GUS histochemical staining and fluorometric assay

The activities of GUS in the Arabidopsis transgenic lines were determined using one-week-old seedlings, leaves from three-week-old plants, flowers, stems and siliques from five-week-old plants. Plant tissues were treated in 90% acetone for 30 min, washed with 50 mM sodium phosphate buffer (pH 7.2) for three times, and then incubated at 37°C overnight in a GUS staining solution (50 mM, pH 7.2), which consisted of 0.1% Triton X-100, 1 mM X-Gluc, 2 mM potassium ferrocyanide, 2 mM potassium ferricyanide and 10 mM ethylene diamine tetraacetic acid (EDTA, pH 8.0). The tissues stained were immersed in 70% ethanol for several hours to remove the chlorophyll before observing under a stereo fluorescence microscope (Discovery V12, Zeiss, Jena, Germany).

For quantitative fluorometric GUS assay, approximate 0.1 g of plant tissues were chilled in liquid nitrogen and ground in GUS extraction buffer (10 mM EDTA, pH 8.0, 50 mM sodium phosphate buffer, pH 7.0, 0.1% β-mercaptoethanol, 0.1% sodium dodecyl sulfonate (SDS), 0.1% Triton X-100 and 20% methanol). The extracts were centrifuged at 4000 ×g for 10 min to produce the supernatants for GUS assays. The protein concentration was determined as described previously [27]. The activity of GUS was determined using 4-methylumbelliferyl-β-D-glucuronide as the substrate following the method described by Jefferson [28]. The reaction was stopped by adding 200 mM Na₂CO₃. Fluorescence was quantified using a FluoroMax-4 fluorescence photometer (HORIBA Jobin Yvon, Paris, France) with the excitation and the emission filters at 365 nm and 455 nm, respectively.