Zymogram analysis of E1 enzyme activity in the leaves. Total soluble protein was extracted from the leaves of six independent T0 transgenic lines (#1 to #6) and wild type (WT) rice plants and separated by SDS-PAGE in gel containing 0.1% carboxymethyl cellulose. After electrophoretic separation (20 μg/lane), the gel was washed and E1 activity stained in situ with Congo Red. The size of the clear zone indicates the degree of activity. Note that no E1 activity was detected in transgenic #4 and wild type plants. Purified E1 protein was obtained by heating (78°C) the leaf soluble protein extract of T0 transgenic line #3-5 for 45 min, and purified E1 protein: (A) 1 μg; (B) 2 μg; (C) 3 μg.