Localization of PP2C19 proteins and effects of light treatments. (a) Confocal fluorescence microscopy images of transgenic pp2c19-1 plants harboring the PP2C19pro::VENUS-PP2C19. Two-day-old etiolated seedlings were stimulated with BL at 0.17 µmol m–2 s–1, and VENUS fluorescence signals were observed with a microscope at the indicated times after the onset of BL irradiation. Scale bars = 20 μm. (b) Immunoblot analysis of PP2C19 proteins in the wild-type (Col-0) seedlings before and after phototropic stimulation. Two-day-old etiolated seedlings were preirradiated with overhead RL irradiation for 2 min at 20 μmol m−2 s−1 2 h before BL irradiation and then irradiated with unilateral BL at 0.17 µmol m–2 s–1 for 3 h. Seedlings were collected before the RL pretreatment (sample D), before the BL irradiation (sample RL), and after the BL irradiation for 3 h (sample BL), as shown in an upper panel. Soluble proteins (soluble) and crude microsomal proteins (microsome) were prepared from collected seedlings, and proteins (2 μg) in each fraction were separated on 6% SDS-PAGE gels, followed by immunoblotting with anti-PP2C19 antibody. The PVDF membranes were stained using a Pierce reversible protein staining kit as a loading control.
