Characterisation of cuticle mechanical properties: analysing stiffness in layered living systems to understand surface buckling patterns

Development of a living organism is a highly regulated process during which biological materials undergo constant change. <i>De novo</i> material synthesis and genetically-regulated changes in mechanical properties of materials are key for organ development. However, few studies have attempted to produce quantitative measurements of the mechanical properties of biological materials during growth. Such quantitative analysis is particularly challenging where the material is layered, and yet layering of materials with different mechanical properties may be essential to morphogenetic pattern formation. This is the case for the <i>Hibiscus trionum</i> flower petal, where buckling of the cuticle on top of the epidermal cell wall forms ridges, producing an iridescent effect. This ridge formation is hypothesised to be due to mechanical instability, which directly depends upon the mechanical properties of the individual layers of cuticle and cell wall. We set out to develop methods to measure the mechanical properties of the surface layers of plant epidermal cells through atomic force microscopy (AFM). To ensure that our results were reproducible and represented the most appropriate combination of experimental parameters, we used the uniaxial tensile tester for ultrathin films (TUTTUT) to provide independent measurement of cuticle stiffness. We explored mechanical properties of the upper cuticle and lower cuticular layer of the epidermal cell surface across growth stages. In addition to offering technical approaches to explore the stiffness of living layered materials, our findings suggest that temporal changes in biological material properties are key to understanding the development of biological surface patterns.