Safranine fluorescent staining of wood cell walls

Safranine is an azo dye commonly used for plant microscopy, especially as a stain for lignified tissues such as xylem. Safranine fluorescently labels the wood cell wall, producing green/yellow fluorescence in the secondary cell wall and red/orange fluorescence in the middle lamella (ML) region. We examined the fluorescence behavior of safranine under blue light excitation using a variety of wood- and fiber-based samples of known composition to interpret the observed color differentiation of different cell wall types. We also examined the basis for the differences in fluorescence emission using spectral confocal microscopy to examine lignin-rich and cellulose-rich cell walls including reaction wood and decayed wood compared to normal wood. Our results indicate that lignin-rich cell walls, such as the ML of tracheids, the secondary wall of compression wood tracheids, and wood decayed by brown rot, tend to fluoresce red or orange, while cellulose-rich cell walls such as resin canals, wood decayed by white rot, cotton fibers and the G-layer of tension wood fibers, tend to fluoresce green/yellow. This variation in fluorescence emission seems to be due to factors including an emission shift toward red wavelengths combined with dye quenching at shorter wavelengths in regions with high lignin content. Safranine fluorescence provides a useful way to differentiate lignin-rich and cellulose-rich cell walls without counterstaining as required for bright field microscopy.     

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.     

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.     

Modelling of the hygroelastic behaviour of normal and compression wood tracheids

Compression wood conifer tracheids show different swelling and stiffness properties than those of usual normal wood, which has a practical function in the living plant: when a conifer shoot is moved from its vertical position, compression wood is formed in the under part of the shoot. The growth rate of the compression wood is faster than in the upper part resulting in a renewed horizontal growth. The actuating and load-carrying function of the compression wood is addressed, on the basis of its special ultrastructure and shape of the tracheids. As a first step, a quantitative model is developed to predict the difference of moisture-induced expansion and axial stiffness between normal wood and compression wood. The model is based on a state space approach using concentric cylinders with anisotropic helical structure for each cell-wall layer, whose hygroelastic properties are in turn determined by a self-consistent concentric cylinder assemblage of the constituent wood polymers. The predicted properties compare well with experimental results found in the literature. Significant differences in both stiffness and hygroexpansion are found for normal and compression wood, primarily due to the large difference in microfibril angle and lignin content. On the basis of these numerical results, some functional arguments for the reason of high microfibril angle, high lignin content and cylindrical structure of compression wood tracheids are supported.     

马尾松正常木与应压木的比较解剖

用光学显微镜和扫描电镜观察比较了马尾松(Pinus massoniana)正常木与应压木的结构差异。研究结果表明:马尾松应压木从早材到晚材呈缓变,管胞在横切面上除生长轮分界处的两侧外均为圆形,管胞之间具明显的胞间隙;管胞的次生壁仅有 S_1和 S_2两层;次生壁上螺纹间隙和螺纹裂隙都很明显,并与 S_2层微纤丝平行。此外,还初步讨论了应压木形成的可能机理。     

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.     

Cavitation of intercellular spaces is critical to establishment of hydraulic properties of compression wood of Chamaecyparis obtusa seedlings

Background and Aims When the orientation of the stems of conifers departs from the vertical as a result of environmental influences, conifers form compression wood that results in restoration of verticality. It is well known that intercellular spaces are formed between tracheids in compression wood, but the function of these spaces remains to be clarified. In the present study, we evaluated the impact of these spaces in artificially induced compression wood in Chamaecyparis obtusa seedlings.Methods We monitored the presence or absence of liquid in the intercellular spaces of differentiating xylem by cryo-scanning electron microscopy. In addition, we analysed the relationship between intercellular spaces and the hydraulic properties of the compression wood.Key Results Initially, we detected small intercellular spaces with liquid in regions in which the profiles of tracheids were not rounded in transverse surfaces, indicating that the intercellular spaces had originally contained no gases. In the regions where tracheids had formed secondary walls, we found that some intercellular spaces had lost their liquid. Cavitation of intercellular spaces would affect hydraulic conductivity as a consequence of the induction of cavitation in neighbouring tracheids.Conclusions Our observations suggest that cavitation of intercellular spaces is the critical event that affects not only the functions of intercellular spaces but also the hydraulic properties of compression wood.     

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.     

Structure–function relationships of four compression wood types: micromechanical properties at the tissue and fibre level

The mechanisms behind compressive stress generation in gymnosperms are not yet fully understood. Investigating the structure–function relationships at the tissue and cell level, however, can provide new insights. Severe compression wood of all species lacks a S3 layer, has a high microfibril angle in the S2 layer and a high lignin content. Additionally, special features like helical cavities or spiral thickenings appear, which are not well understood in terms of their mechanical relevance, but need to be examined with regard to evolutionary trends in compression wood development. Thin compression wood foils and isolated tracheids of four gymnosperm species [Ginkgo biloba L., Taxus baccata L., Juniperus virginiana L., Picea abies (L.) Karst.] were investigated. The tracheids were isolated mechanically by peeling them out of the solid wood using fine tweezers. In contrast to chemical macerations, the cell wall components remained in their original condition. Tensile properties of tissue foils and tracheids were measured in a microtensile apparatus under wet conditions. Our results clearly show an evolutionary trend to a much more flexible compression wood. An interpretation with respect to compressive stress generation is discussed.     

