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Mixture analysis of spruce needle spectra. Based on a reference library the spectra from the True Component Analysis are fit by a mixture alogorithm (see method part). (A) Cuticle top layer (black spectrum) compared with the fit result (yellow), which is a linear combination of a flavonol (Kaempferol 3-rutionside), saturated fatty alcohol (1-Octadecanol), and milled wood lignin (MWL BA-53). The residual error is shown on the right and the Raman images from Figure 4 for better orientation. (B) Spectrum of the cuticle wax sealing of the stoma (black) and the resulting fit (cyan) and reference spectra. (C) The thin epicuticular wax layer spectrum (black) and its fit (blue) and reference spectra. Note that this layer also has incorporated cinnamic acid and flavonol. (D) The first cell wall layer (red) is mainly fit by lignin, but also cinnamic acid and flavonol (see also Supplementary Figure 1).
Source publication
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 isolat...
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Citations
... These two peaks are the characteristic peaks of CaC 2 O 4 .2H 2 O. Moreover, the CaC 2 O 4 .2H 2 O fingerprint peaks of 506, 597, 868, and 910 cm −1 (the vibrational peak of M-O、C-C、O-C = O) and the -OH vibrational peak of H 2 O at 3470 cm −1 also appeared in extracted crystals with tetrahedral and spherical shapes [28][29][30]. It is worth noting that two abnormal Raman spectroscopy bands (2947 cm −1 and 3290 cm −1 ) in the spherical CaC 2 O 4 .2H 2 O, were the stretching vibrational peaks of C-H and -OH bonds, indicating that the spherical druse crystals may contain organic matrix [31]. ...
Background
Calcium oxalate (CaOx) is the most prevalent and widespread biomineral in plants and is involved in protective and/or defensive functions against abiotic stress factors. It is, however, expected that this function has an extremely significant contribution to growth processes in plants bearing large amounts of CaOx, such as cacti growing in desert environment.
Results
In our research, small-sized CaOx crystals (≤ 20 µm) with tetrahedral or spherical shapes were observed to dominate in each epidermal and cortical cell from the tubercles of Mammillaria schumannii, a species from the Cereoideae subfamily, having tubercles (main photosynthetic organs) united with adjacent ones almost into ridges on its stem. Because they have potential significant functions, differential centrifugations after mechanical blending were used to obtain these small-sized CaOx crystals, which extremely tend to adhere to tissue or suspend in solution. And then the combined Scanning Electron Microscope Energy Dispersive System (SEM–EDS) and Raman spectroscopy were further performed to demonstrate that the extracted crystals were mainly CaC2O4·2H2O. Interestingly, spherical druses had 2 obvious abnormal Raman spectroscopy peaks of -CH and -OH at 2947 and 3290 cm⁻¹, respectively, which may be attributed to the occluded organic matrix. The organic matrix was further extracted from spherical crystals, which could be polysaccharide, flavone, or lipid compounds on the basis of Raman spectroscopy bands at 2650, 2720, 2770, and 2958 cm⁻¹.
Conclusions
Here we used a highlightedly improved method to effectively isolate small-sized CaOx crystals dominating in the epidermal and cortical cells from tubercles of Mammillaria schumannii, which extremely tended to adhere plant tissues or suspend in isolation solution. And then we further clarified the organic matrix getting involved in the formation of CaOx crystals. This improved method for isolating and characterizing biomineral crystals can be helpful to understand how CaOx crystals in cacti function against harsh environments such as strong light, high and cold temperature, and aridity.
... Except for the purple pepper (cv.11), all the other sweet peppers also had red fluorescence below the cuticle layer. The cuticle layer acts as a transpiration barrier, sunscreen and first line of defense against pathogens in sweet pepper [24,25]. As noted previously, the blue fluorescence is thought to originate from ferulic acid in the exocarp [12], within in the cuticle layer [11]. ...
Sweet peppers are a popular vegetable with various surface colors, such as green, purple, red, or yellow. To characterize the unique fluorescence properties associated with a broad range of sweet peppers of various colors (14 varieties), a fluorescence spectrofluorometer and imaging were used. The results showed that all cultivars in the experiment had blue fluorescence emissions when excited with light in the UV-A region, while chlorophyll fluorescence could be observed in green peppers. The emitted blue fluorescence originated from the epidermis (cuticle layer). The color distribution of these sweet peppers in the a* and b* color space were compared to the image obtained under white LED light. Yellow and red pepper cultivars have thicker, multiple cuticular wax layers and more distinct maturity stages than other sweet pepper varieties observed. With the establishment of this basic fluorescence database, further applications of fluorescence-based techniques and the unification of evaluation methods for pepper quality will be more easily established.
... Raman imaging is a versatile nondestructive technique that can provide information on the chemical composition of a sample with high spatial resolution. Confocal Raman microscopy has been applied to plants, for example, to study the polysaccharide composition of the cell wall (Gierlinger et al., 2012;Chyli nska et al., 2014;Zeise et al., 2018), lignified tissues (Bock and Gierlinger, 2019), and more recently, cuticles (Littlejohn et al., 2015;Bock et al., 2021;Sasani et al., 2021). ...
