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     

Look back over the studies of lignin biochemistry

The role of the cinnamate pathway in monolignol biosynthesis based on feeding experiments with lignifying plant stems and characterization of the enzymes in the pathway, O-methyltransferase (OMT), cinnamyl alcohol dehydrogenase (CAD), etc. is discussed. Monolignol biosynthesis via metabolic grids according to newly characterized enzymes in the pathway is also reviewed and discussed. The cleavage mechanisms of side chains and aromatic rings by lignin peroxidase and laccase elucidated by using ¹⁸O, ²H, and ¹³C labeled lignin substructure dimers and DHP with ¹⁸O₂ and H₂ ¹⁸O are reviewed. Finally, the prospects of lignin biochemistry in the wood and paper industries are discussed according to the recent progress on gene technology on wood formation and microbial degradation of lignin.     

Coniferyl aldehyde dimers in dehydrogenative polymerization: model of abnormal lignin formation in cinnamyl alcohol dehydrogenase-deficient plants

The enzymatically dehydrogenative polymerization of coniferyl aldehyde and coniferyl alcohol was studied to understand lignins in cinnamyl alcohol dehydrogenase (CAD)-downregulated plants. The sample dimers were prepared by polymerization under three reaction systems (coniferyl alcohol, coniferyl aldehyde, and their combination) with horseradish peroxidase/H₂O₂ under the conditions of limited reaction time. In addition, the residual amount of substrate in each reaction was determined at specified time intervals. In the reaction system of coniferyl aldehyde, the 5-5-type dimer was formed in preference to- and-5 dimers; in the reaction system of coniferyl alcohol the-5 dimer was preferentially formed. Furthermore, it was revealed when quantifying dimers among reaction systems that the total dimer formation capability of coniferyl alcohol clearly surpassed that of coniferyl aldehyde. However, the dimers cross-coupled with coniferyl alcohol and coniferyl aldehyde were formed in amounts not accounted for by the difference seen in dimer formation abilities with the two substrates.Part of this paper was presented at the 49th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1999     

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.     

Electropolymerization of coniferyl alcohol

Electropolymerization of coniferyl alcohol was carried out in an aqueous system (0.2 M NaOH) and in an organic solvent system [CH₂Cl₂/methanol (4:1 v/v) in the presence of 0.2 M LiClO₄] to produce a dehydrogenation polymer (DHP) - artificial lignin. In both systems, the polymerization of coniferyl alcohol was visually confi rmed. In the aqueous system, no dimer was detected in the reaction medium after electropolymerization, suggesting that endwise polymerization occurred on the electrode surface. Thioacidolysis degradation revealed that the obtained polymers had numerous 8-O-4′ linkages. The electropolymerization products obtained in the organic solvent system also had numerous 8-O-4′ linkages; in particular, the polymers obtained in the initial polymerization stage. This was probably because of the limited area available for reaction and the orientation of coniferyl alcohol on the electrode surface controlled the polymerization.     

Monolignol oxidation by xylem peroxidase isoforms of Norway spruce (Picea abies) and silver birch (Betula pendula)

We partially purified peroxidase isoform fractions from xylem extracts of a gymnosperm, Norway spruce (Picea abies (L.) Karst.), and an angiosperm, silver birch (Betula pendula Roth.), to determine the participation of xylem-localized peroxidases in polymerization of different types of lignin in vivo. Several peroxidase fractions varying in isoelectric point values from acidic to basic were tested for their ability to catalyze the oxidation of the monolignols coniferyl alcohol, sinapyl alcohol and p-coumaryl alcohol in vitro. All of the xylem peroxidases extracted from Norway spruce and most of those from silver birch showed the highest rate of oxidation with coniferyl alcohol in the presence of hydrogen peroxide. The exception was an acidic peroxidase fraction (pI 3.60-3.65) from silver birch that exhibited higher oxidation activity for sinapyl alcohol than for coniferyl alcohol. For the xylem enzyme fractions extracted from silver birch, the ability to oxidize the artificial phenolic substrate syringaldazine coincided with high specific activity for sinapyl alcohol. Therefore, we conclude that the acidic, neutral and basic xylem peroxidases of Norway spruce all function in the synthesis of guaiacyl-type lignin, whereas in silver birch the acidic peroxidases preferentially oxidize sinapyl subunits. The latter provides a mechanism for synthesis of guaiacyl-syringyl lignin typical of tracheid cell walls in angiosperm trees.     

