‘Defence lignin’ and hydroxycinnamyl alcohol dehydrogenase activities in wounded Eucalyptus gunnii

To learn more about lignin formation in response to wounding in trees, we adopted two complementary approaches: (1) microscopic and histochemical studies of the wound response in 3.5‐month‐old Eucalyptus gunnii plantlets and (2) biochemical investigations of hydroxycinnamyl alcohol dehydrogenase activities in wounded 6‐year‐old, field‐grown E. gunnii trees. The first approach revealed that a barrier zone was formed in response to wounding in both ground tissues (cortex barrier and pith reaction zone) and vascular tissues. The barrier zone was barely detectable after 24 h but well‐developed 7 days after wounding. Microscopic analyses indicated that the barrier zone was formed by the reinforcement of cell walls with ‘lignin‐like material’ in both ground tissues and vascular tissue, and that, in addition, the lumen of certain xylem cells (vessels and fibres) were blocked by the deposition of polymeric phenolic material. Histochemical characterization revealed that the lignin‐like material (‘defence lignin’) deposited in ground tissue cell walls and xylem cell blockages was poor in syringyl (S‐type) lignin units and therefore differed from the usual mixed guaiacyl–syringyl (G–S) lignin unit composition of E. gunnii developmental lignin. In contrast, S‐type lignin appeared to be deposited in the cell walls of immature developing secondary xylem cells at a stage when the cell walls of comparable cells from unwounded control stems contained lignin poor in syringyl units. The second approach indicated that two different types of cinnamyl alcohol dehydrogenase activity are induced, and apparently regulated differentially, in response to wounding in E. gunnii trees. Coniferyl alcohol dehydrogenase activity was induced immediately and continued to increase throughout the first 15 days of the 17‐day experimental period, while sinapyl alcohol dehydrogenase activity was first detected at 8 days after wounding and continued to increase throughout the experimental period. The biological roles of the two alcohol dehydrogenase activities are discussed in relation to the formation of defence lignin versus developmental lignin in trees.     

Anatomy and lignin distribution of reaction wood in two Magnolia species

Summary Anatomical features of reaction wood formed in two Magnolia species, M. obovata Thunb. and M. kobus DC. which are considered to be among the primitive angiosperms, were observed. In addition, the distribution of guaiacyl and syringyl units of lignins in the cell walls of normal and reaction wood was examined using ultraviolet (UV)- and visible light (VL)- microspectrophotometry coupled with the Wiesner and M?ule reactions. The two Magnolia species formed a tension-like reaction wood without possessing the typical gelatinous layer (G-layer) on the upper side of the inclined stem or branch, in which a radial growth promotion occurred. Compared with the normal wood, the reaction wood had the following anatomical features: (1) the secondary walls of fiber tracheids lacked the S₃ layer, (2) the innermost layer of fiber-tracheid walls showed a small microfibril angle, a fact being similar to the orientation of the microfibril angle of the G-layer in tension wood, and (3) the amounts of lignin decreased in the cell walls of fiber tracheids, especially with great decrease in proportion of guaiacyl units in lignins. In addition, VL-microspectrophotometry coupled with the Wiesner and M?ule reactions adopted in the present study showed potential to estimate the lignin contents in the cell walls and the proportion of guaiacyl and syringyl units in lignins. Received: 15 July 1998     

Reaction wood anatomy and lignin distribution in <Emphasis Type="Italic">Gnetum gnemon</Emphasis> branches

This study investigated the anatomical and chemical characteristics of the reaction wood of a gymnpsperm species, Gnetum gnemon, and discussed on contributing factor for the type of reaction wood in this species. Cell morphology, microfibril angle (MFA) of the S₂ layer and lignin distribution in secondary walls of tracheary elements, and lignin content were examined on three branches. Observations included no G-layer formation, significant decreases in vessel frequency, and altered MFA, and visible-light absorbance after lignin colour reactions in tracheid and fiber tracheid walls on the upper side in almost all samples. These results suggest that reaction wood in G. gnemon was similar to that in ‘tension-wood-like-reaction wood’ in angiosperms. On the other hand, reaction wood showed decrease in the lignin concentration in the fiber tracheid walls compared to the tracheid walls. In addition, the lignin in the tracheid and fiber tracheid walls was originally rich in syringyl units, suggesting that changes in the anatomical and chemical characteristics of secondary xylem due to reaction wood formation might relate to the ratio of the syringyl to guaiacyl units in lignin in the cell walls which function for mechanical support.     