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.     

A differential staining method to identify lignified and unlignified tissues.

We investigated the use of safranin O and astra blue dissolved in ethyl alcohol as differential stains to distinguish between lignified and unlignified tissues in microtome sections of tension and normal wood of sugar and red maple. Normal wood was used as a control for the histochemical analysis. Lignified and unlignified tissues were found in the same section for both tension and normal wood of each species. These results were confirmed in unstained samples using ultraviolet light. Unlignified libriform fibers were detected using both techniques. Libriform fibers did not fluoresce in UV light, although fluorescence was observed in some of the cell corners. The astra blue in ethyl alcohol and the UV wavelength we used differentiated syringyl from guaiacyl lignins. Ethyl alcohol solutions of these dyes provide an effective and reliable method to distinguish lignified and unlignified tissues.     

Immunolocalization of 8-5′ and 8-8′ linked structures of lignin in cell walls of Chamaecyparis obtusa using monoclonal antibodies

Mouse monoclonal antibodies were generated against dehydrodiconiferyl alcohol- or pinoresinol-p-aminohippuric acid (pAHA)-bovine serum albumin (BSA) conjugate as probes that specifically react with 8-5′ or 8-8′ linked structure of lignin in plant cell walls. Hybridoma clones were selected that produced antibodies that positively reacted with dehydrodiconiferyl alcohol- or pinoresinol-pAHA–BSA and negatively reacted with pAHA–BSA and guaiacylglycerol-beta-guaiacyl ether-pAHA–BSA conjugates containing 8-O-4′ linkage. Eight clones were established for each antigen and one of each clone that positively reacted with wood sections was selected. The specificity of these antibodies was examined by competitive ELISA tests using various lignin dimers with different linkages. The anti-dehydrodiconiferyl alcohol antibody reacted specifically with dehydrodiconiferyl alcohol and did not react with other model compounds containing 8-O-4′, 8-8′, or 5-5′ linkages. The anti-pinoresinol antibody reacted specifically with pinoresinol and syringaresinol and did not react with the other model compounds containing 8-O-4′, 8-5′, or 5-5′ linkages. The antibodies also did not react with dehydrodiconiferyl alcohol acetate or pinoresinol acetate, indicating that the presence of free phenolic or aliphatic hydroxyl group was an important factor in their reactivity. In sections of Japanese cypress (Chamaecyparis obtusa), labeling by the anti-dehydrodiconiferyl alcohol antibody was found in the secondary walls of phloem fibers and in the compound middle lamellae, and secondary walls of tracheids. Weak labeling by the anti-pinoresinol antibody was found in secondary walls of phloem fibers and secondary walls and compound middle lamellae of developed tracheids. These labelings show the localization of 8-5′ and 8-8′ linked structure of lignin in the cell walls.     

A differential staining method to identify lignified and unlignified tissues

We investigated the use of safranin O and astra blue dissolved in ethyl alcohol as differential stains to distinguish between lignified and unlignified tissues in microtome sections of tension and normal wood of sugar and red maple. Normal wood was used as a control for the histochemical analysis. Lignified and unlignified tissues were found in the same section for both tension and normal wood of each species. These results were confirmed in unstained samples using ultraviolet light. Unlignified libriform fibers were detected using both techniques. Libriform fibers did not fluoresce in UV light, although fluorescence was observed in some of the cell corners. The astra blue in ethyl alcohol and the UV wavelength we used differentiated syringyl from guaiacyl lignins. Ethyl alcohol solutions of these dyes provide an effective and reliable method to distinguish lignified and unlignified tissues.     

A differential staining method to identify lignified and unlignified tissues

We investigated the use of safranin O and astra blue dissolved in ethyl alcohol as differential stains to distinguish between lignified and unlignified tissues in microtome sections of tension and normal wood of sugar and red maple. Normal wood was used as a control for the histochemical analysis. Lignified and unlignified tissues were found in the same section for both tension and normal wood of each species. These results were confirmed in unstained samples using ultraviolet light. Unlignified libriform fibers were detected using both techniques. Libriform fibers did not fluoresce in UV light, although fluorescence was observed in some of the cell corners. The astra blue in ethyl alcohol and the UV wavelength we used differentiated syringyl from guaiacyl lignins. Ethyl alcohol solutions of these dyes provide an effective and reliable method to distinguish lignified and unlignified tissues.     

