Quantitative GUS Activity Assay

GUS activity was assayed according to Jefferson et al. (1987) with some modifications. Briefly, cotyledon samples were ground in liquid nitrogen and subsequently homogenized in 4-methylumbelliferyl-β-d-glucuronide extraction buffer (approximately 150 mg tissue fresh weight mL^–1) composed of 50 mm HEPES-KOH (pH 7), 5 mm DTT, and 0.5% (w/v) polyvinylpyrrolidone. After centrifugation (13,000g, 20 min, and 4°C), 200-µL aliquots of the supernatant were mixed with 200 µL of GUS assay buffer composed of 50 mm HEPES-KOH (pH 7), 5 mm DTT, 10 mm EDTA, and 2 mm 4-methylumbelliferyl-β-d-glucuronide and incubated at 37°C for 30 min. Subsequently, aliquots of 100 µL were taken from each tube, the reactions were stopped with 2.9 mL of 0.2 m Na₂CO₃ (pH 9.5), and fluorescence was determined using a spectrofluorometer (LS55; Perkin Elmer) with 365-nm excitation and 460-nm emission wavelengths (5-nm bandwidth). Fluorescence was measured at the same instrument settings in all experiments.