Cellular UV Microspectrophotometry Research Articles

Topochemical and light microscopic investigations of non-enzymatic oxidative changes at the initial decay stage of furfuryl alcohol-modified radiata pine (Pinus radiata) degraded by the brown rot fungus Rhodonia placenta

The aim of this study was to visualize non-enzymatic oxidative degradation damages in the initial decay stage of the brown rot fungus Rhodonia placenta degradation in furfuryl alcohol (FA) modified wood cell walls and untreated wood cell walls of radiata pine (Pinus radiata) sapwood. A decay test with small wood blocks (1.5 × 1.5 × 5 mm³) of untreated and furfurylated radiata pine selected from two different furfurylation processes was performed until the first mass loss occurred. The samples were exposed to the brown rot fungus R. placenta, monitored by light microscopy and analyzed topochemically by cellular UV microspectrophotometry (UMSP). The results showed that the FA modification process directly influenced: i) the fungal colonization and hyphal growth, ii) the spectral UV behavior, and iii) degradation patterns of the entire cell wall layers. For the first time, UMSP area scans and selective line scans of individual cell wall regions provide topochemical insights into oxidative degradation at the initial decay stage of furfuryl alcohol-modified P. radiata visualizing oxidative degradation in situ. Knowledge of the initial decay stage of brown rot degradation in FA-modified wood compared to untreated wood extends our understanding of the brown rot decay processes of cell wall compounds.

Characterization of the wood properties of Cedrelinga cateniformis as substitute for timbers used for window manufacturing and outdoor applications

Cedrelinga cateniformis (tornillo) is a timber species of the South American Amazon Basin. In its natural distribution area, the wood has various local uses, such as furniture, art work, door and window frames, and light construction. In order to promote this lesser known species for high valued applications on the international market, wood anatomical, topochemical and physical/mechanical studies were carried out to characterize the wood properties. The topochemical distribution of the lignin and phenolic extractives in the tissue were studied by means of cellular UV microspectrophotometry (UMSP). The results of the structural and topochemical analyses were compared with the interrelation of certain anatomical and subcellular structures as well as the chemical composition with regard to the physical and mechanical properties. The natural durability of the mature heartwood was analyzed according to the European Standards and is resulting in a durability class 1 against basidiomycetes. Based on the findings of the comprehensive investigations concerning physical and biological features, e.g. the dimensional stability and durability, Cedrelinga cateniformis is ideally suited as a substitute for overexploited tropical woods currently used in Europe for wooden window frames and other above ground outdoor applications and thus can contribute to increase the value-added production in Peruvian forests.

Wood anatomical and topochemical analyses to characterize juvenile and adult wood of lesser-known species from Central America (Mexico)

ABSTRACT Anatomical and subcellular characteristics of juvenile and adult wood of seven species (Manilkara zapota (L.) P. Royen, Platymiscium yucatanum Standl., Lonchocarpus castilloi Standl., Roseodendron donnell-smithii (Rose) Miranda, Terminalia buceras (L.) Wright, Tabebuia rosea (Bertol.) DC., Lysiloma latisiliquum (L.) Benth. from Mexico, including a histometric evaluation, were investigated by light microscopy with a digitized image analysis system and by X-ray diffractometry using the SilviScan® system. The topochemical distribution of lignin and phenolic deposits in the tissue was studied by means of cellular UV-microspectrophotometry (UMSP). Extractive contents (acetone/water and water) were determined gravimetrically. The results of the structural and topochemical analyses were compared with the interrelations of certain anatomical and subcellular structures as well as the topochemical composition with regard to the physical and mechanical properties of the timbers investigated. The objective was to provide a detailed cellular and subcellular description of the heartwood of seven lesser-known timbers from Central America. All examined tree species show significant differences between juvenile and adult heartwood. For individual species, however, the differences vary greatly and have to be individually assessed in addition to general trends observed for all studied species. It has been shown that vessel size, fibre length, size of fibre lumina, and height and width of rays, as well as the content of extractives and topochemical composition of the cell walls, are suitable indicators for the differentiation of the two heartwood types. The results also contribute to a better understanding of the wood properties of the investigated timbers in relation to their utilization and added value increase.

