The walnut shell network: 3D visualisation of symplastic and apoplastic transport routes in sclerenchyma tissue.

MAIN CONCLUSION: High symplastic connectivity via pits was linked to the lignification of the developing walnut shell. With maturation, this network lessened, whereas apoplastic intercellular space remained and became relevant for shell drying. The shell of the walnut (Juglans regia) sclerifies within several weeks. This fast secondary cell wall thickening and lignification of the shell tissue might need metabolites from the supporting husk tissue. To reveal the transport capacity of the walnut shell tissue and its connection to the husk, we visualised the symplastic and apoplastic transport routes during shell development by serial block face-SEM and 3D reconstruction. We found an extensive network of pit channels connecting the cells within the shell tissue, but even more towards the husk tissue. Each pit channel ended in a pit field, which was occupied by multiple plasmodesmata passing through the middle lamella. During shell development, secondary cell wall formation progressed towards the interior of the cell, leaving active pit channels open. In contrast, pit channels, which had no plasmodesmata connection to a neighbouring cell, got filled by cellulose layers from the inner cell wall lamellae. A comparison with other nut species showed that an extended network during sclerification seemed to be linked to high cell wall lignification and that the connectivity between cells got reduced with maturation. In contrast, intercellular spaces between cells remained unchanged during the entire sclerification process, allowing air and water to flow through the walnut shell tissue when mature. The connectivity between inner tissue and environment was essential during shell drying in the last month of nut development to avoid mould formation. The findings highlight how connectivity and transport work in developing walnut shell tissue and how finally in the mature state these structures influence shell mechanics, permeability, conservation and germination.

Atypical lignification in eastern leatherwood (Dirca palustris).

Lignin is a complex phenolic biopolymer found mainly in the secondary cell walls of vascular plants, where it contributes to mechanical strength, water conduction, and plant defence. We studied the lignin of eastern leatherwood (Dirca palustris) because this slow-growing woody shrub is known for its flexible stems. Various analytical techniques and microscopy methods were employed to examine the composition and distribution of lignin and structural polysaccharides in leatherwood xylem in comparison with trembling aspen (Populus tremuloides) and white spruce (Picea glauca). We found that leatherwood has low overall levels of lignin, a high syringyl lignin content, and a unique distribution of lignin. Most remarkably, the cell corners and middle lamellae remain unlignified in mature xylem. These findings help explain the flexibility of leatherwood and also call into question the classical model of lignification, which purports that lignin polymerization begins in the cell corners and middle lamellae. This atypical lignification regime vividly illustrates the diversity in plant secondary cell wall formation that abounds in nature and casts leatherwood as a new model for the study of lignin biogenesis.

Vessel-Specific Reintroduction of CINNAMOYL-COA REDUCTASE1 (CCR1) in Dwarfed ccr1 Mutants Restores Vessel and Xylary Fiber Integrity and Increases Biomass.

Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars due to the presence of lignin. To render lignocellulosic biomass a suitable feedstock for the bio-based economy, plants can be engineered to have decreased amounts of lignin. However, engineered plants with the lowest amounts of lignin exhibit collapsed vessels and yield penalties. Previous efforts were not able to fully overcome this phenotype without settling in sugar yield upon saccharification. Here, we reintroduced CINNAMOYL-COENZYME A REDUCTASE1 (CCR1) expression specifically in the protoxylem and metaxylem vessel cells of Arabidopsis (Arabidopsis thaliana) ccr1 mutants. The resulting ccr1 ProSNBE:CCR1 lines had overcome the vascular collapse and had a total stem biomass yield that was increased up to 59% as compared with the wild type. Raman analysis showed that monolignols synthesized in the vessels also contribute to the lignification of neighboring xylary fibers. The cell wall composition and metabolome of ccr1 ProSNBE:CCR1 still exhibited many similarities to those of ccr1 mutants, regardless of their yield increase. In contrast to a recent report, the yield penalty of ccr1 mutants was not caused by ferulic acid accumulation but was (largely) the consequence of collapsed vessels. Finally, ccr1 ProSNBE:CCR1 plants had a 4-fold increase in total sugar yield when compared with wild-type plants.

Significant influence of lignin on axial elastic modulus of poplar wood at low microfibril angles under wet conditions.

