Cloning, identification and expression analysis of ACC oxidase gene involved in ethylene production pathway Zohreh Jafari • Raheem Haddad • Ramin Hosseini • Ghasemali Garoosi Received: 8 April 2012 / Accepted: 8 October 2012 / Published online: 18 October 2012 Ó Springer Science+Business Media Dordrecht 2012 Abstract 1-aminocyclopropane-1-carboxylic acid oxi- dase (ACO) enzyme is a member of the Fe II-dependent family of oxidases/oxygenases which require Fe 2? as a cofactor, ascorbate as a cosubstrate and CO 2 as an activator. This enzyme catalyses the terminal step in the plant sig- naling of ethylene biosynthetic pathway. A 948 bp frag- ment of the ACO1 gene cDNA sequence was cloned from tomato (Lycopersicon esculentum) fruit tissues by using reverse transcriptase-polymerase chain reaction (RT-PCR) with two PCR primers designed according to the sequence of a tomato cDNA clone (X58273). The BLAST search showed a high level of similarity (77–98 %) between ACO1 and ACO genes of other plants. The calculated molecular mass and predicted isoelectric point of LeACO1 were 35.8 kDa and 5.13, respectively. The three-dimensional structure studies illustrated that the LeACO1 protein folds into a compact jelly-roll motif comprised of 8 a-helices, 12 b-strands and several long loops. The cosubstrate was located in a cofactor-binding pocket referred to as a 2-His- 1-carboxylate facial triad. Semi-quantitative RT-PCR analysis of gene expression revealed that the LeACO1 was expressed in fruit tissues at different ripening stages. Keywords ACC oxidase 1 (ACO1)  Active site  Gene expression  Lycopersicon esculentum  Motif Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid ACO ACC oxidase ACS ACC synthase SAM S-adenosylmethionine ORF Open reading frame RT-PCR Reverse transcription polymerase chain reaction Bp Base pair Introduction Ethylene, one of the most important phytohormones, involves in the various aspects of plant growth and development including flower and leaf senescence, sexual development, seed germination, fruit ripening and response to both biotic and abiotic stresses [1]. Ethylene biosynthetic pathway involves in the conversion of S-adenosylmethio- nine (AdoMet) to 1-aminocyclopropane-1-carboxylic acid (ACC) which is synthesized by ACC synthase (ACS) and then converts ACC to ethylene by ACO (1-aminocyclo- propane-1-carboxylic acid oxidase)[2]. ACO enzyme belongs to the family of oxidoreductases [3] which utilize Fe(II) as a cofactor and 2-oxoglutarate (2OG) as a cosub- strate [4], although ACO uses ascorbate as a cosubstrate [5]. ACO requires bicarbonate as an activator and catalyzes the oxidation of ACC to give ethylene, CO 2 , and HCN. It has been revealed that ACO forms a complex with Fe(II) and its active site contains a single Fe(II) matched with three residues [5]. In all isoforms and homolog proteins with ACO such as IPNS and ANS Fe(II)-binding motif and other motifs that are involved in binding the carboxylate of ACC, are well conserved [6]. ACO has been isolated and characterised as an expressed multigene family in many plant species such as white Z. Jafari  R. Haddad (&)  R. Hosseini  G. Garoosi Department of Agricultural Biotechnology, Imam Khomeini International University, P.O. Box 34149-288, Qazvin, Islamic Republic of Iran e-mail: raheemhaddad@yahoo.co.uk 123 Mol Biol Rep (2013) 40:1341–1350 DOI 10.1007/s11033-012-2178-7