Biological and topochemical studies on the resistance of excavated oak piles (Quercus sp.) from a historical bridge in Bavaria

Abstract Archaeological wood of 13 excavated oak piles from five historical bridge generations in Bavaria, dated from 1447 to 1787, was investigated by means of light microscopy (LM) and cellular UV-microspectrophotometry (UMSP) to study the ageing and natural resistance of the excavated wood. Furthermore, the mineral content of the ancient wood was determined to evaluate the impact of mineral inclusions on microbial resistance, tested using a mini block fungal test. The LM and UMSP analyses revealed well preserved cell wall structures and no significant modification of the lignin composition and distribution in predominant parts of the piles. Slight microbial decay caused by soft rot and erosion bacteria was only detectable at the outermost millimetres of individual piles. The fungal mini block tests yielded mass losses of ancient oak samples between 15% and 30%, independent of their analysed mineral content. The results provided evidence that the fungal resistance of the investigated ancient wood is mainly determined from the special soil with its high mineral content.

Improvement of a method for topochemical investigations of degraded furfurylated wood

Today there is an increasing demand for wood protection systems which are based on low environmental impact technologies. The aim of this study was to improve a method for investigating furfurylated Pinus radiata, both in terms of the furfuryl alcohol polymerisation process in the wood cell wall and the detection of initial signs of brown rot degradation. It is an established theory today that brown rot degradation starts with lignin modification in the outermost part of the secondary cell wall and the combined middle lamella. We adapted a method (cellular UV microspectrophotometry, UMSP) which enables direct imaging of the lignin distribution and modification within individual cell wall layers. Furfurylated P. radiata, incubated with Postia placenta, were scanned with monochromatic UV-light at 280 nm. The results showed that USMP is a promising method to study furfurylated wood and early fungal degradation damages in the cell wall of furfurylated wood.

Scanning UV microspectrophotometry as a tool to study the changes of lignin in hydrothermally modified wood

Abstract Thermal modification (TM) of wood has occupied a relatively narrow but stable niche as an alternative for chemical wood protection. There are different technological solutions for TM and not all details of their effects on wood tissue have been understood. The one-stage hydrothermal modification (HTM) at elevated vapour pressure essentially changes the wood’s composition and structure. In the present paper, the changes in three hardwood lignins (alder, aspen, and birch) were observed within the cell wall by means of cellular UV microspectrophotometry. The lignin absorbances in the compound middle lamella (CML) of unmodified wood are 1.7- to 2.0-fold higher than those in the fibre S2 layer. The woods were modified in the temperature range from 140 to 180°C, while in the lower temperature range (140°C/1 h), the UV absorbances are little affected. Essential changes occur in the range of 160–180°C and the UV data reflect these by absorbtion changes, while the absorbances at 278 nm rise with factors around 2 more in the S2 layer than in the CML. The absorbance increments are interpreted as polycondensation reactions with furfural and other degradation products of hemicelluloses with the lignin moiety of the cell wall.

The Massaria disease of plane trees:its wood decay mechanism

Branches of Platanus × hispanica with distinct symptoms of the Massaria disease were investigated by light and transmission electron microscopy and cellular UVmicrospectrophotometry. The samples collected in the city of Mannheim, Germany, were infected in vivo with the fungus Splanchnonema platani and showed various degrees of wood decay. The investigations were focused on the decay pattern of cell walls in the different cells, i.e., fibres, vessels as well as ray and axial parenchyma cells. The following results were obtained. Hyphae of the ascomycete fungus Splanchnonema platani penetrated from cell to cell through the pits and not through the cell wall middle lamella, by the formation of thin perforation hyphae. During this process, the 1–5 μm thick hyphae became narrower without attacking the wall around the pit canal. After penetration through a pit, the hyphae again enlarged to their original diameter. This is true for all pit pairs connecting the various cell types. Late decay stages did not show a decay of cell corner regions and middle lamellae of fibres as well as vessel and parenchyma cell walls. Phenolic deposits in parenchyma cells were still present in severely attacked xylem tissue. These features point to a low lignolytic capacity of the fungus. The frequently found microscopic decay pattern with the formation of oval or spherical cavities in the S2 layer of the secondary wall with an often structurally intact S3 layer is a characteristic of softrot decay. This classification is also supported by the remaining cell corner and middle lamella regions in advanced decay stages. As a consequence of this decay type, branches fracture in a brittle mode.

