Quantification of compression wood severity in tracheids of Pinus radiata D. Don using confocal fluorescence imaging and spectral deconvolution

Confocal fluorescence microscopy was used to examine the spectral characteristics of lignin autofluorescence in secondary cell walls of normal and compression wood from Pinus radiata. Using UV excitation, fluorescence spectra of normal and compression wood sections showed significant differences, especially in the outer secondary cell wall of tracheids, with a shift in maxima from violet to blue wavelengths between normal and compression wood. A comparison of normal wood, mild and severe compression wood, showed that the wavelength shift was intermediate in the mild compression wood compared to the severe compression wood, thus offering the possibility of quantifying the severity by measuring ratios of fluorescence at violet and blue wavelengths. Fluorescence induced by blue light, rather than UV, was less well differentiated amongst wood types. Spectral deconvolution indicated the presence of a minimum of five discrete lignin fluorophores in the cell walls of both normal and compression wood tracheids. Comparison with lignin model compounds suggest that the wavelength shift may correspond in part to increased levels of p-hydroxy type lignin in the compression wood samples. The combination of confocal fluorescence imaging and related spectral deconvolution therefore offers a novel technique for characterising cell wall lignin in situ.     

Localization of cell wall polysaccharides in normal and compression wood of radiata pine: relationships with lignification and microfibril orientation

The distribution of noncellulosic polysaccharides in cell walls of tracheids and xylem parenchyma cells in normal and compression wood of Pinus radiata, was examined to determine the relationships with lignification and cellulose microfibril orientation. Using fluorescence microscopy combined with immunocytochemistry, monoclonal antibodies were used to detect xyloglucan (LM15), β(1,4)-galactan (LM5), heteroxylan (LM10 and LM11), and galactoglucomannan (LM21 and LM22). Lignin and crystalline cellulose were localized on the same sections used for immunocytochemistry by autofluorescence and polarized light microscopy, respectively. Changes in the distribution of noncellulosic polysaccharides between normal and compression wood were associated with changes in lignin distribution. Increased lignification of compression wood secondary walls was associated with novel deposition of β(1,4)-galactan and with reduced amounts of xylan and mannan in the outer S2 (S2L) region of tracheids. Xylan and mannan were detected in all lignified xylem cell types (tracheids, ray tracheids, and thick-walled ray parenchyma) but were not detected in unlignified cell types (thin-walled ray parenchyma and resin canal parenchyma). Mannan was absent from the highly lignified compound middle lamella, but xylan occurred throughout the cell walls of tracheids. Using colocalization measurements, we confirmed that polysaccharides containing galactose, mannose, and xylose have consistent correlations with lignification. Low or unsubstituted xylans were localized in cell wall layers characterized by transverse cellulose microfibril orientation in both normal and compression wood tracheids. Our results support the theory that the assembly of wood cell walls, including lignification and microfibril orientation, may be mediated by changes in the amount and distribution of noncellulosic polysaccharides.     

Use of monoclonal antibodies to detect Mn(II)-peroxidase in birch wood degraded by Phanerochaete chrysosporium

Summary A monoclonal antibody (Mab) produced to purified Mn(II)-peroxidase was visualized on and within cell corners of birch wood degraded by Phanerochaete chrysosporium using colloidal gold immuno-transmission electron microscopy techniques. Labelling of the fungal cell membrane and cell wall was also observed. The same Mab was used to visualize the penetration of extracellular fungal metabolite extracts, infiltrated into previously decayed wood. Binding of antibodies to the lignin-rich cell corner region of the middle lamella in wood decayed by P. chrysosporium was observed in sectioned wood blocks and in wood infiltrated with crude extracellular extracts from P. chrysospirium liquid cultures. When a control monoclonal antiserum, produced to extracellular metabolites of Postia (Poria) placenta and cross-reactive with fungal cellulase, was used in labelling, the cellulose rich region of the wood cell walls were labelled. Labelling in the middle lamella cell corners was only noted in what has been described as nonor poorly lignified cell corner regions. Offprint requests to: G. Daniel     

