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The ethylene biosynthesis pathway controls flower senescence. Previous studies have shown that Arabidopsis ETHYLENE-OVERPRODUCER1 (ETO1) interacts specifically with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases (ACSs), the rate-limiting enzymes of ethylene biosynthesis. The ethylene biosynthesis pathway controls flower senescence in petunias (Petunia hybrida Juss.). However, the role of ETO1-like genes (EOLs) during flower senescence has not been investigated. Here, two full-length petunia EOL cDNAs, PhEOL1 and PhEOL2, were isolated. RT-PCR assays indicated that the expression of PhEOL1 and PhEOL2 increased after exogenous ethylene treatment. The VIGS-mediated silencing of PhEOL1 accelerated flower senescence and produced more ethylene than the control condition, whereas the silencing of PhEOL2 did not. Notably, the effects caused by PhEOL1 suppression were not enhanced by PhEOL2 suppression in corollas. In addition, the expression of two petunia type 2 PhACS genes increased during flower senescence and after ethylene treatment. A yeast two-hybrid assay showed that PhEOL1 interacts with both PhACS2 and PhACS3. It is possible that PhEOL1 is involved in flower senescence by interacting with type 2 PhACSs in petunias.