usW. Wood blocks were also cut from the usW lignin linkages (Akiyama et al. 2003; Akiyama et al. 2005; and lsW of branches of P. merkusii, P. falcataria, and M. azedarach.

Table 1: Woody species and their type of reaction wood (RW) and lignin.

Woody species (common name) Class Sample Diameterc (cm) Place of harvestd RW typee Lignin typef

Pinus merkusii (Sumatran pine) Gymnosperm Stema 30 Bogor CW G Branch 13 Bogor CW G Gnetum gnemon Gymnosperm Stemb 22 Bogor CW GS Branchb 7 Bogor CW GS Eusideroxylon zwageri (ulin) Angiosperm Branch 13 Bogor CW GS Melia azedarach Angiosperm Stem 27 Bogor TW GS Branch 4 Bogor TW GS Avicennia sp. Angiosperm Stem 28 Bengkulu TW GS Paraserianthes falcataria Angiosperm Stema 26 Bogor TW GS Branch 12 Bogor TW GS 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. D. S. Nawawi et al.: Reaction wood lignins of E. zwageri and four other species 13

a b (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 suspen- sion at a rate of 0.5 l min−1 for 2 h, and the suspension was stirred ced 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 col- Figure 1: Cross sections of wood disks obtained from the leaning umn was eluted with water until an eluent pH of approximately 7–8 stem or branch of different woody species: (a) Pinus merkusii, (b) was reached, and the total volume eluted was adjusted to 100 ml. Eusideroxylon zwageri, (c) Melia azedarach, (d) Avicennia sp., and An aliquot of the eluent (2 ml) was dried at low pressure at 40°C. (e) Paraserianthes falcataria. Arrows in the pictures indicate the pith The residue was trimethylsilylated with dimethyl sulfoxide (DMSO) position. (300 μl), hexamethyldisilazane (200 μl), and trimethylchlorosilane (100 μl) at 60°C for 30 min. The upper layer, which contains the prod- uct, was then subjected to GC-FID using a Shimadzu 17A with an IC-1 Each wood block was ground in a Wiley mill to obtain 40- to column to determine the yields of erythronic acid (E) and threonic 60-mesh wood meals, which were pre-extracted with ethanol/ben- acid (T) in the ozonation product. The erythro ratio is defined as the zene mixture (1:2, v/v) for 8 h in a Soxhlet apparatus and were then E/(E + T) ratio. 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. Results and discussion 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% H SO solution (5 ml) 2 4 GH-lignin-CW for 3 h at room temperature (r.t.). The mixture was diluted with deion- ized water to obtain a 3% H SO solution, which was then autoclaved 2 4 Eccentric growth was observed in the lsW of the 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 P. merkusii leaning stem (Figure 1), similar to CW of pine 105°C overnight and then weighed. The concentration of acid-soluble and other coniferous species (Timell 1986; Donaldson­ lignin in the filtrate was determined by measuring the UV absorption and Knox 2012). Uncondensed-type NBO products −1 −1 of the filtrate at 205 nm with the absorption coefficient 110 l g cm of the P. merkusii CW were mostly G-type products (Swan 1965). (V = V + V ) and a smaller amount of H-type prod- The proportions of S and G units in lignin were evaluated ald acid = + through the alkaline NBO method (Chen 1992) with minor modifica- ucts (H Hald Hacid; Table 2). The amounts of both tions as described by Akiyama et al. (2005). Wood meals (40 mg), lignin and H-type products were higher in the lsW than 2 M NaOH solution (4 ml), and nitrobenzene (0.25 ml) were sealed in the usW, typical of CW (Timell 1986; Lapierre et al. in a 10-ml stainless-steel autoclave and heated to 170°C for 2 h. The 1988; ­Fukushima and Terashima 1991; Yeh et al. 2006; reaction mixture was cooled with ice water, and the internal stand- ­Nanayakkara et al. 2009). Figure 2 shows the distribu- ard (IS), ethyl vanillin (3-ethoxy-4- hydroxybenzaldehyde; 6 μmol), tions of lignin and NBO products around the periphery was added. The mixture was then washed three times with CH2Cl2 (15 ml) and acidified with 2 M HCl solution. The acidified aqueous of the stem. The relative yield of H-type products relative + layer was extracted three times with CH2Cl2 (20 ml) and once with to the total NBO products [defined as H/(H V) ratio] was diethyl ether (15 ml). The organic layers were combined, washed <2% in the usW, reaching up to 14% toward the lsW. It with water (20 ml), and dried over Na SO . After filtration, the solu- 2 4 is noteworthy that all data from P. merkusii show a high ° tion was dried at 30 C at low pressure. The residue was trimethylsi- correlation between lignin content and p-hydroxyphenyl lylated with bis(trimethylsilyl)acetamide (100 μl) at 100°C for 10 min 2 = and then analyzed by gas chromatography (GC-FID) with a Shimadzu ratios (Figure 3a, R 0.98). 17A instrument equipped with an IC-1 column. The yields of syringal- The pine CW lignin was further characterized by ozo- dehyde (Sald) and syringic acid (Sacid), vanillin (Vald) and vanillic acid nation according to Akiyama et al. (2002). The content of 14 D. S. Nawawi et al.: Reaction wood lignins of E. zwageri and four other species

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).

