Peeling back the layers: Raman imaging reveals microchemistry of tomato cuticle during development

The cuticle is a protective sheath that covers the aerial epidermal surface of terrestrial plants. It prevents dehydration, protects tissue from ultraviolet (UV) damage, and forms a first line of defense against pathogens. The cuticle is largely composed of cutin, a waxy, water-repellent substance, as well as associated polysaccharides and aromatic compounds (Yeats and Rose, 2013; Fich et al., 2016; Philippe et al., 2020). Much previous research concerning the biochemical composition of cuticles derives from destructive bulk studies that provide little or no spatial information. Thus, while we understand the bulk composition of the cuticle, there is a huge knowledge gap concerning the spatial distribution of its various components. 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; Chylińska 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). In work published in Plant Physiology, González Moreno et al. (2022) applied Raman imaging to the tomato (Solanum lycopersicum) fruit cuticle to illustrate changes in the microchemistry as the fruit develops. To get an overview of the chemical components, the authors scanned small regions of isolated cuticles from different developmental stages of the fruit (8–55 days post anthesis/flowering). For each growth stage, they determined an averaged Raman spectra stage that serves as a “molecular fingerprint” with bands unique to different chemical compounds. By comparing the intensity of key cuticle compounds in the spectra they found that cutin and phenolic acids co-occur early in cuticle development, while flavonoids increase sharply during ripening. Next, they visualized the spatial distribution of the prominent compounds of the cuticle. It is quite challenging to extract robust and interpretable information from Raman spectral data of a dynamic composite structure like the cuticle. To tackle this, the authors used the enrichment of compounds in the cuticle as the fruit develops to extract an almost “pure” component spectra for cutin, phenolic acids, and flavonoids. With this they were able to draw a chemical map of cuticle from select developmental stages (Figure 1A). Tomato fruit cuticle microchemistry visualized by Raman imaging. A, Image of an isolated tomato cuticle cross-section 40 d after anthesis/flowering (40 daa) with the pegs (raised areas) marked by an arrow, and the chemical map of cuticle components—phenolic acids (outer edge and centre of pegs in turquoise), cutin (blue), and flavonoids (along inner surface in yellow). B, Schematic models of the microchemistry of the tomato cuticle during development. Cutin is present across the entire cuticle width in all the stages of development (texture). (i) Early stage (8–12 daa), cuticle enriched in phenolic acids; (ii) Growing phase (15–35 daa), phenolic acids were only located in the outer region of the cuticle and centre of pegs; (iii) During ripening, phenolic acids preserved their initial location and extend throughout the entire cuticle width and flavonoids showed a gradient with higher deposition toward the inner side of the cuticle (stripes). Modified from González Moreno et al. (2022). What about polysaccharides in the cuticle? Polysaccharides make up about 25%–30% of the cuticle, but their fine distribution within the cuticle is unknown. The major polysaccharides cellulose, hemicellulose, and pectin are weak Raman scatterers and often get masked by stronger signals from lipids and aromatics. In the study by González Moreno et al., strong Raman bands for carbohydrates were not obvious given the presence of strongly scattering phenolics in the cuticle regardless of growth stage. However, using a mixture analysis that applies an iterative matching algorithm to a reference library, they were able to see the distribution of cellulose and pectin in the pegs (spiky raised areas inside the fruit) that occur at the younger stages and that become sites of flavonoid accumulation as the fruit develops. Based on these findings, González Moreno et al. present three stages in the development of the tomato fruit with distinct microchemistry in the cuticle (Figure 1B). In the early stages of fruit growth, phenolic acids accumulate on the outer-edge of the cuticle. As the fruit develops and expands in size, the accumulation of phenolic acids extends into the center of the pegs. With the onset of fruit ripening, flavonoids begin to accumulate on the inner-side of the cuticle and spread throughout with strongest accumulation in the areas complementary to phenolic acids when the fruit is ripe (Figure 1B). Cutin shows uniform distribution within the cuticle irrespective of growth stage. Interestingly, flavonoids start to accumulate in the areas where cellulose and pectins are present as the fruit ripens, supporting the idea that flavonoids act as fillers that restrict the molecular movement of cutin chains, which could potentially alter the mechanical property of the cuticle (Domínguez et al., 2012). This is further reinforced by the observation that cutin crosslinking increases by 20% in the fruit during ripening (Philippe et al., 2020). An interesting picture begins to emerge where phenolics and flavonoids form a link between cutin and the polysaccharide domain in the cuticle. It seems plausible that each organ customizes its cuticular composition to perform a specialized function by modulating its phenolic components. Advances in instrumentation, growing spectral databases, and improved algorithms make Raman imaging a valuable technique to fill the gaps and answer these questions. Particularly when combined with other complementary techniques, such as Raman-Atomic Force Microscopy (AFM), the chemical properties of the sample can be easily linked to the surface structure. The “pure” spectra of cuticle components are a valuable addition to the Raman spectral database. They enable the extension of the Raman imaging approach to study the cuticular structure of wild relatives of tomato to understand its evolution. The findings can be extended to other commercially valuable fruits to improve shelf-life in the market and improve protection against pathogens and UV-damage. Conflict of interest statement. The author has no conflicts of interest to declare.