Extracellular peroxidase activity at the hyphal tips of the white-rot fungus Phanerochaete crassa WD1694

The white-rot fungus Phanerochaete crassa WD1694 was cultivated and peroxidase activity staining was performed to determine the sites at which the extracellular peroxidase reaction actually occurs in vivo. Although the ligninolytic peroxidases were found in the culture filtrates, the culture medium did not show a color reaction. However, a particularly strong color reaction was observed on the hyphal tips. Visible spectra and absorbance of the staining were analyzed by microspectrophotometry, and the catalytic rates of the peroxidase reaction at the hyphal tips were calculated. The estimated catalytic rate of the peroxidase reaction at the hyphal tips peaked at 794 μM/min, expressed as the consumption rate of H₂O₂, on day 3 of the cultivation. Analysis of the extracellular enzyme eluted with 0.1% Tween 80 from the mycelium revealed that manganese peroxidase accounted for 89% of all the peroxidase activity measured. The results clearly showed the existence of the concentrated manganese peroxidase reaction around the hyphal tips of the organism.     

Stereochemistry and biosynthesis of 8-O-4′ neolignans in Eucommia ulmoides: diastereoselective formation of guaiacylglycerol-8-O-4′-(sinapyl alcohol) ether

Stereochemistry and biosynthesis of guaiacylglycerol-8-O-4′-(sinapyl alcohol) ether (GGSE), an 8-O-4′ neolignan, which consists of coniferyl and sinapyl alcohol moieties, in Eucommia ulmoides were investigated. Four 8-O-4′ neolignans, GGSE, syringylglycerol-8-O-4′-(coniferyl alcohol) ether (SGCE), guaiacylglycerol-8-O-4′-(coniferyl alcohol) ether (GGCE), and syringylglycerol-8-O-4′-(sinapyl alcohol) ether (SGSE), were synthesized. Their erythro and threo diastereomers were separated through acetonide derivatives, intermediates of the synthesis, and identified by means of nuclear magnetic resonance (NMR) spectroscopy. All of the erythro-acetonide derivatives have larger coupling constants (ca 9 Hz) for the C₇-H resonances than those of the threo ones (1.5–2 Hz). In the case of the four 8-O-4′ neolignans, the C₇-H coupling constants of the threo-isomers are not smaller than those of the erythro ones. GGSE isolated previously from this plant was identified as the erythro isomer by comparison of the ¹³C-NMR data with synthetic erythro-GGSE and threo-GGSE and the other 8-O-4′ neolignans mentioned as above. Administration of a mixture of [8-¹⁴C]coniferyl alcohol and [8-¹⁴C]sinapyl alcohol to excised shoots of E. ulmoides was carried out and the incorporation of ¹⁴C into erythro-[¹⁴C]GGSE was found to be higher than that in threo-[¹⁴C]GGSE. The occurrence of diastereoselective formation of erythro-GGSE by cross coupling of coniferyl and sinapyl alcohols is suggested.Part of this paper was presented at the 47th Lignin Symposium, Fukuoka, October 2002 and the 53rd Annual Meeting of the Japan Wood Research Society, Fukuoka, April 2003     

Substituent effects of 3,5-disubstituted p-coumaryl alcohols on their oxidation using horseradish peroxidase–H2O2 as the oxidant