Spectrofluorometric studies of the lipid probe, nile red

We found that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, can be applied as a fluorescent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry (J. Cell. Biol. 1985. 100: 965-973). To understand the selectivity of the staining, we examined the fluorescence properties of nile red in the presence of organic solvents and model lipid systems. Nile red was found to be both very soluble and strongly fluorescent in organic solvents. The excitation and emission spectra of nile red shifted to shorter wavelengths with decreasing solvent polarity. However, the fluorescence of nile red was quenched in aqueous medium. Nile red was observed to fluoresce intensely in the presence of aqueous suspensions of phosphatidylcholine vesicles (excitation maximum: 549 nm; emission maximum: 628 nm). When neutral lipids such as triacylglycerols or cholesteryl esters were incorporated with phosphatidylcholine to form microemulsions, nile red fluorescence emission maxima shifted to shorter wavelengths. Serum lipoproteins also induced nile red fluorescence and produced spectral blue shifts. Nile red fluorescence was not observed in the presence of either immunoglobulin G or gelatin. These results demonstrate that nile red fluorescence accompanied by a spectral blue shift reflects the presence of nile red in a hydrophobic lipid environment and account for the selective detection of neutral lipid by the dye. Nile red thus serves as an excellent fluorescent lipid probe.     

Visualization of halos in the epidermal cell wall of Allium cepa caused by Colletotrichum dematium f. circinans and Botrytis allii using fluorochromes

Halos were detected with epifluorescence microscopy around penetration sites of Colletotrichum dematium f. circinans and Botrytis allii in onion epidermal cell walls as areas of less intense fluorescence or negatively stained areas in fluorescing cell walls following treatments with berberin sulphate and acridine orange but not with brilliant sulphaflavine (which stained the cell wall), ninhydrin, dansylchloride, or analine blue. Since pectin, pectic acid, avacil (microcrystaline cellulose super fine), filter paper, and Sephadex G-100–120 fluoresced with acridine orange and berberin sulphate, it was inferred that the halos were negatively stained or appeared as areas with less intense fluorescence because enzymes from these pathogens degraded cell wall pectin and cellulose at the point of penetration. Spores of both pathogens fluoresced when stained with brilliant sulphaflavine, acridine orange, ninhydrin, and dansylchloride. These stains and berberin sulphate caused germ tubes, appressoria, and primary infection mycelia to fluoresce. Nuclei in these fungal structures fluoresced when stained with acridine orange and brilliant sulphaflavine.     

Localization of dibenzodioxocin substructures in lignifying Norway spruce xylem by transmission electron microscopy–immunogold labeling

The lignification process in mature Norway spruce [Picea abies (L.) H. Karsten] xylem cell walls was studied using transmission electron microscopy (TEM)–immunogold detection with a polyclonal antibody raised against a specific lignin substructure, dibenzodioxocin. The study reveals for the first time the exact location of this abundant eight-ring structure in the cell wall layers of wood. Spruce wood samples were collected in Southern Finland at the time of active growth and lignification of the xylem cell walls. In very young tracheids where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown at all. During secondary cell wall thickening, the dibenzodioxocin structure was more abundant in the secondary cell wall layers than in the middle lamella. The highest number of gold particles revealing dibenzodioxocin was in the S2+S3 layer. Statistically significant differences were found in the frequency of gold particles present in various cell wall layers. For comparison, wood sections were also cut with a cryomicrotome for light and fluorescence microscopy.     

Evidence of fungal activity in silicified gymnosperm wood from the Eocene of southern Patagonia (Argentina)

Evidence of fungal activity expressed as typical decay patterns is described from silicified podocarpaceous wood from the Eocene of Patagonia, Argentina. Decay features consist of tracheids of the secondary xylem that are degraded, resulting in thin-celled, lignin-free, translucent, circular to elliptical areas, some of which have cells devoid of all cell wall components including lignin, hemicellulose, and cellulose, and other areas that show only partial simultaneous decay of all cell wall layers. These patterns conform to the white rot and its variant white pocket rot decay patterns produced by basidiomycetes and ascomycetes in gymnosperm and angiosperm wood in modern terrestrial ecosystems. Coagulated opaque bodies in the lumen of some cells and enlarged secondary walls may represent host reactions to infection or remains of metabolic products of fungal enzymatic activity. Similar decay patterns and reaction features have been described from fossil woods ranging in age from the Devonian to the present. This record expands the fossil record of wood rot fungi and underscores their importance as drivers of biological cycles in ancient terrestrial ecosystems.     