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     

“I-214杨”心材、边材木质素的红外光谱、质子和碳-13核磁共振波谱特征研究

对从杨树心、边材提取的磨木木质素进行了元素分析和红外光谱（FTIR）质子和碳－13核磁共振波谱（^1H,^13C NMR)等化学特征研究。研究结果表明：杨树心、边材木质素的经验式分别为C9H7．16O2．38（OCH3）1．99和C9H8．61O2．73（OCH3）1．33。心材木质素甲氧基含量28．16％,比边材高8．73％。两种木质素均具有典型阔叶材的特征,化学结构类型基本一致,碳骨架结构基本相同,但化学官能团和键型的组成上存在差异。     

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.     

Formation and chemical structures of acid-soluble lignin II: reaction of aromatic nuclei model compounds with xylan in the presence of a counterpart for condensation,and behavior of lignin model compounds with guaiacyl and syringyl nuclei in 72% sulfuric acid

To elucidate the formation and chemical structures of water-soluble material in acid-soluble lignin (ASL), lignin aromatic nuclei model compounds of creosol (I) and 5-methoxycreosol (II) were reacted with xylose or xylan in the presence of apocynol as a counterpart for condensation in 72% sulfuric acid (SA). The reaction of I gave mainly condensation product. However, the condensation reaction of II with apocynol was suppressed because of steric hindrance from the methoxyl group, and II yielded a C-xyloside after refluxing in 3% SA together with condensation products. To obtain information on CHCl₃-soluble material in ASL, model compounds of arylglycerol--aryl ethers with guaiacyl (VIII) and syringyl (X) nuclei were treated by the Klason procedure. VIII gave only insoluble polymerized product, while X gave insoluble polymerized product and CHCl₃-soluble low molecular weight products, which were dissolved in 3% SA. These results prove earlier views that water-soluble material in ASL consists of condensation products formed from syringyl lignin and monosaccharide units in hemicellulose. In addition, the CHCl₃-soluble material in ASL appears to be composed of low molecular weight degradation products from SA treatment of Klason lignin with the syringyl nucleus.Part of this report was presented at the 51st Annual Meeting of the Japan Wood Research Society, Tokyo, April 2001 and at the 47th Lignin Symposium, Fukuoka, October 2002, and was reviewed in Mokuzai Gakkaishi (2002) 48:55–62     

Fermentation characteristics of vinegar residue and some natural materials

1 Introduction Being the world’s largest agricultural country, China has over one billion tons of cellulosic wastes from farms and forests every year. With petroleum prices and the number of automobiles ever increasing in China, there will be more and more needs for alterna tive fuel sources. Using corn to make ethanol is only a part of the potential from agriculture. If cellu lose-containing material like straw and cornstalks could be converted into liquid fuels, it could assist the nation…     

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.     

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.     

At5g54160 gene encodes <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> 5-hydroxyconiferaldehyde <Emphasis Type="Italic">O</Emphasis>-methyltransferase

The function of an Arabidopsis thaliana gene, At5g54160 annotated as a caffeic acid O-methyltransferase CAOMT gene was characterized. The recombinant enzyme of this gene (AtOMT1) catalyzed the O-methylation of phenylpropanoid and flavonoid substrates. The specificity constants (k _cat/K _m) for 5-hydroxyconiferaldehyde (5-HCAld) and quercetin were both 0.11 μM⁻¹·min⁻¹. On the other hand, lignins of At5g54160-knockout Arabidopsis mutants lacked syringyl units. In addition, we showed that the gene silencing also resulted in significant accumulation of caffeyl alcohol (CaAlc). These results strongly suggested that At5g54160 gene is involved in syringyl lignin synthesis for the methylation of both 5-hydroxyconiferaldehyde and 3,4-dihydroxyphenyl compound(s). Part of this work was presented at the Annual Meeting of Japan Society for Bioscience, Biotechnology, and Agrochemistry, March 24–27, 2007     

Acetic acid pulping of wheat straw under atmospheric pressure

Atmospheric acetic acid pulping of wheat straw was carried out. Pulping conditions and their effects on pulp properties were investigated in detail, and a comparison between acetic acid (AcOH) pulp and soda-anthraquinone (AQ) pulps of wheat straw was made of the chemical composition, strength, and fiber morphology of the pulps. Wheat straw was successfully pulped and fractionated into pulp (cellulose), acetic acid lignin, and sugars (monosaccharides from hemicellulose), making it easy to utilize them. It was found that among the pulping conditions the dosage of H₂SO₄ as catalyst was the most notable, and the extent and rate of delignification could be controlled by varying the amount of the catalyst. The results also showed that acetic acid pulp was quite different from soda-AQ pulp. About 70% of the ash or 90% of the silica in wheat straw were kept in AcOH pulp. The ash might function as filler and be beneficial to the printability of paper. It was known that many epidermal cells existed in AcOH pulp in bundles or in single cells. These ash-rich nonfiber cells seemed to hinder the bonding between fibers. AcOH pulp had lower strength than soda-AQ pulp, which might result mainly from the chemical damage of fibers caused by acid, not from the depolymerization of cellulose.Part of this paper was presented at the 65th Pulp and Paper Research Conference of Japan TAPPI, Tokyo, June 1998     

