Hydrolyzable tannins are incorporated into the endocarp during sclerification of the water caltrop Trapa natans.

The water caltrop (Trapa natans) develops unique woody fruits with unusually large seeds among aquatic plants. During fruit development, the inner fruit wall (endocarp) sclerifies and forms a protective layer for the seed. Endocarp sclerification also occurs in many land plants with large seeds; however, in T. natans, the processes of fruit formation, endocarp hardening, and seed storage take place entirely underwater. To identify potential chemical and structural adaptations for the aquatic environment, we investigated the cell-wall composition in the endocarp at a young developmental stage, as well as at fruit maturity. Our work shows that hydrolyzable tannins-specifically gallotannins-flood the endocarp tissue during secondary wall formation and are integrated into cell walls along with lignin during maturation. Within the secondary walls of mature tissue, we identified unusually strong spectroscopic features of ester linkages, suggesting that the gallotannins and their derivatives are cross-linked to other wall components via ester bonds, leading to unique cell-wall properties. The synthesis of large amounts of water-soluble, defensive aromatic metabolites during secondary wall formation might be a fast way to defend seeds within the insufficiently lignified endocarp of T. natans.

Wood Deformation Leads to Rearrangement of Molecules at the Nanoscale.

Wood, as the most abundant carbon dioxide storing bioresource, is currently driven beyond its traditional use through creative innovations and nanotechnology. For many properties the micro- and nanostructure plays a crucial role and one key challenge is control and detection of chemical and physical processes in the confined microstructure and nanopores of the wooden cell wall. In this study, correlative Raman and atomic force microscopy show high potential for tracking in situ molecular rearrangement of wood polymers during compression. More water molecules (interpreted as wider cellulose microfibril distances) and disentangling of hemicellulose chains are detected in the opened cell wall regions, whereas an increase of lignin is revealed in the compressed areas. These results support a new more "loose" cell wall model based on flexible lignin nanodomains and advance our knowledge of the molecular reorganization during deformation of wood for optimized processing and utilization.

Strong, Shape-Memory Lignocellulosic Aerogel via Wood Cell Wall Nanoscale Reassembly.

Polymer shape-memory aerogels (PSMAs) are prospects in various fields of application ranging from aerospace to biomedicine, as advanced thermal insulators, actuators, or sensors. However, the fabrication of PSMAs with good mechanical performance is challenging and is currently dominated by fossil-based polymers. In this work, strong, shape-memory bio-aerogels with high specific surface areas (up to 220 m2/g) and low radial thermal conductivity (0.042 W/mK) were prepared through a one-step treatment of native wood using an ionic liquid mixture of [MTBD]+[MMP]-/DMSO. The aerogel showed similar chemical composition similar to native wood. Nanoscale spatial rearrangement of wood biopolymers in the cell wall and lumen was achieved, resulting in flexible hydrogels, offering design freedom for subsequent aerogels with intricate geometries. Shape-memory function under stimuli of water was reported. The chemical composition and distribution, morphology, and mechanical performance of the aerogel were carefully studied using confocal Raman spectroscopy, AFM, SAXS/WAXS, NMR, digital image correlation, etc. With its simplicity, sustainability, and the broad range of applicability, the methodology developed for nanoscale reassembly of wood is an advancement for the design of biobased shape-memory aerogels.

Method for High-Yield Hydrothermal Growth of Silica Shells on Nanoparticles.

Coating processes are commonly used in materials science to protect a core or modify material properties. We describe a hydrothermal coating process using TEOS (tetraethyl orthosilicate), a widely used precursor for silica coatings, on three representative template materials (carbon nanotubes, silica, and polystyrene nanoparticles) with different properties and shapes. We compare the efficiency of previously published protocols for silica coatings at room temperature and atmospheric pressure with the hydrothermal process at 160 °C and 3 bar. The hydrothermal method achieves higher yields and thicker silica coatings with the same amount of precursor when compared to the conventional way, thus offering higher effectiveness. Furthermore, the hydrothermal coating process yields more homogeneous shells with a higher density, making hydrothermal coating the method of choice when mechanical integrity and low permeability of the coating are required.

Raman imaging reveals in-situ microchemistry of cuticle and epidermis of spruce needles.

