4 methylumbelliferyl glucuronide

GUS staining was carried out as described by Jefferson with some modifications [48] (link). Various tissues from transgenic Arabidopsis were immersed in X-Gluc solution (1 mg/ml X-Gluc, 100 mM sodium phosphate buffer [pH 7.0], 10 mM EDTA, 1 mM potassium ferricyanide, 1 mM potassium ferrocyanide, 1% TritonX-100, 100 µg/mL chloramphenicol, 20% methanol) and incubated for 16–24 h at 37°C, and then immersed in 70% ethanol for 3 to 4 times.
For fluorometric measurement of GUS activity, plants were ground to a fine powder and then suspended in 1 ml GUS extraction buffer (50 mM sodium phosphate, pH 7.0; 0.1% Triton X-100; 10 mM β-mercaptoethanol; 10 mM EDTA and 0.1% sarcosyl (v/v)). The supernatant, after being centrifuged at 12,000 g for 20 minute at 4°C, was assayed for GUS activity with 4-methyl umbelliferyl glucuronide (Sigma) substrate using an F-4500 fluorescence spectrophotometer at the excitation/emission wavelengths of 365/455 nm. The protein concentrations were quantified according to Gallagher and the GUS enzyme activity was expressed as nmols of 4-methylumbelliferone produced per mg protein per minute [49] . In this study, all materials were collected from three individual transgenic lines for GUS staining and measurement of GUS activity.

To assay GUS activity, Arabidopsis leaves were harvested and stored at -80°C before use. Frozen leaves were ground in extraction buffer (50 mM pH 7 sodium phosphate, 10 mM EDTA, 0.1 Sarkosyl, 0.1 M Triton X-100, and 10 mM β-mercaptoethanol). The homogenate was centrifuged at 12,000 × g for 15 min (4°C), then the supernatant was used for GUS activity assays. GUS activity was assayed by using 10 μL extract and 4-methyl-umbelliferyl-glucuronide (Sigma) as a substrate. The protein concentration of the extracts was determined utilizing bovine serum albumin as a standard protein according to the assay described by Bradford (1976) (link). Fluorescence was measured in a fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The excitation wavelength was 365 nm and the emission wavelength 455 nm. Each assay was repeated three times. The data presented were collected from at least three independent experiments.