Wood is extensively used as a construction material. Despite increasing knowledge of its mechanical properties, the contribution of the cell-wall matrix polymers to wood mechanics is still not well understood. Previous studies have shown that axial stiffness correlates with lignin content only for cellulose microfibril angles larger than around 20°, while no influence is found for smaller angles. Here, by analysing the wood of poplar with reduced lignin content due to down-regulation of CAFFEOYL SHIKIMATE ESTERASE, we show that lignin content also influences axial stiffness at smaller angles. Micro-tensile tests of the xylem revealed that axial stiffness was strongly reduced in the low-lignin transgenic lines. Strikingly, microfibril angles were around 15° for both wild-type and transgenic poplars, suggesting that cellulose orientation is not responsible for the observed changes in mechanical behavior. Multiple linear regression analysis showed that the decrease in stiffness was almost completely related to the variation in both density and lignin content. We suggest that the influence of lignin content on axial stiffness may gradually increase as a function of the microfibril angle. Our results may help in building up comprehensive models of the cell wall that can unravel the individual roles of the matrix polymers.

Unravelling the impact of lignin on cell wall mechanics: a comprehensive study on young poplar trees downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD).

Lignin engineering is a promising tool to reduce the energy input and the need of chemical pre-treatments for the efficient conversion of plant biomass into fermentable sugars for downstream applications. At the same time, lignin engineering can offer new insight into the structure-function relationships of plant cell walls by combined mechanical, structural and chemical analyses. Here, this comprehensive approach was applied to poplar trees (Populus tremula × Populus alba) downregulated for CINNAMYL ALCOHOL DEHYDROGENASE (CAD) in order to gain insight into the impact of lignin reduction on mechanical properties. The downregulation of CAD resulted in a significant decrease in both elastic modulus and yield stress. As wood density and cellulose microfibril angle (MFA) did not show any significant differences between the wild type and the transgenic lines, these structural features could be excluded as influencing factors. Fourier transform infrared spectroscopy (FTIR) and Raman imaging were performed to elucidate changes in the chemical composition directly on the mechanically tested tissue sections. Lignin content was identified as a mechanically relevant factor, as a correlation with a coefficient of determination (r²) of 0.65 between lignin absorbance (as an indicator of lignin content) and tensile stiffness was found. A comparison of the present results with those of previous investigations shows that the mechanical impact of lignin alteration under tensile stress depends on certain structural conditions, such as a high cellulose MFA, which emphasizes the complex relationship between the chemistry and mechanical properties in plant cell walls.

The effect of altered lignin composition on mechanical properties of CINNAMYL ALCOHOL DEHYDROGENASE (CAD) deficient poplars.

MAIN CONCLUSION: CAD-deficient poplars enabled studying the influence of altered lignin composition on mechanical properties. Severe alterations in lignin composition did not influence the mechanical properties. Wood represents a hierarchical fiber-composite material with excellent mechanical properties. Despite its wide use and versatility, its mechanical behavior has not been entirely understood. It has especially been challenging to unravel the mechanical function of the cell wall matrix. Lignin engineering has been a useful tool to increase the knowledge on the mechanical function of lignin as it allows for modifications of lignin content and composition and the subsequent studying of the mechanical properties of these transgenics. Hereby, in most cases, both lignin composition and content are altered and the specific influence of lignin composition has hardly been revealed. Here, we have performed a comprehensive micromechanical, structural, and spectroscopic analysis on xylem strips of transgenic poplar plants, which are downregulated for cinnamyl alcohol dehydrogenase (CAD) by a hairpin-RNA-mediated silencing approach. All parameters were evaluated on the same samples. Raman microscopy revealed that the lignin of the hpCAD poplars was significantly enriched in aldehydes and reduced in the (relative) amount of G-units. FTIR spectra indicated pronounced changes in lignin composition, whereas lignin content was not significantly changed between WT and the hpCAD poplars. Microfibril angles were in the range of 18°-24° and were not significantly different between WT and transgenics. No significant changes were observed in mechanical properties, such as tensile stiffness, ultimate stress, and yield stress. The specific findings on hpCAD poplar allowed studying the specific influence of lignin composition on mechanics. It can be concluded that the changes in lignin composition in hpCAD poplars did not affect the micromechanical tensile properties.

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.

The molecular structure within dislocations in Cannabis sativa fibres studied by polarised Raman microspectroscopy.

