Lignin characteristics of Abies beshanzuensis, a critically endangered tree species

As one of the major components of plant cell walls, lignin structural features are closely related with taxonomical and genetic classification of plants. In this study, the structural features of lignin of Abies beshanzuensis were investigated. Abies firma, which is genetically the closest species to A. beshanzuensis, Cryptomeria japonica, a typical gymnosperm tree species, and Phyllostachys pubescens (bamboo), which includes p-hydroxyphenyl nuclei in arylglycerol-β-aryl intermonomer linkages, were also analyzed to compare lignin features with those of A. beshanzuensis. The lignin content of A. beshanzuensis (39.2%) was significantly higher than that of A. firma (33.7%). The high value may be due to the adaptation of A. beshanzuensis to environmental stresses in surviving the Riss glacial period. Alkaline nitrobenzene oxidation, ozonation, acidolysis, and 1H nuclear magnetic resonance spectra showed that the structural features of lignin of A. beshanzuensis were similar to those of A. firma, which is genetically the closest species of A. beshanzuensis. The results of this study suggest that A. firma would be a suitable mother tree species for grafting A. beshanzuensis on the basis of their lignin characteristics.     

Lignification and peroxidase in tension wood ofEucalyptus viminalis seedlings

Seedlings ofEucalyptus viminalis were grown for 50 days with their stems bent so tension wood would form. Every 10 days the lignin content, monomeric composition, and peroxidase activity in the tension wood were compared with those in the lower side (opposite wood) and in vertically grown controls. The lignin content in the developing tension wood started to decrease after 10 days of bending and kept decreasing for 50 days, whereas those in control plants and opposite wood remained almost unchanged. The yields of syringaldehyde from tension wood by nitrobenzene oxidation increased, and consequently the syringyl/ guaiacyl ratio of the lignin was higher in tension wood than in opposite wood and control plants. The peroxidase ionically bound to the cell walls (IPO) catalyzed oxidation of guaiacol and syringaldazine. The syringaldazineoxidizing activity of IPO from tension wood increased, whereas the activities of IPO from opposite wood and control plants did not show any marked change. In tension wood the increase in syringaldazine-oxidizing activity of IPO was consistent with an increase in the syringaldehyde yield. This suggests that IPO contributes to syringyl lignin deposition as other enzymes involved in the monolignol biosynthesis do in tension wood formation.This study was presented at the 50th Annual Meeting of the Japan Wood Research Society, Kyoto, April 2000     

Ready chemical conversion of acid hydrolysis lignin into water-soluble lignosulfonate III: Successive treatment of acid hydrolysis lignin and a lignin model compound by phenolation and arylsulfonation*

The chemical conversion of red pine sulfuric acid lignin (Klason lignin) (SAL) as an acid hydrolysis lignin sample to water-soluble arylsulfonates of lignin derivation (i.e., phenolized SAL) was investigated. Treatment of phenolized SAL with chlorosulfonic acid followed by alkali hydrolysis gave water-soluble sulfonated products with a sulfonic acid group on their aromatic nuclei quantitatively. The products possess 2.0 SO₃Na/C₉ C₆. In contrast, the content of sulfuric acid group in sulfonated SAL was only 0.33C₉. Chlorosulfonation of 1-guaiacyl-l-p-hydroxyphenylethane as a phenolized guaiacyl lignin model compound revealed that the sulfonyl chloride group was introduced at thepara position of an aromatic methoxyl group, theortho position of a phenolic hydroxyl group, or both.     

Decomposition behavior of woody biomass in water-added supercritical methanol

The chemical conversion of Japanese beech (Fagus crenata Blume) in water-added supercritical methanol was studied for a wide range of water content using a batch-type reaction vessel to obtain chemicals from lignocellulosics. It was consequently found that addition of water enhanced the decomposition of wood cell wall components; cellulose, hemicelluloses, and lignin. In cases of high water content, however, it resulted in low solubility of lignin-derived products causing an increase in the mass of the residue. The water content was thus optimized to be around 10% (v/v) for the decomposition of wood. Concomitantly, the yields and selectivity of the chemicals from wood could be regulated by the addition of water, especially for the lignin-derived products. As a result, the monomeric compounds of lignin, coniferyl alcohol and sinapyl alcohol, were recovered as their γ-methyl ethers in the presence of water in higher yields than those obtained without addition of water.     

