A simple method for the isolation of leaf epidermis from graminaceous species for studying stomatal physiology

Brachypodium distachyon has been gaining traction as the model annual species for economically important temperate grasses such as wheat and barley. Methods for tissue culture and genetic transformation have been developed for this model grass as a resource for functional genomics and reverse genetics. More recently, B. distachyon has been used to elucidate the underlying mechanisms regulating stomatal development in monocotyledons. However, there is a paucity of methods for facilitating the use of B. distachyon for studying stomatal physiology. We developed a simple and easy method for the isolation of intact leaf epidermis from B. distachyon with minimal mesophyll contamination. Our results showed that stomatal guard cells remain viable in the isolated leaf epidermis and that stomata can open in the presence of potassium chloride and light, and that this opening response can be inhibited by abscisic acid in a dose-dependent manner. Additionally, we also showed that the method we have developed can be used for isolating leaf epidermis from a range of graminaceous species with minimal mesophyll contamination, including Triticum aestivum, T. macha, T. uratu, and T. aethiopicum, Hordeum vulgare, Avena sativa, and Aegilops tauschii.

Automated summary

Brachypodium distachyon has been established as a model plant for studying stomatal development and physiology, and a new method for isolating leaf epidermis from this plant and other grass species with minimal mesophyll contamination has been developed. Researchers have demonstrated that stomatal guard cells in the isolated leaf epidermis remain viable, and that stomatal opening and closing responses can be induced and inhibited by specific treatments.