Figure 5.

IQM1 directly promotes the enzyme activity of CAT2. A, DAB staining images of leaves from 4-week-old wild-type and iqm1-1 mutant plants. B, Relative DAB staining intensity detected by Photoshop software. C, Catalase activity of 4-week-old wild-type and iqm1-1 mutant plants after 3 d of inoculation with B. cinerea. The catalase activity is presented as units per mg of protein. D, In vitro assays of catalase activity. Ten micrograms of purified His-IQM1 or His-CaM5 protein was added to 10 µg of His-CAT2. The mixture was incubated at 4°C overnight, and then CAT2 enzyme activity was tested. All experiments were repeated at least three times. The data are means ± sd of three biological replicates. Asterisks represent significant differences (Student’s t test, **, P < 0.01). E, Pull-down assay confirming that CAT2 interacts with CaM5 in a Ca²⁺-dependent manner. Immobilized GST-CAT2 was incubated with purified His-CaM5 in the presence of 1 mm Ca²⁺ or 5 mm EGTA (a Ca²⁺ chelator) and eluted for immunoblot analysis with anti-His antibody (top) and anti-GST antibody (bottom).