Characterization of lignin in situ by photoacoustic spectroscopy

Photoacoustic spectroscopy is a recently developed nondestructive analytical technique that provides ultraviolet, visible, and infrared absorption spectra from intensely light scattering, solid, and/or optically opaque materials not suitable for conventional spectrophotometric analysis. In wood and other lignocellulosics, the principal ultraviolet absorption bands, in the absence of photosynthetic pigments, arise from the aromatic lignin component of the cell walls. Photoacoustic spectra of extracted lignin fragments (milled wood lignin) and synthetic lignin-like polymers contain a single major absorption band at 280 nanometers with an absorption tail extending beyond 400 nanometers. Photoacoustic spectra of pine, maple, and oak lignin in situ contain a broad primary absorption band at 300 nanometers and a longer wavelength shoulder around 370 nanometers. Wheat lignin in situ, on the other hand, exhibits two principle absorption peaks, at 280 nanometers and 320 nanometers. The presence of absorption bands at wavelengths greater than 300 nanometers in intact lignin could result from (a) interacting, nonconjugated chromophores, or (b) the presence of more highly conjugated structural components formed as the result of oxidation of the polymer. Evidence for the latter comes from the observation that, on the outer surface of senescent, field-dried wheat culms (stems), new absorption bands in the 350 to 400 nanometer region predominate. These new bands are less apparent on the outer surface of presenescent wheat culms and are virtually absent on the inner surface of either senescent or presenescent culms, suggesting that the appearance of longer wavelength absorption bands in senescent wheat is the result of accumulated photochemical modifications of the ligin polymer. These studies also demonstrate photoacoustic spectroscopy to be an important new tool for the investigation of insoluble plant components.     

ANATOMY OF A LIGNITIZED WOOD FROM SENFTENBERG

A piece of lignitized wood from the Miocene of Senftenberg, Germany, was identified as a type of compression wood, and was studied anatomically, as groundwork, in an investigation on cell wall cellulose and lignin. The wood showed affinity to modern Cryptomeria and Taxodium. In each growth ring the early wood was compacted. By contrast, cellular structure in the late wood had been retained to a high degree. Massive compaction of the early wood most likely occurred during the initial stages of sedimentation prior to mineralization. Numerous cell wall deformations in late wood tracheids apparently originated from compressive forces applied to the wood during sedimentation.     

Effects of Flooding, Tilting of Stems, and Ethrel Application on Growth, Stem Anatomy and Ethylene Production of Pinus densiflora Seedlings

Flooding of soil, tilting of seedlings, application of ethrelto stems, and combinations of these treatments, variously alteredthe rate of growth and stem anatomy of 2-year-old Pinus densifloraseedlings. Either flooding or tilting increased stem diametergrowth and induced formation of abnormal xylem. Whereas floodingdecreased the rate of dry weight increment of roots and needlesand increased growth of bark tissues, tilting of stems did not.However, tilting decreased the rate of height growth, stimulatedtracheid production, and induced formation of well-developedcompression wood with rounded, thick-walled tracheids, witha high lignin content but without an S3 layer in the tracheidwall. Ethylene appeared to have an important regulatory rolein stimulating growth of bark tissues as shown by thicker barkin flooded seedlings or those treated with ethrel. Ethyleneappeared to have a less important role in regulating formationof compression wood. Flooding increased the ethylene contentsof stems and induced formation of rounded, thick-walled tracheids.However, these tracheids lacked such features of well-developedcompression wood tracheids as a thick S2 layer, high lignincontent, and absence of an S3 layer. Furthermore, applicationof ethrel to vertical stems greatly increased their ethylenecontents but did not induce formation of well-developed compressionwood. Furthermore, ethrel application blocked development ofcertain characteristics of compression wood when applied totilted seedlings. For example an S3 wall layer was absent intracheids of tilted seedlings but present in tracheids of tilted,ethrel-treated seedlings. Also lignification of tracheids wasincreased on the under side of tilted stems, but reduced intilted, ethrel-treated seedlings, further de-emphasizing a directrole of ethylene in the formation of compression wood. Ethreltreatment induced formation of longitudinal resin ducts in thexylem whereas flooding or tilting of stems did not. Key words: Pinus densiflora, xylogenesis, reaction wood, compression wood, lignification, ethrel, ethylene     

Tetrachromacy in a butterfly that has eight varieties of spectral receptors

This paper presents the first evidence of tetrachromacy among invertebrates. The Japanese yellow swallowtail butterfly, Papilio xuthus, uses colour vision when foraging. The retina of Papilio is furnished with eight varieties of spectral receptors of six classes that are the ultraviolet (UV), violet, blue (narrow-band and wide-band), green (single-peaked and double-peaked), red and broad-band classes. We investigated whether all of the spectral receptors are involved in colour vision by measuring the wavelength discrimination ability of foraging Papilio. We trained Papilio to take nectar while seeing a certain monochromatic light. We then let the trained Papilio choose between two lights of different wavelengths and determined the minimum discriminable wavelength difference Deltalambda. The Deltalambda function of Papilio has three minima at approximately 430, 480 and 560nm, where the Deltalambda values approximately 1nm. This is the smallest value found for wavelength discrimination so far, including that of humans. The profile of the Deltalambda function of Papilio can be best reproduced by postulating that the UV, blue (narrow-band and wide-band), green (double-peaked) and red classes are involved in foraging. Papilio colour vision is therefore tetrachromatic.