Polarised Raman micrsospectroscopy was employed to study the molecular structure within dislocations (slip planes) in the cell walls of Hemp fibre cells (Cannabis sativa (L.)). It was found that the cellulose microfibrils within dislocations have a different orientation than in the surrounding cell wall, and that the cellulose in the transition zones between a large dislocation and the surrounding wall may have yet another orientation. Furthermore, cellulose orientation seemed to be less uniform within dislocations than in the surrounding cell wall.

G-fibres in storage roots of Trifolium pratense (Fabaceae): tensile stress generators for contraction.

Root contraction has been described for many species within the plant kingdom for over a century, and many suggestions have been made for mechanisms behind these contractions. To move the foliage buds deeper into the soil, the proximal part of the storage root of Trifolium pratense contracts by up to 30%. Anatomical studies have shown undeformed fibres next to strongly deformed tissues. Raman imaging revealed that these fibres are chemically and structurally very similar to poplar (Populus) tension wood fibres, which are known to generate high tensile stresses and bend leaning stems or branches upright. Analogously, an almost pure cellulosic layer is laid down in the lumen of certain root fibres, on a thin lignified secondary cell wall layer. To reveal its stress generation capacities, the thick cellulosic layer, reminiscent of a gelatinous layer (G-layer) in tension wood, was selectively removed by enzymatic treatment. A substantial change in the dimensions of the isolated wood fibre bundles was observed. This high stress relaxation indicates the presence of high tensile stress for root contraction. These findings indicate a mechanism of root contraction in T. pratense (red clover) actuated via tension wood fibres, which follows the same principle known for poplar tension wood.

Functional plant cell wall design revealed by the Raman imaging approach.

Using the Raman imaging approach, the optimization of the plant cell wall design was investigated on the micron level within different tissue types at different positions of a Phormium tenax leaf. Pectin and lignin distribution were visualized and the cellulose microfibril angle (MFA) of the cell walls was determined. A detailed analysis of the Raman spectra extracted from the selected regions, allowed a semi-quantitative comparison of the chemical composition of the investigated tissue types on the micron level. The cell corners of the parenchyma revealed almost pure pectin and the cell wall an amount of 38-49% thereof. Slight lignification was observed in the parenchyma and collenchyma in the top of the leaf and a high variability (7-44%) in the sclerenchyma. In the cell corners and in the cell wall of the sclerenchymatic fibres surrounding the vascular tissue, the highest lignification was observed, which can act as a barrier and protection of the vascular tissue. In the sclerenchyma high variable MFA (4°-40°) was detected, which was related with lignin variability. In the primary cell walls a constant high MFA (57°-58°) was found together with pectin. The different plant cell wall designs on the tissue and microlevel involve changes in chemical composition as well as cellulose microfibril alignment and are discussed and related according to the development and function.

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.

Cellulose microfibril orientation of Picea abies and its variability at the micron-level determined by Raman imaging.

The functional characteristics of plant cell walls depend on the composition of the cell wall polymers, as well as on their highly ordered architecture at scales from a few nanometres to several microns. Raman spectra of wood acquired with linear polarized laser light include information about polymer composition as well as the alignment of cellulose microfibrils with respect to the fibre axis (microfibril angle). By changing the laser polarization direction in 3 degrees steps, the dependency between cellulose and laser orientation direction was investigated. Orientation-dependent changes of band height ratios and spectra were described by quadratic linear regression and partial least square regressions, respectively. Using the models and regressions with high coefficients of determination (R(2) > 0.99) microfibril orientation was predicted in the S1 and S2 layers distinguished by the Raman imaging approach in cross-sections of spruce normal, opposite, and compression wood. The determined microfibril angle (MFA) in the different S2 layers ranged from 0 degrees to 49.9 degrees and was in coincidence with X-ray diffraction determination. With the prerequisite of geometric sample and laser alignment, exact MFA prediction can complete the picture of the chemical cell wall design gained by the Raman imaging approach at the micron level in all plant tissues.

Raman Imaging of Plant Cell Walls.

