Pavement Cell-Size and -Shape Analysis.

The area and lobe number of pavement cells of mixed developmental stages were measured as follows. Confocal microscopy raw images from four different third leaves were preprocessed in order to enhance cell-wall signal using the following ImageJ functions: subtract background and enhance contrast. Images were segmented using CellSeT (66). Stomata were then filled with background. Area was measured for each cell using the analyze particles function in ImageJ. Lobe number was estimated automatically using the skeletonize function and the plugin analyze skeleton. Cell-area values were log-transformed to reduce skewness and statistics was performed using the Wilcoxon test in R software. Experiments were always repeated three times in three independent biological replicates, which showed the same patterns. Only the data from one representative experiment are presented in the figures, and n values in the figure legends represent the total number of cells analyzed in that one particular experiment.