Cell corner middle lamella in hydroids of dendroid moss Hypnodendron menziesii gametophyte is prominently thickened: a proposed role in the mechanical support function.

MAIN CONCLUSION: Significantly thickened corner middle lamella of the hydroid cell wall in the stipe of dendroid moss Hypnodendron menziesii has a mechanical support function. The hydroid cell walls of the erect stipe of Hypnodendron menziesii were investigated using light microscopy (LM), transmission electron microscopy (TEM), and TEM-immunogold labeling in support of the proposed biomechanical function for the highly thickened cell corner middle lamellae. The statistical analyses of dimensions of hydroid cell and wall parameters revealed a strong positive correlation between the area of hydroid cell and (i) the hydroid cell walls adhering to thick corner middle lamella, (ii) the area of the thick cell wall at hydroid corners, and (iii) the maximum thickness of cell wall at hydroid corners. The total area of the thick cell wall at the hydroid corners concomitantly increased with the area of the hydroid cell wall adhering to the middle lamella, and with the increased number of hydroids surrounding a reference hydroid. The results suggest that markedly thickened middle lamellae of the hydroid cell wall in Hypnodendron likely function by preventing hydroid cells from collapsing under the tensile forces generated from the transpirational pull on the water column. The specific localization of (1→4)- ß-D-galactan and (1,5)-α-L-arabinan in the interface region of the hydroid cell wall and the thick middle lamella is consistent with these cell wall components being involved in the mechanical strengthening of the interface through firm adhesion as well as elasticity, ensuring the structural stability of this cell wall region, which may be prone to delamination/fracturing from the various internal and external pressures imposed. The copious presence of homogalacturonan in the thick middle lamella may further enhance the strength and flexibility of hydroid cell walls.

Physiological and anatomical responses to drought stress differ between two larch species and their hybrid.

KEY MESSAGE: Hybrid saplings were more reactive to soil water deficit than Japanese and European larch. European larch had hydraulically safer wood and anisohydric behavior, Japanese and hybrid larch showed isohydric strategy. ABSTRACT: Deciduous larch species could be an alternative to evergreen conifers in reforestation, but little is known about drought sensitivity of their saplings. The effect of an experimental drought on hydraulics and quantitative wood anatomy was tested on saplings of European larch (EL, Larix decidua), Japanese larch (JL, Larix kaempferi) and their hybrid (HL). Across species, biomass, transpiration rate and relative water content were higher in controls than in drought stressed trees, but transpiration efficiency was lower. JL had the highest transpiration efficiency under drought, and EL the lowest, coinciding with slower growth of EL. Wood of EL formed before drought was hydraulically safer as shown by higher wall/lumen ratio and lower pit cavity area. EL neither had a significant increase in transpiration efficiency nor a reduction in transpiration rate under drought, suggesting that the stomata remained open under soil water deficit. HL saplings were the most reactive to water shortage, indicated by intra-annual density fluctuations and a decrease in relative water content of the sapwood. Significant reduction in transpiration by HL suggested a higher stomatal sensitivity, while the same leaf surface area was maintained and radial growth was still similar to its best parent, the JL. The latter showed a significantly lower leaf surface area under drought than controls. EL, with its hydraulically safer wood, followed an anisohydric behavior, while JL and HL revealed an isohydric strategy. Altogether, our results suggest species dependent acclimations to drought stress, whereby HL followed the strategy of JL rather than that of EL. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00468-021-02129-4.

Heteromannans are the predominant hemicelluloses in the gametophytic stem of the umbrella moss Hypnodendron menziesii and occur in the walls of all cell types.

MAIN CONCLUSION: Heteromannans are the predominant hemicelluloses in the gametophytic stem of the moss Hypnodendron menziesii and occur in the walls of all cell types Little is known about the cell-wall polysaccharides of mosses. Monosaccharide analysis of cell walls isolated from the stem of the umbrella moss Hypnodendron menziesii was consistent with heteromannans, probably galactoglucomannans, being the predominant hemicellulosic polysaccharides in the walls. Immunofluorescence and immunogold microscopy with the monoclonal antibody LM21, specific for heteromannans, showed that these polysaccharides were present in the walls of all stem cell types. These cell types, except the hydroids, have secondary walls. Experiments in which sections were pre-treated with 0.1 M sodium carbonate and with the enzyme pectate lyase indicated that the heteromannans have O-acetyl groups that limit LM21 binding and the cell walls contain pectic homogalacturonan that masks detection of heteromannans using LM21. Therefore, to fully detect heteromannans in the cell walls, it was essential to use these pre-treatments to remove the O-acetyl groups from the heteromannans and pectic homogalacturonan from the cell walls. Fluorescence microscopy experiments with a second monoclonal antibody, LM22, also specific for heteromannans, showed similar results, but the binding was considerably weaker than with LM21, possibly as a result of subtle structural differences in the epitopes of the two antibodies. Although heteromannans occur abundantly in the cell walls of many species in basal lineages of tracheophytes, prior to the present study, research on the distribution of these polysaccharides in the walls of different cell types in mosses was confined to the model species Physcomitrium patens.

