A study of lignification in loblolly pine tracheids by the SEM-EDXA technique

Summary The lignification process in different morphological regions of loblolly pine tracheids was studied by the SEM-EDXA technique. Prior to S₂ layer formation, lignification was initiated in the cell corner middle lamella and compound middle lamella regions. Subsequently a rapid lignin deposition was observed in both regions, whereas secondary wall lignification was a more gradual process and initiated when the middle lamella lignin concentration was approximately 50% of maximum. Within the secondary wall, the S₁ layer is lignified first. Then, lagging just behind cell wall formation, lignification of the S₂ layer is initiated adjacent to the S₁ layer and extends toward the lumen. Finally, the S₃ layer lignified. Upon completion of lignification, the cell walls had a higher concentration of lignin in both the S₁ and S₃ layers than in the S₂ layer.This Paper is an excerpt from the Ph.D. dissertation of Shiro Saka     

Mechanical constraints on lignin deposition during lignification

Summary The ultrastructure of lignifying cell walls in Pinus radiata D.Don was investigated using potassium permanganate staining and transmission electron microscopy. Lignin deposition occurred at numerous discrete sites within various cell wall regions, suggesting the presence of some initiating agent at these sites. In the middle lamella region, lignin deposition occurred by addition of protolignin monomers to spherical particles of lignin. Lignification was completed by expansion of these spherical particles, initially forming irregular patterns of lignification followed by infilling of adjacent areas. In contrast, lignification in the secondary wall occurred by deposition of protolignin monomers onto the ends of expanding lignin lamellae between cellulose microfibrils leading to greatly elongated patches of lignin due to the greater rate of deposition along the microfibril axis compared to that across it. It is concluded that the cellulose matrix in which lignin deposition occurs, in the secondary wall, can exert a mechanical influence which limits the rate of lignin deposition in the direction perpendicular to the microfibril axis.     

Chemical characterization of tissue fractions from the middle lamella and secondary wall of black spruce tracheids

Summary Elemental and functional group analyses were carried out on tissue fractions from the secondary wall and middle lamella of black spruce tracheids. The secondary wall lignin was found to contain 1.7 times as much methoxyl per C₉ as the middle lamella lignin, indicating a substantial proportion of unmethylated para-hydroxyphenylpropane residues in the middle lamella. The content of carbonyl groups was at least three times larger in middle lamella lignin than in secondary wall lignin. The carboxyl content of the middle lamella was found to be about three times as large as that of the secondary wall. Elemental analyses show a higher carbon and lower oxygen content in the middle lamella than in the secondary wall.     

The distribution of lignin in sprucewood as determined by ultraviolet microscopy

The distribution of lignin in black spruce has been determined quantitatively by the study of 0.5 m transverse sections in a UV microscope. The average lignin concentration in the compound middle lamella was about twice that in the secondary wall. The lignin concentration of the middle lamella at the cell corners of adjacent tracheids was nearly four times that in the secondary wall but the volume of the secondary wall was much greater than the volume of the middle lamella. Thus, for earlywood, 72% of the total lignin was in the secondary wall leaving only 28% in the compound middle lamella and cell corner middle lamella regions. The corresponding values for latewood were 82% and 18% respectively. Use of oblique longitudinal sections of 0.1 m thick permitted the resolution of the compound middle lamella. The lignin concentration in the true middle lamella was found to be equal to that in the cell corner middle lamella and the primary wall lignin content to be about twice that in the secondary wall.

