Seasonal movement of chloroplasts in mesophyll cells of two Picea species.

Cold acclimation in evergreen conifers of temperate zone is associated with seasonal structural changes of mesophyll cells. Photoprotective reactions include the movement of chloroplasts from summer position when they are located along the cell walls to winter arrangement with their aggregation in one part of the cell. Special spatial arrangement of mesophyll in Picea species with chloroplasts located along the two opposite cell walls causes the very specific pattern of chloroplast movement. To reveal the intracellular apparatus involved in the seasonal organelle position changes, 3D reconstruction of mesophyll cell structure was applied. Two Picea species, P. obovata and P. pungens, from two geographic regions were studied in a 3-year course. The involvement of small transparent vacuoles in the development of cytoplasmic strands penetrating through the central vacuole and connecting two opposite lateral sides of the cell was shown. The nucleus and cytoplasmic organelles including chloroplasts move inwards the strand forming the cytoplasmic conglomerate enclosed by the vacuole at the cell center. Two Picea species have distinct differences in spatial organization of winter mesophyll cells and in structural events leading to its formation. Analysis of Picea species from two geographic regions over 3 years of monitoring reveals dependence of seasonal organelle movement on the dynamics of temperature decline in autumn.

Finite element simulation based-on atomic force microscopy and nanoindentation for spruce wood microstructure analysis.

Spruce wood (picea abis) has been widely used as structural element, from buildings to musical instruments, due to its outstanding mechanical performances. The main stem transverse section exhibits growth rings formed by periodic fringes patterns, which are constituted by lamellae-tracheid arrangements. In order to improve the understanding of each wood microstructure role, the morphology and crystallinity of earlywood and latewood fibers were examined mainly using scanning electron microscopy, atomic force microscopy, and X-ray difracction. Moreover, measurements of effective elastic modulus and hardness were obtained by nanoindentation tests using a Berkovich indenter in order to confirmed increase in compactness of the wood microstructures. The results indicate that variations in mechanical properties values can be associated with well defined microstructural performances for each characteristic fiber type, where those that belong to latewood fiber showed the most improved behaviors. A finite element simulation of a lamellar-tracheids arrangement was carried out in order to clarify its stiffness and elastic deformation capabilities, as relevant factors contributing to the successful adaptation of picea abis colonies to harsh conditions habitats as well as for its construction applications of string instruments.

A close-up view of the wood cell wall ultrastructure and its mechanics at different cutting angles by atomic force microscopy.

MAIN CONCLUSION: AFM measurements on spruce sample cross-sections reveal that the structural appearance of the S2 layer changes from a network structure to a concentric lamellar texture depending on the cutting angle. The structural assembly of wood constituents within the secondary cell wall has been subject of numerous studies over the last decades, which has resulted in contradicting models on the spatial arrangement and orientation of the wood macromolecules. Here, we use multichannel atomic force microscopy by means of quantitative imaging, to gain new insights into the macromolecular assembly. Cross-sections of spruce wood, which had been cut at different angles ranging from 0° to 30° were investigated. Strikingly, depending on the cutting angle, the structural appearance of the S2 layer changed from a network-like structure to a distinct concentric lamellar texture. This makes us conclude that the often visualized lamellar organization of the secondary cell wall is not the consequence of a continuous inherent ring pattern, but rather a result of the specific surface cross-section appearance of cellulose aggregates at larger cutting angles. By analyzing the recorded force distance curves in every pixel, a nano-mechanical characterization of the secondary cell wall was conducted. Substantially lower indentation modulus values were obtained compared to nanoindentation values reported in the literature. This is potentially due to a smaller interaction volume of the probe with a by far less deep indentation.

Histology and cell wall biochemistry of stone cells in the physical defence of conifers against insects.