Oak wood inhabiting fungi and their effect on lignin studied by UV microspectrophotometry

Decayed-wood samples were collected from a naturally-infected bridge made of Quercus robur. Fruiting bodies of the white-rot basidiomycetes Hymenochaete rubiginosa and Stereum hirsutum were sampled. The white-rot fungus Fuscoporia ferrea and the secondary saprobiont Mycena galericulata were identified from the rotten wood by means of rDNA-ITS sequencing. The topochemistry of lignin degradation within individual cell-wall layers was determined by cellular UV-microspectrophotometry (UMSP) at 278 nm wavelength. Increased delignification occurred in compound middle lamella regions (CML), secondary wall layers of fibres (S2), longitudinal and ray parenchyma cells as well in vessels. The highest lignin content at initial and medium decay was recorded in the CML, whereas in advanced decay secondary wall layers of the vessels exhibited the highest content of residual lignin. In all stages of degradation, the S2 layers of fibres had the lowest lignin amount.

Steam pretreatment for enzymatic hydrolysis of poplar wood: comparison of optimal conditions with and without SO2 impregnation

Abstract Steam refining of non-debarked poplar wood with SO2 impregnation prior to steaming was investigated as pretreatment for enzymatic hydrolysis. Pretreatment conditions were varied in the range of 170°C–220°C, 3–30 min and 0.7–2.5% SO2 according to a factorial design. Predicted steaming conditions for highest carbohydrate yields after enzymatic hydrolysis were at 200°C, 15 min, and 2.5% SO2. The yield of glucose and xylose from control tests under these conditions was 43% representing an increase of 9% compared to results of former experiments without SO2 impregnation. Investigations on lignin extracted from the fibers revealed no distinct differences between pretreatment with and without SO2. No sulfonation occurred by the impregnation with SO2. Topochemical analyses of the fibers by cellular UV microspectrophotometry (UMSP) showed an inhomogeneous lignin distribution within the S2 of fibers after pretreatment without SO2 and local depositions of high UV-absorbing substances in the lumina of fibers and parenchyma cells. The lignin distribution of fiber cell walls after pretreatment with SO2 was more homogeneous with a preserved fiber network and only little amounts of deposited phenolic compounds in the lumina. Therefore, it might be concluded that the expulsion of lignin hinders the enzymes in accessing the cellulose.

A Structural and Compositional Analysis of Intervessel pit Membranes in the Sapwood of some Mangrove Woods

Intervessel pits are prominent wall structures involved in the water transport mechanism of land plants. The role of their intra-tree variation in the regulation of water transport, however, remains enigmatic. The hypothesis was tested that pit membrane thickness and degree of impregnation with phenolic substances increase along the stem axis with increasing tension on the water column as an adaptation to the higher risk for cavitation. Wood samples were taken at different heights from the mangrove tree Rhizophora mucronata growing at Gazi Bay (Kenya). Additional samples were taken along the stem radius to distinguish height from age effect, and from six other mangrove species growing in the same forest. Intervessel pit membranes were studied via transmission and scanning electron microscopy and cellular UV-microspectrophotometry. The hypothesis of pit membrane thickness and composition as a static adaptation to the hydrostatic conditions during vessel differentiation could be refuted. Instead, our findings point to a more dynamic pit membrane appearance with seasonal changes in thickness and chemical composition.