BACKGROUND: The cuticle is a protective layer playing an important role in plant defense against biotic and abiotic stresses. So far cuticle structure and chemistry was mainly studied by electron microscopy and chemical extraction. Thus, analysing composition involved sample destruction and the link between chemistry and microstructure remained unclear. In the last decade, Raman imaging showed high potential to link plant anatomical structure with microchemistry and to give insights into orientation of molecules. In this study, we use Raman imaging and polarization experiments to study the native cuticle and epidermal layer of needles of Norway spruce, one of the economically most important trees in Europe. The acquired hyperspectral dataset is the basis to image the chemical heterogeneity using univariate (band integration) as well as multivariate data analysis (cluster analysis and non-negative matrix factorization). RESULTS: Confocal Raman microscopy probes the cuticle together with the underlying epidermis in the native state and tracks aromatics, lipids, carbohydrates and minerals with a spatial resolution of 300 nm. All three data analysis approaches distinguish a waxy, crystalline layer on top, in which aliphatic chains and coumaric acid are aligned perpendicular to the surface. Also in the lipidic amorphous cuticle beneath, strong signals of coumaric acid and flavonoids are detected. Even the unmixing algorithm results in mixed endmember spectra and confirms that lipids co-locate with aromatics. The underlying epidermal cell walls are devoid of lipids but show strong aromatic Raman bands. Especially the upper periclinal thicker cell wall is impregnated with aromatics. At the interface between epidermis and cuticle Calcium oxalate crystals are detected in a layer-like fashion. Non-negative matrix factorization gives the purest component spectra, thus the best match with reference spectra and by this promotes band assignments and interpretation of the visualized chemical heterogeneity. CONCLUSIONS: Results sharpen our view about the cuticle as the outermost layer of plants and highlight the aromatic impregnation throughout. In the future, developmental studies tracking lipid and aromatic pathways might give new insights into cuticle formation and comparative studies might deepen our understanding why some trees and their needle and leaf surfaces are more resistant to biotic and abiotic stresses than others.

A Guide to Elucidate the Hidden Multicomponent Layered Structure of Plant Cuticles by Raman Imaging.

The cuticle covers almost all plant organs as the outermost layer and serves as a transpiration barrier, sunscreen, and first line of defense against pathogens. Waxes, fatty acids, and aromatic components build chemically and structurally diverse layers with different functionality. So far, electron microscopy has elucidated structure, while isolation, extraction, and analysis procedures have revealed chemistry. With this method paper, we close the missing link by demonstrating how Raman microscopy gives detailed information about chemistry and structure of the native cuticle on the microscale. We introduce an optimized experimental workflow, covering the whole process of sample preparation, Raman imaging experiment, data analysis, and interpretation and show the versatility of the approach on cuticles of a spruce needle, a tomato peel, and an Arabidopsis stem. We include laser polarization experiments to deduce the orientation of molecules and multivariate data analysis to separate cuticle layers and verify their molecular composition. Based on the three investigated cuticles, we discuss the chemical and structural diversity and validate our findings by comparing models based on our spectroscopic data with the current view of the cuticle. We amend the model by adding the distribution of cinnamic acids and flavonoids within the cuticle layers and their transition to the epidermal layer. Raman imaging proves as a non-destructive and fast approach to assess the chemical and structural variability in space and time. It might become a valuable tool to tackle knowledge gaps in plant cuticle research.

Raman imaging of Micrasterias: new insights into shape formation.

The algae Micrasterias with its star-shaped cell pattern is a perfect unicellular model system to study morphogenesis. How the indentations are formed in the primary cell wall at exactly defined areas puzzled scientists for decades, and they searched for chemical differences in the primary wall of the extending tips compared to the resting indents. We now tackled the question by Raman imaging and scanned in situ Micrasterias cells at different stages of development. Thousands of Raman spectra were acquired from the mother cell and the developing semicell to calculate chemical images based on an algorithm finding the most different Raman spectra. Each of those spectra had characteristic Raman bands, which were assigned to molecular vibrations of BaSO4, proteins, lipids, starch, and plant cell wall carbohydrates. Visualizing the cell wall carbohydrates revealed a cell wall thickening at the indentations of the primary cell wall of the growing semicell and uniplanar orientation of the cellulose microfibrils to the cell surface in the secondary cell wall. Crystalline cellulose dominated in the secondary cell wall spectra, while in the primary cell wall spectra, also xyloglucan and pectin were reflected. Spectral differences between the indent and tip region of the primary cell wall were scarce, but a spectral mixing approach pointed to more cellulose fibrils deposited in the indent region. Therefore, we suggest that cell wall thickening together with a denser network of cellulose microfibrils stiffens the cell wall at the indent and induces different cell wall extensibility to shape the lobes.