Conifers possess an array of physical and chemical defences against stem-boring insects. Stone cells provide a physical defence associated with resistance against bark beetles and weevils. In Sitka spruce (Picea sitchensis), abundance of stone cells in the cortex of apical shoots is positively correlated with resistance to white pine weevil (Pissodes strobi). We identified histological, biochemical and molecular differences in the stone cell phenotype of weevil resistant (R) or susceptible (S) Sitka spruce genotypes. R trees displayed significantly higher quantities of cortical stone cells near the apical shoot node, the primary site for weevil feeding. Lignin, cellulose, xylan and mannan were the most abundant components of stone cell secondary walls, respectively. Lignin composition of stone cells isolated from R trees contained a higher percentage of G-lignin compared with S trees. Transcript profiling revealed higher transcript abundance in the R genotype of coumarate 3-hydroxylase, a key monolignol biosynthetic gene. Developing stone cells in current year apical shoots incorporated fluorescent-tagged monolignol into the secondary cell wall, while mature stone cells of previous year apical shoots did not. Stone cell development is an ephemeral process, and fortification of shoot tips in R trees is an effective strategy against insect feeding.

Plant material features responsible for bamboo's excellent mechanical performance: a comparison of tensile properties of bamboo and spruce at the tissue, fibre and cell wall levels.

BACKGROUND AND AIMS: Bamboo is well known for its fast growth and excellent mechanical performance, but the underlying relationships between its structure and properties are only partially known. Since it lacks secondary thickening, bamboo cannot use adaptive growth in the same way as a tree would in order to modify the geometry of the stem and increase its moment of inertia to cope with bending stresses caused by wind loads. Consequently, mechanical adaptation can only be achieved at the tissue level, and this study aims to examine how this is achieved by comparison with a softwood tree species at the tissue, fibre and cell wall levels. METHODS: The mechanical properties of single fibres and tissue slices of stems of mature moso bamboo (Phyllostachys pubescens) and spruce (Picea abies) latewood were investigated in microtensile tests. Cell parameters, cellulose microfibril angles and chemical composition were determined using light and electron microscopy, wide-angle X-ray scattering and confocal Raman microscopy. KEY RESULTS: Pronounced differences in tensile stiffness and strength were found at the tissue and fibre levels, but not at the cell wall level. Thus, under tensile loads, the differing wall structures of bamboo (multilayered) and spruce (sandwich-like) appear to be of minor relevance. CONCLUSIONS: The superior tensile properties of bamboo fibres and fibre bundles are mainly a result of amplified cell wall formation, leading to a densely packed tissue, rather than being based on specific cell wall properties. The material optimization towards extremely compact fibres with a multi-lamellar cell wall in bamboo might be a result of a plant growth strategy that compensates for the lack of secondary thickening growth at the tissue level, which is not only favourable for the biomechanics of the plant but is also increasingly utilized in terms of engineering products made from bamboo culms.

Isolation and characterization of cellulose nanocrystals from spruce bark in a biorefinery perspective.

The present study reports for the first time the isolation of cellulose fibers and cellulose nanocrystals (CNCs) from the bark of Norway spruce. The upgrading of bark cellulose to value-added products, such as CNCs, is part of the "bark biorefinery" concept. The removal of non-cellulosic constituents was monitored throughout the isolation process by detailed chemical composition analyses. The morphological investigation of the CNCs was performed using AFM and showed the presence of nanocrystals with an average length of 175.3 nm and a diameter of 2.8 nm, giving an aspect ratio of around 63. X-ray diffraction (XRD) analyses showed that the crystallinity index increased with successive treatments to reach a final value greater than 80% for CNCs. The thermal degradation of the isolated bark CNCs started at 190 °C. Spruce bark appeared to be a new promising industrial source of cellulose fibers and CNCs.

Supernumerary (B) chromosomes in populations of Picea abies (L.) H. Karst. from Western Rhodopes (Bulgaria).

Investigations on B chromosomes found for the first time for Picea abies (L.) H. Karst. have been conducted. Seeds of Picea abies from two populations of Western Rhodopes (Bulgaria) located at the southern border of species range, and protected according to Bern Convention and EC Habitat Directive were collected for this study. Mixoploidy was detected in some germinating seeds of Picea abies. It was found that metaphase cells of germinating seeds contain 0-4 B chromosomes of both metacentric and submetacentric types. The variability of B chromosomes number and their occurrence was observed. Along with B chromosomes, some chromosome aberrations such as fragments and ring chromosomes were revealed in metaphase cells of Picea abies from studied populations. The possible adaptive role of B chromosomes presence for Picea spp. is discussed.