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Zusammenfassung Die Verteilung des Lignins in Fichtenholz wurde quantitativ durch Untersuchung von 0,5 m dicken Querschnitten unter dem UV-Mikroskop bestimmt. Die mittlere Ligninkonzentration war in der Mittelschicht etwa doppelt so hoch wie in der Sekundärwand. Die Ligninkonzentration der Mittelschicht war in den an die Tracheiden anstoßenden Zellecken annähernd viermal höher als in der Sekundärwand, wogegen das Volumen der Sekundärwand wesentlich größer war als das der Mittelschicht. Dagegen befand sich beim Frühholz 72% des gesamten Lignins in der Sekundärwand und nur 28% fanden sich in der Mittelschicht selbst und in ihren Zelleckbereichen. Die entsprechenden Werte für Spätholz betragen 82% bzw. 18%. Die Anwendung von schrägen Längsschnitten von 0,1 m Dicke erlaubte die Auflösung der Mittelschicht. Die Ligninkonzentration in der Mittllamelle war gleich groß wie in der in den Zellzwickeln befindlichen Mittellamelle und der Ligningehalt der Primärwand war etwa doppelt so groß wie derjenige in der Sekundärwand.

     

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.     

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).     

Topochemistry of delignification in Douglas-fir wood with soda,soda-anthraquinone and karft pulping as determined by SEM-EDXA

Summary Delignification studies on 0.5 m sections of Douglas-fir earlywood tracheids pulped by soda, soda-anthraquinone (soda/AQ) and kraft pulping processes were performed by determining bromine concentrations in various morphological regions with SEM-EDXA technique. Soda/AQ pulping was much more selective in removing lignin from the middle lamella regions than either soda or kraft pulping. However, up to 50% delignification, more lignin was removed from the secondary wall by soda or kraft, compared to soda/AQ pulping. The kinetics of lignin removal in the various morphological regions were established. Addition of AQ and sodium sulfide resulted in an earlier transition from a slow initial to a rapid bulk delignification, particularly in the middle lamella, and in an enhanced bulk delignification in the secondary wall. Anthraquinone was also found to promote residual delignification in the secondary wall, where sodium sulfide was not effective. The opposite was observed for the bulk delignification in the middle lamella, where only sodium sulfide addition improved the rate significantly. The great differences observed in the bulk delignification rates between middle lamella and secondary wall in soda pulping as well as their response to additives suggest structural differences between middle lamella and secondary wall lignins.Paper No. 6712 in the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina. The paper, an excerpt from the Ph. D. dissertation of Shiro Saka, was selected for the second place Wood Award for 1980 cosponsored by Forest Products Research Society and the Kirby Forest Industries, Inc., Houston, Texas, and presented at the American Chemical Society Annual Meeting, Las Vegas, Nevada, August, 1980     

Comparative studies on lignin distribution by UV microscopy and bromination combined with EDXA

Summary In order to elucidate a previously reported discrepancy in the ratio of the lignin concentration in the middle lamella to that in the secondary wall as determined by ultraviolet (UV) microscopy and bromination combined with EDXA, the ultraviolet absorptivity of the lignin and the lignin reactivity towards bromination were compared for black spruce wood (Picea mariana Mill.). In addition, UV microscopy and EDXA techniques were applied to the determination of lignin distribution in the tracheids in order to establish the relationship between the two techniques. The results indicated that, although the ultraviolet absorptivity in different morphological regions is essentially the same, the secondary wall lignin was 1.70 times more reactive towards bromination than the middle lamella lignin. By applying the value of 1.70 as a correction to the EDXA results, the estimated lignin distribution by EDXA was in fairly good agreement with that from UV microscopy.The authors would like to thank Dr. J.-F. Revol and Dr. M. Tsuji for their assistance during this study     

