Histochemical Assay for β-Glucuronidase Activity

β-Glucuronidase activity in the roots and shoots of ENA1 promoter::GUS transgenic plants was determined using a histochemical assay, as described previously (Inoue et al., 2003 (link); Nozoye et al., 2011 (link), 2015 (link)). Subcellular localization of ENA1 in onion epidermal cells or rice roots was observed as described previously (Nozoye et al., 2011 (link), 2015 (link)). FM-64 (1 mM; Molecular Probes) was used for staining the plasma membranes by incubation of rice roots section on glass slides at room temperature. Confocal images of rice roots were acquired with a laser scanning microscope (LSM Pascal and LSM 780, Zeiss, Germany) equipped with a 10×/0.45 M27 objective. Excitation/emission wavelengths of 488 nm/490–540 nm or ∼515 nm/640 nm were used for detection of GFP or FM4-64 fluorescence, respectively.