The consumption rates of three monolignols (p-coumaryl, coniferyl, and sinapyl alcohols) and eight analogues using horseradish peroxidase (HRP)–H₂O₂ as an oxidant were measured and compared with the anodic peak potentials thereof measured with cyclic voltammetry. 3-Monosubstituted p-coumaryl alcohols, i.e., 3-methoxy-, 3-ethoxy-, 3-n-propoxy-, and 3-n-butoxy-p-coumaryl alcohols, had faster reaction rates than p-coumaryl alcohol. This is most probably due to the electron-donating effect of alkoxyl groups. However, the reaction rates gradually decreased with an increase in the molecular weight of the alkoxyl groups. Furthermore, t-butoxyl group, which is a very bulky substituent, caused an extreme reduction in the reaction rate, even though its electron-donating effect was almost the same as that of other alkoxyl groups. The reaction rates of 3,5-disubstituted p-coumaryl alcohols, especially 3,5-dimethyl-p-coumaryl alcohol, were very low compared with 3-monosubstituted p-coumaryl alcohols. These results suggest that there are three main factors of hindrance during the approach of monolignols to the active site of HRP. First, from the results of 3-monoalkoxy-p-coumaryl alcohols, it was suggested that the volume of substituents could decrease their oxidation rates. Second, from the results of 3,5-disubstituted p-coumaryl alcohols, it was suggested that local steric hindrance by the amino residues quite near the heme decreased the oxidation rates. Third, from the results of the substrates with hydrophobic substituents at their 3,5-positions, we suggested that hydrophilicity near heme would decrease their oxidation rates.     

Efficacy of wood preservatives formulated from okara with copper and/or boron salts

As a substitute for high-cost copper azole (CuAz) and alkaline copper quaternary (ACQ) wood preservatives, alternative wood preservatives were formulated with okara, which is an organic waste from the production of tofu, and copper chloride and/or borax. Each preservative was used in treatment of wood blocks in a reduced-pressure method to measure its treatability. The treated wood blocks were placed in hot water for 3 days to examine the stability of the preservatives against hot-water leaching. The preservatives successfully penetrated into wood blocks, probably due to the use of ammonium hydroxide as a dissociating agent. However, the stability of okara-based preservatives dropped as the concentration of acid in the solutions used for hydrolysis of okara increased. The treatability and leachability of the preservatives were not affected by hydrolysis temperature but were negatively affected by the addition of borax. Leached wood blocks treated with okara-based preservatives and exposed to decay fungi Gloeophyllum trabeum and Postia placenta over 12 weeks showed good decay resistance. Okara-based wood preservatives can protect wood against fungal attack as effectively as CuAz, and have potential for use as environmentally friendly wood preservatives.     

Strength,decay and termite resistance of oriented kenaf fiberboards

The development of oriented fiberboards made from kenaf (Hibiscus cannabinus L.) and their suitability as a construction material has been investigated. Three different types of boards consisting of five layers with individual orientations were prepared using a combination of low molecular weight and high molecular weight phenol-formaldehyde (PF) resin for impregnation and adhesion purposes. Additional boards with the same structure were prepared using high molecular weight PF resin only. The mechanical properties of the boards have been examined as well as their resistance against fungal decay and termite attack. All kenaf fiberboards showed elevated mechanical properties compared with medium-density fiberboard made from wood fibers, and showed increased decay and termite resistance. Differences in the decay and termite resistance between the board types were caused by the presence of the low molecular weight PF resin for the impregnation of the fibers. No significant difference was found for the mechanical properties. The effect of the PF resin for impregnation was much clearer in fungal decay resistance than for termite resistance; however, fiber orientation had no effect on both decay and termite resistance of the specimens.     