Recent progress in the chemistry and topochemistry of compression wood

Summary A review of the chemistry and topochemistry of compression wood with 200 references. Compression wood contains on the average 30% cellulose, 35–40% lignin, 10% galactan, 9% galactoglucomannan, 8% xylan, and 2% of a 1,3-glucan (laricinan). The cellulose is less crystalline, and the xylan has fewer arabinose side chains than in normal wood. The lignin is composed of guaiacylpropane and p-hydroxyphenylpropane units. It is more condensed, has a higher proportion of carbon-carbon bonds, and contains fewer arylglycerol--aryl ether structures than a normal conifer lignin. The ray cells and the primary wall of the tracheids have the same chemical composition in normal and compression woods. The galactan is largely located in the outer region of the secondary wall. Only 5–10% of the lignin in compression wood tracheids is extracellular. The middle lamella is less lignified than in normal wood, while the S₁ and inner S₂ layers have a lignin concentration of 30–40% which is twice as high as in normal wood. The lignin content of the S₂ (L) layer is equal to or higher than that of the intercellular region along the wall. The review is concluded with a brief reference to areas where present information is incomplete or lacking.A portion of an Academy Lecture of the International Academy of Wood Science, presented at the International Symposium on Wood and Pulping Chemistry (Ekmandagarna 1981), held in Stockholm, Sweden, June 9–12, 1981. Reprints of the unabridged review, published under the title Recent Progress in the Chemistry, Ultrastructure, and Formation of Compression Wood in the preprints of the symposium (SPCI Report 38, Vol. 1, p. 99–147) are available from the author. I wish to express my gratitude to my colleague Professor Robert A. Zabel for generous travel assistance     

Lignin distribution in birch (Betula verrucosa) as determined by mercurization with SEM- and TEM-EDXA

Summary A new technique suitable for determination of lignin distribution in hardwoods has been developed. The technique is based on the mercurization of the aromatic nucleus of the lignin and a concomitant determination of the mercury content by the SEM- or TEM-EDXA technique. An incorporation of one mole mercury per mole lignin (C₉-unit) was obtained for the birch lignin. The concentration of lignin was found to be about 3 times higher in the cell corners of the middle lamella than in the secondary wall of the birch fibers. The cell wall of the vessels and the ray cells were almost equal in lignin concentration and had about 1.5–1.6 times higher concentration than the secondary wall of the fibers. No specific mass loss of the organically bound mercury during the electron beam bombardment was found under the conditions used in this work.     

Lignification in cell cultures of Pinus radiata: activities of enzymes and lignin topochemistry

Enzymatic and topochemical aspects of lignification were studied in a Pinus radiata D. Don cell culture system that was induced to differentiate tracheary elements and sclereids with lignified secondary cell walls. The activities of the lignin-related enzymes phenylalanine ammonia lyase (PAL; EC 4.3.1.5) and cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) increased concomitantly with cell differentiation, indicating that the increase in enzyme activity was related to lignification of the cell walls and was not induced by stress. This result also indicates that PAL and CAD are suitable markers for tracheary element differentiation in coniferous gymnosperms. To further characterize lignification in this cell culture system, cellular UV-microspectrophotometry and thioacidolysis were employed. Typical UV-absorption spectra of lignin were obtained from the secondary cell walls of the tracheary elements and sclereids and from the compound middle lamella connecting differentiated cells, and the presence of lignin was confirmed by thioacidolysis. Certain aspects of lignin topochemistry in the cell walls of the tracheary elements were similar to cell walls of P. radiata wood, such as the high lignin concentration in the compound middle lamella connecting adjacent cells and the lower lignin concentration in the secondary cell walls. Therefore, the P. radiata cell culture system appears to be well suited to study the formation of lignified secondary cell walls in coniferous gymnosperms.     

Lignin deposition during earlywood and latewood formation in Scots pine stems

Lignin deposition at consecutive secondary wall thickening stages of early and late xylem cells during annual ring wood formation in Scots pine (Pinus sylvestris L.) stems was studied. Lignin patterns, isolated by thioglycolic acid method, consisted of alcohol-soluble (LTGA-I) and alkali-soluble (LTGA-II) fractions. The sum of two fractions, being the total lignin content, gradually increased in the course of lignification. However, the increments of lignin amount at each development stage of early and late tracheids were different. The intensity of lignin deposition increased in the course of earlywood tracheid maturation and decreased toward the end of latewood cell differentiation. The deposition of two lignin fractions in each layer of forming wood also occurred oppositely. The increment of LTGA-I descended, whereas that of LTGA-II increased from the beginning to the end of early xylem lignification. In contrast, LTGA-I increment dropped, whereas LTGA-II rose during late xylem lignification. Gel permeation chromatography showed that the lignins, formed at the beginning of lignification, were more homogeneous and had higher molecular weight compared with the lignins at the end of cell differentiation. Besides, the content of cellulose, estimated as the residue after lignin isolation, and of cell wall substances, presented as cell wall cross-section areas, at consecutive maturation stages of early and late xylem cells have been found to be different. The data show that lignin deposition occurred in different conditions and with opposite dynamics during early and late xylem formation.     

