*4.10. Ultra-structure and Flow Cytometry Analysis*

The ultramorphology of the leaf was investigated according to our previous study [82]. The H2O2 was detected according to the method of Sheteiwy et al. [82]. Briefly, the roots were stained with 5 μM dichlorodihydrofluorescein diacetate for 15 min, and then washed with excess 20 mM sodium phosphate buffer (pH 6.1) to stop the reaction. The changes in ΔΨm were analyzed using a tetramethylrhodamine methyl ester assay kit (Immunochemistry Technologies, Bloomington, IN, USA) and imaged using a laser confocal scan microscope (Zeiss LSM 780, Zeiss, Germany). Then, nuclear isolation was performed according to the method of Hu et al. [99]. The root samples were cut into small pieces and then fixed with nucleus isolation buffer [10 mM MgSO4, 50 mM KCl, 5 mM Hepes, 1 mg/mL dithiothreitol (Sigma, St. Louis, MI, USA) and 0.2% Triton X-100] and filtered through a 33 mm nylon mesh. The nuclei were fixed in 4% paraformaldehyde for 30 min and were then precipitated (200 g, 10 min, 4u C) and re-suspended in the isolation buffer.
