Cloning of an Additional cDNA for the Alternative Oxidase in Tobacco

Abstract

The alternative oxidase is a cyanide-insensitive terminal oxidase found in a variety of organisms although it is best characterized in plants. It branches from the Cyt chain at the level of ubiquinone and does not pump protons and is thus non-energy-conserving. In thermogenic floral appendages it plays a clear role in the volatilization of compounds to attract insects for pollination, but its role in nonthermogenic plants is unclear (Moore and Siedow, 1991). Induction of the alternative oxidase at the gene level has been characterized in a number of studies with compounds that inhibit the Cyt chain. Additionally, aging of potato slices, treatment with ethylene in fruits and storage tissue, cold treatment in tobacco (Nicofiana fobacum) and wheat, and salicylic acid treatment of Sauromatum gut ta tum have a11 been shown to induce the alternative oxidase (Day et al., 1995). At the biochemical level allosteric stimulation by pyruvate and the oxidation reduction state of the protein have been shown to be important determinants of activity (Millar et al., 1993; Umbach and Siedow, 1993). We have isolated and sequenced a cDNA clone from tobacco for the alternative oxidase. The predicted protein shows high identity with alternative oxidase from other species (Table I). However, when it was compared with the recently published sequence from tobacco (Vanlerberghe and McIntosh 1994), it showed significant differences, having 93 and 95% identity at the nucleic acid and protein levels, respectively (Vanlerberghe and McIntosh, 1994). The gene sequenced in this study is 209 bp shorter at the 5‘ end compared to that of Vanlerberghe and McIntosh (1994); additionally, there are 47 base pair differences in the open reading frame, which translate into 14 differences in amino acids. At the 3’ end there are 13 base pairs different between the two clones. The differences between these two tobacco clones may be accounted for by varietal differences between the two studies, since we used cv SRl versus Bright Yellow, which was used by Vanlerberghe and McIntosh (1994). Alternatively, the differences in sequence may indicate a second gene for the alternative oxidase in tobacco. This possibility is supported by the following: (a) N. tabacum