Confocal laser scanning microscopy elucidation of the micromorphology of the leaf cuticle and analysis of its chemical composition

Abstract

Electron microscopy techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) have been invaluable tools for the study of the micromorphology of plant cuticles. However, for electron microscopy, the preparation techniques required may invariably introduce artefacts in cuticle preservation. Further, there are a limited number of methods available for quantifying the image data obtained through electron microscopy. Therefore, in this study, optical microscopy techniques were coupled with staining procedures and, along with SEM were used to qualitatively and quantitatively assess the ultrastructure of plant leaf cuticles. Leaf cryosections of Triticum aestivum (wheat), Zea mays (maize), and Lupinus angustifolius (lupin) were stained with either fat-soluble azo stain Sudan IV or fluorescent, diarylmethane Auramine O and were observed under confocal laser scanning microscope (CLSM). For all the plant species tested, the cuticle on the leaf surfaces could be clearly resolved in many cases into cuticular proper (CP), external cuticular layer (ECL), and internal cuticular layer (ICL). Novel image data analysis procedures for quantifying the epicuticular wax micromorphology were developed, and epicuticular waxes of L. angustifolius were described here for the first time. Together, application of a multifaceted approach involving the use of a range of techniques to study the plant cuticle has led to a better understanding of cuticular structure and provides new insights into leaf surface architecture.

Cuticular Waxes of Arabidopsis thaliana Shoots: Cell-Type-Specific Composition and Biosynthesis

“…The occurrence of very low amounts of compounds at the high end of the homolog distribution suggested that they are formed merely as by-products of the normal wax biosynthesis machinery rather than through dedicated processes specific to their chain lengths. Characterization of enzymes involved in wax precursor elongation in respective species may reveal whether, in these cases, single FAEs indeed form both the ubiquitous chain lengths and the C 35+ homologs [ 2 , 6 , 23 , 39 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 ].…”

Tristearin as a Model Cuticle for High-Throughput Screening of Agricultural Adjuvant Systems

“…The aim of the first part of this study was to determine whether tristearin is a suitable model cuticle in comparison with the extracted cuticle wax. , Ficus macrophylla was selected as the nominal source for the cuticle wax as the tree is evergreen, readily available in suitable quantities required for extraction, and the cuticle of other members of the moraceae family have been readily studied . The second aspect of this study was the use of tristearin films to rank the likely trends in efficacy of different adjuvant types (fatty acid ester and mineral oil-based adjuvants) and their major components.…”

Stocky1, a Novel Gene Involved in Maize Seedling Development and Cuticle Integrity

“…Sections of about 20–30 µm were collected and stained for 2 min in Calcofluor White M2R, 0.1%, in water, to facilitate the visualization of leaf structure. The sections were gently washed with distilled water, transferred onto microscope slides and post-stained with Auramine-O 0.1% in 50 mM Tris/HCL at pH 7.2, following the protocol from Nadiminti et al, 2015 [ 41 ]. After staining, sections were gently washed with distilled water and mounted with 30% glycerol, and the coverslips sealed with nail polish.…”