Induction of Conjugation and Zygospore Cell Wall Characteristics in the Alpine Spirogyra mirabilis (Zygnematophyceae, Charophyta): Advantage under Climate Change Scenarios?

Extreme environments, such as alpine habitats at high elevation, are increasingly exposed to man-made climate change. Zygnematophyceae thriving in these regions possess a special means of sexual reproduction, termed conjugation, leading to the formation of resistant zygospores. A field sample of Spirogyra with numerous conjugating stages was isolated and characterized by molecular phylogeny. We successfully induced sexual reproduction under laboratory conditions by a transfer to artificial pond water and increasing the light intensity to 184 µmol photons m-2 s-1. This, however was only possible in early spring, suggesting that the isolated cultures had an internal rhythm. The reproductive morphology was characterized by light- and transmission electron microscopy, and the latter allowed the detection of distinctly oriented microfibrils in the exo- and endospore, and an electron-dense mesospore. Glycan microarray profiling showed that Spirogyra cell walls are rich in major pectic and hemicellulosic polysaccharides, and immuno-fluorescence allowed the detection of arabinogalactan proteins (AGPs) and xyloglucan in the zygospore cell walls. Confocal RAMAN spectroscopy detected complex aromatic compounds, similar in their spectral signature to that of Lycopodium spores. These data support the idea that sexual reproduction in Zygnematophyceae, the sister lineage to land plants, might have played an important role in the process of terrestrialization.

Oak wood drying: precipitation of crystalline ellagic acid leads to discoloration.

Oak heartwood usually darkens during and after drying. This darkening can be heterogeneous, leaving noncolored areas in the wood board. These light discolorations have been linked to heterogeneous distribution of tannins, but compelling evidence on the microscale is lacking. In this study Raman and fluorescence microscopy revealed precipitations of crystalline ellagic acid, especially in the ray cells but also in lumina, cell corners and cell walls in the non-colored areas (NCA), which also had higher density. In these denser areas free water is longer present during drying and leads to accumulation of hydrolyzed tannins. When eventually falling dry, these tannins precipitate irreversible as non-colored ellagic acid and are not available for chemical reactions leading to darkening of the wood. Therefore, pronounced density fluctuations in wood boards require adjusting the drying and processing parameters so that water domains and ellagic acid precipitations are avoided during drying.

Cell wall characteristics during sexual reproduction of Mougeotia sp. (Zygnematophyceae) revealed by electron microscopy, glycan microarrays and RAMAN spectroscopy.

Mougeotia spp. collected from field samples were investigated for their conjugation morphology by light-, fluorescence-, scanning- and transmission electron microscopy. During a scalarifom conjugation, the extragametangial zygospores were initially surrounded by a thin cell wall that developed into a multi-layered zygospore wall. Maturing zygospores turned dark brown and were filled with storage compounds such as lipids and starch. While M. parvula had a smooth surface, M. disjuncta had a punctated surface structure and a prominent suture. The zygospore wall consisted of a polysaccharide rich endospore, followed by a thin layer with a lipid-like appaerance, a massive electron dense mesospore and a very thin exospore composed of polysaccharides. Glycan microarray analysis of zygospores of different developmental stages revealed the occurrence of pectins and hemicelluloses, mostly composed of homogalacturonan (HG), xyloglucans, xylans, arabino-galactan proteins and extensins. In situ localization by the probe OG7-13AF 488 labelled HG in young zygospore walls, vegetative filaments and most prominently in conjugation tubes and cross walls. Raman imaging showed the distribution of proteins, lipids, carbohydrates and aromatic components of the mature zygospore with a spatial resolution of ~ 250 nm. The carbohydrate nature of the endo- and exospore was confirmed and in-between an enrichment of lipids and aromatic components, probably algaenan or a sporopollenin-like material. Taken together, these results indicate that during zygospore formation, reorganizations of the cell walls occured, leading to a resistant and protective structure.

Twist and lock: nutshell structures for high strength and energy absorption.