NBO yieldd Ozonationf (μmol g−1) (μmol g−1) Sampling Lignin RW sample position contentc (%) H V H/(H + V)e (%) E + T E/(E + T)g (%)

Stem (disk 1)a usWb 28.1 37 1948 1.9 1033 49.3 lsWb 33.9 231 1625 10.7 930 51.9 Stem (disk 2)a usWb 27.5 38 1931 1.9 955 49.5 lsWb 34.9 215 1519 12.4 802 53.7 Stem (disk 3)a usWb 28.2 34 1865 1.8 912 49.7 lsWb 35.7 236 1440 14.1 711 53.5 Branch usWb 27.7 36 1959 1.8 955 49.9 lsWb 31.4 130 1720 7.1 928 51.1 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.

a

b

c ratio Erythro

Figure 2: Structural differences in Pinus merkusii lignins within the reaction wood (GH-lignin-CW): (a) lignin content, (b) p-hydroxy- phenyl 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 Figure 3: Relationships between p-hydroxyphenyl (H)/guaiacyl (G) ­vanillin and vanillic acid; E, erythronic acid; T, threonic acid. 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) β -O-4 structures and their erythro/threo ratios were evalu- yields of ozonation products vs. H. H/(H + V), H-ratio; E/(E + T), erythro ated from the yields of E and T, which were released from ratio; H, total of p-hydroxybenzaldehyde and p-hydroxybenzoic acid; the side-chain of the β-O-4 structures by ozonation. In V, total of vanillin and vanillic acid; E, erythronic acid; T, threonic acid. D. S. Nawawi et al.: Reaction wood lignins of E. zwageri and four other species 15 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 struc- tures 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 corre- sponding 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 propor- tion 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 gymno- sperm 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 cor- relation (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 conse- quence 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. Figure 4: Formation of the erythro and threo forms of β-O-4 struc- For the erythro form to be produced in excess during tures by addition of water to quinone methide intermediates. lignin biosynthesis, stereo-preferential water addition must occur to the β-O-4-bonded quinone methide (QM) the erythro/threo ratio was 50:50 in gymnosperm lignins intermediate (Figure 4). The QM intermediate is formed as (containing mainly G-lignin). As indicated by the experi- a result of the β-O-4 coupling reaction between a monol- mental data, the erythro configuration is preferentially ignol radical and a radical on a growing lignin polymer. formed in the CW of P. merkusii, depending on the pres- Water can be added to either face of the planar QM. This ence of H-units. However, the mechanism of this reaction step produces two isomeric forms of the β-O-4 structures, is still unknown. 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 GS-lignin-CW to a G-type QM did not promote the erythro form, although in case of S-type QMs, the formation of the erythro config- Eusideroxylon zwageri differed from the other three uration was dominating (Brunow et al. 1993). In addition, angiosperms as its branch (13 cm Ø) showed eccentric 16 D. S. Nawawi et al.: Reaction wood lignins of E. zwageri and four other species

Table 3: Lignin content, yields of nitrobenzene oxidation (NBO) products, and ozonation products obtained from the reaction woods of Eusideroxylon zwageri, Melia azedarach, Avicennia sp., and Paraserianthes falcataria (GS-lignin-CW and GS-lignin-TW).

NBO yielde Ozonation (μmol g−1) yieldg (μmol g−1) Sampling Lignin Wood species RW sample position contentd (%) H V S S/(S + V)f (%) E + T E/(E + T)h (%)

Eusideroxylon zwageri Branch usWb 40.6 14 1299 325 20.0 965 54.3 lsWb 40.8 12 1401 175 11.1 861 53.0 Melia azedarach Stem usWb 30.7 9 1125 784 41.1 1300 61.7 lsWb 31.9 10 1297 635 32.9 1066 59.3 Branch usWc 29.9 8 1066 1031 49.2 1196 64.4 lsWc 30.7 10 1171 775 39.8 1047 61.8 Avicennia sp. Stem usWb 27.9 12 472 1992 80.9 1271 72.7 lsWb 28.4 11 528 1905 78.3 1306 71.9 Paraserianthes falcataria Stem (disk 1)a usWb 29.8 22 984 1370 58.0 1329 67.1 lsWb 29.2 22 976 1465 60.0 1325 68.1 Stem (disk 2)a usWb 28.1 28 1024 1256 55.0 1357 66.2 lsWb 28.8 19 978 1454 59.7 1424 67.6 Stem (disk 3)a usWb 28.6 23 995 1219 55.0 1253 66.6 lsWb 29.2 22 950 1330 58.2 1425 67.2 Branch usWc 18.2 14 778 1365 63.7 1173 67.9 lsWc 28.7 11 975 1339 57.9 1351 66.9 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.