Out-of-plane orientation of cellulose elementary fibrils on spruce tracheid wall based on imaging with high-resolution transmission electron microscopy.

MAIN CONCLUSION: A 3D model of the tracheid wall has been proposed based on high-resolution cryo-TEM where, in contrast to the current understanding, the cellulose elementary fibrils protrude from the cell wall plane. The ultrastructure of the tracheid walls of Picea abies was examined through imaging of ultrathin radial, tangential and transverse sections of wood by transmission electron microscopy and with digital image processing. It was found that the elementary fibrils (EFs) of cellulose were rarely deposited in the plane of the concentric cell wall layers, in contrast to the current understanding. In addition to the adopted concept of longitudinal fibril angle, EFs protruded from the cell wall plane in varying angles depending on the layer. Moreover, the out-of-plane fibril angle varied between radial and tangential walls. In the tangential S2 layers, EFs were always out-of-plane whereas planar orientation was typical for the S2 layer in radial walls. The pattern of protruding EFs was evident in almost all axial and transverse images of the S1 layer. Similar out-of-plane orientation was found in the transverse sections of the S3 layer. A new model of the tracheid wall with EF orientation is presented as a summary of this study. The outcome of this study will enhance our understanding of the elementary fibril orientation in the tracheid wall.

Synchrotron X-ray micro-tomography imaging and analysis of wood degraded by Physisporinus vitreus and Xylaria longipes.

Incubation of Norway spruce with Physisporinus vitreus and sycamore with Xylaria longipes results in reduction in density of these wood species that are traditionally used for the top and bottom plate of a violin, which follows by enhanced acoustic properties. We used Synchrotron X-ray micro-tomography, to study the three-dimensional structure of wood at the micro-scale level and the alterations of the density distribution after incubation with two white-rot fungi. Micro-tomography data from wood treated at different incubation periods are analyzed and compared with untreated (control) specimens to determine the wood density map and changes at the cell-wall level. Differences between the density of early- and latewood, xylem ray and around bordered pits in both Norway spruce and sycamore are studied. Three-dimensional hyphal networks of the P.vitreus and Xylaria longipes hyphae are visualized inside the cell lumina and their significance on the density of the early- and latewood cells after different incubation periods are discussed. The study illustrates the utility of X-ray micro-tomography for both qualitative and quantitative studies of a wide variety of biological systems and due to its high sensitivity, small structural changes can be quantified.

Cell structural changes in the mesophyll of Norway spruce needles by elevated ozone and elevated temperature in open-field exposure during cold acclimation.

The effects of elevated ozone (1.4× ambient) and temperature (ambient +1.3 °C) alone and in combination were studied on the needle cell structure of soil-grown Norway spruce seedlings in the late growing season and winter. Temperature treatment continued over winter and lengthened the snow-free period. Elevated temperature caused microscopic changes related to photosynthesis (decreased chloroplast size and increased number), carbon storage (reduced starch and increased cytoplasmic lipids) and defence (decreased mitochondrial size and proportion per cytoplasm, increased peroxisomes and plastoglobuli, altered appearance of tannins). The results suggest increased oxidative stress by elevated temperature and altered allocation of limited carbon reserve to defence. The number of peroxisomes and plastoglobuli remained high in the outer cells of needles of ozone-exposed seedlings but decreased in the inner cells. This may indicate defence allocation to cells close to the stomata and surface, which are experiencing more oxidative stress. Ozone reduced winter hardiness based on seasonal changes in chloroplast shape and location in the cells. The effects of ozone became evident at the end of the growing season, indicating the effect of cumulative ozone dose or that the seedlings were vulnerable to ozone at the later phases of winter hardening. Elevated temperature increased cellular damage in early winter and visible damage in spring, and the damage was enhanced by ozone. In conclusion, the study suggests that modest air temperature elevation increases stress at the cell structural level in spruce seedlings and is enhanced by low ozone elevation. Future climatic conditions where snow cover is formed later or is lacking but temperatures are low can increase the risk of severe seedling damage, and current and future predicted ozone concentrations increase this risk.

