Structure and ethylene-induced expression of the 1-aminocyclopro-pane-1-carboxylate oxidase gene in mung bean ( Vigna radiate L.)

Previously we isolated two closely related 1-aminocyclopropane-i-carboxylate (ACC) oxidase cDNAs, pVR-ACOl, and pVR-ACO 2 , from mung bean seedlings. Ethylene caused a marked increase in the accumulation of VR-ACO1 transcript, whereas the ethylene-induction of VR-ACO 2 was much lower in the hypocotyl and root tissues. In the current study, genomic Southern blot analysis confirmed the existence of two ACC oxidase genes in the mung bean genome. To gain information concerning the structure of the ethylene-responsive VR-ACO1 gene as well as to investigate its promoter sequence, we also obtained and sequenced the genomic clone encoding VR-ACO1. The VR-ACO1 gene consists of four exons interrupted by three introns whose junctions are in agreement with the consensus intron/exon borders of plant genes. Individual sizes of the three introns are 100, 413, and 205 bp. The coding region of VR-ACO1 comprises 951 bp encoding a protein of 317 amino acid residues, with a predicted molecular mass of 36 kDa. The major start site of transcription is located 66 nucleotides upstream from the 5’-end of the coding region of the VR-ACO1 gene; the putative TATA box is found at the 27th nucleotide upstream from this start site. The nuclear run-on transcription assay indicated that ethylene induces the expression of VR-ACO1 at the transcriptional level in mung bean hypocotyls. Comparing the sequence of the 5’-flanking region of VR-ACO1 with known ethylene responsive ele-ments (EREs) revealed two copies of sequence (TTCAA) that displays a high degree of identity with the primary ethylene responsive element (PERE) found in the CST1, E4 , and ERF1 genes. In addition, the promoter contains three octanucleotide sequence motifs reminiscent of the EIN3 binding site that exists in Arabidopsis ERF1 and tobacco ERF2. We also discuss the possible molecular mechanism of regulation of ACC oxidase gene expression by ethylene in mung bean seedlings.