growth of the lsW (Figure 1). The lignin content of the GS-lignin-CW of the usW of both species showed higher lsW is higher than that of the usW. The RW of this species syringyl and erythro ratios than those of the lsW, although is atypical, similar to that found in some angiosperms the S-units were more abundant in G. gnemon lignin (Kučera and Philipson 1977; Kučera and Philipson 1978; [S/(S + V) ≈ 46–55%] than in E. zwageri lignin. 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). GS-lignin-TW The NBO products from the E. zwageri wood meal primarily consisted of S-type products (S = Sald + Sacid) Similar to the TW of angiosperms (Pilate et al. 2004; and G-type products and included only <1% of H-type Nakagawa et al. 2012), P. falcataria, M. azedarach, and products (Table 3). The syringyl ratio [S/(S + V)] from the Avicennia sp. showed eccentric growth of the usW of their branch lsW was 11% (0.12 as S/V ratio), which is lower leaning stems. With the exception of P. falcataria, the than the ratios of 16 other woody angiosperm species lignin content of their usW tended to be lower than that of (15% or more) reported by Akiyama et al. (2005). There- their lsW. The S/G distributions in M. azedarach and Avi- fore, E. zwageri lignin may be regarded as a G-rich GS- cennia sp. were also similar to those seen in most stems lignin. The syringyl ratio of E. zwageri CW increased with TW (Timell 1969; Akiyama et al. 2003): the syringyl around the periphery of the stem, reaching 20% toward ratio of the usW (the TW side) was higher than that of the usW (Figure 5a). The erythro ratio of the β-O-4 struc- the lsW (Figure 5c and d). Examination of the three wood ture [erythro/(erythro + threo)] of the usW was also disks taken from the P. falcataria tree at three different higher than that of the lsW. The erythro ratio distribu- heights above ground did not show a distribution pattern tion within the wood disk is correlated with the S-ratio of syringyl ratios (Figure 5e). distribution with 0.92 R2 between E/(E + T) and S/(S + V). The GS-lignin-TW species (P. falcataria, M. azedar- These E. zwageri CW data resemble to those of the CW ach, and Avicennia sp.) contained β-O-4 structures with of the gymnosperm G. gnemon (Nawawi et al. 2016). The more erythro form than threo form (Table 3; Figure 5c D. S. Nawawi et al.: Reaction wood lignins of E. zwageri and four other species 17

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). and d). The TW of the usW of M. azedarach and Avicennia S and erythro ratios in GS-lignin-CW and TW sp. showed a proportion of the erythro form higher than species that of the lsW, similar to the patterns of the GS-lignin- CW in E. zwageri and G. gnemon and the patterns of the Data from the four GS-lignin species examined in this TW of Liriodendron tulipifera L. (Akiyama et al. 2003). The study and from G. gnemon cover a wide range of S ratios distribution of the erythro ratio is positively correlated and lignin contents; however, the range within each with the syringyl ratio distribution for the disks with TW woody species is narrow (Figure 6a). The lignin content (R2 = 0.89 for M. azedarach­ , R2 = 0.47 for Avicennia sp.; tended to decrease with increasing S ratios, although the Figure 5c and d). Even though P. falcataria TW did not relationship is not strong (Figure 6a, R2 = 0.66). In contrast, show clear distribution patterns of erythro and syringyl the erythro ratio increased from the lsW toward the usW, ratios, these ratios are positively correlated (R2 = 0.50, together with an elevated syringyl ratio, regardless of the 0.82, 0.66 for disks taken from 0.5, 1.5, and 2.5 m above type of reaction wood (TW or CW). The S and erythro ratios ground, respectively). for all the RW samples containing GS-lignin (GS-lignin-TW 18 D. S. Nawawi et al.: Reaction wood lignins of E. zwageri and four other species

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 relation- ship 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, , 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 angio- sperm) 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 pat- terns 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 Figure 6: Relationships between syringyl/guaiacyl composition, CW (GH-lignin-CW) was higher than that in the usW and lignin content, and the erythro and threo forms of β-O-4 structures was correlated with proportion of H-units in the lignin. 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, Acknowledgments: This work was supported by the Grant- and (c) yields of ozonation products vs. syringyl ratio. Syringyl ratio, in-Aid for Scientific Research (17208015 and 15K14767) 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; from the Ministry of Education, Culture, Sports, Science, T, threonic acid. Experimental data for G. gnemon are from Nawawi and Technology of Japan (MEXT). et al. (2016). and -CW) are highly correlated with R2 = 0.96 (Figure 6b). References The higher erythro ratio of the usW is the consequence of Aguayo, M.G., Quintupill, L., Castillo, R., Baeza, J., Freer, J., higher abundance of the erythro form of β-O-4 structures ­Mendonca, R.T. (2010) Determination of differences in ana- instead of lower abundance of the threo form (Figure 6c). tomical and chemical characteristics of tension and opposite In G-lignins, the β-O-4 structures consist of approxi- wood of 8-year old Eucalyptus globulus. Maderas-Cien. Tecnol. mately equal amounts of the erythro and threo forms, as 12:241–251. D. S. Nawawi et al.: Reaction wood lignins of E. zwageri and four other species 19

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