Stomatal Trait Analysis Protocol

Twelve stomatal traits were analysed as described by Liu et al. [26 (link)], Mak et al. [37 (link)] and O’Carrigan et al. [36 (link)]. The parameters were aperture length (AL), aperture width (AW), aperture width/length (AWL), stomatal pore area (SA), guard cell length (GCL), guard cell width (GCW), guard cell volume (GCV), subsidiary cell length (SCL), subsidiary cell width (SCW), subsidiary cell volume (SCV), stomatal density (SD) and stomatal index (SI). For these measurements, the third fully expanded leaves were collected from the glasshouse and placed on tissue paper soaked in a stabilising solution (50 mM KCl, 5 mM Na⁺-MES, pH 6.1) in Petri dishes. Abaxial epidermal strips were then peeled and mounted on slides using a measuring solution (10 mM KCl, 5 mM Ca²⁺-MES, pH 6.1). Quick peeling and mounting was important to ensure stomatal images were true representations of the stomata found naturally on the whole plant in the glasshouse. Stomatal imaging was conducted using a CCD camera (NIS-F1 Nikon, Tokyo, Japan) attached to a microscope (Leica Microsystems AG, Solms, Germany). All images were analysed using Nikon NIS Element imaging software (Nikon, Tokyo, Japan) and measured with Image J software (NIH, USA).