Quantifying Heterochromatin Dynamics in Plant Roots

Stack images of the roots (1 µm section) were acquired using a ×40 oil objective with a NIKON A1R + confocal microscope. Tile-scanning (4 × 1 tiles) was used to ensure the imaging of the whole meristem and the elongation zone of the roots. The Z Project tool (Projection type: Sum slices) was used to sum the fluorescence intensity of the pixels corresponding to each nucleus present in the epidermal layer. The background fluorescence was subtracted for each color channel and the fluorescent intensity of chromocenters and nuclei was measured as the integrity density of a determined ROI. In all cases, independent measurements were taken for each color channel. Data acquisition and processing were done using FIJI. The Relative Heterochromatin Fraction (RHF) was calculated using the formula RHF = [Ac × (Ʃ Ic − Ib)] / [An × (In − Ib)]. Ac: the total chromocenters area; Ic: fluorescence intensity of chromocenter; Ib: fluorescence intensity of the background; An: area of the nucleus; In: fluorescence intensity of the nucleus.