Studies on opposite wood in conifers part II: Histology and ultrastructure

Summary A study has been made of the histology and ultrastructure of opposite wood in Larix laricina, Picea rubens, and Pinus resinosa. The width of the growth rings varied considerably, in one case from 0.1–1.0 mm, with the wide rings containing a much higher proportion of latewood than the narrow ones. The earlywood tracheids were square in outline and more regularly arranged than in normal wood. In the latewood they were sometimes irregular and distorted. The S₃ layer in the tracheids was 0.2 m thick in the earlywood and 0.4–0.8 m in the latewood, as compared to a thickness in normal wood of 0.1–0.2 m in both zones. The S₃ was often buckled in the latewood and was terminated towards the lumen by a spiral thickening. The cell wall structure of the tracheid pit border was described. Normal coniferous wood might be regarded as an intermediate between opposite wood and compression wood.This paper is dedicated to Dean Edwin C. Jahn in honor of his 70th birthday.     

Characterization of cellulose in compression and opposite woods of a Pinus densiflora tree grown under the influence of strong wind

Summary Structural factors in a Pinus densiflora tree grown under the influence of strong wind were measured. No difference for cellulose molecules was noticed between compression and opposite wood, but the was somewhat lower in the region where the compression wood was concentrated. The degree of crystallinity of cellulose was 45–50% in compression wood, about 50% in normal wood, and 50–60% in opposite wood. The crystallinity decreased with increasing height above the ground. The maximum point of crystallographic b-axis (fiber axis) orientation distribution for cellulose crystallites in compression wood was located at 30°, in normal wood at 25° and in opposite wood at 0°. The cellulose crystallite dimension in the transverse direction was 3.2 nm, corresponding to four cellulose unit cells, a value that was almost constant throughout the wood. In the longitudinal direction, there were large differences in cellulose crystallite dimensions between compression and opposite woods. In compression wood the cellulose crystallite dimensions was 12 nm corresponding to 11–12 cellulose unit cells. In opposite wood it was 17–32.5 nm corresponding to 17–32 cellulose unit cells. These structural factors were apparently affected by the environmental conditions, and the mechanical properties of the wood were influenced by these factors. Opposite wood had longer crystallites, a higher degree of crystallinity and a better orientation distribution of cellulose crystallites in the longitudinal direction. Compression wood, on the other hand, had shorter crystallites, a lower degree of crystallinity and a large angle between the stem and the direction of the crystallites.     

Seasonal changes in lignin distribution during tracheid development in Pinus radiata D. Don

Summary Lignin distribution in developing tracheids of Pinus radiata was studied throughout the growth' season using quantitative interference microscopy. The pattern of lignification remained constant although the number of lignifying cells varied reaching a maximum in summer. Lignification of the secondary wall of latewood tracheids was incomplete at the onset of winter. Each stage of lignification was preceded by deposition of carbohydrates with lignification of the middle lamella starting after S1 formation and lignification of the secondary wall starting after S3 formation. Lignification of the middle lamella was completed before the start of lignin deposition in the secondary wall. In one of the trees examined, the secondary wall lignified concurrently with the middle lamella and this was associated with a low lignin concentration in the middle lamella of mature cells. The secondary wall reached a mature lignin concentration of 21–22% v/v except in one specimen containing severe compression wood which reached 28% v/v. The cell corner middle lamella reached a mature lignin concentration of 74–87% v/v.     

Studies on compression wood—part VII: Distribution of lignin in normal and compression wood of tamarack [Larix laricina (Du Roi) K. Koch]