Characterizing Fungal Decay of Beech Wood: Potential for Biotechnological Applications.

The biotechnological potential of nine decay fungi collected from stored beech logs at a pulp and paper factory yard in Northern Iran was investigated. Beech blocks exposed to the fungi in a laboratory decay test were used to study changes in cell wall chemistry using both wet chemistry and spectroscopic methods. Pleurotus ostreatus, P. pulmonarius, and Lentinus sajor-caju caused greater lignin breakdown compared to other white-rot fungi, which led to a 28% reduction in refining energy. Trametesversicolor caused the greatest glucan loss, while P. ostreatus and L. sajor-caju were associated with the lowest losses of this sugar. Fourier transform infrared spectroscopy (FTIR) analyses indicated that white-rot fungi caused greater lignin degradation in the cell walls via the oxidation aromatic rings, confirming the chemical analysis. The rate of cellulose and lignin degradation by the T.versicolor and Pleurotus species was high compared to the other decay fungi analyzed in this study. Based on the above information, we propose that, among the fungi tested, P. ostreatus (27.42% lignin loss and 1.58% cellulose loss) and L. sajor-caju (29.92% lignin loss and 5.95% cellulose loss) have the greatest potential for biopulping.

Comparison of the Decay Behavior of Two White-Rot Fungi in Relation to Wood Type and Exposure Conditions.

Fungal wood decay strategies are influenced by several factors, such as wood species, moisture content, and temperature. This study aims to evaluate wood degradation characteristics of spruce, beech, and oak after exposure to the white-rot fungi Pleurotusostreatus and Trametesversicolor. Both fungi caused high mass losses in beech wood, while spruce and oak wood were more resistant to decay. The moisture content values of the decayed wood correlated with the mass losses for all three wood species and incubation periods. Combined microscopic and chemical studies indicated that the two fungi differed in their decay behavior. While T. versicolor produced a decay pattern (cell wall erosion) typical of white-rot fungi in all wood species, P. ostreatus caused cell wall erosion in spruce and beech and soft-rot type I (cavity formation) decay in oak wood. These observations suggest that P. ostreatus may have the capacity to produce a wider range of enzymes/radicals triggered by the chemical composition of wood cell walls and/or local compositional variability within the cell wall.

A novel approach for FE-SEM imaging of wood-matrix polymer interface in a biocomposite.

Understanding the interface between polymer and biomass in composite products is important for developing high performance products, as the quality of adhesion at the interface determines composite properties. For example, with greater stiffness compared to polymer matrix, such as that of high density polyethylene, the wood component enhances stiffness of wood-polymer composites, provided there is good adhesion between composite components. However, in composites made from wood flour (wood particles) and synthetic resins it is often difficult to clearly resolve particle-matrix interfaces in the conventionally employed microscopy method that involves SEM examination of fractured faces of composites. We developed a novel approach, where composites made from high density polyethylene and wood flour were examined and imaged with a FE-SEM (field emission scanning electron microscope) in transverse sections cut through the composites. Improved definition of the interface was achieved using this approach, which enabled a more thorough comparison to be made of the features of the interface between wood particles and the matrix in composites with and without a coupling agent, as it was possible to clearly resolve the interfaces for particles of all sizes, from large particles consisting of many cells down to tiny cell wall fragments, particularly in composites that did not incorporate the coupling agent used to enhance particle adhesion with the matrix polymer. The method developed would be suitable particularly for high definition SEM imaging of a wide range of composites made combining wood and agricultural residues with synthetic polymers.

MRT letter: high resolution SEM imaging of nano-architecture of cured urea-formaldehyde resin using plasma coating of osmium.