Detection of Lignin Peroxidase and Xylanase by Immunocytochemical Labeling in Wood Decayed by Basidiomycetes

The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra-and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay. Lignin peroxidase was localized at sites within the cell wall where electron-dense areas of the lignified cell wall layers remained. In wood decayed by Trametes versicolor, lignin peroxidase was located primarily along the surface of eroded cell walls. No lignin peroxidase was evident in brown-rotted wood, but slight labeling occurred within hyphal cells. Use of polyclonal antisera to xylanase followed by immunogold labeling showed intense labeling on fungal hyphae and surrounding slime layers and within the woody cell wall, where evidence of degradation was apparent. Colloidal-gold-labeled xylanase was prevalent in wood decayed by all fungi used in this study. Areas of the wood with early stages of cell wall decay had the greatest concentration of gold particles, while little labeling occurred in cells in advanced stages of decay by brown or white rot fungi.     

Characterization of Palo Podrido, a Natural Process of Delignification in Wood

Chemical and morphological changes of incipient to advanced stages of palo podrido, an extensively delignified wood, and other types of white rot decay found in the temperate forests of southern Chile were investigated. Palo podrido is a general term for white rot decay that is either selective or nonselective for the removal of lignin, whereas palo blanco describes the white decayed wood that has advanced stages of delignification. Selective delignification occurs mainly in trunks of Eucryphia cordifolia and Nothofagus dombeyi, which have the lowest lignin content and whose lignins have the largest amount of β-aryl ether bonds and the highest syringyl/guaiacyl ratio of all the native woods included in this study. A Ganoderma species was the main white rot fungus associated with the decay. The structural changes in lignin during the white rot degradation were examined by thioacidolysis, which revealed that the β-aryl ether-linked syringyl units were more specifically degraded than the guaiacyl ones, particularly in the case of selective delignification. Ultrastructural studies showed that the delignification process was diffuse throughout the cell wall. Lignin was first removed from the secondary wall nearest the lumen and then throughout the secondary wall toward the middle lamella. The middle lamella and cell corners were the last areas to be degraded. Black manganese deposits were found in some, but not all, selectively delignified samples. In advanced stages of delignification, almost pure cellulose could be found, although with a reduced degree of polymerization. Cellulolytic enzymes appeared to be responsible for depolymerization. A high brightness and an easy refining capacity were found in an unbleached pulp made from selectively delignified N. dombeyi wood. Its low viscosity, however, resulted in poor resistance properties of the pulp. The last stage of degradation (i.e., decomposition of cellulose-rich secondary wall layers) resulted in a gelatinlike substance. Ultrastructural and chemical analyses of this substance showed the matrix to have no microfibrillar structure characteristic of woody cell walls but to still be rich in glucan.     

Screening Wood Decayed by White Rot Fungi for Preferential Lignin Degradation

A screening procedure in which scanning electron microscopy was used indicated that 26 white rot fungi selectively removed lignin from various coniferous and hardwood tree species. Delignified wood from field collections had distinct micromorphological characteristics that were easily differentiated from other types of decay. The middle lamella was degraded, and the cells were separated from one another. Secondary cell wall layers that remained had a fibrillar appearance. Chemical analyses of delignified wood indicated that the cells were composed primarily of cellulose. Only small percentages of lignin and hemicellulose were evident. Delignified wood was not uniformly distributed throughout the decayed wood samples. White-pocket and white-mottled areas of the various decayed wood examined contained delignified cells, but adjacent wood had a nonselective removal of lignin where all cell wall components had been degraded simultaneously. This investigation demonstrates that selective delignification among white rot fungi is more prevalent than previously realized and identifies a large number of fungi for use in studies of preferential lignin degradation.     