Summary The distribution of lignin has been studied in tracheids and ray cells of normal and compression wood of tamarack [Larix laricina (Du Roi) K. Koch]. The three layers in the secondary wall of normal wood tracheids are lignified to approximately the same extent, and previous evidence that the S ₃ layer should contain a higher proportion of lignin than the other regions has not been confirmed. The lignin follows closely the orientation of the cellulose microfibrils in all three layers. Compared to the tracheids, the ray cells contain a denser network of lignin in their secondary wall.Only a small proportion of the total lignin in compression wood tracheids is present in the compound middle lamella. The thick S ₁ layer is only slightly lignified; the orientation of the lignin in this region is that of the transversely oriented, lamellated microfibrils. The outer portion of S ₂ consists largely of lignin but also contains lamellae of cellulose microfibrils which probably have the same helical orientation as the microfibrils in the inner part of S ₂. The latter region, which contains the helical cavities, consists of lamellae of cellulose microfibrils which are uniformly encrusted with lignin. The ray cells in compression wood appear to be lignified to the same extent as in normal wood. Transverse sections of the cells reveal a lateral orientation of the lignin. The orientation of the cellulose microfibrils in the S ₂ layer of the first-formed springwood tracheids of compression wood is the same as in the cells which are formed later. It is suggested that for ease of reference, the outer, lignin-rich layer in compression wood tracheids be referred to as the S ₂(L) layer.

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Zusammenfassung Im Druckholz und im normalen Holz von Tamarack (Larix laricina (Du Roi) K. Koch) wurde die Verteilung des Lignins in Tracheiden und Markstrahlzellen untersucht. Die drei Schichten der Sekundärwand in den Tracheiden normalen Holzes werden in nahezu demselben Umfange lignifiziert. Frühere Feststellungen, daß die S ₃-Schicht einen höheren Ligningehalt erreicht als andere Zellwandbereiche, konnten also nicht bestätigt werden. Das Lignin folgt sehr genau der Orientierung der Cellulose-Mikrofibrillen aller drei Schichten. Im Vergleich zu den Tracheiden erfahren die Sekundärwände der Markstrahlzellen eine stärkere Ligninauskleidung.Nur ein geringer Prozentsatz des gesamten Lignins der Druckholztracheiden befindet sich in der Mittellamelle. Die dicke S ₁-Schicht ist nur wenig lignifiziert. Die Orientierung des Lignins in diesem Bereich entspricht den transversal orientierten, lamellierten Mikrofibrillen. Der äußere Teil der S ₂-Schicht enthält sehr viel Lignin, daneben aber auch Lamellen von Cellulose-Mikrofibrillen, die wahrscheinlich dieselbe spiralige Orientierung besitzen wie die Mikrofibrillen des inneren Teiles der S ₂-Schicht. Der letzterwähnte Bereich, der spiralige Kavitäten enthält, weist Lamellen von Cellulose-Mikrofibrillen auf, in welche gleichmäßig Lignin eingelagert ist. Die Markstrahlzellen des Druckholzes erscheinen ebenso stark lignifiziert wie die Markstrahlzellen des Normalholzes. Querschnitte durch diese Zellen lassen die laterale Orientierung des Lignins erkennen. Die Orientierung der Cellulose-Mikrofibrillen in der S ₂-Schicht der zuerst gebildeten Frühholztracheiden des Druckholzes ist dieselbe wie in jenen Zellen, die später ausgeformt werden. Es wird vorgeschlagen, daß zur eindeutigeren Kennzeichnung die äußere ligninreiche Schicht der Druckholztracheiden als S ₂(L)-Schicht bezeichnet wird.

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The authors wish to express their gratitude to Messrs. A. K. Bentum, D. C. Jones, and B. W. Simson for technical assistance. They are also thankful to Dr. D. A. I. Goring, McGill University, Montreal, Canada, for valuable discussions and for making available to them important, unpublished information. This investigation was supported by the United States Department of Agriculture, Forest Service, through Forest Service Research Grant No. 1, which is hereby gratefully acknowledged.     

Significance of laricinan in compression wood tracheids

Summary After a -D-1,3-linked glucan had been isolated from compression wood, identified, and named laricinan, other researchers suggested that it occupied the helical cavities in the S₂ layer of those tracheids. They postulated that the glucan was responsible for the capacity of compression wood to generate the large forces associated with reorientation of displaced stems and branches, and also caused its severe shrinkage with drying.Analyses herein indicate that it is improbable that such a glucan could be the primary factor responsible for those characteristics of compression wood. An alternative significance is proposed, namely that its presence strengthens the already well-supported deduction that the helical cavities have a schizogenous origin.     