Raman imaging is a microspectroscopic approach revealing the chemistry and structure of plant cell walls in situ on the micro- and nanoscale. The method is based on the Raman effect (inelastic scattering) that takes place when monochromatic laser light interacts with matter. The scattered light conveys a change in energy that is inherent of the involved molecule vibrations. The Raman spectra are thus characteristic for the chemical structure of the molecules and can be recorded spatially ordered with a lateral resolution of about 300 nm. Based on thousands of acquired Raman spectra, images can be assessed using univariate as well as multivariate data analysis approaches. One advantage compared to staining or labeling techniques is that not only one image is obtained as a result but different components and characteristics can be displayed in several images. Furthermore, as every pixel corresponds to a Raman spectrum, which is a kind of "molecular fingerprint," the imaging results should always be evaluated and further details revealed by analysis (e.g., band assignment) of extracted spectra. In this chapter, the basic theoretical background of the technique and instrumentation are described together with sample preparation requirements and tips for high-quality plant tissue sections and successful Raman measurements. Typical Raman spectra of the different plant cell wall components are shown as well as an exemplified analysis of Raman data acquired on the model plant Arabidopsis. Important preprocessing methods of the spectra are included as well as single component image generation (univariate) and spectral unmixing by means of multivariate approaches (e.g., vertex component analysis).

In situ FT-IR microscopic study on enzymatic treatment of poplar wood cross-sections.

The feasibility of Fourier transform infrared (FT-IR) microscopy to monitor in situ the enzymatic degradation of wood was investigated. Cross-sections of poplar wood were treated with cellulase Onozuka RS within a custom-built fluidic cell. Light-optical micrographs and FT-IR spectra were acquired in situ from normal and tension wood fibers. Light-optical micrographs showed almost complete removal of the gelatinous (G) layer in tension wood. No structural and spectral changes were observed in the lignified cell walls. The accessibility of cellulose within the lignified cell wall was found to be the main limiting factor, whereas the depletion of the enzyme due to lignin adsorption could be ruled out. The fast, selective hydrolysis of the crystalline cellulose in the G-layer, even at room temperature, might be explained by the gel-like structure and the highly porous surface. Young plantation grown hardwood trees with a high proportion of G-fibers thus represent an interesting resource for bioconversion to fermentable sugars in the process to bioethanol.

Biomineralization of strontium and barium contributes to detoxification in the freshwater alga Micrasterias.

The unicellular model alga Micrasterias denticulata inhabits acid peat bogs that are highly endangered by pollutants due to their high humidity. As it was known from earlier studies that algae like Micrasterias are capable of storing barium naturally in form of BaSO4 crystals, it was interesting to experimentally investigate distribution and sequestration of barium and the chemically similar alkaline earth metal strontium. Additionally, we intended to analyze whether biomineralization by crystal formation contributes to diminution of the generally toxic effects of these minerals to physiology and structure of this alga which is closely related to higher plants. The results show that depending on the treatment differently shaped crystals are formed in BaCl2 and Cl2Sr exposed Micrasterias cells. Modern microscopic techniques such as analytical TEM by electron energy loss spectroscopy and Raman microscopy provide evidence for the chemical composition of these crystals. It is shown that barium treatment results in the formation of insoluble BaSO4 crystals that develop within distinct compartments. During strontium exposure long rod-like crystals are formed and are surrounded by membranes. Based on the Raman signature of these crystals their composition is attributed to strontium citrate. These crystals are instable and are dissolved during cell death. During strontium as well as barium treatment cell division rates and photosynthetic oxygen production decreased in dependence of the concentration, whereas cell vitality was reduced only slightly. Together with the fact that TEM analyses revealed only minor ultrastructural alterations as consequence of relatively high concentrated BaCl2 and Cl2Sr exposure, this indicates that biomineralization of Sr and Ba protects the cells from severe damage or cell death at least within a particular concentration range and time period. In the case of Sr treatment where ROS levels were found to be elevated, hallmarks for autophagy of single organelles were observed by TEM, indicating beginning degradation processes.

Following laser induced changes of plant phenylpropanoids by Raman microscopy.

Raman microscopy is a powerful imaging technique for biological materials providing information about chemistry in context with microstructure. A 532 nm laser is often used as excitation source, because high spatial resolution and signal intensity can be achieved. The latter can be controlled by laser power and integration time, whereby high power and long times give good signal to noise ratio. However, most biological materials absorb in the VIS range and fluorescence masking the signal or even sample degradation might be hindering. Here, we show that on lignified plant cell walls even very short integration times and low laser powers induce a change in the ratio of the lignin bands at 1660 and 1600 cm-1. Time series on lignin model compounds revealed this change only in aromatic molecules with two OH-groups, such as coniferyl alcohol. Therefore, we conclude that monolignols are present in the cell wall and responsible for the observed effect. The solvent selectivity of the changes points to a laser induced polymerization process. The results emphasize how crucial careful adjustment of experimental parameters in Raman imaging of biological materials is and show the potential of time series and repeated imaging to get additional insights (e.g. monolignols).

