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

## TL;DR

The paper explores how mechanical properties of layered plant cell surfaces change during growth, influencing surface patterns like iridescent ridges.

## Contribution

The study introduces methods to measure stiffness in living layered biological materials using AFM and TUTTUT.

## Key findings

- Temporal changes in cuticle stiffness correlate with surface pattern formation.
- Layered cuticle properties are critical for mechanical instability and ridge formation.
- AFM and TUTTUT provide reproducible measurements of plant epidermal stiffness.

## Abstract

Development of a living organism is a highly regulated process during which biological materials undergo constant change. De novo 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 Hibiscus trionum 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.

Development of a living organism is a highly regulated process during which biological materials undergo constant change.