Treatment of poplar callus with ferulic and sinapic acids I: incorporation and enhancement of lignin biosynthesis

Ferulic acid (FA), tetradeuteroferulic acid (DFA), sinapic acid (SA), or heptadeuterosinapic acid (DSA) was exogenously supplied to poplar (Populus alba L.) callus. Administration of FA or SA increased the lignin content of the callus to about twice that of the control callus. Gas chromatographic analysis of the alkali hydrolysate of the cell wall residue revealed that only a trace amount of SA was bound to the cell wall, and the amount of FA was less than 2% of the total callus lignin. Thioacidolysis of the DFA-treated callus indicated that DFA is effectively converted to both coniferyl and sinapyl alcohols and then incorporated into the corresponding lignin. Incorporation of DSA into syringyl lignin or guaiacyl lignin was not observed, but yields of syringyl lignin thioacidolysis products were markedly increased by DSA treatment of the callus. These results suggest that SA may not be a precursor of sinapyl alcohol and syringyl lignin per se, but it may induce or enhance the biosynthesis of syringyl lignin in poplar callus.     

Characteristics of senescent straw cell walls of dwarf, semidwarf, and normal strains of rice (Oryza sativa) plants

A normal variety of rice (Oryza sativa L.cv. Taichung 65, T65c), its isogenic dwarf line (T65d ₁), and a semidwarf variety of a different line (Oryza sativa L.cv. IR8, IR8) were studied. The results were compared with those of an isogenic dwarf line (Rh _i) of wheat straw, which was previously reported. Expression of the dwarf gene,d ₁, on the chemical composition and the structural features of lignin present in rice internodes differs from that in an isogenic dwarf line of wheat. The differences include the lignin content, total yield of alkaline nitrobenzene oxidation products, and distribution of wall-bound hydroxycinnamic acids. There was, however, no difference in the syringyl/ guaiacyl nuclei (S/V) molar ratio and neutral sugar composition. The lignin composition of rice straw cell walls, particularly that of the dwarf variety, contained more of the condensed structure and fewer syringyl nuclei than lignin in wheat straw cell walls. It is suggested that crosslinking between lignin and polysaccharides by ester-ether bridges via ferulic acid contributes to the mechanical properties of the cell walls of rice straw. Thus the chemical and structural characteristics of lignin in rice straw differ to some extent from those of other temperate grasses, such as wheat (Triticum aestivum) and phalaris (Phalaris aquatica), as reported previously. This can probably be attributed to the water environment of rapidly growing rice seedlings, but it also depends on the genetic variety of the rice plant.     

Effects of <Emphasis Type="Italic">Ips typographus</Emphasis> (L.) damage on litter quality and decomposition rates of Oriental Spruce [<Emphasis Type="Italic">Picea Orientalis</Emphasis> (L.) Link.] in Hatila Valley National Park,Turkey

This study investigated the effects of Ips typographus (L.) damage on initial litter quality parameters and subsequent decomposition rates of oriental spruce tree species [Picea orientalis (L.) Link]. The needle litter was collected from highly damaged, moderately damaged and control stands on two aspects (north and south) and two slope position (top and bottom) on each aspect. The litter was analyzed for initial total carbon, lignin and nutrient (nitrogen, phosphorus, potassium, calcium, magnesium and manganese) concentrations. The variability in nitrogen and calcium concentrations and ratios of C:N, lignin:N and lignin:Ca was significantly affected by the insect damaged levels. While nitrogen concentrations in needle litter increased with increasing insect damage (and consequently the ratios of C:N and lignin:N decreased), calcium concentrations decreased (and consequently the ratio of lignin:Ca increased). Aspect and slope positions explained most of the variability in carbon, lignin, phosphorus, potassium, magnesium and manganese concentrations and lignin:P ratio between all studied stands. Litter decomposition was studied in the field using the litterbag technique. The litter from highly damaged stands showed highest decomposition rates followed by moderately damaged and control stands. The mass loss rates were significantly positively correlated with initial nitrogen concentration and negatively with C:N and lignin:N ratios. The effects of microclimate resulting from canopy damage on litter decomposition was also examined at the same time using standard litter with the same litter quality parameters, but they showed no significant differences among the insect damage levels indicating that alteration of the litter quality parameters produced by I. typographus damage played a more important role than altered microclimate in controlling needle litter decomposition rates. However, changes in microclimate factors due to topography influenced decomposition rates.     

