Ultrastructure of Plant Leaf Cuticles in relation to Sample Preparation as Observed by Transmission Electron Microscopy

Abstract

The leaf cuticular ultrastructure of some plant species has been examined by transmission electron microscopy (TEM) in only few studies. Attending to the different cuticle layers and inner structure, plant cuticles have been grouped into six general morphological types. With the aim of critically examining the effect of cuticle isolation and preparation for TEM analysis on cuticular ultrastructure, adaxial leaf cuticles of blue-gum eucalypt, grey poplar, and European pear were assessed, following a membrane science approach. The embedding and staining protocols affected the ultrastructure of the cuticles analysed. The solubility parameter, surface tension, and contact angles with water of pure Spurr's and LR-White resins were within a similar range. Differences were however estimated for resin : solvent mixtures, since Spurr's resin is combined with acetone and LR-White resin is mixed with ethanol. Given the composite hydrophilic and lipophilic nature of plant cuticles, the particular TEM tissue embedding and staining procedures employed may affect sample ultrastructure and the interpretation of the results in physicochemical and biological terms. It is concluded that tissue preparation procedures may be optimised to facilitate the observation of the micro- and nanostructure of cuticular layers and components with different degrees of polarity and hydrophobicity.

The presence of cutan limits the interpretation of cuticular chemistry and structure: Ficus elastica leaf as an example

“…The internal ultrastructure of F. elastica adaxial leaf cuticle has been described as having a faintly lamellate outer region and a mainly reticulate inner region (Holloway , Gouret et al , Kim ). The electron‐lucent lamellae of the inner region were not previously described for this species, but for the leaf cuticle of other species, either in the most external area (Viougeas et al , Guzmán et al ) or throughout such region (Wattendorff and Holloway , Riederer and Schönherr ).…”

“…As an example, the increased electron‐dense spots or ‘reticulum’ along the SED cuticle transversal section may be because of the presence of superficial material with higher polarity (e.g. polysaccharides and mineral elements) and consequently, with higher affinity for TEM aqueous stains (Guzmán et al ). Furthermore, the Si deposits of F. lyrata leaf cuticle had an intrinsic electron density (Davis ).…”

Cuticle Structure in Relation to Chemical Composition: Re-assessing the Prevailing Model

“…When analyzing the leaf cuticle of pear ( Pyrus communis ), Norris and Bukovac (1968) used the term cuticle to ‘include all the layers that can be separated from the underlying cellulose cell wall.’ However, many plant cuticles from different species and organs cannot be isolated from the underlying tissues as intact layers of significant size (e.g., Gouret et al, 1993 ; Fernández et al, 2014a ; Guzmán et al, 2014b ).…”

“…Nevertheless, the author highlighted the heterogeneity in plant cuticle structure and the need to consider each species individually to avoid oversimplifications and generalizations. Moreover, structural differences can be observed within the same organ, species and even within a cuticle section analyzed by TEM, hence making it risky to establish broad conclusions ( Figure 3C ; Guzmán et al, 2014b ). In this regard, the assignment of the cuticle of a number of species to one or other morphological type may vary, for example, according to the interpretation of the authors ( Jeffree, 2006 ), or to the sample preparation procedure used for TEM observation ( Guzmán et al, 2014b ).…”

Absorption and translocation of Zn foliar fertilisers

“…The waxes are composed of mostly mixtures of C20 -C40 n-alcohols, n-aldehydes, n-alkanes, and very long chain fatty acids. Phenolics are principally cinnamic acids and flavonoids, while polysaccharides in the cuticle are similar to those found in the plant cell wall, which mainly consist of cellulose, hemicellulose, or pectin (Caffall et al, 2009, Guzmán et al, 2014c. Based on their histochemical staining and presumed chemical composition, Yeats et al (2013a) classified cuticles into two domains from the internal to the external surface : (1) cuticular layer: a cutin-rich domain embedded with polysaccharides, intracuticular waxes (waxes deposited within the cutin matrix), and phenolics; and (2) cuticle proper: an overlying layer that is less abundant in polysaccharides, but enriched in intracuticular waxes and epicuticular waxes (waxes accumulated on its surface as epicuticular wax crystals, or films), with phenolics also embedded in cutin.…”