Detection and measurement of necrosis in plants.

Necrosis plays a fundamental role in plant physiology and pathology. When plants or plant cell cultures are subjected to abiotic stress they initiate rapid cell death with necrotic morphology. Likewise, when plants are attacked by pathogens, they develop necrotic lesions, the reaction known as hypersensitive response. Great advances in the understanding of signaling pathways that lead to necrosis during plant-pathogen interaction have been made in the last two decades using Arabidopsis thaliana as a model plant. Further understanding of these signaling pathways, as well as those regulating the execution phase of necrotic cell death per se would require a robust set of readout assays to detect and measure necrosis in various plant model systems. Here we provide description of such assays, beginning from electron microscopy, as the "gold standard" to diagnose necrosis. This is followed by two groups of biochemical and cytochemical assays used by our group to detect and quantify mitochondrial dysfunction and the loss of protoplast integrity during necrosis in Arabidopsis plants and cell suspension cultures of both Arabidopsis and Norway spruce.

Direct fluorination applied to wood flour used as a reinforcement for polymers.

Direct fluorination was applied to wood flour in order to improve its compatibility with polymers and thus enhance the properties of wood-polymer composites. Fourier-transform infrared spectra and (19)F solid-state nuclear magnetic resonance results underlined a successful covalent grafting of fluorine atoms onto the wood chemical structure. No physical damage of the wood particles was observed during scanning electron microscopy analysis. The thermal behaviour of the wood flour was also studied by thermogravimetric analysis. The hydrophilic property changes of wood flour were examined by evaluating the water content and the rate of water uptake of samples under different relative humidity conditions. A decrease in the wood flour water content was noted after fluorination. All these studies tend to prove the efficiency of this treatment on wood hydrophilia.

Computational fluid dynamics models of conifer bordered pits show how pit structure affects flow.

• The flow of xylem sap through conifer bordered pits, particularly through the pores in the pit membrane, is not well understood, but is critical for an understanding of water transport through trees. • Models solving the Navier-Stokes equation governing fluid flow were based on the geometry of bordered pits in black spruce (Picea mariana) and scanning electron microscopy images showing details of the pores in the margo of the pit membrane. • Solutions showed that the pit canals contributed a relatively small fraction of resistance to flow, whereas the torus and margo pores formed a large fraction, which depended on the structure of the individual pit. The flow through individual pores in the margo was strongly dependent on pore area, but also on the radial location of the pore with respect to the edge of the torus. • Model results suggest that only a few per cent of the pores in the margo account for nearly half of the flow and these pores tend to be located in the inner region of the margo where their contribution will be maximized. A high density of strands in outer portions of the margo (hence narrower pores) may be more significant for mechanical support of the torus.

Distribution of lignin and its coniferyl alcohol and coniferyl aldehyde groups in Picea abies and Pinus sylvestris as observed by Raman imaging.

Wood cell wall consists of several structural components, such as cellulose, hemicelluloses and lignin, whose concentrations vary throughout the cell wall. It is a composite where semicrystalline cellulose fibrils, acting as reinforcement, are bound together by amorphous hemicelluloses and lignin matrix. Understanding the distribution of these components and their functions within the cell wall can provide useful information on the biosynthesis of trees. Raman imaging enables us to study chemistry of cell wall without altering the structure by staining the sample or fractionating it. Raman imaging has been used to analyze distributions of lignin and cellulose, as well as the functional groups of lignin in wood. In our study, we observed the distribution of cellulose and lignin, as well as the amount of coniferyl alcohol and aldehyde groups compared to the total amount of lignin in pine (Pinus sylvestris) and spruce (Picea abies) wood samples. No significant differences could be seen in lignin and cellulose distribution between these samples, while clear distinction was observed in the distribution of coniferyl alcohols and coniferyl aldehyde in them. These results could provide valuable insight on how two similar wood species control biosynthesis of lignin differently during the differentiation of cell wall.

Effect of steam treatment on the properties of wood cell walls.

