Leaf epidermal profiling as a phenotyping tool for DNA methylation mutants

Phenotypic evaluation of epigenetic mutants is mainly based on the analysis of plant growth and morphological features. However, there are cellular level changes that are not visible to the naked eye and require analysis with higher resolution techniques. In this study, we carried out a phenotypic characterisation of several Arabidopsis thaliana hypomethylation mutants by quantitative image analysis combined with flow cytometry. This phenotyping approach permitted identification of abnormalities at the cellular level in mutants with wild-type morphology at the organ level. Morphometry of adaxial leaf epidermis revealed variations in the size and number of pavement cells, and the density and distribution of stomata in the analysed second rosette leaves from the mutants studied. A direct correlation between DNA ploidy status and leaf pavement cell size in wild type and mutant leaves was observed. Recognition of hidden phenotypic variations could facilitate the identification of key genetic loci underlying the phenotypes caused by modifications of DNA methylation. Thus, this study outlines an easy and fast phenotyping strategy that can be used as a reliable tool for characterisation of epigenetic mutants at the cellular level.