Differences in biosyntheses of guaiacyl and syringyl lignins in woods

Summary Metabolic differences in the formation of guaiacyl and syringyl lignins were explained in terms of the different functions of O-methyltransferases and reducing enzymes which participate in methylation and reduction of the hydroxycinnamic acid intermediates in the biosynthetic pathway of these two types of lignins. Sinapyl alcohol was dehydrogenated with peroxidase and H₂O₂ under various reaction conditions. Chemical properties of the dehydrogenation polymers (DHPs) formed were characterized, and the possible occurrence of syringyl lignin in hardwood was discussed. DHP and dimers of p-coumaryl alcohol were also characterized and discussed in relation to the formation of grass lignin which contains p-hydroxyphenyl propane as an additional lignin monomer.The authors are indebted to Messrs. Y. Nakamura and H. Kuroda in this Division and Mr. T. Yamasaki at Kagawa University for their cooperation in the course of these investigations     

Characterization of lignin-derived products from various lignocellulosics as treated by semi-flow hot-compressed water

To elucidate the decomposition behaviors of lignin from different taxonomic groups, five different lignocellulosics were treated with hot-compressed water (230 °C/10 MPa/15 min) to fractionate lignins into water-soluble portions, precipitates, and insoluble residues. The lignin-derived products in each fraction were characterized and compared. The delignification of monocotyledons [nipa palm (Nypa fruticans) frond, rice (Oryza sativa) straw, and corn (Zea mays) cob] was more extensive than that achieved for Japanese cedar (Cryptomeria japonica, gymnosperm) and Japanese beech (Fagus crenata, dicotyledon angiosperm). The water-soluble portions contained lignin monomers like coniferyl alcohol and phenolic acids, while the precipitates contained higher molecular weight lignin with high content of ether-type linkages. Lignin in the insoluble residues was rich in condensed-type structures. In all five lignocellulosics, ether-type linkages were preferentially cleaved, while condensed-type lignin showed resistance to hot-compressed water. In the monocotyledons, lignin–carbohydrate complexes were cleaved and gave lignins that had higher molecular weights than those eluted from the woods. These differences would facilitate the delignification in monocotyledons. Such information provides useful information for efficient utilization of various lignocellulosics.     

Relations between various extracted basic densities and wood chemical components in Eucalyptus globulus

Relations between various extracted basic densities and wood chemical components were investigated by their within-tree variations in Eucalyptus globulus for assistance in the prediction of the properties of wood or wood-derived products. Extraneous compounds affect the relations between various basic densities and wood chemical components such as holocellulose and the lignin syringyl/guaiacyl ratio. We also discuss the relation of various densities, the molar composition of neutral sugars constituting hemicellulose, and fiber morphology.     

The lamination influence on properties of agro-based particleboard

Abstract

Response surface methodology (RSM) based on a three-level, three-variable central composite rotatable design was applied to evaluate the effects of the parameters such as ratio of rice straw (Oryza sativa)/poplar (Populus deltoids) wood particle bonded with urea-formaldehyde resin in panels, species of wood veneer coating these panels and amount of adhesive in glue line on the modulus of rupture (MOR), internal bonding (IB), and thickness swelling (TS) of panels. Mathematical model equations were derived by computer simulation programming to optimize the properties of the particleboard. These equations that are second-order response functions representing MOR, IB, and TS were expressed as functions of three operating parameters of panel properties. Predicted values were found to be in a good agreement with experimental values (R ² values of 0.96, 0.98 and 0.98 for MOR, IB, and TS, respectively). This study has shown that the RSM could efficiently be applied for modeling panel properties. It was found that the variables affected the properties of panels. Straw usage up to 30% in the mixture did not cause a significant decrease in MOR, IB, and TS. Using beech veneer and 190 g/m² glue line had the highest MOR and lowest TS.     