Ultrastructure of the innermost surface of differentiating normal and compression wood tracheids as revealed by field emission scanning electron microscopy

The ultrastructure of the innermost surface of Cryptomeria japonica differentiating normal wood (NW) and compression wood (CW) was comparatively investigated by field emission electron microscopy (FE-SEM) combined with enzymatic degradation of hemicelluloses. Cellulose microfibril (CMF) bundles were readily observed in NW tracheids in the early stage of secondary cell wall formation, but not in CW tracheids because of the heavy accumulation of amorphous materials composed mainly of galactans and lignin. This result suggests that the ultrastructural deposition of cell wall components in the tracheid cell wall differ between NW and CW from the early stage of secondary cell wall formation. Delignified NW and CW tracheids showed similar structural changes during differentiating stages after xylanase or β-mannanase treatment, whereas they exhibited clear differences in ultrastructure in mature stages. Although thin CMF bundles were exposed in both delignified mature NW and CW tracheids by xylanase treatment, ultrastructural changes following β-mannanase treatment were only observed in CW tracheids. CW tracheids also showed different degradation patterns between xylanase and β-mannanase. CMF bundles showed a smooth surface in delignified mature CW tracheids treated with xylanase, whereas they had an uneven surface in delignified mature CW tracheids treated with β-mannanase, indicating that the uneven surface of CMF bundles was related to xylans. The present results suggest that ultrastructural deposition and organization of lignin and hemicelluloses in CW tracheids may differ from those of NW tracheids.     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange.

Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and muscopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracelluar dye contents as low as 5 X 10(-16) mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

A combined FT-IR microscopy and principal component analysis on softwood cell walls

A combined FT-IR microscopy and principle component analysis was used to investigate chemical variations between softwood species as well as types of wood cell walls; latewood tracheids, earlywood tracheids and earlywood ray parenchyma cells. The method allowed us to detect small spectral differences between cell types rather than species and to predict characteristic chemical components of each cell type. The method enabled information to be obtained which allowed a evaluation of the polysaccharide composition even in lignified woody plant cell walls.     

Changes in the arrangement of cellulose microfibrils associated with the cessation of cell expansion in tracheids

 The relationship between the cessation of cell expansion and formation of the secondary wall was investigated in the early-wood tracheids of Abies sachalinensis Masters by image analysis and field emission scanning electron microscopy. The area of the lumen and the length of the perimeter of the lumen of differentiating tracheids increased from the cambium towards the xylem. These increases had just ceased in the case of tracheids closest to the cambium in which birefringence was first detected by observations with a polarizing light microscope. Cellulose microfibrils (MFs) deposited on the innermost surfaces of radial walls were not well ordered during the expansion of cells, but well ordered MFs were deposited at the subsequent stage of cell wall formation. The first well ordered MFs were oriented in an S-helix. The well ordered MFs had already been deposited at the tracheids where birefringence was first detected under the polarizing light microscope. These results indicate that the deposition of the well ordered MFs, namely, the formation of the secondary wall, begins before the cessation of cell expansion of tracheids. Therefore, it seems that the expansion of tracheids is restricted by the deposition of the secondary wall because the cell walls become rigid simultaneously with the development of the secondary wall and, therefore, the yield point of cell walls exceeds the turgor pressure of the cell. Received: 3 July 1996 / Accepted: 24 September 1996     

Fluorescence microscopy of viable mast cells stained with different concentrations of acridine orange

Summary Freshly harvested rat peritoneal mast cells were stained with different concentrations of acridine orange, a metachromatic fluorochrome known to form complexes with chromatin and mucopolysaccharides. Fluorescence metachromasia was observed in cytoplasmic granules in cell populations with intracellular dye contents as low as 5×10^–16 mole per cell, one-half decade lower than required to produce metachromatic staining of the nucleus. Cytoplasmic granules did not stain uniformly throughout the cell; some granules exhibited red fluorescence and others green. As the amount of acridine orange uptake per cell was increased, cytoplasmic fluorescence became uniformly red and nuclear fluorescence gradually changed from green to yellow.     

