GUS Staining, MUG Assay, and Microscopic Analysis

Transgenic plants harboring pIPT1-GUS were used for histochemical detection of GUS activity (Robatzek and Somssich, 2001). Plant tissues were infiltrated in GUS staining solution containing 50 mm NaPO₄, 0.5 mm K₃Fe(CN)₆, 0.5mM K₄Fe(CN)₆, 0.1% Triton X-100, 5 mm EDTA, and 1 mg mL⁻¹ X-Gluc, incubated at 37°C overnight, and destained several times in 70% ethanol and then photographed using a Leica M165C Stereo microscope (Leica Microsystems, http://www.leica-microsystems.com). Columbia-0, ∆761, ∆40-63, and ∆64-87 were grown on 1/2 Murashige and Skoog medium for 3 d, and 20 seedlings for each sample were harvested for quantitative measurement of GUS activity (4-methylumbelliferyl-beta-d-glucuronide, MUG assays) according to Hornitschek et al., 2012. Three transgenic lines for each construct were used for MUG assays, and the experiment was repeated three times. Reproductive organs with GFP or YFP reporter were observed using a Leica TCS SP5 confocal laser scanning microscope or Leica DM6000 epifluorescence microscope equipped with a GFP or YFP filter set. To evaluate GFP signal intensity, all samples were photographed with the same parameter settings. At least 20 pollen grains were used for statistical analysis of GFP signal, which was performed by ImageJ analysis software from the US National Institutes of Health (http://rsbweb.nih.gov/ij) according to Burgess et al. (2010).