Assignment of new cDNA for maize chloroplastic CuZn-superoxide dismutase(SOD-1 and structural characterization of sod-1 genes

A cDNA encoding chloroplastic CuZn-SOD (SOD-1 from maize revealed an unusual deduced amino acid sequence (Kernodle and Scandalios 2001 compared to those in other plants. To elucidate whether a typical type of chloroplastic CuZn-SOD is also present in addition to this unique SOD, we purified and characterized SOD-1, and cloned its cDNA and genomic genes. The purified SOD-1 from maize leaves was a homodimer of 32 kDa, and exhibited an N-terminal amino acid sequence characteristic of the chloroplastic type of CuZn-SOD. A SOD-1 cDNA of 890 bp was obtained by combination of RT-PCR and RACE. Although the cDNA showed incomplete ORF, the deduced amino acid sequence contained a partial chloroplast transit peptide and a complete sequence of mature protein (154 residues that was identical to the N-terminal sequence of the purified protein up to the 35th residue. The amino acid sequence of mature SOD-1 was 95% identical to that of rice chloroplastic CuZn-SOD. Thus, it is clear that the previously reported“SOD-1 cDNA”from maize is not the cDNA responsible for SOD-1. Genomic SOD-1 gene (sod-1 was obtained by long PCR for the central region and gene-walking method for the 5'-upstream and 3'-downstream regions, and was sequenced for 3.9kbp. The sod-1 gene was allelic, termed sod-1a and sod-1b, and contained 8 exons and 7 introns, which is the same as the chloroplastic CuZn-SOD gene in rice. The Cu-responsive element (TNNNGCTG and a sequence similar to the target sequence of Arabidopsis thaliana microRNA 398 that regulates CuZn-SOD gene expression in response to Cu availability were observed upstream near the TATA box in the promoter region and in the 4th exon of maize sod-1, respectively.