Genetic variation of Norway spruce clones regarding their natural durability, physical and chemical properties

Genetic variation of ten Norway spruce (Picea abies (L.) Karst.) clones regarding their decay resistance against brown rot fungi, as well as physical and chemical properties of clones were investigated. 31- year-old spruce clones: 26, 31, A10, A15, A7, B10, B15, B6, V7, V9 were selected across Latvia. The stem diameters of spruce clones varied between 13.0 and 20.9 cm. The wood density of clones ranged from 361 to 443 kg/m3. Klason lignin content, depending on the clone, was between 27.0%-28.9%. Cellular UV microspectrophotometry of the non-infected tracheids displayed the typical lignin distribution with highest absorbance values in the cell corners (abs280 nm 0.80) and compound middle lamellae (abs280 nm 0.48), while secondary wall showed lower lignin absorbance values (abs280 nm0.29 - 0.35). The deposition of phenolic extractives in ray parenchyma and epithelial cells of resin canals were emphasized by a significantly higher UV-absorbance (abs280 nm 0.68 to 0.78) when compared to the cell wall associated lignin. The content of acetone-soluble extractives of spruce clones varied between 1.1% - 1.8%. The x-value (natural durability) for all spruce clones after exposure to C. puteana and P. placentawas >0.90 (durability class 5, not durable). Most of clones after degradation by G. trabeum had x-value > 0.90 with exception of clones B15 and V9 that showed x-value ≤ 0.90 (durability class 4, slightly durable). Natural durability of spruce clones did not correlate with stem diameter, density, content of lignin and extractives.

Effect of Physisporinus vitreus on wood properties of Norway spruce. Part 1: Aspects of delignification and surface hardness

Abstract The white rot fungus Physisporinus vitreus is currently tested for several biotechnological applications such as permeability improvement of refractory wood species or the optimization of the acoustic properties of wood for violins. The enzymatic activity of P. vitreus results in the degradation of pit membranes and simultaneous alterations of the tracheid cell wall structure in wood of Norway spruce [Picea abies (L.) Karst]. By this means, selective delignification and simultaneous degradation occur in the latewood tracheids at short incubation times. To study the delignification of individual cell wall layers in latewood tracheids, cellular UV-microspectrophotometry was applied to wood of Norway spruce that had been incubated for between 3 and 9 weeks. By means of this technique, the progressing delignification was demonstrated in the latewood tracheid secondary walls. Moreover, local delignification in close proximity to hyphal tunneling, cavities, and notches was evident. Additionally, the mechanical changes were measured (a) at the macroscopic level by Brinell hardness test and (b) at the cellular level by nanoindentation. Brinell hardness was significantly reduced with increasing incubation time which was attributed to the partial delignification. Unlike Brinell tests, results from nanoindentation tests did not show a clear effect of fungal activity because of the material heterogeneity and the high spatial resolution of this technique. The present study provides methodological approaches for the investigation of wood-fungus interactions and contributes to a better understanding of the characteristics of wood decay at the subcellular level caused by the white rot fungus P. vitreus. Moreover, it establishes the basis for a subsequent chemical analysis, for which the results will be the topic of a second paper in this series.

Topochemical investigations of cell walls in developing xylem of beech (Fagus sylvatica L.)

Abstract Formation and lignification of xylem cells in mature beech (Fagus sylvatica L.) trees growing in a forest site in Slovenia (46° N, 14°40′ E, 400 m a.s.l.) were studied on the cellular and subcellular level. Samples containing the cambial zone and developing xylem were taken from six beech trees every week throughout the 2006 vegetation period. Cell wall thickening and lignification in individual cell wall layers and cell types were determined by light microscopy, cellular UV-microspectrophotometry and transmission electron microscopy, respectively. Cell division started between the 18th and the 24th of April 2006. Lignification began in the newly formed xylem tissue on the 2nd of May. After 1 month, the developing earlywood portion contained fully differentiated vessels with completed wall deposition and lignification, and differentiated fibres and axial parenchyma became visible after 2 months. At the end of cambial cell division on the 9th August, the differentiation of the most recently formed fibres in the terminal zone of the growth ring continued for approximately 4 weeks. This indicates that the process of lignification in earlywood is slower than in latewood. The high temporal resolution of the investigated processes and the combination of the above-mentioned microscopic techniques provides a detailed insight into the process of cell wall thickening and lignification of woody tissue in beech.