Lignin biochemistry: Biosynthesis and biodegradation

Summary Lignin biosynthesis via shikimate-cinnamate pathways in plants, and the biosynthetic differences of guaiacyl-and syringyl lignins between gymnosperms and angiosperms have been elucidated by tracer experiments using ¹⁴C labeled precursors and the following enzyme reactions. The formation of guaiacyl lignin but not syringyl lignin in gymnosperms was attributed to the following factors; absence of ferulate-5-hydroxylase, poor affinity of O-methyltransferase toward 5-hydroxyferulate, and lack of activation and/or reduction of sinapatc. A mechanism of lignin-carbohydrate complexes formation in wood cell walls was elucidated based on the reaction of the quinone methide of guaiacylglycerol--guaiacyl ether with sugars, and the analysis of DHP-polysaccharide complexes.The main cleavage mechanisms of side chains and aromatic rings of lignin model compounds and synthetic lignin (DHP) by white-rot fungi and their enzymes, lignin peroxidase and laccase have been elucidated using ²H, ¹³C and ¹⁸O-labeled lignin substructure dimcrs with ¹⁸O₂ and H₂ ¹⁸O. Side chains and aromatic rings of these substrates were cleaved via aryl cation radical and phenoxy radical intermediates, in reaction mediated only by lignin peroxidase/H₂O₂ and laccase/O₂.Academy Lecture presented at the Cellucon 88 in Japan, International Symposium on New Functionalisation Developments in Cellulosics and Wood, held in Kyoto, Japan, November 28 to December 1, 1988This paper is a conclusion of our investigations on the biosynthesis and microbial degradation of lignin for 30 years in the Research Section of Lignin Chemistry, Wood Research Institute, Kyoto University. The author is greatly indebted to Drs. M. Shimada, F. Nakatsubo, T. Yamasaki, H. Ohashi, M. Tanahashi, Y. Nakamura, H. Kuroda, H. Kutsuki, T. Katayama, Y. Kamaya, T. Umezawa and Messrs. H. Fushiki, M. Ohta, A. Noguchi, H. Namba, T. Habe, S. Kawai, S. Yokota, and T. Hattori for their kind cooperation in these investigations. These invstigations were supported in part by Grant-in-Aid Nos. 548047, 57480058, 59760124, 60760130, 60440015, 61760142, 61560193, 62790250 for Scientific Research, and 1980 Grant-in-Aid for Environmental Science (R-33-8), No. 403064 from the Ministry of Education, Science and Culture of Japan, and a 1980 Weyerhaeuser grant     

Wettability changes of wheat straw treated with chemicals and enzymes

A study was conducted to test wettability changes of the wheat straw treated with different methods for the preparation of wheat straw particle board. The wheat straws were separately sprayed with two chemicals (0.6% NaOH, 0.3% H2O2) and three enzymes (lipase, xylanase, cellulase). The contact angle between water and the surface of wheat straw was measured and the spreading-penetration parameters (K-values) were also calculated with wetting model. The surfaces of treated wheat straw and control sample were scanned by means of Micro-FTIR, and their peaks arrangements were analyzed. The surface morphologies of treated wheat straw and control sample were also observed by SEM. Chemical etching was found on the exterior surfaces of the straws treated separately with 0.6% NaOH and 0.3% H2O2; furthermore, the spreading-penetration parameters (K-values) of the distilled water on the exterior surfaces of the treated wheat straw along the grain were higher than that of control. The wettability of exterior surfaces of the wheat straws treated separately with lipase, xylanase and cellulose were improved after treating for seven days, and among the three enzymes treatments, the lipase treatment showed best result. The lipase treatment and NaOH treatment were determined as better methods for improving the wettability of wheat straw surfaces. However, in the economic aspect, NaOH treatment was more practical and easier in the pretreatment for the manufacture of straw particle board.     

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.     

Studies on the lignin of the bamboo species Phyllostachys makinoi Hay.

Summary Milled wood lignin (MWL) isolated from Phyllostachys makinoi was investigated by chemical and physical methods. The bamboo lignin is rich in syringyl units which is indicated by a high methoxyl content and respective bands in the IR spectrum. The distinct absorption bands for aromatic ring vibrations are attributed to final p-coumaryl ester groups. In the UV spectrum the extinction maximum in the range of 280 nm is shifted to longer wavelengths compared to wood lignin. With NaOH and TFA mainly an arabinoxylan with a Xyl: Ara ratio of 14:1 was extracted from bamboo saw dust. The lignin proportion in the NaOH extract, which derives mainly from the secondary walls has a lower methoxyl content, the lignin proportion in the TFA extract, which derives from the compound middle lamellae has a higher methoxyl content compared to MWL.