Nanoarchitecture of cured urea-formaldehyde (UF) resins was examined with a field-emission scanning electron microscope (FE-SEM) after coating samples with osmium, which is considered to produce particles of considerably smaller size compared to other metal coatings used in SEM studies. This method enabled comparison of the nanoarchitecture of UF resins of low (1.0) and high (1.6) formaldehyde/urea (F/U) mole ratios to be made, based on imaging of extremely small size particles as part of UF resin architecture, not described before. Imaging revealed presence of relatively large globular particles (148.084-703.983 nm size range) as well as smaller substructures (28.004-39.604 nm size range) as part of the architecture of 1.0-mole UF resin. Globular particles were also present in 1.6 mole UF resin, but of considerably smaller size (14.760-50.269 nm). The work presented demonstrates usefulness of osmium coating in unraveling the intricacies of the nanostructural organization of cured UF resins, prompting wider application of this immensely useful but grossly underutilized metal coating type in high resolution SEM examination of biological and materials samples.

Correlative light and scanning electron microscopy of the same sections gives new insights into the effects of pectin lyase on bordered pit membranes in Pinus radiata wood.

Bordered pits are structures in the cell walls of softwood tracheids which permit the movement of water between adjacent cells. These structures contain a central pit membrane composed of an outer porous ring (margo) and an inner dense and pectin-rich disc (torus). The membrane is overarched on each side by pit borders. Pits may be aspirated, a condition where the torus seals against the pit border, effectively blocking the pathway between cells. In living trees this maintains overall continuity of water conduction in xylem by sealing off tracheids containing air. Drying of timber results in further pit aspiration, which reduces wood permeability to liquid treatment agents such as antifungal chemicals. One possible way to increase permeability is by treating wood with pectin lyase to modify or remove the torus. The effectiveness of this treatment was initially evaluated using light microscopy (LM) of toluidine blue stained wood. Pectic material is coloured pink-magenta with this stain, and loss of this colour after treatment has been interpreted as indicating destruction of the torus. However, correlative light (LM) and scanning electron (SEM) microscopic observations of identical areas of toluidine blue stained sections revealed that many unstained pits had intact but modified tori when viewed with SEM. These observations indicate that LM alone is not sufficient to evaluate the effects of pectin lyase on pit membranes in wood. Combining LM and SEM gives more complete information.

Visualising impregnated chitosan in Pinus radiata early wood cells using light and scanning electron microscopy.

Chitosan, a deacetylated product of an abundant naturally occurring biopolymer chitin, has been used in a range of applications, particularly in food and health areas, as an antimicrobial agent. In the work reported here Pinus radiata wood was impregnated with chitosan as an environmentally compatible organic biocide (Eikenes et al., 2005a,b) to protect wood against wood deteriorating microorganisms and to thus prolong the service life of wooden products. We developed sample preparation techniques targeted to visualise impregnated chitosan within wood tissues using light microscope and field-emission scanning electron microscope (FE-SEM). Sections were viewed with the light microscope without staining with a dye as well as after staining with the dye toluidine blue. Light microscopy was also undertaken on sections that had been stained with 1% aqueous osmium tetroxide (OsO(4)). For SEM observations, the sections were treated with OsO(4) and then examined with the FE-SEM, first in the secondary electron imaging mode (SEI) and then in the backscattered electron imaging (BEI) mode, imaging the same areas of a section in both SEI and BEI modes. The preparation techniques employed and the combined use of light and scanning electron microscopy provided valuable complementary information, revealing that chitosan had penetrated into the cavities (cell lumens, intercellular spaces) of all sizes present within wood tissues and had also impregnated early wood cell walls. The information obtained is discussed in relation to its importance in further development of chitosan formulations and refinement of impregnation technologies to optimise chitosan impregnation into and distribution within wood tissues as well as in assessing chitosan efficacy.

Chitosan-mediated changes in cell wall composition, morphology and ultrastructure in two wood-inhabiting fungi.

The effect of chitosan on cell wall deposition was investigated in the two wood-inhabiting fungal species Trichoderma harzianum (CBS 597.91) and Sphaeropsis sapinea (NZFS 2725). The study used three independent analytical techniques to quantify chitin in the fungal mycelium. A colorimetric method for the detection of D-glucosamine was compared with two gas chromatography-mass spectroscopy (GC-MS) methods employing alditol acetates analysis and pyrolysis. The latter used a stable-isotope-labelled internal standard, d(3)-N-acetyl glucosamine. At least in the case of S. sapinea, the study provided evidence of an increase in the chitin content in the mycelium due to chitosan treatment, indicating that chitosan treatment affected cell wall deposition. Electron microscopy techniques showed alteration in surface morphology and cell wall texture due to chitosan treatment. The implications of these results are discussed with a view to analysing possible mechanisms for growth inhibitory effects of chitosan on fungal hyphae.