Combined histochemistry and autofluorescence for identifying lignin distribution in cell walls

Histological staining methods commonly used for detecting cellulose and lignin in cell walls were combined with epifluorescence microscopy to visualize differences in lignification between and within cellular elements. We tested our approach on sections of one-year-old branches of Fraxinus ornus L., Myrtus communis L., Olea europaea L., Pistacia lentiscus L. and Rhamnus alaternus L., containing both normal and tension wood. Sections were subjected to various staining techniques, viz. safranin O, safranin O/fast green FCF, and alcoholic solutions of safranin O/astra blue, according to the commonly accepted protocols. Stained and unstained sections were compared using both light and epifluorescence microscopy. Safranin O with or without counterstaining hid the strong fluorescence of vessel walls, cell corners and middle lamellae allowing the secondary wall fibers to fluoresce more clearly. Epifluorescence microscopy applied to stained sections showed more cell wall details than autofluorescence of unstained sections or white light microscopy of counterstained sections. This simple approach proved reliable and valuable for detecting differences in lignification in thick sections without the need for costly equipment.     

Enhancing aniline blue fluorescent staining of cell wall structures.

Prior staining with the periodic acid-Schiff reaction, toluidine blue O, Congo red, or Calcofluor White M2R New, or reduction by NaBH4 do not interfere with aniline blue-induced fluorescence of sieve plates, new cell walls, pit fields or tracheids in compression wood of conifers. Detail of such fluorescent structures is improved by these treatments because of increased contrast, reduced flare, and a quenching of autofluorescence.     

Enhancing Aniline Blue Fluorescent Staining of Cell Wall Structures

Prior staining with the periodic acid-Schiff reaction, toluidine blue O, Congo red, or Calcofluor White M2R New, or reduction by NaBH₄ do not interfere with aniline blue-induced fluorescence of sieve plates, new cell walls, pit fields or tracheids in compression wood of conifers. Detail of such fluorescent structures is improved by these treatments because of increased contrast, reduced flare, and a quenching of autofluorescence.     

Combined histochemistry and autofluorescence for identifying lignin distribution in cell walls

Histological staining methods commonly used for detecting cellulose and lignin in cell walls were combined with epifluorescence microscopy to visualize differences in lignification between and within cellular elements. We tested our approach on sections of one-year-old branches of Fraxinus ornus L., Myrtus communis L., Olea europaea L., Pistacia lentiscus L. and Rhamnus alaternus L., containing both normal and tension wood. Sections were subjected to various staining techniques, viz. safranin O, safranin O/fast green FCF, and alcoholic solutions of safranin O/astra blue, according to the commonly accepted protocols. Stained and unstained sections were compared using both light and epifluorescence microscopy. Safranin O with or without counterstaining hid the strong fluorescence of vessel walls, cell corners and middle lamellae allowing the secondary wall fibers to fluoresce more clearly. Epifluorescence microscopy applied to stained sections showed more cell wall details than autofluorescence of unstained sections or white light microscopy of counterstained sections. This simple approach proved reliable and valuable for detecting differences in lignification in thick sections without the need for costly equipment.     

A rapid decrease in temperature induces latewood formation in artificially reactivated cambium of conifer stems

Background and Aims Latewood formation in conifers occurs during the later part of the growing season, when the cell division activity of the cambium declines. Changes in temperature might be important for wood formation in trees. Therefore, the effects of a rapid decrease in temperature on cellular morphology of tracheids were investigated in localized heating-induced cambial reactivation in Cryptomeria japonica trees and in Abies firma seedlings. Methods Electric heating tape and heating ribbon were wrapped on the stems of C. japonica trees and A. firma seedlings. Heating was discontinued when 11 or 12 and eight or nine radial files of differentiating and differentiated tracheids had been produced in C. japonica and A. firma stems, respectively. Tracheid diameter, cell wall thickness, percentage of cell wall area and percentage of lumen area were determined by image analysis of transverse sections and scanning electron microscopy. Key Results Localized heating induced earlier cambial reactivation and xylem differentiation in stems of C. japonica and A. firma as compared with non-heated stems. One week after cessation of heating, there were no obvious changes in the dimensions of the differentiating tracheids in the samples from adult C. japonica. In contrast, tracheids with a smaller diameter were observed in A. firma seedlings after 1 week of cessation of heating. Two or three weeks after cessation of heating, tracheids with reduced diameters and thickened cell walls were found. The results showed that the rapid decrease in temperature produced slender tracheids with obvious thickening of cell walls that resembled latewood cells. Conclusions The results suggest that a localized decrease in temperature of stems induces changes in the diameter and cell wall thickness of differentiating tracheids, indicating that cambium and its derivatives can respond directly to changes in temperature.