Waterproofing in Arabidopsis: Following Phenolics and Lipids In situ by Confocal Raman Microscopy

Abstract

Waterproofing of the aerial organs of plants imposed a big evolutionary step during the colonization of the terrestrial environment. The main plant polymers responsible of water repelling are lipids and lignin, which play also important roles in the protection against biotic/abiotic stresses, regulation of flux of gases and solutes, and mechanical stability against negative pressure, among others. While the lipids, non-polymerized cuticular waxes together with the polymerized cutin, protect the outer surface, lignin is confined to the secondary cell wall within mechanical important tissues. In the present work a micro cross-section of the stem of Arabidopsis thaliana was used to track in situ the distribution of these non-carbohydrate polymers by Confocal Raman Microscopy. Raman hyperspectral imaging gives a molecular fingerprint of the native waterproofing tissues and cells with diffraction limited spatial resolution (~300 nm) at relatively high speed and without any tedious sample preparation. Lipids and lignified tissues as well as their effect on water content was directly visualized by integrating the 1299, 1600, and 3400 cm−1 band, respectively. For detailed insights into compositional changes of these polymers vertex component analysis was performed on selected sample positions. Changes have been elucidated in the composition of lignin within the lignified tissues and between interfascicular fibers and xylem vessels. Hydrophobizing changes were revealed from the epidermal layer to the cuticle as well as a change in the aromatic composition within the cuticle of trichomes. To verify Raman signatures of different waterproofing polymers additionally Raman spectra of the cuticle and cutin monomer from tomato (Solanum lycopersicum) as well as aromatic model polymers (milled wood lignin and dehydrogenation polymer of coniferyl alcohol) and phenolic acids were acquired.

Synchrotron FTIR and Raman spectroscopy 1 ectroscopy provide unique spectral fingerprints for Arabidopsis floral stem vascular tissues

“…To further identify Raman shifts associated with different xylem cell types, a CART-based classification method was applied on the 1000-1800 cm -1 Raman shift range, which includes the predominant constituents of the xylem cell wall (Prats Mateu et al ., 2016; Özparpucu et al ., 2017). The CART model was built on the calibration dataset (80% of the total dataset), without a variable selection step, and the resulting classification tree shows that only four Raman shifts are sufficient to distinguish the three different cell wall types (Fig.…”

“…Based on the literature, the 1093 and 1372 cm -1 shifts are related to cellulose (Chylinska et al ., 2014; Özparpucu et al ., 2017), while the 1743 cm -1 is assigned to the υ(C=O) ester in pectins or hemicelluloses compounds (Chylinska et al ., 2014). The 1606 and 1618 cm -1 Raman shifts have been reported to describe lignin bands (Prats Mateu et al ., 2016). The performance of the model produced from VF spectra (for both calibration and validation datasets) was similar to the previous model.…”

“…In addition to IR microspectroscopy, Raman microspectroscopy is commonly used to study plant cell wall composition (Gierlinger et al ., 2012; Gierlinger, 2017) including that of xylem cell types (i.e. Arabidopsis thaliana (Prats Mateu et al ., 2016); Cucumis sativus (Zeise et al ., 2018); Malus sp. : (Horbens et al ., 2014); Populus sp .…”

“…Regarding the plant model Arabidopsis thaliana , only a few studies have investigated the compositions of cell walls in the floral stem (Schmidt et al ., 2010; Prats Mateu et al ., 2016) despite the important physiological role of this plant organ. The floral stem, which supports the flowers and the fruits, is also a major contributor to lifetime carbon gain (Earley et al ., 2009), representing 40% of a plant’s total biomass.…”

Topochemical Correlation between Carbohydrates and Lignin in Eucommia ulmoides Cell Wall from Tissue to Cell Level

“…In addition, sample isolation difficulties arise when small cell wall areas or single layers are of interest because they have to be carefully excised. Advances in confocal Raman microscopy and imaging have tackled this problem in a noninvasive way to provide chemical and structural information in situ with a high spatial resolution (< 0.5 μm) (Agarwal et al 2011;Richter et al 2011;Prats et al 2016). Both lignin and carbohydrates can be mapped simultaneously by selecting the Raman bands that are specific to these cell wall components.…”