Nutshells achieve remarkable properties by optimizing structure and chemistry at different hierarchical levels. Probing nutshells from the cellular down to the nano- and molecular level by microchemical and nanomechanical imaging techniques reveals insights into nature's packing concepts. In walnut and pistachio shells, carbohydrate and lignin polymers assemble to form thick-walled puzzle cells, which interlock three-dimensionally and show high tissue strength. Pistachio additionally achieves high-energy absorption by numerous lobes interconnected via ball-joint-like structures. By contrast, the three times more lignified walnut shells show brittle LEGO-brick failure, often along the numerous pit channels. In both species, cell walls (CWs) show distinct lamellar structures. These lamellae involve a helicoidal arrangement of cellulose macrofibrils as a recurring motif. Between the two nutshell species, these lamellae show differences in thickness and pitch angle, which can explain the different mechanical properties on the nanolevel. Our in-depth study of the two nutshell tissues highlights the role of cell form and their interlocking as well as plant CW composition and structure for mechanical protection. Understanding these plant shell concepts might inspire biomimetic material developments as well as using walnut and pistachio shell waste as sustainable raw material in future applications.

Antifungal stilbene impregnation: transport and distribution on the micron-level.

The transition from the living water-transporting sapwood to heartwood involves in many tree species impregnation with extractives. These differ in amount and composition, and enhance resistance against bacteria, insects or fungi. To understand the synthesis, transport and impregnation processes new insights into the biochemical processes are needed by in-situ methods. Here we show the extractive distribution in pine (Pinus sylvestris) microsections with a high lateral resolution sampled in a non-destructive manner using Confocal Raman Microscopy. Integrating marker bands of stilbenes and lipids enables to clearly track the rapid change from sapwood to heartwood within one tree ring. The higher impregnation of the cell corner, compound middle lamella, the S3 layer and pits reveals the optimization of decay resistance on the micron-level. Furthermore, deposits with changing chemical composition are elucidated in the rays and lumen of the tracheids. The spectral signature of these deposits shows the co-location of lipids and pinosylvins with changing ratios from the living to the dead tissue. The results demonstrate that the extractive impregnation on the micro- and nano-level is optimized by a symbiotic relationship of lipids and pinosylvins to enhance the tree's resistance and lifetime.

Multivariate unmixing approaches on Raman images of plant cell walls: new insights or overinterpretation of results?

BACKGROUND: Plant cell walls are nanocomposites based on cellulose microfibrils embedded in a matrix of polysaccharides and aromatic polymers. They are optimized for different functions (e.g. mechanical stability) by changing cell form, cell wall thickness and composition. To reveal the composition of plant tissues in a non-destructive way on the microscale, Raman imaging has become an important tool. Thousands of Raman spectra are acquired, each one being a spatially resolved molecular fingerprint of the plant cell wall. Nevertheless, due to the multicomponent nature of plant cell walls, many bands are overlapping and classical band integration approaches often not suitable for imaging. Multivariate data analysing approaches have a high potential as the whole wavenumber region of all thousands of spectra is analysed at once. RESULTS: Three multivariate unmixing algorithms, vertex component analysis, non-negative matrix factorization and multivariate curve resolution-alternating least squares were applied to find the purest components within datasets acquired from micro-sections of spruce wood and Arabidopsis. With all three approaches different cell wall layers (including tiny S1 and S3 with 0.09-0.14 µm thickness) and cell contents were distinguished and endmember spectra with a good signal to noise ratio extracted. Baseline correction influences the results obtained in all methods as well as the way in which algorithm extracts components, i.e. prioritizing the extraction of positive endmembers by sequential orthogonal projections in VCA or performing a simultaneous extraction of non-negative components aiming at explaining the maximum variance in NMF and MCR-ALS. Other constraints applied (e.g. closure in VCA) or a previous principal component analysis filtering step in MCR-ALS also contribute to the differences obtained. CONCLUSIONS: VCA is recommended as a good preliminary approach, since it is fast, does not require setting many input parameters and the endmember spectra result in good approximations of the raw data. Yet the endmember spectra are more correlated and mixed than those retrieved by NMF and MCR-ALS methods. The latter two give the best model statistics (with lower lack of fit in the models), but care has to be taken about overestimating the rank as it can lead to artificial shapes due to peak splitting or inverted bands.