Large-scale histological analysis of leaf mutants using two simple leaf observation methods: identification of novel genetic pathways governing the size and shape of leaves

Observations of cellular organization are essential in understanding the mechanisms underlying leaf morphogenesis. These observations require several preparative steps, such as fixation and clearing of organs, and such procedures are time-consuming and labor-intensive for large-scale analyses. Thus, we have developed simple methods for the observation of leaf epidermal and mesophyll cells. To visualize the epidermis, a gel cast was made of the leaf surface, which was then observed under a light microscope. To visualize the leaf mesophyll cells, leaves were immersed in a solution containing Triton X-100, briefly centrifuged, and then viewed under a light microscope. These methods allowed us to conduct a histological phenome analysis for a large number of known and newly isolated leaf-shape/size mutants of Arabidopsis thaliana by measuring various parameters, including cell number, size, and distribution of cells within a leaf blade. Mutants showed changes in leaf size caused by specific increases or decreases in the number and/or size of cells. In addition, altered cell distributions in the leaf blade were observed, resulting from increases or decreases in the number of cells along the proximo-distal or medio-lateral axis, or recruitment of cells along a particular axis at the expense of other leaf parts. These results provide a phenomic view of the cellular behavior involved in organ size control and leaf-shape patterning.