Rapid accumulation of hydrogen peroxide in cucumber roots due to exposure to low temperature appears to mediate decreases in water transport.

Water transport across root systems of young cucumber (Cucumis sativus L.) seedlings was measured following exposure to low temperature (LT, 8-13 degrees C) for varying periods of time. In addition, the amount of water transported through the stems was evaluated using a heat-balance sap-flow gauge. Following LT treatment, hydrogen peroxide was localized cytochemically in root tissue by the oxidation of cerium (III) chloride. The effects of hydrogen peroxide on the hydraulic conductivity of single cells (Lp) in root tissues, and on the H+-ATPase activity of isolated root plasma membrane, have been worked out. Cytochemical evidence suggested that exposure of roots to LT stress caused a release of hydrogen peroxide in the millimolar range in the vicinity of plasma membranes. In response to a low root temperature (8 degrees C), the hydraulic conductivity of the root (Lp(r)) decreased by a factor of 4, and the half-times of water exchange increased by a factor of 5-6. Decreasing root temperatures from 25-13 degrees C increased the half-times of water exchange in a cell by a factor of 6-9. The measurement of axial water transport with a heat-balance sap-flow gauge showed that only a small amount of water was transported when 8 degrees C was imposed on cucumber roots. Lp and the H+-ATPase activity of the isolated root plasma membrane were very sensitive to externally applied hydrogen peroxide at a concentration of 1-16 mM. These observations suggest that the accumulation of hydrogen peroxide appears to mediate decreases in water transport in cucumber roots under low temperature.

Simple and rapid methods for SEM observation and TEM immunolabeling of rubber particles.

We developed a method involving air-drying of a rubber suspension after fixation in glutaraldehyde-tannic acid and postfixation in osmium tetroxide for SEM observation. For TEM immunolabeling the suspension was air-dried after osmium-only fixation. Whereas conventional methods failed to satisfactorily stabilize rubber particles, the methods described here proved successful in preserving their integrity.

The micromorphology and protein characterization of rubber particles in Ficus carica, Ficus benghalensis and Hevea brasiliensis.

Rubber biosynthesis takes place on the surface of rubber particles. These particles are surrounded by a monolayer membrane in which the rubber transferase is anchored. In order to gain better insight into whether rubber particles from different plant species share common structural characteristics, the micromorphology of rubber particles from Ficus carica, Ficus benghalensis, and Hevea brasiliensis was examined by electron microscopy. Rubber particles of all three species were spherical in shape, and the size of rubber particles of H. brasiliensis was much smaller than those of F. carica and F. benghalensis. In addition, investigations were undertaken to compare the cross-reactivity of the antibody raised against either the H. brasiliensis small rubber particle protein (SRPP) which is suggested to be involved in rubber biosynthesis, or the cis-prenyltransferase (CPT) which has an activity similar to rubber transferase. Both western analysis and TEM-immunogold labelling studies showed that rubber particles of F. carica and F. benghalensis do not contain the SRPP. None of the rubber particles in F. carica, F. benghalensis and H. brasiliensis contained the CPT, suggesting that the CPT itself could not catalyse the formation of high molecular weight rubber. These results indicate that rubber particles in the three different plant species investigated share some degree of similarity in architecture, and that the SRPP and CPT themselves are not the core proteins necessary for rubber biosynthesis.

Chilling root temperature causes rapid ultrastructural changes in cortical cells of cucumber (Cucumis sativus L.) root tips.

Examination of root tips from cucumber (Cucumis sativus L.) seedlings grown at 8 degrees C for varying periods ranging from 15 min to 96 h, showed marked changes in the ultrastructure of cortical cells within only 15 min of exposure. Greater parts of the cortex were affected with longer periods of exposure, but the sequence of morphological changes in cell components was similar to that found for the roots exposed for 15 min. The effect of chilling injury included alterations in cell walls, nuclei, ER, mitochondria, plastids, and ribosomes. The extent of alterations varied greatly among cells, moderate to severe alterations to cell components being observable among adjoining cells. The measurements of root pressure using the root pressure probe showed a sudden, steep drop in the root pressure in response to lowering of the temperature of the bathing solution from 25 degrees C to 8 degrees C. These observations are discussed in the light of the information available on the ultrastructural and biochemical characteristics of the effect of cold exposure in chilling-sensitive plants.