Planta (1996)198:197-201 Planta 9 Springer-Verlag1996 The alternative oxidase is encoded in a multigene family in soybean James Whelan*, A. Harvey Millar, David A. Day Division of Biochemistry and Molecular Biology, Faculty of Science, and The Cooperative Research Centre for Plant Science, Australian National University, Canberra, ACT 0200, Australia. Received: 6 March 1995/Accepted: 12 May 1995 Abstract. The copy number of the alternative oxidase gene, Aox, was investigated in soybean (Glycine max L.) using a Polymerase chain reaction (PCR) approach to amplify fragments from soybean genomic DNA. The primers used were based on absolutely conserved regions of Aox cDNA clones from a variety of plant species and the yeast Hansenula anomala. After subcloning of the 170-bp PCR products, 12 individual colonies were se- quenced. Eleven plasmids yielded inserts representing three sequences in the ratio 4: 3: 4 (Aoxl-3). The sequence of Aoxl was 100% identical at the nucleic acid level to the published full-length eDNA from soybean. The other two sequences were 60-75% identical to Aoxl and to each other at the nucleic acid and protein levels. Similar analy- sis of Nicotiana tabacum L. revealed an additional gene copy with high homology to the soybean Aox2 sequence. Genomic DNA from soybean cut with Hind III and probed with the full-length Aoxl yielded a single positive band of 6.5 kb; when the same genomic blot was probed with a mixture of all three PCR fragments, three bands of 9 kb, 6.5 kb and 3 kb were detected. Reverse transcrip- tion-PCR performed on total RNA from various soybean tissues, followed by hybridisation with the three Aox se- quences individually, revealed differential expression of the Aox genes between cotyledons and leaves. It is sugges- ted that soybean contains a multigene Aox family. The implication of this for the understanding of alternative oxidase expression and regulation in plant tissues is discussed. Key words: Alternative oxidase - Gene family - Glycine - Mitochondrion *Present address: Department of Biochemistry, University of West- ern Australia, Nedlands WA 6009, Australia Abbreviations: AOX (Aox)= alternative oxidase protein (gene); bp = base pairs; PCR = polymerase chain reaction; RT-PCR = reverse transcription-PCR Correspondence to: D. A. Day; FAX: 61(6)2490313; E-mail: david.day@anu.edu.au Introduction The alternative oxidase is a cyanide-insensitive, hy- droxamic acid-sensitive terminal oxidase found in the mitochondria of a variety of organisms. The alternative oxidase branches from the cytochrome chain at the ubiquinone side of complex III and is non-energy conserv- ing. Therefore ATP is not synthesised when succinate or external NADH are used as sources of reductant and only one coupling site is used in the case of NAD-linked sub- strates (Moore and Siedow 1991). The degree to which this apparently energy-wasteful pathway is engaged in plants varies greatly: it can account for most of the respiration in thermogenic aroid spadices but for only a few percent of that in potato tubers (Moore and Siedow 1991). There have been several recent advances in our under- standing of the regulation of the alternative oxidase. Um- bach and Siedow (1993) showed that the oxidation state of the oxidase itself determined its capacity, with oxidation leading to inactivation. Millar et al. (1993) showed that pyruvate can activate the alternative oxidase several-fold in a substrate-independent manner and thus the level of engagement of the alternative oxidase may have been substantially underestimated in previous studies. Sub- sequently, it has been shown that activation with pyruvate allows the alternative oxidase to be active at low ubiquinol levels and thus it may compete for electrons with the cytochrome chain (Umbach et al. 1994; Hoef- nagel et al. 1995). This is in contrast to the previously held idea that the alternative oxidase could only operate as an overflow mechanism when the cytochrome chain was ef- fectively saturated (Laties 1982; Lambers 1985). The con- tribution of the alternative oxidase to plant respiration may, therefore, be more pronounced than previously thought. Synthesis of the alternative oxidase can be induced by a number of treatments in plants and fungi. For example, it is induced when the cytochrome chain is blocked by inhibitors and this is thought to be related to the genera- tion of superoxide (Mclntosh 1994). In addition to the characterised role in the flowering of thermogenic species, the alternative oxidase has been shown to be induced by