Intervessel Pit Structure and Histochemistry of Two Mangrove Species as Revealed by Cellular UV Microspectrophotometry and Electron Microscopy: Intraspecific Variation and Functional Significance

Intervessel pits play a key role in trees' water transport, lying at the base of drought-induced embolism, and in the regulation of hydraulic conductivity via hydrogels bordering pit canals. Recently, their microstructure has been the focus of numerous studies, but the considerable variation, even within species and the histochemistry of pit membranes, remains largely unexplained. In the present study, intervessel pits of the outermost wood were examined for Avicennia marina, of dry and rainy season wood separately for Rhizophora mucronata. The thickness of the pit membranes was measured on transmission electron micrographs while their topochemical nature was also analyzed via cellular UV microspectrophotometry. Pit membranes of R. mucronata were slightly thicker in dry season wood than in rainy season wood, but their spectra showed for both seasons a lignin and a yet unidentified higher wavelength absorbing component. It was suggested to be a derivative of the deposits, regularly filling pit canals. The vestures of A. marina chemically resembled pit membranes rather than cell walls.

A comprehensive analysis of the relation of cellulose microfibril orientation and lignin content in the S2 layer of different tissue types of spruce wood (Picea abies (L.) Karst.)

Abstract A possible relation between cellulose microfibril angle and lignin content in the S2 layer was investigated by X-ray diffraction and cellular UV microspectrophotometry on spruce tissues [Picea abies] with different structural features and chemical composition. A strong correlation was not found, neither for the individual tissue types nor for the compiled data of all tissues. As the data did not confirm the findings in former studies, further examinations are necessary concerning a possible general interrelation between microfibril angle and lignin content.

Topochemical investigation on tension wood fibres of Acer spp., Fagus sylvatica L. and Quercus robur L.

Abstract Topochemical detection of lignin and phenolic compounds was carried out at subcellular level in tension wood fibres of maple (Acer spp.), beech (Fagus sylvatica) and oak (Quercus robur) by means of cellular UV-microspectrophotometry (UMSP) and confocal Raman microscopy. UMSP field scans at 278 nm revealed the presence of aromatic compounds in the gelatinous layer (G-layer) in tension wood of all three species. Characterisation of the detected substances by point measurements from 240 nm to 400 nm revealed a plausible relationship to by-products from the lignin biosynthesis pathway. Raman spectra, recorded from different regions within the G-layer, showed a high affinity to spectra of lignins. The degree of aromatic compounds in the G-layer of oak tension wood fibres was approximately three-fold higher than that in maple and beech. Moreover, the oak G-layer showed an accumulation of aromatic compounds towards the cell lumen up to 50% higher than that detected in the secondary cell wall. UMSP and Raman microscopy are considered valuable complementary methods for topochemical investigation on a subcellular level.

Anatomy, Cell Wall Structure and Topochemistry of Water-Logged Archaeological wood aged 5,200 and 4,500 years

Evaluating the state of deterioration of water-logged archaeological wood is necessary in order to select treatments for its conservation and storage, particularly in the case of valuable archaeological artefacts. For this purpose archaeological wood of ash (Fraxinus sp.) and oak (Quercus sp.) buried in water-logged conditions at prehistoric settlements on the Ljubljansko barje (Ljubljana moor), Slovenia, aged approx. 5,200 and 4,500 years, was investigated by means of light microscopy (LM), transmission electron microscopy (TEM) and cellular UV-microspectrophotometry (UMSP). LM and TEM revealed that the ash wood aged 5,200 years was the least preserved. The secondary walls of fibres, vessels and parenchyma cells were considerably thinner than in normal wood, indicating distinct degradation. TEM and UMSP additionally revealed strong delignification of the remaining parts of the secondary walls of all cell types. The compound middle lamellae appeared structurally intact, but had lower UV-absorbance than normal wood of the same species. The cell corners were topochemically unchanged, as shown by high analogue UV-absorbance. The UV-absorbance maxima at a wavelength of 278 nm corresponded to those of hardwood lignins. The oak heartwood was generally better preserved than the ash wood. Within each species, the 4,500- year-old samples generally appeared better preserved than those 5,200 years old.

