Distribution of lignin and lignin precursors in differentiating xylem of Japanese cypress and poplar

Lignin is an integral component of the cell wall of vascular plants. The mechanism of supply of lignin precursors from the cytosol into the cell wall of differentiating xylem has not yet been elucidated. The present study showed that a certain amount of coniferyl alcohol glucoside (coniferin) occurred in the differentiating xylem of Japanese cypress (Chamaecyparis obtusa), as previously reported in gymnosperms. Coniferin content peaked in the early stages of secondary wall formation and decreased during lignification. In contrast to gymnosperms, coniferin content was limited in the differentiating xylem of poplar (Populus sieboldii × Populus grandidentata). Moreover, coniferyl alcohol was not detected in all specimens. In the differentiating xylem of poplar, a higher amount of sinapyl alcohol occurred than glucoside (syringin). However, the phloem contained syringin and not sinapyl alcohol. The sinapyl alcohol content in the xylem peaked in the cells with ceasing cell wall formation, and decreased gradually towards the boundary of the annual ring, where the lignin content kept increasing. Sinapyl alcohol in the differentiating xylem of poplar may be used for the lignification of the xylem.     

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     

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.     

‘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.     

Sinapyl alcohol-specific peroxidase isoenzyme catalyzes the formation of the dehydrogenative polymer from sinapyl alcohol

Two peroxidases, CWPO-A and CWPO-C, were isolated from the cell walls of poplar (Populus alba L.) callus culture. The cationic CWPO-C showed a strong preference for sinapyl alcohol over coniferyl alcohol as substrate. Thus, the monolignol utilization of CWPO-C is unique compared with other peroxidases, including anionic CWPO-A and horseradish peroxidase (HRP). CWPO-C polymerized oligomeric sinapyl alcohol (S-oligo) and sinapyl alcohol, producing a polymer of greater molecular weight. In contrast, HRP, which is specific to coniferyl alcohol, produced sinapyl alcohol dimers, rather than catalyzing polymerization. Adding coniferyl alcohol as a radical mediator in the HRP-mediated reaction did not result in S-oligo polymerization. This report shows that CWPO-C is an isoenzyme specific to sinapyl alcohol that polymerizes oligomeric lignols. Its catalytic activity toward oligomeric lignols may be related to the lignification of angiosperm woody plant cell walls.Part of this study was presented at the 45th Lignin Symposium, Matsuyama, October 2000     

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.     

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.     

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     

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.     

Formation and chemical structures of acid-soluble lignin I: sulfuric acid treatment time and acid-soluble lignin content of hardwood

To elucidate the formation mechanism of acidsoluble lignin (ASL) formed in the Klason lignin determination, beech wood meals were treated with sulfuric acid (SA) under various conditions, and the ASL solution was extracted with CHC1₃. The results indicated the following: (1) wood components yielding ASL are dissolved in 72% SA during the initial stage; (2) the quantity of ASL is highest during the initial stage, then decreases with prolonged time of 72% SA treatment and finally reaches a constant value; (3) soluble lignin prepared by 72% SA treatment and subsequent standing in 3% SA again yield insoluble Klason lignin and ASL after boiling in 3% SA; and (4) about half the amount of ASL is dissolved in CHC1₃. The foregoing suggest that wood components yielding ASL are dissolved in 72% SA at the beginning and finally change to ASL after being subjected to depolymerization, hydrolysis, and other reactions. ASL may thus be composed of low-molecular-weight degradation products and hydrophilic derivatives of lignin.     

Origin of free radicals produced from the syringyl end groups in lignins

Enzyme lignin was prepared from reed; it was acid-hydrolyzed and reduced with NaBH₄. Spin concentrations of radicals produced by oxidation of the lignins with K₃[Fe(CN)₆] or H₂O₂ were determined from electron spin resonance spectra. The radicals were observed by the oxidation of sinapyl alcohol. It was found that the two radicals observed by the oxidation of dioxane lignins were deprotonated 2,6-dimethoxyp-benzosemiquinone and 6-hydroxy-2-methoxy-p-benzosemiquinone, and that they were produced from syringyl end groups. The production rate of radicals from syringyl end groups possessing an-carbonyl group was greater than that possessing an-hydroxyl group. It was suggested that stilbene-type syringyl end groups were produced from phenylcoumaran moieties in lignins by acidolysis. The radicals produced from guaiacyl end groups were not observed by the oxidation process.     