Application of the amount of oxygen consumption to the investigation of the oxidation mechanism of lignin during oxygen-alkali treatment

The dioxygen consumption by kraft lignin and several lignin model compounds during oxygen-alkali treatments were directly analyzed using a dioxygen fl owmeter. The average dioxygen consumption by 200 g of kraft lignin was about 3 moles. Because this value was as much as those obtained for monomeric phenolic lignin model compounds, guaiacol and vanillyl alcohol, it was postulated that not only phenolic but also nonphenolic moieties in kraft lignin are extensively oxidized. The dioxygen consumption by 0.5 moles (one equivalent of aromatic units) of a dimeric lignin model compound, guaiacylglycerol-β-guaiacyl ether (GG), was also similar to that for 1 mole of guaiacol and vanillyl alcohol, regardless of the type of the aromatic moiety, which supports the above postulation. The most plausible mechanism for the oxidation of nonphenolic moieties is the oxidation of side chains of residual β-O-4 substructures by active oxygen species. By this mechanism, nonphenolic moieties in kraft lignin and GG are converted into corresponding phenolic moieties, and the oxidation by dioxygen progresses. Part of this article was presented at the 13th International Symposium on Wood, Fiber, and Pulping Chemistry (13th ISWFPC), Auckland, New Zealand, May 2005     

Determination of nitrobenzene oxidation products by GC and1H-NMR spectroscopy using 5-iodovanillin as a new internal standard

The nitrobenzene oxidation method was modified to obtain more reproducible data and more structural information about lignin, not only by gas chromatography (GC) but also by proton nuclear magnetic resonance (¹H-NMR) spectroscopy for quantitative determination of the oxidation products and to simplify the procedures. The nitrobenzene oxidation mixture was directly extracted after acidification without preextraction of by-products. The direct extraction made the extractive step easy and gave reproducible data. 5-Iodovanillin was selected as a new internal standard. The reason for this selection was that 5-iodovanillin did not exist in the nitrobenzene oxidation products from any plant species and had an aldehyde group whose peak did not overlap with the other aldehyde peaks on an¹H-NMR spectrum. Thus, the use of 5-iodovanillin enabled us to quantifyp-hydroxybenzaldehyde, vanillin, and syringaldehyde in oxidation products on the basis of¹H-NMR analysis as well as GC. Furthermore, more information about the condensed structure of lignin was derived by comparing the¹H-NMR and GC analyses.Part of this work was presented at the 42nd Annual Meeting of the Lignin Symposium, Sapporo, October 1997     

Antisense expression of a caffeic acidO-methyltransferase ofStylosanthes humilis in transgenic poplar: Effect of expression onO-methyltransferase activity and lignin composition

Poplar (Populus tremula) was transformed with a construct carrying an antisense caffeic acidO-methyltransferase (COMT) cDNA (pOMT8) from a tropical pasture legume,Stylosanthes humilis. pOMT8 shows 83% overall homology to the corresponding COMT gene (pPCLA) of poplar. Of the 200 putatively-transformed plants regenerated on selective media after co-cultivation of poplar stem explants withAgrobacterium tumefaciens harbouring a CaMV 35S-antisensepOMT8 construct, a subset of 20 plants were randomly chosen for further analysis. PCR and Southern blot analysis demonstrated the stable integration of T-DNA into the genome of these plants. Antisense expression ofpOMT8 resulted in reductions in total COMT activity in the majority of the transgenic plants with the lowest total COMT activities (61–70% of untransformed control plants) being observed in four transgenic plants. The composition of lignin in transgenic plants was also changed, as detected by reductions in the content of syringyl units using infrared spectroscopy. However, no changes were found in the amount of insoluble lignin in transgenic plants as compared to untransformed control plants. These results indicate the potential of thepOMT8 gene to partially suppress COMT activity and modify the composition of lignin in transgenic poplar. This work was partly supported by General Management of Turkish Pulp and Paper Mills.     

