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).