Effects of mechanical damage and herbivore wounding on H2O2 metabolism and antioxidant enzyme activities in hybrid poplar leaves

The changes of hydrogen peroxide (H₂O₂) metabolism and antioxidant enzyme activities in a hybrid poplar (Populus simonii × P. pyramidalis ‘Opera 8277’) in response to mechanical damage (MD) and herbivore wounding (HW) were investigated to determine whether H₂O₂ could function as the secondary messenger in the signaling of systemic resistance. Results show that H₂O₂ was generated in wounded leaves through MD and HW treatments and systemically in unwounded leaves around the wounded leaves. The activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were also enhanced. However, the H₂O₂ accumulation and antioxidant enzyme activities were inhibited in MD leaves through the pretreatment with DPI (which is a specific inhibitor of NADPH oxidase). The results of this study suggest that H₂O₂ could be systemically induced by MD and HW treatments, and H₂O₂ metabolism was closely related to the change in SOD, APX and CAT activities. A high level of antioxidant enzymes could decrease membrane lipid peroxidation levels and effectively induce plant defense responses. Foundation project: This research is supported by the Key Science Program of the Sate Forestry Administration of China (2006–59), and the National Key Project of Scientific and Technical Supporting Programs Funded by Ministry of Science & Technology of China (2006BAD01A15; 2006BAD24B04). Biography: AN Yu (1982–), female, Postgraduate in College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, P. R. China.     

Fungal–copper interactions in wood examined with large field of view synchrotron-based X-ray fluorescence microscopy

ABSTRACT

The goal of this study was to demonstrate how synchrotron-based X-ray fluorescence microscopy (XFM) can be used to better understand the mechanisms of copper tolerance in wood decay fungi. Copper is a major component in commercial wood preservatives as it is toxic to many wood decay fungi. However, certain fungi are copper tolerant and can attack preservative-treated wood, resulting in structural damage to treated wood members. Here we used large-field XFM to visualize six different elements (K, Ca, Mn, Fe, Cu, and Zn) in the mycelia and wood inoculated with four different species of brown rot wood decay fungi. Wood blocks were partially dipped into a solution of copper sulfate, exposed to fungi in malt extract agar petri dish assays for nine weeks, and then imaged and compared to blocks that were partially dipped in water. The blocks were imaged immediately adjacent to an end-matched control that was placed in malt extract agar petri dish assays for 9 weeks, but not exposed to the fungi so that the differences in the elemental distributions could be directly compared. The colonized wood and mycelia were rich in K, Ca, Mn, and Fe; however, the elements and the spatial distribution in the mycelia and wood differed across fungal species. The most interesting results were the maps showing the copper distribution. While three of the four fungi grew on the copper-rich region of the wood, only one species, Fibroporia radiculosa, dramatically reduced the copper concentration in the region of fungal growth.     

Characterization of host–fungus interactions among wood decay fungi associated with Khaya senegalensis (Desr.) A. Juss (Meliaceae) in Singapore

Tree pruning creates wounds that are amenable for wood decay fungi colonization. To characterize the dynamic host–fungus interactions at this location in Senegal mahogany (Khaya senegalensis), in vitro and in vivo pathogenicity tests were conducted with wood decay fungi associated with this tropical tree species. Fomitiporella caryophylii, Hymenochaete murina and Phellinus noxius isolates were included in this experiment following their frequent isolation from Senegal mahogany pruning wounds. The evaluated isolates demonstrated unique host interactions in laboratory tests that suggest equally divergent prognoses for living Senegal mahoganies affected by these fungi. Although all evaluated fungal isolates successfully breached naturally induced reaction zones, P. noxius alone caused significant mass loss to incubated wood blocks. In addition, P. noxius caused extensive wood decay after inoculation in living hosts, successfully illustrating Koch's postulates for this host–fungus relationship. The wood decay ability, invasiveness and facultative parasitism demonstrated by P. noxius suggest its dominant role in wood decay columns below pruning wounds on living Senegal mahoganies. These results highlight the importance of characterizing specific host–fungus interactions and their implications for wood decay severity below pruning wounds in living trees.     