... The special lenticel type "Klappenventile" (cap valve), found in some conifers (Neger & Kupka 1920) and in aerial roots of Philodendron (Neger 1922), might allow sealing of the periderm under the impact of drought stress and eventual bark shrinkage, since the layer of densely packed sclereids ("sclerophelloids") is without intercellular spaces (Fig. 3f). Combining cutting-edge techniques such as Raman spectroscopy (e.g., Gierlinger et al. 2012;Bock et al. 2021) and micro-CT (e.g., Earles et al. 2016;Cuneo et al. 2018;Crouvisier-Urion et al. 2019) will help to answer questions regarding the opening and closing mechanisms of lenticels. ...
Lenticels can be defined as pores that are the entrance of a continuous aeration system from the atmosphere via the living bark to the secondary xylem in the otherwise protective layers of the periderm. Most work on lenticels has had an anatomical focus but the structure-function relationships of lenticels still remain poorly understood. Gas exchange has been considered the main function of lenticels, analogous to the stomata in leaves. In this perspective review, we introduce novel ideas pertaining to lenticel functions beyond gas exchange. We review studies on lenticel structure, as this knowledge can give information about structure-function relationships. The number of species investigated to-date is low and we provide suggestions for staining techniques for easy categorization of lenticel types. In the follow-up sections we review and bring together new hypotheses on lenticel functioning in the daily “normal operation range”, including regulative mechanisms for gas exchange and crack prevention, the “stress operation range” comprising flooding, drought and recovery from drought and the “emergency operation range”, which includes infestation by insects and pathogens, wounding and bending. We conclude that the significance of dermal tissues and particularly of lenticels for tree survival has so far been overlooked. This review aims to establish a new research discipline called “ Phytodermatology ”, which will help to fill knowledge gaps regarding tree survival by linking quantitative and qualitative lenticel anatomy to tree hydraulics and biomechanics. A first step into this direction will be to screen more species from a great diversity of biomes for their lenticel structure.
... The sclerenchymatic fibers surrounding the vascular tissue had the highest lignin levels and potentially acting as a barrier and protection of the vascular tissue (Richter, Mussig, & Gierlinger, 2011). In spruce needles, Raman imaging was used to follow the transition of aromatic components (lignin, cinnamic acids, flavonoids) from the cuticle to the epidermal layer to get a better understanding of plant surfaces (Bock, Felhofer, Mayer, & Gierlinger, 2021;Sasani et al., 2021). ...
Spectral imaging technologies simultaneously record spectral and spatial information about plant tissues in a noninvasive way. Differences in techniques result in different selection rules and spatial resolutions. This article introduces the basic principles of Raman, Fourier-transform Infrared (FTIR) and autofluorescence imaging and finally compares their strength and drawbacks. The methods result in spectral datasets as bases for image generation. Spectral preprocessing together with univariate and multivariate data analysis approaches are essential for informative lignin imaging and analysis and therefore also briefly illustrated and discussed. Examples of imaging lignin and other aromatic components in a broad range of plant tissues show the potential as well as the limitations of microspectroscopic imaging.
Kalmia procumbens is a prostrate, alpine shrub growing above tree line. Owing to its growth form, periodic overheating risk is regular. Along with a gradual increase in mean air temperature, climate change is predicted to increase the threat imposed to plants, especially those that grow in endangered ecosystems. Elucidating plant leaf cuticle composition, along with its properties and thermal tolerance, is essential to predict species adaptation to environmental changes. In K. procumbens leaves, along with the cuticle, stomata are only located on the abaxial surface, surrounded by trichomes, while on the adaxial surface, we observed microscopic trichomes essentially made out of flavonoids. Thin (12-14 ⴗm) leaf sections were prepared using a cryo-microtome. Sections were placed on microscopy glass slides with a drop of distilled water, covered with microscopy coverslips, and sealed with nail polish. Linear polarized (0°) 785 nm and 532 nm lasers, were used for Raman imaging experiments. Based on the Raman spectra, chemical images were generated, and components visualized within the anatomical structures. Similar like in other leaf cuticles (P. abies, Arabidopsis thaliana), flavonoids and phenolic acids were located together with lipids in the different layers of the cuticle. In K. procumbens additionally triterpenoids were found in the cuticle as well as in the trichomes on the lower side, whereas in the adaxial surface tiny trichomes flavonoids dominated. The different chemical composition may reflect environmental adaptations to the ecosystem, particularly offering tolerance to freezing, drought and protecting against UV rays.
As a crop quality sensor, Raman spectroscopy has been consistently proposed as one of the most promising and non-destructive methods for qualitative and quantitative analysis of plant substances, because it can measure molecular structures in a short time without requiring pretreatment along with simple usage. The sensitivity of the Raman spectrum to target chemicals depends largely on the wavelength, intensity of the laser power, and exposure time. Especially for plant samples, it is very likely that the peak of the target material is covered by strong fluorescence effects. Therefore, methods using lasers with low energy causing less fluorescence, such as 785 nm or near-infrared, are vigorously discussed. Furthermore, advanced techniques for obtaining more sensitive and clear spectra, like surface-enhanced Raman spectroscopy, time-gated Raman spectroscopy or combination with thin-layer chromatography, are being investigated. Numerous interpretations of plant quality can be represented not only by the measurement conditions but also by the spectral analysis methods. Up to date, there have been attempted to optimize and generalize analysis methods. This review summarizes the state of the art of micro-Raman spectroscopy in crop quality assessment focusing on secondary metabolites, from in vitro to in vivo and even in situ, and suggests future research to achieve universal application.