Distribution of lignin in normal and compression wood of Pinus taeda L.

Summary The distribution of lignin in normal and compression wood of loblolly pine (Pinus taeda L.) has been studied by the technique of lignin skeletonizing. Hydrolysis of the wood carbohydrates with hydrofluoric acid left normal wood tracheids with a uniform distribution of lignin in the S1 and S2 cell wall layers. However, the S3 region of both earlywood and latewood tracheids consistently retained a dense network of unhydrolyzable material throughout, perhaps lignin.Lignin content in compression wood averaged about 7% more than in normal wood and appears to be concentrated in the outer zone of the S2 layer. The inner S2 region, despite helical checking, is also heavily lignified. The S1 layer, although thicker than normal in compression wood tracheids, contains relatively little lignin.Ray cells, at least in normal wood, appear to be lignified to the same extent, if not more so in certain cases, than the longitudinal tracheids. Other locations where lignin may be concentrated include initial pit border regions and the membranes of bordered pits.This report is a detailed excerpt from the Ph. D. dissertation of R. A. P. Financial support provided by the College of Forestry at Syracuse University and the National Defense Education Act is hereby gratefully acknowledged.     

Delignification of cell walls of <Emphasis Type="Italic">Chamaecyparis obtusa</Emphasis> during alkaline nitrobenzene oxidation

To clarify the behavior of whole lignins in wood cell walls during alkaline nitrobenzene oxidation, the delignification process from cell walls in normal and compression woods of Chamaecyparis obtusa Endl. (Cupressaceae) was observed using ultraviolet and transmission electron microscopies. The lignin content conspicuously decreased to around 10% after 35min in normal wood. The lignin content in compression wood finally leveled off at aroumd 10% after 50min. In gel filtration of oxidation products in ethyl acetate, a high molecular weight fraction was prominent in extracts from the early stage of the reaction. As the oxidation progressed, the high molecular weight fraction became less prominent in both normal and compression wood. Changes in the weights of cell wall residues during reaction indicated that approximately half of the components other than lignin were also removed from the cell walls. This shows that the majority of lignin with relatively high molecular weight is removed from the cell walls together with polysaccharides in the early stage of the reaction and that further oxidative degradation occurs in solution in later stages. Only a small amount of the lignin with low molecular weight could be analyzed by gas chromatography.Parts of this report were presented at the 47th (Kochi, April 1997) and 48th (Shizuoka, April 1998) Annual Meetings of the Japan Wood Research Society, and at the Lignin Symposium, Sapporo, October 1997     

Effects of quenching on relaxation properties of wet wood

A new relaxation property is discussed on the basis of creep behavior of wet wood specimens pretreated with heating at various temperatures followed by quenching. The treated samples showed more marked relaxation than that of an untreated sample. The relationship between relaxation time and heating history was represented by an equation ln() = –( _f – k ₁)T + [ln( _g) + k ₂], where ln() is the logarithmic relaxation time of wet samples after quenching, T is the difference between the heating temperature and the glass transition temperature (T _g), ln( _g) is the logarithmic relaxation time at T _g, is a constant, _f is the coefficient of thermal bulk expansion, and k ₁ and k ₂ are constants. It was concluded from the analysis of experimental results that the change in the relaxation property caused by heating and the following quenching is due to the temporary free volume created by freezing of molecular chain motion of wood components, most probably lignin, during quenching.This work was presented at the 52nd Annual Meeting of Japan Wood Research Society, Gifu, April 2002     

Helical thickenings and helical cavities in normal and compression woods of Taxus baccata