Reaction of manganese peroxidase ofBjerkandera adusta with synthetic lignin in acetone solution

The reaction of manganese peroxidase (MnP) of the white-rot fungusBjerkandera adusta with synthetic lignin dehydrogenation polymer, DHP) in acetone medium was investigated. Gel-permeation chromatography of the DHP treated by MnP demonstrated depolymerization of syringyl DHP in the reaction mixture containing 70% acetone; moreover, concomitant repolymerization occurred to give highly polymerized products. Guaiacyl DHP was only repolymerized by MnP in the same acetone solution without giving degradation products. Addition of ascorbic acid to reaction mixtures containing acetone resulted in preferential depolymerization of syringyl DHP.Part of this report was presented at the meeting of Kansai Branch, Japan Society for Bioscience, Biotechnology, and Agrochemistry in Kagawa, October 1996     

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.     

Heterogeneity in formation of lignin

Summary The formation of lignin in the cell wall of compression wood of Pinus thunbergii was examined by selective radio-labeling of specific structural units in the lignin and visualization of the label in the different morphological regions by microautoradiography. Deposition of lignin in the tracheid cell wall of compression wood occurred in the order: p-hydroxyphenyl, guaiacyl and syringyl lignin, which is the same order as observed in normal wood. However, the period of lignification in the compression wood was quite different from those of normal and opposite woods. The p-hydroxyphenyl units were deposited mainly in the early stage of cell wall formation in compound middle lamella in normal and opposite woods, while in compression wood, they were formed in both the compound middle lamella and the secondary wall. The most intensive lignification was observed during the formation of the S₂ layer, proceeding from the outer to inner S₂ layers for a long period in compression wood. In the normal or opposite woods, in contrast, the lignification became active after formation of S₃ had begun, then proceeded uniformly in the secondary wall and ended after a short period.A part of this report was originally presented at the 1989 International Symposium on Wood and Pulping Chemistry at Raleigh, NC, U.S.A.     

Stereochemistry and biosynthesis of (+)-lyoniresinol,a syringyl tetrahydronaphthalene lignan in <Emphasis Type="Italic">Lyonia ovalifolia</Emphasis> var. <Emphasis Type="Italic">elliptica</Emphasis> II: feeding experiments with <Superscript>14</Superscript>C labeled precursors

To clarify the biosynthetic pathway for syringyl lignans, especially syringyl tetrahydronaphthalene lignans and formation of the C2–C7′ linkage, production of (+)-lyoniresinol (LYR) and its predicted intermediates [syringaresinol (SYR), 5,5′-dimethoxylariciresinol (DMLR), and 5,5′-dimethoxysecoisolariciresinol (DMSLR)] in Lyonia ovalifolia var. elliptica was investigated by means of feeding experiments with radiolabeled precursors. Following individual administration of l-[U-¹⁴C]phenylalanine (Phe), [8-¹⁴C]sinapyl alcohol (SA), and [8,8′-¹⁴C]SYR to excised young shoots of L. ovalifolia and their subsequent metabolism, free [¹⁴C]lignans and [¹⁴C]lignan glycosides were extracted with methanol from stems and leaves and were divided into ethyl acetate-soluble fractions (lignans) and aqueous fractions (lignan glycosides), respectively. Using a combination of xylanase, cellulase, and β-glucosidase, the glycosides were hydrolyzed to liberate [¹⁴C]lignans as aglycones. l-[U-¹⁴C]Phe was incorporated into (+)-[¹⁴C]SYR [stem 0.38%, 8% enantiomeric excess (e.e.)], (−)-[¹⁴C]SYR (leaves 2.75%, 72% e.e.), (+)-[¹⁴C]DMLR (stem 0.07%, 18% e.e. and leaves 0.009%, 58% e.e.), (−)-[¹⁴C]DMSLR (stem 0.03%, 46% e.e. and leaves 0.05%, 20% e.e.), (+)-[¹⁴C]LYR (leaves 0.013%, 22% e.e.) and glycosides of (+)-[¹⁴C]LYR (stem 0.036%, 50% e.e.) in 24h. Based on the percent incorporation and enantiomeric composition of the lignans, the biosynthetic pathway of (8R,8′R)-(+)-LYR was proposed as follows: a nonselective dehydrogenative dimerization of sinapyl alcohol yields (±)-SYR, which is reduced with low specificity to give (8R,8′R)-(+)-DMLR. This is cyclized to directly give (+)-LYR as well as reduced again to (8R,8′R)-(−)-DMSLR. Although further transformation of (−)-DMSLR also leads to the formation of (+)-LYR, cyclization could be a main pathway for (+)-LYR biosynthesis. This report was presented at the IAWPS 2005 International Symposium on Wood Science and Technology, Yokohama, November 2005     

