Fluorometric GUS enzymatic assay
A fluorometric GUS enzymatic assay for measuring GUS activity in pepper plant extract was performed as described previously (RA, TA, & MW, 1987). Leaf samples used for GUS assay were harvested and then frozen with liquid nitrogen, and then were ground into fine powder using a pestle and mortar. The samples were lysed in extraction buffer (50 mM phosphate buffer, pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.1% sodium lauryl sarcosine, and 10 mM β-mercaptoethanol), and then collect the supernatant by centrifugation. Aliquots of the extracts (100 μl) were added to 1 ml of assay buffer (extraction buffer containing 1 mM MU), pre-warmed, and incubated at 37 ℃. After 0, 5, and 20 min of incubation, 100 μl samples were removed and placed in 1.9 ml of stop buffer (200 μM sodium carbonate). Fluorescence was measured using a Multi Detection Microplate Reader (Bio-TEK Synergy™ HT, Bad Friedrichshall, Germany). The total protein content in plant extracts was estimated by the Bradford method using BSA as a standard (MM, 1976).