Effects of bark stripping by sika deer, Cervus nippon, on wind damage to coniferous trees in subalpine forest of central Japan

We evaluated the effect of bark stripping by sika deer (Cervus nippon), and subsequent wood decay, on tree fall in a coniferous forest on Mt Ohdaigahara in central Japan from July to September 2006. This valuable primeval coniferous forest is declining because of bark stripping. Broken trunks of Abies homolepis and Picea jezoensis var. hondoensis had more serious decay and larger bark-stripping wounds on the trunk than standing trees, suggesting that bark stripping causes trunk decay and results in broken trunks and uprooting by typhoons.     

Structure of small lignin fragment retained in water-soluble polysaccharide extracted from sugi MWL isolation residue

In order to analyze the structural features of small lignin fragments that are closely associated to polysaccharides, lignin carbohydrate complex (LCC) with low lignin content was extracted with water from the residual wood meal (sugi, Cryptomeria japonica) of milled wood lignin (MWL) isolation. This LCC exhibited almost the same neutral sugar composition as those extracted by other LCC solvents (dimethylformamide, dimethylsulfoxide, and others) but the lignin content was only 5.3%, which was much lower than others. Gel filtration chromatography demonstrated that lignin in this LCC was found together with carbohydrates at the higher molecular weight region, but after the treatment with carbohydrate-degrading enzymes the apparent molecular weight of both lignin and carbo-hydrates decreased significantly. Using a mild alkaline treatment, the apparent molecular weight of lignin also decreased while that of polysaccharide was not affected. These data indicated that lignin in this LCC is present as small fragments attached to high molecular weight polysaccharide at least partly by alkali-unstable linkages. Structural analysis by ozonation method revealed that the lignin in this LCC was lower in erythro/threo ratio of -O-4 structure and relatively richer in the threo type structure carrying C-aryl linkages at -position (-5 and/or -1) than other lignin fractions present in MWL, LCCs extracted by other solvents, and their extraction residues. It was suggested that the chemical structure of lignin closely associated to carbohydrates was different from that of the main part of lignin.This paper was presented in part at the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, Japan, April 2002, at the 47th Lignin Symposium, Gifu, Japan, October 2002, and at the 12th International Symposium on Wood and Pulping Chemistry, Madison, USA, June 2003     

Structural conversion of the lignin subunit at the cinnamyl alcohol stage in Eucalyptus camaldulensis

The lignin biosynthetic pathway in Eucalyptus camaldulensis was investigated by feeding stems with deuterium-labeled precursor. Pentadeutero[,-D₂ OCD₃] coniferyl alcohol was synthesized and supplied to shoots of E. camaldulensis, and incorporation of the labeled precursor into lignin was traced by gas chromatography-mass spectrometry. In addition to the direct incorporation of labeled precursor into the guaiacyl unit, a pentadeuterium-labeled syringyl unit was detected. This finding indicates that the -deuterium atoms in the hydroxymethyl group of labeled coniferyl alcohol remain intact during modification of the aromatic ring. The relative level of trideuterium-labeled syringyl monomer (the result of conversion via the cinnamic acid pathway) was negligible, suggesting that the pathway at the monolignol stage is used for conversion of exogenously supplied precursor. Our results provide conclusive evidence of a novel alternative pathway for generation of lignin subunits at the monolignol stage even in plants that do not accumulate coniferin in lignifying tissues.     

