ProACO4-GUS expression and RT-PCR analysis revealed that ACO4 is predominantly expressed in shoots of Arabidopsis seedlings under light conditions. ACO4-overexpressed mutant 35S-ACO4 produced more ethylene relative to the wild-type, which resulted in reduced growth of Arabidopsis seedlings. The abnormal growth of seedlings recurred after the application of Co2+ ions, suggesting that ACO4 is a functional ACO necessary to regulate the growth and development of Arabidopsis seedlings. Exogenously-applied brassinosteroids (BRs) inhibited the expression of ACO4, and an enhanced ACO4 expression was found in det2, a BR-deficient mutant. Additionally, expression of ACO4 was decreased in bzr1-D (a BZR1-dominant mutant), implying that BR signaling negatively regulates ACO4 expression via BZR1 in Arabidopsis. In the intergenic region of ACO4, four E-boxes and a BR regulatory element (BRRE) are found. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that BZR1 binds directly to the BRRE in the putative promoter region of ACO4. By binding of BZR1 to BRRE, less ethylene was produced, which seems to regulate the growth and development of Arabidopsis seedlings.
Keywords: ACC oxidase 4; Arabidopsis thaliana; BZR1; Brassinosteroids; ethylene production.