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     

Biological performance,water absorption,and swelling of wood treated with nano-particles combined with the application of Paraloid B72<Superscript>®</Superscript>

We evaluated fungal decay and mold resistance, leaching, and water absorption of nano-compounds and Paraloid B72^® (PB72) in treated wood specimens to develop new methods of consolidation by combining nano-particles and consolidants. Scots pine wood specimens were treated with dispersions of nano-CuO, nano-ZnO, nano-B₂O₃, nano-TiO₂, and nano-CeO₂. PB72 treatments of nano-particle-treated wood specimens were then carried out by either vacuum or immersion for 24 h. Previously, decayed wood specimens were also consolidated with the nano-compounds and PB72. PB72 treatments reduced element release from treated wood specimens. Nearly all nano-compounds + PB72 treatments increased the biological performance of treated wood specimens against decay fungi tested. PB72-only treated wood specimens had the highest weight losses in decay tests. No improvements were obtained in mold resistance tests when the nano-compounds and PB72 were combined. In nano-compound-only treatments, unleached specimens showed slightly lower water absorption values compared to untreated control specimens. Incorporation of PB72 into nano-compound-treated wood specimens resulted in considerably lower water absorption and volumetric swell. In previously decayed specimens treated with the nano-compounds and PB72 solution, water absorption after 2-h immersion declined compared to control specimens.     

Evaluation of Schizophyllum commune Fr. potential for biodegradation of lignin: A light microscopic analysis

Lignin biodegradation potential of Schizophyllum commune Fr. is studied by using sound wood blocks of Ailanthus excelsa, Azadirachta indica, Tectona grandis, Eucalyptus sp. and Leucaena leucocephala. Initially, in vitro wood decay test showed minor weight loss, but it became rapid after one month. After 120 days of incubation, weight loss was minimum in T. grandis (24.05%) whereas it was maximum in A. excelsa (34.44%). Treated test blocks were characterised by enlargement of pits on ray cell wall, formation of additional boreholes in rays, separation of fibres and cell wall thinning and formation of ‘U’-shape notches. Fungal hyphae moved through the xylem cell lumen, and intercellular spaces formed in response to separation of fibres. Hyphae traverse in adjacent cell through the cell wall pits or by making additional boreholes. In all the species studied, xylem fibres and parenchyma (axial and ray) cells were more susceptible while vessels were resistant to fungal attack. In advanced stage of decay, fibres and axial parenchyma lost their rigidity while vessel walls showed uneven thinning. In the tension wood, G-fibres remained unaffected initially but loosening and separation of gelatinous layer facilitated fungal action and showed similar pattern of cell wall deterioration. Among the wood of different species studied, Tectona was more resistant whereas Ailanthus was more susceptible to fungal attack.     

Interactions between Scytalidium species and brown- or white-rot basidiomycetes in birch wood

Summary Growth and subsequent decay by seven basidiomycetes in birch wood blocks was inhibited by three Scytalidium isolates. Scytalidium initially colonized the surface of the blocks and gradually overgrew the basidiomycetes. In individual wood blocks from 11 Scytalidium-basidiomycete paired treatment combinations, the basidiomycete was not inhibited throughout the entire wood block. These wood blocks demonstrated interspecific interactions and antagonism between the different fungi. The white-rot fungi responded to isolates of Scytalidium by occluding xylem cells with masses of hyphae, forming pseudosclerotial plates in the zone of initial interaction. Scytalidium appeared to gain access into portions of wood colonized by the basidiomycetes only after substantial decay had resulted by the wood decay fungus.     

Colonization patterns and topochemical aspects of sap streak caused by Stereum sanguinolentum in Norway spruce

Fresh sapwood blocks of Norway spruce (Picea abies) were exposed to 12- and 60-day attack by Stereum sanguinolentum in pure culture and were examined with respect to structural, topochemical and colour alterations. Final stages of degradation by 5. sanguinolentum were studied on naturally decayed spruce wood after 1 year of fungal growth. The development of discoloration in the main anatomical directions of wood was assessed. Furthermore, microspectrophotometrical studies on the decomposition of cell wall polyphenols were performed. Initial signs of sap streak appeared already 8 days after fungal infection. Within the three-dimensional decay pattern, mycelial aggregations associ-ated with the ray tissue proved to be the major cause of discoloration. UV extinction measurements taken at 280 nm of different parts of cell wall transverse sections revealed a specific pattern of lignin distribution and its derivatives during fungal degradation.