Structural characteristics of lignin in primitive pteridophytes: <Emphasis Type="Italic">Selaginella</Emphasis> species

The lignin chemical structures of eight species of the Selaginella family, which are primitive vascular plants, were characterized by alkaline nitrobenzene oxidation, acidolysis, and ozonation. Selaginella involvens, Selaginella tamariscina, and Selaginella remotifolia were collected from the University Forest in Chiba, the University of Tokyo, Japan, and Selaginella biformis, Selaginella pennata, S. involvens, Selaginella chrysorrhizos, and unidentified Selaginella species (Selaginella sp.) were collected from northern Thailand. Lignin of all Selaginella species examined in this study was rich in syringyl nuclei. It was confirmed that a considerable portion of syringyl nuclei of Selaginella lignin formed syringylglycerol-β-aryl ether intermonomer linkages. The major diastereomer of arylglycerol-β-aryl ether intermonomer linkages of Selaginella lignins was the erythro-form exhibiting angiosperm lignin characteristics. In addition, lignins of S. involvens, S. tamariscina, and S. remotifolia collected from the University Forest in Chiba, the University of Tokyo, Japan, were isolated according to Björkman’s procedure, and structural features of the lignins were spectrometrically analyzed. It was confirmed that lignin of Selaginella species, which are primitive pteridophytes, was typical guaiacyl-syringyl type as well as being similar to angiosperm lignin.     

Structural changes in lignin of tropical woods during digestion by termite, <Emphasis Type="Italic">Cryptotermes brevis</Emphasis>

Wood samples of apitong (Dipterocarpus grandiflorua) and ilang-ilang (Ilang-Ilang C. dadloyi) and feces of termites [Cryptotermes brevis (Walker)] fed on these woods were collected from University of the Philippines, Los Baňos. Lignin of each sample was isolated by Björkman’s procedure. There was no significant difference in ¹H nuclear magnetic resonance (NMR) spectra or in the methoxyl content between Björkman lignins from original woods and termite feces. Differences were detected in the contents of aliphatic and unconjugated phenolic hydroxyl groups, suggesting minor structural changes of lignin during digestion by termites. In addition, the ratio of syringyl to guaiacyl nuclei of Björkman lignin from termite feces determined by ¹H NMR spectra was higher than those from the original woods. The molar ratio of syringyl to guaiacyl nuclei of termite feces was higher than those from the original woods as determined by alkaline nitrobenzene oxidation. These results suggest that the structural changes of lignin in the termite gut are due to the insignificant formation of C-C linkages in guaiacyl nuclei. It was concluded that there were minor changes in the structural features of lignin under mostly anaerobic conditions, in contrast to the significant changes that occur through biological modification under aerobic conditions.     

The monomer composition controls the Σ<Emphasis Type="Italic">β-O</Emphasis>-4/Σ<Emphasis Type="Italic">O</Emphasis>-4 end monomer ratio of the linear lignin fraction

Lignins are cell wall phenolic heteropolymers that result from the oxidative coupling of three monolignols bearing p-coumaryl (H), coniferyl (G), and sinapyl (S) units, in a reaction mediated by peroxidases. Here, we report the existence of a relationship between the Σβ-O-4/ΣO-4 end monomer ratio of the linear lignin fraction, released through the specific cleavage of the alkyl ether linkages by thioacidolysis, and the G/S ratio of lignins, when this was estimated in differentially evolved vascular land plants. Most importantly, in the case of angiosperms, Gnetales, and lycopods, the Σβ-O-4/ΣO-4 end monomer ratio was apparently predictable from the proportions at which the G and S units were mixed. In the case of G lignins (present in basal gymnosperms and ferns), the Σβ-O-4/ΣO-4 end monomer ratio decayed exponentially to increase the O-4-linked dihydroconiferyl alcohol (DHCA) content. The results obtained suggest that the Σβ-O-4/ΣO-4 end monomer ratio of the linear lignin fraction depends intimately on the lignin monomer composition, and, therefore, on the chemical nature of the radicals derived from three monolignols (coniferyl, dihydroconiferyl, and sinapyl alcohols), whose gain have been finely tuned during land plant evolution.     