Steam treatment is a hygrothermal method of potential industrial significance for improving the dimensional stability and durability of wood materials. The steaming results in different chemical and micromechanical changes in the nanostructured biocomposite that comprise a wood cell wall. In this study, spruce wood ( Picea abies Karst.) that had been subjected to high-temperature steaming up to 180 °C was examined, using imaging Fourier Transform Infrared (FT-IR) microscopy and nanoindentation to track changes in the chemical structure and the micromechanical properties of the secondary cell wall. Similar changes in the chemical components, due to the steam treatment, were found in earlywood and latewood. A progressive degradation of the carbonyl groups in the glucuronic acid unit of xylan and a loss of mannose units in the glucomannan backbone, that is, a degradation of glucomannan, together with a loss of the C═O group linked to the aromatic skeleton in lignin, was found. The development of the hygroscopic and micromechanical properties that occurred with an elevation in the steam temperature correlated well with this pattern of degradation in the constituents in the biocomposite matrix in the cell wall (hemicellulose and lignin).

Mitochondrial dynamics and its responds to proteasome defection during Picea wilsonii pollen tube development.

Tip growth of pollen tubes is essential for higher plant sexual reproduction and has been proposed to be highly regulated by the ubiquitin/proteasome pathway (UPP). The dynamics of mitochondria and the functions of the UPP on mitochondrial dynamics during pollen tube development are still poorly understood. In the present study, using real-time laser scanning and transmission electron microscope, it was revealed that mitochondria in Picea wilsonii, are either ellipsoid or filamentous with various lengths. Time-lapse images indicated that the two forms of mitochondria interconvert frequently through opposite process of fusion and fission. Examination of mitochondrial morphology during four key stages of in vitro pollen tube development revealed a link between mitochondrial remodeling and the process of pollen tube elongation. We also report that MG132, a specific proteasome inhibitor, not only strongly disturbed the mitochondrial remodeling but also significantly reduced mitochondrial membrane potential during pollen tube development. This finding provides new insight into the function of the proteasome in tip growth of pollen tubes.

Distribution of (1->4)-beta-galactans, arabinogalactan proteins, xylans and (1->3)-beta-glucans in tracheid cell walls of softwoods.

Polysaccharides were located in the walls of normal and compression wood tracheids of Pinus radiata (radiata pine), Picea sitchensis (Sitka spruce) and Picea abies (Norway spruce) by transmission electron microscopy using immunogold labelling with monoclonal antibodies to (1-->4)-beta-galactan (LM5), (1-->3)-beta-glucan, arabinogalactan proteins (AGPs) (MAC207) and heteroxylans (LM10 and LM11). In fully differentiated compression wood tracheids, (1-->4)-beta-galactan was found in the S2((L)) layer and, to a smaller extent, at the interface between the compound middle lamella and the S1 layer. (1-->4)-beta-Galactan appeared to be displaced from, or modified in, the S1 layer during cell wall formation. (1-->3)-beta-Glucan (callose) was confined to the helical cavities in the inner S2 layer of severe compression wood. MAC207 AGP glycan epitope was found exclusively in the S1 and S3 layers of normal wood tracheids and in the S1 and inner S2 layers of compression wood tracheids. Binding of LM10, which specifically recognizes unsubstituted or low-substituted xylans, occurred at similar locations to the MAC207 epitope, whereas binding of LM11, which recognizes more highly substituted as well as unsubstituted xylans, occurred throughout the tracheid walls with the exception of the primary wall. Immunogold labelling showed that the different wall layers of softwood tracheids have different polysaccharide compositions which change abruptly during cell wall formation.

Gabaculine alters plastid development and differentially affects abundance of plastid-encoded DPOR and nuclear-encoded GluTR and FLU-like proteins in spruce cotyledons.

