GUS Staining

Histochemical detection of GUS activity was performed with 5-bromo-4-chloro-3-indolyl b-d-GlcA (X-gluc) as the substrate. Plant tissues were first prefixed in ice-cold 90% (v/v) acetone for 20 min, then washed three times with GUS staining buffer (without X-gluc) before incubation in X-gluc solution [1 mm X-gluc, 50 mm NaPO4, pH 7, 1 mm K₃Fe(CN)₆, 1 mm K₄Fe(CN)₆, and 0.05% (w/v) Triton X-100] under a vacuum for 10 min at room temperature, then incubated overnight at 37°C. Chlorophyll was removed using several changes of 70% (v/v) ethanol and the tissues were photographed.