Tree-root systems and herbaceous species-characteristics under tree species introduced into grazing lands in subhumid Cameroon

The effect of tree species on the characteristics of the herbaceous stratum, during the first five years of a fallow, was evaluated in the North of Cameroon (average annual temperature 28.2 °C, total annual rainfall 1050 mm). Treatments included a natural grazed herbaceous fallow, a natural ungrazed herbaceous fallow and three planted tree fallows (Acacia polyacantha Willd. ssp. campylacantha (Hochst. ex A. Rich.), Senna siamea Lam. and Eucalyptus camaldulensis Dehnh.), which were protected against grazing. Because tree species influenced light interception in different ways, as well as having different root patterns, they had different effects on the herbaceous stratum in terms of species composition and biomass. The grazed herbaceous fallow maintained the greatest species richness. Protection against grazing or the introduction of tree species associated with the absence of grazing induced both a progressive evolution to a particular species composition. The ungrazed herbaceous fallow consisted mainly of Andropogon gayanus Kunth, which provided the greatest biomass (8 t dry matter ha^–1 at the end of the fallow period). E. camaldulensis provided little shade and the lowest fine root mass in the top layer allowing the growth of A. gayanus and thus a greater herbaceous biomass (3.5 t DM ha^–1) than that found under the other tree species. Under the heavy shade of A. polyacantha, the herbaceous stratum consisted mainly of annual Pennisetum spp. (2.2 t DM ha^–1) and showed the greatest N concentration (1.3%), probably due to N₂ fixation by the tree species. After the fourth year, despite the relatively open tree canopy, S. siamea, which showed the highest fine root mass, had a strong depressive effect on the herbaceous stratum. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ultrastructure of the S2 layer in relation to lignin distribution inPinus radiata tracheids

The ultrastructure of the S₂ layer in relation to its lignin distribution was examined using transmission electron microscopy in the tracheids ofPinus radiata. The S₂ layer had a striated appearance at low magnification. Observations at higher magnifications showed lignin to be distributed inhomogeneously in this layer, appearing as a mosaic of electron-dense and electron-lucent regions. These regions are scattered, showing a pattern of often interconnecting sinuous features in a predominantly radial profile. The significance of these features of the S₂ layer is discussed, particularly in relation to the available information from recent ultrastructural observations on the appearance of cellulose microfibrils and the pattern of their distribution in the S₂ layer using rapid freeze-deep etching in conjunction with transmission electron microscopy. Predictions are made as to the likely distribution and arrangement of cellulose microfibrils in the S₂ layer based on the pattern of lignin distribution observed in this layer.     

Leaf quality of selected hedgerow species at two canopy ages in the derived savanna zone of West Africa

Prunings in hedgerow intercropping systems are a potential source of organic matter and their quality is an important characteristic driving decomposition and nutrient release. To determine the potential range of residue characteristics and the impact of canopy age on those, selected characteristics of Leucaena leucocephala, Gliricidia sepium, and Senna siamea hedgerow leaf residues were determined for young (5–10 weeks) and old (39–47 weeks) hedgerow canopies in six sites, representative in terms of soil for the derived savanna zone of West-Africa. The N content of the Leucaena and Senna residues decreased with age (from 4.8 to 3.9% and from 4.1 to 3.0%, respectively, while the N content of the Gliricidia residues remained constant (3.9%). The P content of all species decreased with age (from 0.30 to 0.20%, on average). The lignin content of the Leucaena residues increased, and their polyphenol content decreased with age, while for the Senna residues only an increase in polyphenol content with canopy age was evident. Neither the lignin nor the polyphenol content of the Gliricidia leaf residues was markedly affected by canopy age. For all species, the P and ADF content were positively correlated with the ash and lignin content, respectively. The N content of the Leucaena and Senna residues was positively correlated with their P content while the lignin content of the same species was negatively correlated with the polyphenol content. Because of the low variation in certain residue characteristics between sites, the presented data and equations could potentially be used to reasonably estimate those characteristics if no other information is available. For other characteristics, such as polyphenol contents, measurements are preferable in view of the relatively high variation encountered.This revised version was published online in November 2005 with corrections to the Cover Date.