Organization and structure of the 1-aminocyclopropane-1-carboxylate oxidase gene family from Petunia hybrida

Abstract

In this paper we present the structural analysis of the 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family from Petunia hybrida. Southern blot analysis and restriction endonuclease mapping showed that two cloned regions of the petunia genome contained sequences highly homologous to a previously isolated ACC oxidase cDNA clone. Nucleotide sequencing of these two regions of the genome showed that each contained two tandemly arranged genes designated ACO1, ACO2, ACO3 and ACO4. Comparison of the nucleotide sequences of the cloned genomic regions with the cDNA clone pPHEFE indicated that ACO1 encoded the transcript in 4 exons interrupted by 3 introns. The other three members of the petunia ACC oxidase gene family shared identical intron numbers and positions with ACO1 and their exons were greater than 80% homologous. Nucleotide substitutions and deletions in the ACO2 gene indicate that it likely represents a pseudogene. Overall homology between ACO1 and ACO2 indicates that this gene cluster arose by a more recent duplication event than the gene duplication giving rise to the ACO3 and ACO4 cluster. The 5-flanking sequences share little overall homology between members of this gene family. However, sequences which likely make up the core promoter of these genes including the TATA box are highly homologous. RNA-based PCR amplification of ACC oxidase cDNAs from ethylene-treated corollas and wounded leaves revealed transcripts for ACO1, ACO3 and ACO4 indicating that a least three of these genes are transcriptionally active. The proteins encoded by ACO1, ACO3 and ACO4 share more than 90% identity with one another and more than 70% identity with ACC oxidases from other species. The ACC oxidase proteins share significant sequence homology with other enzymes that require Fe(II) and ascorbate for catalytic activity.