Tip in-light on: Advantages, challenges, and applications of combining AFM and Raman microscopy on biological samples.

Scanning probe microscopies and spectroscopies, especially AFM and Confocal Raman microscopy are powerful tools to characterize biological materials. They are both non-destructive methods and reveal mechanical and chemical properties on the micro and nano-scale. In the last years the interest for increasing the lateral resolution of optical and spectral images has driven the development of new technologies that overcome the diffraction limit of light. The combination of AFM and Raman reaches resolutions of about 50-150 nm in near-field Raman and 1.7-50 nm in tip enhanced Raman spectroscopy (TERS) and both give a molecular information of the sample and the topography of the scanned surface. In this review, the mentioned approaches are introduced, the main advantages and problems for application on biological samples discussed and some examples for successful experiments given. Finally the potential of colocated AFM and Raman measurements is shown on a case study of cellulose-lignin films: the topography structures revealed by AFM can be related to a certain chemistry by the colocated Raman scan and additionally the mechanical properties be revealed by using the digital pulsed force mode. Microsc. Res. Tech. 80:30-40, 2017. © 2016 Wiley Periodicals, Inc.

Imaging of plant cell walls by confocal Raman microscopy.

Raman imaging of plant cell walls represents a nondestructive technique that can provide insights into chemical composition in context with structure at the micrometer level (<0.5 µm). The major steps of the experimental procedure are described: sample preparation (embedding and microcutting), setting the mapping parameters, and finally the calculation of chemical images on the basis of the acquired Raman spectra. Every Raman image is based on thousands of spectra, each being a spatially resolved molecular 'fingerprint' of the cell wall. Multiple components are analyzed within the native cell walls, and insights into polymer composition as well as the orientation of the cellulose microfibrils can be gained. The most labor-intensive step of this process is often the sample preparation, as the imaging approach requires a flat surface of the plant tissue with intact cell walls. After finishing the map (acquisition time is ∼10 min to 10 h, depending on the size of the region of interest and scanning parameters), many possibilities exist for the analysis of spectral data and image generation.

Insights into the chemical composition of Equisetum hyemale by high resolution Raman imaging.

Equisetaceae has been of research interest for decades, as it is one of the oldest living plant families, and also due to its high accumulation of silica up to 25% dry wt. Aspects of silica deposition, its association with other biomolecules, as well as the chemical composition of the outer strengthening tissue still remain unclear. These questions were addressed by using high resolution (<1 microm) Confocal Raman microscopy. Two-dimensional spectral maps were acquired on cross sections of Equisetum hyemale and Raman images calculated by integrating over the intensity of characteristic spectral regions. This enabled direct visualization of differences in chemical composition and extraction of average spectra from defined regions for detailed analyses, including principal component analysis (PCA) and basis analysis (partial least square fit based on model spectra). Accumulation of silica was imaged in the knobs and in a thin layer below the cuticula. In the spectrum extracted from the knob region as main contributions, a broad band below 500 cm(-1) attributed to amorphous silica, and a band at 976 cm(-1) assigned to silanol groups, were found. From this, we concluded that these protrusions were almost pure amorphous, hydrated silica. No silanol group vibration was detected in the silicified epidermal layer below and association with pectin and hemicelluloses indicated. Pectin and hemicelluloses (glucomannan) were found in high levels in the epidermal layer and in a clearly distinguished outer part of the hypodermal sterome fibers. The inner part of the two-layered cells revealed as almost pure cellulose, oriented parallel along the fiber.

Tensile and compressive stresses in tracheids are induced by swelling based on geometrical constraints of the wood cell.

Plants are able to pre-stress their tissues in order to actuate their organs. Here, we demonstrate with two tissue types of the secondary xylem of conifers (normal wood and compression wood of spruce (Picea abies)) that either tensile or compressive stresses can develop in the longitudinal direction during the swelling of the cell wall. This dramatic difference appears to be due mostly to differences in cell geometry and cellulose fibril orientation. A mechanical model was developed to demonstrate swelling experiments with the help of sodium iodide experiments. The reversal of longitudinal extension can be predicted, based on the orientation of the (nearly inextensible) cellulose fibrils and the shape of the cell.