No abstract

Confocal Raman microscopy was used to illustrate changes of molecular composition in secondary plant cell wall tissues of poplar (Populus nigra 3 Populus deltoids) wood. Two-dimensional spectral maps were acquired and chemical images calculated by integrating the intensity of characteristic spectral bands. This enabled direct visualization of the spatial variation of the lignin content without any chemical treatment or staining of the cell wall. A small (0.5 mm) lignified border toward the lumen was observed in the gelatinous layer of poplar tension wood. The variable orientation of the cellulose was also characterized, leading to visualization of the S1 layer with dimensions smaller than 0.5 mm. Scanning Raman microscopy was thus shown to be a powerful, nondestructive tool for imaging changes in molecular cell wall organization with high spatial resolution.

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.

To gain a better understanding on structure, chemical composition and properties of plant cells, tissues and organs several microscopic, chemical and physical methods have been applied during the last years. However, a knowledge gap exists about the location, quantity and structural arrangement of molecules in the native sample or what happens on the molecular level when samples are chemically or mechanically treated or how they respond to mechanical stress. These questions need to be answered to optimise utilization of plants in food industry and pharmacy and to understand structure-function relationships of plant cells to learn from natures unique. Advances in combining microscopy with Raman spectroscopy have tackled this problem in a non-invasive way and provide chemical and structural informationin situwithout any staining or complicated sample preparation. In this review the different Raman techniques (e.g. near infrared Fourier Transform Raman spectroscopy (NIR-FT), resonance Raman spectroscopy, surface-enhanced Raman spectroscopy) are briefly described before approaches in plant science are summarised. Investigations on structural cell wall components, valuable plant substances, metabolites and inorganic substances are included with emphasis on Raman imaging. The introduction of the NIR-FT-Raman technique led to many applications on green plant material by eliminating the problem of sample fluorescence. For mapping and imaging of whole plant organs (seeds, fruits, leaves) the lateral resolution (~10μm) of the NIR-FT technique is adequate, whereas for investigations on the lower hierarchical level of cells and cell walls the high resolution gained with a visible laser based system is needed. Examples on high resolution Raman imaging are given on wood cells, showing that changes in chemistry and orientation can be followed within and between different cell wall layers having dimensions smaller than 1 μm. In addition imaging the distribution of amorphous silica is shown on horsetail tissue, including an area scan from a cross section as well as a depth profiling within a silica rich knob of the outer stem wall.

Larch heartwood is appreciated for its good mechanical properties, its colour and its texture, and it is often used outdoors because of its natural durability (decay resistance). In this study the colour of larch heartwood was studied in relation to extractives and decay resistance, with the aim to estimate durability of larch heartwood from its colour. On a total of 293 trees colour in the CIE L*a*b* space (L* lightness, a* red/green axis, b* yellow/blue axis), extractives content (acetone and hot-water extractives, amount of phenolics) and the brown-rot decay resistance were determined. For calculating the relative decay resistance (x), mass loss after inoculation for 16 weeks with two fungi [Coniophora puteana (Schum.ex.Fr.) Karst., Poria placenta (Fr.) Cke, European standard EN 113] of larch heartwood samples was compared to Scots pine (Pinus sylvestris L) sapwood reference samples (EN 350-1). Different species [Japanese larch (Larix kaempferi Lamb.), Hybrid larch (Larix deciduax L. kaempferi) and European larch (L. decidua Mill.)], provenances and age classes (38-year, >150-year) were included. Japanese larch heartwood turned out to be significantly more reddish (higher a*-values) compared to the European larch provenances. Reddishness of the hybrids was intermediate. The red hue (+a*) was strongly correlated with the amount of phenols (r=0.84) and decay resistance (r=0.63) and therefore suitable for prediction of both parameters. The results suggest that colour measurements of larch heartwood could be of benefit in tree breeding programs and for an optimised utilization of larch timber.

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° 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 (R2 > 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° to 49.9° 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 spectra were acquired in situ during tensile straining of mechanically isolated fibers of spruce latewood. Stress-strain curves were evaluated along with band positions and intensities to monitor molecular changes due to deformation. Strong correlations (r = 0.99) were found between the shift of the band at 1097 cm(-1) corresponding to the stretching of the cellulose ring structure and the applied stress and strain. High overall shifts (-6.5 cm(-1)) and shift rates (-6.1 cm(-1)/GPa) were observed. After the fiber failed, the band was found on its original position again, proving the elastic nature of the deformation. Additionally, a decrease in the band height ratio of the 1127 and 1097 cm(-1) bands was observed to go hand in hand with the straining of the fiber. This is assumed to reflect a widening of the torsion angle of the glycosidic C-O-C bonding. Thus, the 1097 cm(-1) band shift and the band height ratio enable one to follow the stretching of the cellulose at a molecular level, while the lignin bands are shown to be unaffected. Observed changes in the OH region are shown and interpreted as a weakening of the hydrogen-bonding network during straining. Future experiments on different native wood fibers with variable chemical composition and cellulose orientation and on chemically and enzymatically modified fibers will help to deepen the micromechanical understanding of plant cell walls and the associated macromolecules.

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.