Summary The longitudinal tracheids in compression wood of Taxus baccata contain helical thickenings but no helical cavities. The thickenings are as frequent and well developed and have the same ropelike appearance as in normal wood of this species. They are an integral part of the S₃ in normal and of the S₂ in compression wood and have the same orientation as the innermost microfibrils in these layers. Except for the absence of cavities and presence of thickenings, compression wood tracheids of Taxus baccata possess all the anatomical features typical of such cells, including a rounded outline, intercellular spaces, a thick S₁ layer, a highly lignified S₂ (L) layer, and no S₃ layer. Pronounced compression wood of Pseudotsuga menziesii contains helical cavities but no helical thickenings. Thickenings and cavities seem to be mutually exclusive in Pseudotsuga and Taxus.This investigation was carried out under the McIntire-Stennis Program, Cooperative State Research Service. I am indebted to Mr. A. Rezanowich of the Pulp and Paper Research Institute of Canada for kindly providing the scanning electron micrographs.     

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.     

Structure and function of flexure wood in Abies fraseri

Wood produced during flexure in one-year-old leaders of Abies fraseri (Pursh) Poir. (Fraser fir) was analyzed anatomically and radio-densitometrically. More xylem cells were produced in stems subjected to flexing than in stems that were not flexed. The lumens of tracheids produced in response to flexure were smaller than the lumens of tracheids in normal wood. This was manifest as an increase in the cell wall area/cell lumen area ratio. Microfibril orientation in flexure-induced wood approached the less extreme values found in compression wood. The growth ring composed of flexure-induced wood also had a greater density than normal wood. Compression wood, as defined by cellular characteristics observed in transverse section, was absent in flexed stems. Detailed analysis of the anatomical structure, wood density and biomechanical properties of flexure-induced wood indicated that it has more in common with compression wood than with normal wood.     

Application of the SB distribution to the prediction of concomitant wood properties

Summary It is frequently the case that multiple strength properties of structural wood members are simultaneously needed in design or research. A method has been developed to predict the probability distribution of concomitant material properties of wood from a knowledge of some correlated, nondestructive material property. The method developed in this study is based on properties of the univariate S_B distribution and the bivariate S_BB distribution. The technique involves a priori knowledge of the correlation relationship between a nondestructive parameter and the strength parameter for the two concomitant properties in question. A relationship is developed between the two nondestructive parameters and a simulation procedure is presented to predict either concomitant property from a single nondestructive measurement. The results showed that when a single parameter was predicted directly from a correlated variable, the simulated and experimental distributions were very similar (average error 3–4%). This result confirms previous findings. For the prediction of the concomitant property from an indirect relationship with another parameter, the absolute value of the average error was about 13%.     

The mechanism of the Mäule colour reaction introduction of methylated syringyl nuclei into softwood lignin

Summary Ezo spruce (Picea jezoensis) wood meal and milled wood lignin were successively reduced with sodium borohydride, methylated with methanol-HCl, oxidized with Fremy's salt, reduced with sodium dithionite, and methylated with diazomethane. Permanganate oxidation of the treated milled wood lignin showed that 0.08–0.1/C9 units of 3,4,5-trimethoxyphenyl groups were introduced into the softwood lignin. Although hardwood meal (beech, Fagus crenata) methylated with diazomethane gave a purple-red colour with the Mäule test, the treated softwood meal gave only a dark brown colour. The aromatic nuclei of lignin were broken down by the Mäule treatment. The consumption of permanganate by treated softwood lignin was higher than by hardwood lignin, which suggests that the guaiacyl nuclei were broken down severely. It is proposed that the purple-red colour obtained from methylated hardwood lignin with the Mäule colour test is generated by reaction of syringyl groups which were liberated by -ether cleavage under the permanganate oxidation conditions.     

