Figure 4.

H₂O₂ Is Required for ABA-Induced Inhibition of PP45.

(A) and (B) Effects of pretreatments with DMTU and DPI on the expression of PP45 (A) and on the activity of PP45 (B) in rice leaves exposed to ABA. The rice seedlings were either untreated (Control) or pretreated with 5 mM DMTU or 100 μM DPI for 4 h and then exposed to 100 μM ABA for 30 min (A) or 90 min (B).

(C) and (D) The expression of PP45 (C) and the activity of PP45 (D) in rbohB/E mutant. The rice seedings were treated with 100 μM ABA for 90 min, and the relative expression levels of PP45 and the activity of PP45 were analyzed by real-time quantitative PCR and by Ser/Thr phosphatase assay, respectively.

(E) H₂O₂ directly inhibits the activity of PP45 in vitro. The in vitro-expressed PP45-His protein was incubated with various concentrations of H₂O₂. The graph shown the activity of PP45 as measured by the Ser/Thr phosphatase assay. The protein level of OPP45 after treatment was determined by IB analysis (bottom). The phosphatase activity of PP45 without H₂O₂ treatment was set to 100%.

(F) The inactivation of PP45 by H₂O₂ is reversed by TCEP and DTT. Recombinant PP45 protein was incubated with 0.1 mM H₂O₂ for 30 min, followed by different concentrations of the reducing agents TCEP or DTT for another 30 min. The activity of PP45 was measured by the Ser/Thr phosphatase assay. All experiments were repeated at least three times with similar results. Values are means ± sem of three independent experiments. Means denoted by the same letter did not significantly differ at P < 0.05 according to Duncan’s multiple range test.