The potential of Raman microscopy and Raman imaging in plant research

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.</jats:p

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

Structure, function, and application of self-healing adhesives from mistletoe viscin

Berries from the European Mistletoe (Viscum album) possess a sticky tissue called viscin that facilitates adhesion and germination onto host trees. Recent studies of viscin have demonstrated its adhesive capacity on a range of natural and synthetic surfaces including wood, skin, metals, and plastic. Yet, the underlying mechanisms remain poorly understood. Here, an investigation of the adhesive performance of mistletoe viscin is performed, demonstrating its hygroscopic nature and ability to self-heal following adhesive failure. It is identified that adhesion originates from a water-soluble adhesive component that can be extracted, isolated, and characterized independently. Lap shear mechanical testing indicates that the mistletoe adhesive extract (MAE) outperforms native viscin tissue, as well as gum arabic and arabinogalactan—common plant-based adhesives. Furthermore, humidity uptake experiments reveal that MAE can reversibly absorb nearly 100% of its mass in water from the atmosphere. In-depth spectroscopic and mass spectrometry investigations reveal a composition consisting primarily of an atypical arabinogalactan, with additional sugar alcohols. Finally, several proof-of-concept applications are demonstrated using MAE for hygro-responsive reversible adhesion between various surfaces including skin, plastic, PDMS, and paper, revealing that MAE holds potential as a biorenewable and reusable adhesive for applications in cosmetics, packaging, and potentially, tissue engineering

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° 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

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

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 μm) 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

A millennium-long 'Blue Ring' chronology from the Spanish Pyrenees reveals severe ephemeral summer cooling after volcanic eruptions

Abstract ‘Blue Rings’ (BRs) are distinct wood anatomical anomalies recently discovered in several tree species from different sites. While it is evident that they are associated with a cooling-induced lack of cell wall lignification, BRs have yet to be evaluated systematically in paleoclimate studies. Here, we present a continuous wood anatomical assessment of 31 living and relict pine samples from a high-elevation site in the central Spanish Pyrenees that span the period 1150–2017 CE at annual resolution. While most BR years coincide with cold summer temperatures and many BRs follow large volcanic eruptions, some were formed during overall warm summers. We also see a differential response between eruptions: the Samalas eruption is followed by 80% BRs in 1258, but only a modest signal is evident after the 1815 Tambora eruption, and there are no wood anatomical effects of the Laki eruption in 1783–1784. Apparently linked to a cluster of tropical eruptions in 1695 and 1696 CE, 85% BRs occurred in 1698. This new wood anatomical evidence is corroborated by the record of sulphur deposition in polar ice cores, and corresponds with catastrophic famine and unprecedented mortality in Scotland. The extremely rare occurrence of consecutive BRs in 1345 and 1346 marks the onset and spread of the Black Death, Europe’s most devastating plague pandemic. In their ability to capture severe ephemeral cold spells, as short as several days or weeks, BR chronologies can help to investigate and understand the impacts of volcanism on climate and society.</jats:p