Synthesis of 5-aminolevulinic acid (ALA) represents a rate limiting step in the tetrapyrrole biosynthetic pathway, and is regulated by metabolic feedback control of glutamyl-tRNA reductase (GluTR) activity. The FLU protein has been attributed to this regulation. Later in the biosynthetic pathway, reduction of protochlorophyllide (Pchlide), catalyzed by protochlorophyllide oxidoreductase (POR), ensures another important regulatory step in the chlorophyll biosynthesis. In the present work, we investigated the expression and cellular abundance of nuclear-encoded and plastid-encoded proteins involved in ALA synthesis and Pchlide reduction in Norway spruce (Picea abies L. Karst.) as a representative of plant species with high ability to synthesize chlorophyll in the dark. Using dark-grown, light/dark-grown and gabaculine-treated seedlings, we demonstrated that gabaculine-impaired etiochloroplast and chloroplast development has no negative effect on GluTR accumulation in the cotyledons. However, in contrast to control plants, the relative amount of GluTR was similar both in the dark-grown and light/dark-grown gabaculine-treated seedlings. We identified a partial sequence of the FLU-like gene in Norway spruce, and using antibodies against the FLU-like protein (FLP), we showed that FLP accumulated mostly in the dark-grown control seedlings and gabaculine-treated seedlings. In contrast to nuclear-encoded GluTR and FLP, accumulation of plastid-encoded light-independent POR (DPOR) was sensitive to gabaculine treatment. The levels of DPOR subunits were substantially lower in the light/dark-grown control seedlings and gabaculine-treated seedlings, although the corresponding genes chlL, chlN and chlB were expressed. Since we analyzed the samples with different plastid types, plastid ultrastructure and physiological parameters like Pchlide and chlorophyll contents, in vivo chlorophyll fluorescence and photosynthetic efficiency of the seedlings were characterized. Apart from etiochloroplast-specific accumulation of the DPOR subunits, we described, in some detail, additional specific features of chlorophyll biosynthesis in the spruce seedlings that differ from those known in angiosperms.

Fine structural quantification of drought-stressed Picea abies (L.) organelles based on 3D reconstructions.

Ultrastructural investigations of cells and organelles by transmission electron microscopy (TEM) usually lead to two-dimensional information of cell structures without supplying exact quantitative data due to the limited number of investigated ultrathin sections. This can lead to misinterpretation of observed structures especially in context of their three-dimensional (3D) assembly. 3D investigations and quantitative morphometric analysis are therefore essential to get detailed information about the arrangement and the amount of subcellular structures inside a cell or organelle, respectively, especially when the plant sample was exposed to environmental stress. In the present research, serial sectioned chloroplasts, mitochondria, and peroxisomes from first year spruce needles (Picea abies (L.) Karst.) were 3D reconstructed and digitally measured using a computer-supported image analysis system in order to obtain a detailed quantitative characterization of complete cell organelles including precise morphological data of drought-induced fine structural changes. In control plants, chloroplast volume was composed of 56% stroma, 15% starch, 27% thylakoids, and 2% plastoglobules. In drought-stressed chloroplasts, the relative volume of both the thylakoids and the plastoglobules significantly increased to 37% and 12%, respectively. Chloroplasts of stressed plants differed from control plants not only in the mean thylakoid and plastoglobules content but also in the complete lack of starch grains. Mitochondria occurred in variable forms in both control and stressed samples. In stressed plants, mitochondria showed a significant smaller mean volume which was only 81% when compared with the control organelles. Peroxisomes were inconspicuous in both samples and their volume did not differ between control and drought-stressed samples. The present study shows that specific subcellular structures are subject to significant quantitative changes during drought stress of spruce needles giving a detailed insight in adaptation processes of the investigated cell organelles.

Impact of drying on wood ultrastructure observed by deuterium exchange and photoacoustic FT-IR spectroscopy.

The impact of drying on the ultrastructure of fresh wood was studied by deuterium exchange coupled with FT-IR analysis. This fundamental investigation demonstrated that water removal leads to irreversible alterations of the wood structure, namely, supramolecular rearrangements between wood polymers. The deuteration of fresh wood was shown to be fully reversible by a subsequent exposure of the deuterated sample to water (reprotonation). Therefore, the presence of any OD groups in deuterated and then dried wood samples after reprotonation is a clear indicator of reduced accessibility. The extent of changes was affected by drying temperature and relative humidity. Application of this methodology for the evaluation of chemical pulp sample (reference material) resulted in similar response, only more pronounced. Two hypothetical alternatives were proposed for accessibility reduction in dried wood: (i) irreversible aggregation of cellulose microfibrils and (ii) irreversible stiffening of the hemicellulose/lignin matrix that extensively swells when exposed to water.