Probing the Molecular Structure and Orientation of the Leaf Surface of Brassica oleracea L. by Polarization Modulation-Infrared Reflection-Absorption Spectroscopy

Abstract

The surface of most aerial plant organs is covered with the cuticle, a membrane consisting of a variety of organic compounds, including waxes, cutin (a polyester) and polysaccharides. The cuticle serves as the multifunctional interface between the plant and the environment, and plays a major role in protecting plants against various environmental stress factors. Characterization of the molecular arrangements in the intact cuticle is critical for the fundamental understanding of its physicochemical properties; however, this analysis remains technically challenging. Here, we describe the nondestructive characterization of the intact cuticle of Brassica oleracea L. leaves using polarization modulation-infrared (IR) reflection-absorption spectroscopy (PM-IRRAS). PM-IRRAS has a probing depth of less than several hundreds of nanometers, and reveals the crystalline structure of the wax covering the cuticle surface (epicuticular wax) and the nonhydrogen-bonding character of cutin. Combined analysis using attenuated total reflection-IR spectra suggested that hemicelluloses xylan and xyloglucan are present in the outer cuticle region close to the epicuticular wax, whereas pectins are dominant in the inner cuticle region (depth of ≤2 μm). PM-IRRAS can also determine the average orientation of the cuticular molecules, as indicated by the positive and negative spectral peaks. This unique advantage reveals the orientational order in the intact cuticle; the hydrocarbon chains of the epicuticular wax and cutin and the backbones of hemicelluloses are oriented perpendicular to the leaf surface. PM-IRRAS is a versatile, informative and easy-to-use technique for studying plant cuticles because it is nondestructive and does not require sample pretreatment and background measurements.

Foliar water and solute absorption: an update

“…Based on von Mohl's (1842on von Mohl's ( , 1847 hypotheses, the cuticle has been traditionally understood as a lipid-rich layer which is independent of the epidermal cell wall underneath. However, recent studies showed the presence of cell wall polysaccharides in the leaf cuticle of several species (Guzm an et al, 2014a,b;Hama et al, 2017Hama et al, , 2019 and also in tomato (Solanum lycopersicum) fruit cuticles (Segado et al, 2016(Segado et al, , 2020Philippe et al, 2020a). The cuticle may hence be interpreted as a specialised part of the primary cell wall, somehow analogous to a lignified secondary or a suberised cell wall (Niklas et al, 2017).…”

Sequestration of Exogenous Volatiles by Plant Cuticular Waxes as a Mechanism of Passive Associational Resistance: A Proof of Concept

“…Beyond doubt, the experimental conditions in this study were highly artificial, and several factors should be considered for an adequate interpretation of our observations. First, epicuticular waxes form complex crystal structures (Hama et al, 2019), in particular tubular crystal-like structures that consist mainly of nonacosan-derivatives. These tubular structures easily re-build from wax extracts (Jetter and Riederer, 1995), and nonacosane and triacontane were major compounds in the P. praecox wax preparations (Figure 2).…”

“…However, the outer surface of the above-ground part of the plants represents a major battleground for plant-microbe interactions (Junker and Tholl, 2013). These surfaces are covered by a matrix collectively designated as (epi)cuticular waxes (Buschhaus and Jetter, 2011): complex mixtures of hydrophobic compounds such as long-chain esters-compounds chemically considered as waxes (Bruice, 2006)-and other lipophilic compounds such as saturated aliphatic hydrocarbon chains of at least 20 carbons, pentacyclic triterpenoids, and phenylpropanoids (Vogg et al, 2004;Kunst and Samuels, 2009;Buschhaus and Jetter, 2011;Hama et al, 2019). Thus, due to the lipophilic nature of these epicuticular waxes, it has been proposed that endogenous VOCs can accumulate in the epicuticular wax layers of plants (Widhalm et al, 2015).…”

Genome-wide association study for maize leaf cuticular conductance identifies candidate genes involved in the regulation of cuticle development

“…Other polysaccharides, hemicelluloses such as xylan and xyloglucan, were found close to the surface of the cuticle, within the cuticle proper of several species (Guzmán et al . 2014; Hama et al . 2019).…”