Topochemical investigation on phenolic deposits in the vessels of afzelia (Afzelia spp.) and merbau (Intsia spp.) heartwood

Abstract The topochemical distribution of phenolic deposits in the vessels of afzelia (Afzelia spp.) and merbau (Intsia spp.) heartwood was investigated by means of cellular UV microspectrophotometry (UMSP) to characterise the chemical composition and synthesis by pit membrane-associated enzymes. UV absorbance spectra of the deposits attached to the vessel walls of merbau are characterised by a distinct maximum at a wavelength of 368 nm representing the UV absorbance of pure robinetin (C15H10O7). Deposits in the vessels of afzelia display a typical spectrum of kaempferol (C15H10O2) with two distinct maxima at 270 and 350 nm. The pit membranes and pit canals of associated vessel and parenchyma cells are impregnated by these compounds. These results verify the assumption that the synthesis of deposits in afzelia and merbau is regulated by pit membrane-associated enzymes.

Comparison of maize brown-midrib isogenic lines by cellular UV-microspectrophotometry and comparative transcript profiling

The molecular mechanisms underlying cell wall digestibility in maize (Zea mays L.) have been studied in three sets of maize brown-midrib isogenic lines in the genetic background of inbreds 1332 (1332 and 1332 bm3), 5361 (5361 and 5361 bm3), and F2 (F2, F2 bm1, F2 bm2, and F2 bm3). Two complementary approaches, SSH (suppression subtractive hybridization) and microarray-based expression profiling, were used to isolate and identify candidate genes in isogenic lines for bm mutants. Metabolic pathway analysis revealed that transcriptional events caused by altering the expression of a single bm gene involve all metabolic and signaling pathways. 53 ESTs were differentially expressed in all three isogenic bm3 comparisons, whereas 32 ESTs were consistently differentially expressed in different bm isogenic lines in F2 background. About 70% ESTs isolated by SSH were not present on the unigene microarray, demonstrating the usefulness of the SSH procedure to identify genes related to cell wall digestibility. Together with lignin analysis by cellular UV-microspectrophotometry, expression profiling in isogenic bm lines proved to be useful to understand alterations at the sub-cellular and molecular level with respect to lignin composition. The down-regulation of COMT affected the expression of CCoAOMT genes and caused a reduced content both of G and S units in bm3 mutants.

Lignification and Cell Wall Thickening in Nodes of Phyllostachys viridiglaucescens and Phyllostachys nigra

Bamboos are among the most important plants in the world. The anatomical structure and mechanical properties of the culm internode are well documented. Fewer details are available of the culm node. The aim of this study was a topochemical investigation on lignification and cell wall thickening in developing and maturing bamboo nodes. The deposition sequence and distribution of lignin structural units and cell wall thickening in different anatomical regions of the node of Phyllostachys viridiglaucescens and Phyllostachys nigra are discussed. Cell wall thickening and lignification are investigated in the outer part of the nodal region and in the diaphragm of developing and maturing P. nigra culms and in maturing culms of P. viridiglaucescens of different age classes. The lignification during ageing was studied topochemically by means of cellular UV microspectrophotometry. A combination of light microscopy and image analysis techniques were used to measure cell wall thickness. The fibre and parenchyma cell wall thickness does not significantly increase during ageing. In the diaphragm, the cell walls are thinner and the cell diameter is larger than in the outer part of the node. In shoots, the lignin content in the epidermis, hypodermis and in both fibre and parenchyma cells of the diaphragm is relatively low compared with older culms. The fibre and parenchyma cells of the diaphragm have higher values of p-coumaric and ferulic acids than fibre and parenchyma cells of the outer part of the node. It was hypothesized that the combination of more hydroxycinnamic acids and of thinner cell walls in combination with higher cell diameters (lower density and lower stiffness) in the diaphragm than in the outer part of the node may play an important role in the biomechanical function of the node by acting as a spring-like joint to support the culm by bending forces.