Alternative oxidase from mango (Mangifera indica, L.) is differentially regulated during fruit ripening

Alternative oxidase is a respiratory-chain component of higher plants and fungi that catalyzes cyanide-resistant oxygen consumption. The activity of a alternative oxidase has been detected during ripening in several climacteric fruit including mango (Mangifera indica L.). Synthetic oligonucleotides, corresponding to conserved regions of the Sauromatum guttatum and Arabidopsis thaliana nucleotide sequences, were used as primers for polymerase chain reaction to amplify genomic DNA extracted from mango leaves. The 623-bp fragment was found to encode an open reading frame of 207 amino acids showing high identity to the S. guttatum enzyme. Using this fragment to screen a ripe mango mesocarp cDNA library, one full-length cDNA clone, designated pAOMI.1, was obtained that contained an open reading frame encoding a polypeptide of 318 amino acids. The predicted amino-acid sequence exhibited 62, 64 and 68% identity to the S. guttatum, soybean, and A. thaliana enzymes respectively, indicating that this cDNA encodes a mango homologue of the alternative oxidase. Gel blot hybridization showed that pAOMI.1 is likely to be encoded by a single-copy gene. The 1.6 kb-transcript was induced during mango fruit ripening although the transcript was clearly detectable in unripe and developing fruit. Antibodies raised against the S. guttatum enzyme recognized three bands of approximately 27, approximately 33 and approximately 36 kDa from mitochondrial mango proteins. Two of the bands were detectable before ripening and increase in ripe fruit, the other band (27 kDa) was barely present in unripe fruit but accumulated during ripening. The clone pAOMI.1 was able to complement an Escherichia coli hemA mutant deficient in cytochrome-mediated aerobic respiration. This is the first report on the analysis of alternative oxidase at the molecular level during the ripening of a climacteric fruit.