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     

Distribution of lignin in normal and tension wood

Summary The distribution of lignin in normal and tension wood of four hardwood species has been studied by examination in the electron microscope of the lignin skeletons remaining after removal of the polysaccharides with hydrofluoric acid. In normal wood fibers, the S₁ had a higher lignin concentration than the S₂ layer, which was not as highly lignified as in conifer tracheids. Vessels had a high concentration of lignin in both normal and tension wood, while the extent of lignification of the parenchyma was variable.In tension wood fibers, the S₁ and S₂ layers were highly lignified. A thick, unlignified G-layer was often associated with an extremely thin S₂ layer with a high concentration of lignin. In both normal and tension wood, the lignin had the same orientation as the cellulose micro-fibrils in the different cell wall layers. The results confirm the earlier conclusion that, in the species investigated, the same amount of lignin is present in gelatinous as in normal fibers. Evidently, the lignification mechanism operates normally in the non-gelatinous layers of the fibers, as well as in the vessels and in the parenchyma of tension wood.

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Zusammenfassung Die Ligninverteilung im Normalholz und im Druckholz von vier Laubhölzern wurde untersucht. Die Ligningerüste, die nach der Entfernung der Polysaccharide durch Fluorwasser-stoffsäure übrigblieben, wurden im Elektronenmikroskop beobachtet. In den Normalholzfasern hatte die S₁-eine höhere Ligninkonzentration als die S₂-Schicht, die weniger lignifiziert war als in den Koniferentracheiden. Die Gefäße hatten eine hohe Ligninkonzentration in sowohl Normal-als in Zugholz, während der Lignifizierungsgrad der Parenchymzellen variierte.In den Zugholzfasern waren die S₁- und S₂-Schichten völlig lignifiziert. Eine dicke, unlignifizierte G-Schicht war oft mit einer außerordentlich dünnen S₂-Schicht, die eine hohe Ligninkonzentration zeigte, verbunden. Sowohl im Normal- wie auch im Zugholz besaß das Lignin dieselbe Orientierung wie die Cellulosemikrofibrillen in den verschiedenen Zellwandschichten. Die Ergebnisse bestätigen den früheren Schluß, daß in den hier untersuchten Laubhölzern in den gelatinösen und in den normalen Fasern dieselbe Ligninmenge vorliegt. Offenbar läuft der Mechanismus der Lignifizierung in den S₁- und S₂-Schichten der gelatinösen Fasern des Zugholzes normal ab.

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This investigation was supported by the United States Department of Agriculture, Forest Service, through Forest Service Research Grant No. 1, which is hereby gratefully acknowledged.     

Lignin distribution in spruce (Picea abies) determined by mercurization with SEM-EDXA technique

Summary The lignin distribution between the middle lamella and the cell wall of spruce fibers has been determined by a new technique based on a mercurization of the lignin and a concomitant determination of mercury by the SEM-EDXA technique. The ratio of lignin in the middle lamella at the cell corners to the lignin in the secondary wall was 2.5±0.6 for latewood and 2.4±0.6 for earlywood. This gives a lignin content of 55–58% in the true middle lamella in the cell corners. The reactivity to mercuric acetate of different wood elements was determined in separate experiments. Fractions enriched in ray cells, middle lamella, and compression wood all reacted at the same rate as the whole wood; about one mole of mercury was incorporated per mole of lignin (C₉-unit).     

Estimation of the biomass of fine roots and mycorrhizal fungi: a case study in a Japanese red pine (Pinus densiflora) stand

As part of a study on soil carbon flow in forest ecosystems, the biomass of fine roots (2.0mm in diameter) and root-associated fungi, including ectomycorrhizal fungi, were estimated in the summer season in 1998 at a Pinus densiflora (Japanese red pine) stand in western Japan. Fine roots of pine were classified into three categories: class I roots (0.5–2.0mm in diameter), long class II roots (long roots with diameter 0.5mm; II_L), and short class II roots (short roots with diameter 0.5mm; II_S). Total biomass of fine roots (I + II_L + II_S) at this stand was estimated to be 91.0gm^–2, about 23% of which was class II roots (II_L + II_S). Ergosterol, which is a component of fungal membranes, was analyzed to estimate the biomass of root-associated fungi in roots. In the upper soil layers (from the surface to 13.4cm in depth), ergosterol contents in the class I, II_L and II_S roots were in the ranges 43.1–82.2, 126.1–196.3 and 271.2–321.0µgg^–1 root DW, respectively. The ergosterol content was converted to fungal biomass using the median (minimum–maximum) value of ergosterol concentration reported for ectomycorrhizal fungi. Root-associated fungal biomass in this stand was estimated to be 2.0 (0.5–9.6) gm^–2. The data suggest the biomass of ectomycorrhizal fungi in the P. densiflora stand is small compared with that in other forest ecosystems.     