Reactivity of syringyl quinone methide intermediates in dehydrogenative polymerization I: high-yield production of synthetic lignins (DHPs) in horseradish peroxidase-catalyzed polymerization of sinapyl alcohol in the presence of nucleophilic reagents

It is known that the conventional dehydrogenative polymerization of sinapyl alcohol (S-alc) gave syringyl synthetic lignins (S-DHPs), but in extremely low yields. In this article, to examine the contribution of syringyl quinone methide intermediates (S-QM) on S-DHP production, horseradish peroxidase (HRP)-catalyzed dehydrogenative polymerization of S-alc was carried out in the presence of nucleophilic reagents that promote the rearomatization of S-QM. First, the HRP-catalyzed polymerization of sinapyl alcohol γ-O-β-D-glucopyranoside (isosyringin, iso-S), which allows us to monitor the polymerization process in a homogeneous aqueous phase, was utilized for screening of a nucleophile used as an S-QM scavenger. Monitoring of iso-S polymerization in the presence of various nucleophilic reagents by UV spectroscopy and gel permeation chromatography with photodiode array detection (GPC-PDA) revealed a high ability of azide ion to convert oligomeric S-QM efficiently to S-DHP. Accordingly, azide ion was utilized as an S-QM scavenger in HRP-catalyzed polymerization of S-alc, which resulted in high-yield production of S-DHPs (∼83%), as expected. The ¹H-, ¹³C-, and 2D-HSQC NMR investigations on the resulting S-DHPs clearly demonstrated that azide ion efficiently performed nucleophilic additions to the C-α of S-QM during the polymerization. These results provide experimental proof that the low reactivity of S-QM with nucleophiles (such as water, phenolic, and aliphatic hydroxyl groups) in the conventional polymerization system critically impedes the production of S-DHPs from S-alc.     

“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。     

Main Anatomy Characteristics in Cell Wall and Lignin Distribution of Bamboo Culms(Pseudosasa amabilis)

The anatomy characteristics of cell wall and lignin distribution in different anatomical regions for 12-month-old bamboo species Pseudosasa amabilis was investigated.The lignin distribution was studied by means of optical microscope and confocal laser scanning microscope and the color reaction. In addition,the distribution of lignin in different tissues(fiber,parenchyma and vessel) was investigated by visible-light microspectrophotometry coupled with the Wiesner and Maule reaction.The cell walls of different tissues were all lignified and the lignin was widely distributed in different tissues, in which there was a difference between tissues and anatomical regions in lignin content.Guaiacyl lignin and syringyl lignin unit could be found in cell wall of fiber,parenchyma and vessel through Wiesner and Maule reaction and the absorbance peak of spectrum. There was no regular variation in lignin content with different radial location of culms and different location in fiber strands.The lamellation of the fiber cell wall was generally had as alternating broad and narrow layers with alternating low and high concentration of lignin.