Heterogeneity of lignin concentration in cell corner middle lamella of white birch and black spruce

Summary Raman microprobe spectroscopy was used to study the concentration of lignocellulosics in the cell corner middle lamella. Spectra obtained from 1.6 m regions, from 30 cell corner middle lamellae of both birch and spruce, showed the presence of lignin. However, the relative concentration of lignin to cellulose varied considerably. These results corroborate the view expressed in previous reports of the need for caution in using the lignin concentration values of cell corner middle lamella as a internal reference for studying the variation of lignin concentration in other morphological regions of the cell wall, such as secondary cell wall layers.The use of trade or firm names in this publication is for reader information and does not imply endorsement by the U.S. Department of Agriculture of any product or service     

Heterogeneity in formation of lignin—XI: An autoradiographic study of the heterogeneous formation and structure of pine lignin

Summary Selective labeling of p-hydroxyphenyl-, guaiacyl-and syringylpropane moieties in protolignin was achieved by administration of corresponding ³H-labeled monolignol glucosides to differentiating xylem of pine. The growing process of the protolignin macromolecule in the specific morphological region was visualized by application of high resolution microautoradiography to the selectively labeled wood tissue.p-Hydroxyphenyl lignin is formed mainly in the compound middle lamella and cell corner in an early stage of cell wall differentiation. There are two peaks of deposition of guaiacyl lignin in the compound middle lamella at an early stage and in the secondary wall at a late stage. The content of condensed guaiacyl units is higher in the middle lamella than in the secondary wall lignin. Syringyl lignin is formed mainly in the inner layer of the secondary wall in a late stage as a minor structural moiety. During the formation of the cell wall, protolignin grows under definite biological regulations to a heterogeneous macromolecule which consists of various structural moieties arranged in a regular manner. The origin of the heterogeneous structure was explained as a result of the biogenesis of protolignin in the cell wall.     

S3 lignin concentration in radiata pine tracheids

Summary Lignin concentration in the S3 layer of the tracheid wall of radiata pine was determined by making porosity measurements of hydrolysed cell walls using interference microscopy, with appropriate corrections for swelling. The mean S3 lignin concentration was approximately 53% v/v. The degree of swelling of different wall layers as a result of hydrolysis was proportional to the lignin concentration in that layer with values of 1.645 for the S2 layer, 1.357 for the S3 layer and 1.053 for the compound middle lamella.     

Cell wall fracture properties in relation to lignin distribution and cell dimensions among three genetic groups of radiata pine

Summary The position of fracture within the cell wall on split tangential longitudinal surfaces of air dry wood was quantified for three genetic groups of Pinus radiata D. Don. Differences in the position of fracture within the cell wall among these groups of trees were compared with lignin distribution and cell wall dimensions. In the control trees equivalent to typical New Zealand grown radiata pine, fracture occurred predominantly between the middle lamella and S1 layers as well as within the S1 layer, producing large numbers of fines on the fracture surface. In the open pollinated NZ850–55 group, fracture occurred predominantly between the S1 and S2 layers as well as within the S1 layer, producing fewer fines on the surface. In the NZ850–55 x Guadalupe group, fracture occurred in a similar fashion to the open pollinated group except for a greater number of transwall fractures exposing the cell lumen on the fracture surface. The differences in fracture behaviour between the control and genetically select groups are attributed to reduced lignification at the S1/S2 boundary in the genetically select trees. Observed differences in both the type of fracture and its location were unrelated to cell dimensions. These observations are discussed in relation to the observed differences in thermomechanical pulping properties exhibited by these groups of trees.     