The occurrence of p-hydroxyphenylpropane units in the middle-lamella lignin of spruce (Picea abies)

Summary A sieving technique has been developed for the separation of middle-lamella fragments. The middle-lamella fraction as well as the whole wood and compression wood from Picea abies have been analysed by nitrobenzene oxidation and acidolysis in order to determine the content of p-hydroxyphenylpropane units in the middle-lamella lignin. These analyses revealed only traces of p-hydroxyphenylpropane units in the whole wood and in the middle-lamella fraction but considerable amounts were found in compression-wood lignin. This points to the fact that middle-lamella lignin is of guaiacyl nature and that earlier results reporting high proportions of p-hydroxyphenylpropane units in the middle lamella-lignin may be due to the inclusion of compression wood in the fraction studied. The acidolysis experiments further indicate that the middle-lamella lignin has fewer uncondensed -0-4 aryl ether structures than the whole wood lignin.The skilful technical assistance of Mrs. Britta Samuelsson and Mr. Johan Lindberg is highly appreciated. The author also thanks Dr. Knut Lundquist for supplying some of the reference compounds, and Dr. Hanne-Lise Hardell for help with the microscopic pictures     

Chemical and physical changes required for producing dimensionally stable wood-based composites

Summary The springback of compressed wood caused by built-up internal stresses results in excessive thickness swelling of wood-based composites when exposed to moisture. Steam pretreatment can cause partial hydrolysis of hemicellulose for both hardwoods and softwoods which markedly increases the compressibility of wood and in turn significantly reduces the build-up of internal stresses in composites during hot pressing. This steam pretreatment process is a very effective method for producing dimensionally stable wood-based composites. Mild steam pretreatments (e.g. 3 to 4 min at 1.55 MPa) cause a significant reduction of the water insoluble xylan content in hardwoods and the amount of xylan, mannan and galactan in softwoods without any apparent changes in the cellulose or lignin content.The authors wish to thank G. Bastien and B. S. Lethbridge for technical assistance. W. Schwald is grateful to the Fonds zur Förderung der wissenschaftlichen Forschung, Vienna, Austria for granting an Erwin-Schrödinger scholarship (Proj. No. J0128C)     

Analysis of the structure of lignin-carbohydrate complexes by the specific13C tracer method

In the present study the specifically¹³C-enriched lignin precursors of biosynthesis (i.e., coniferin-[side chain–¹³C], coniferin-[side chain-–¹³C] and coniferin-[side chain-–¹³C]) were synthesized and administered exogeneously to ginkgo shoots (Ginkgo biloba L.) to obtain¹³C-enriched lignin-carbohydrate complexes (LCCs). The specifically¹³C-enriched LCCs were isolated from the newly formed xylem of ginkgo shoots administered with the¹³C-enriched precursors and degraded by enzymes. Lignin-rich fractions, so called enzyme-degraded LCCs (EDLCCs), were obtained. By determining their¹³C-NMR spectra, information related to the chemical structure of lignin building units and linkages between phenylpropane units of lignin and carbohydrates were obtained. It was found that these precursors were incorporated in natural lignin successfully. Three lignin-carbohydrates linkages (i.e., ether type, ester type, ketal type) were found at the C -position of the side chain of phenylpropane units in ginkgo LCC. No lignin-carbohydrate bond at the C- or C-position of the lignin side chain was observed in the¹³C-NMR spectra of the¹³C-enriched LCCs. This fact indicates that a specific¹³C tracer technique can be useful in NMR study of the chemical structure of LCCs.Part of this paper was presented at the 5th Pacific Polymer Conference, Kyongju, Korea, October 1997