Lignification in poplar tension wood lignified cell wall layers

The lignification process in poplar tension wood lignified cell wall layers, specifically the S(1) and S(2) layers and the compound middle lamella (CML), was analysed using ultraviolet (UV) and transmission electron microscopy (TEM). Variations in the thickness of the gelatinous layer (G-layer) were also measured to clarify whether the lignified cell wall layers had completed their lignification before the deposition of G-layers, or, on the contrary, if lignification of these layers was still active during G-layer formation. Observations using UV microscopy and TEM indicated that both UV absorbance and the degree of potassium permanganate staining increased in the CML and S(1) and S(2) layers during G-layer formation, suggesting that the lignification of these lignified layers is still in progress during G-layer formation. In the context of the cell-autonomous monolignol synthesis hypothesis, our observations suggest that monolignols must go through the developing G-layer during the lignification of CML and the S(1) and S(2) layers. The alternative hypothesis of external synthesis (in the rays) does not require that monolignols go through the G-layer before being deposited in the CML, or the S(1) and S(2) layers. Interestingly, the previous observation of lignin in the poplar G-layer was not confirmed with the microscopy techniques used in the present study.     

Relation between growth stress and lignin concentration in the cell wall: Ultraviolet microscopic spectral analysis

Lignin content in the cell wall was investigated to examine its relation with growth stress, using an ultraviolet microscopic spectrum analyzer. Although a weak correlation existed between the growth stress and lignin concentration in the compound middle lamella, it was believed that the compound middle lamella did not contribute to compressive growth stress generation as there was no correlation between growth stress and lignin concentration in the cell corner part of the intercellular layer. In the secondary wall, larger compressive growth stress was associated with higher lignin concentration especially in the outer part. This finding confirms that lignin contributes positively to the generation of compressive longitudinal growth stresses in the compression wood and more substantially in the outer part of the secondary wall. This experimental result strongly supports our hypothesis of growth stress generation given by the model.This paper was presented at the International Academy of Wood Science Meeting at Vancouver, Canada, July 1997     

Ultraviolet microspectrophotometric investigation of the distribution of lignin in Prunus jamasakura differentiated on a three-dimensional clinostat

To examine the effect of gravity on lignin content and deposition in plant cells, we used ultraviolet (UV) microspectrophotometry and chemical methods to investigate the secondary xylem of Prunus jamasakura grown on a three-dimensional (3D) clinostat, which simulates microgravity. The stem of the 3D-clinostat specimens elongated with bending and the width of their secondary phloem increased. The UV absorbance of the 3D-clinostat specimens at 278 nm was higher than that of the control specimens, which were grown on the ground, in the wood fiber cell corner middle lamella, compound middle lamella, and fiber secondary wall; the UV absorbance in the vessel secondary wall did not differ between the specimens. The lignin content in the stem, including the bark, of the 3D-clinostat specimens, as determined using an acetyl bromide method, was less than that of the control specimens. In the specimens that differentiated on a 3D clinostat, the amount of lignin in the wood fibers increased, while the proportion of the lignified xylem in the stem decreased relative to control values.     

Lignin distribution across tracheid cell walls of poorly lignified wood from deformed copper deficient Pinus radiata (D. Don)

Summary Lignin topochemistry of tracheid walls from a deformed, copper deficient Pinus radiata (D. Don) tree was examined by linescan and point analyses using a Scanning Electron Microscope and Energy Dispersive Spectrometry. Both opposite and compression wood had abnormal lignin distributions compared to those observed in normal wood from a straight tree. Lignin contents in the compound middle lamella were lower than lignin contents in the secondary wall in both opposite and compression wood tracheids.One of us (G. D.) held a Commonwealth Forestry Postgraduate Research Award during this study. The research was supported in part by a grant from the Reserve Bank of Australia Rural Credits Development Fund, the Pine Fund, and members of the forest industry