Plant Cell, 7issueand OrganCulture 42: 121-127, 1995. 121 © 1995 KluwerAcademicPublishers. Printedin the Netherlands. Mutagenesis and selection for oligomycin resistance in soybean (Glycine max L. Merr) suspension culture cells Elizabeth A. Grabau, Regina Hanlon & Adam Pesce Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0331, Virginia, USA Received21 September1994;accepted in revisedform15March1995 Key words: ATPase, cross-resistance, mutagenesis, oligomycin, RNA editing Abstract Soybean suspension culture cells were subjected to mutagenesis with ethyl methane sulfonate and cells resistant to the ATP synthase inhibitor oligomycin were recovered. Suspension cultures showed high levels of resistance over a period of 17 months after mutagenesis. The level of resistance to oligomycin decreased during prolonged growth in the absence of the selection agent. Although mitochondrial inheritance of the resistance phenotype was not tested, this observation is consistent with segregation of resistant and sensitive mitochondria in progeny cells. Cross resistance to venturicidin was also observed which suggested the possible involvement of the mitochondrial ATP synthase subunit 9 gene, however, sequences of ATP synthase subunit 9 cDNA clones from resistant cells were identical to the wild type sequence. In addition, Southern blot analyses of DNA from wild type and resistant cultures did not identify rearrangements. These results indicated that resistance to oligomycincould not be directly attributed to a mutation in the primary gene sequence, an alteration in the RNA editing pattern of transcripts, or gross rearrangements in the region containing and directly adjacent to the ATP synthase subunit 9 gene in the mitochondrial DNA from resistant cultures. Introduction The antibiotic oligomycin binds to the membrane por- tion of the mitochondrial ATP synthase complex (F0- portion) to inhibit proton translocation. Oligomycin resistance in Saccharomyces cerevisiae resulted from mutations in the mitochondrially-encoded genes for the ATP synthase subunit 6 (atp6) or subunit 9 (atp9). Mutations in the yeast atp6 gene were primarily non- sense or frameshift mutations, suggesting that atp6 can accommodate amino acid substitutions at many locations without affecting the functional characteris- tics of the enzyme (John et al. 1986). Many of the oligomycin resistant mutants in the yeast atp9 gene were nucleotide substitutions in the coding region resulting in amino acid alterations (Ooi et al. 1985a). A subset of oligomycinresistant atp9 mutants in yeast also showed cross resistance to the antibiotic ventu- ricidin, another ATP synthase inhibitor (Ooi et al. 1985b). Mutant studies have allowed speculation about the folding and orientation of the ATP synthase subunit 9 polypeptide (ATP9) in the mitochondrial membrane and possible contact sites of ATP9 with ATP syn- thase inhibitors (Ooi et al. 1985b). Mitochondrially- inherited oligomycin resistant mutants in mammalian cells are due to a nucleotide substitution in the atp6 gene and do not exhibit cross resistance to venturi- cidin (Breen et al. 1986). In mammals and Neurospora crassa the functional ATP9 polypeptide is encoded by a nuclear gene and imported into the mitochondrion (Nagley, 1988). Resistance to oligomycin in higher plants has been demonstrated in Nicotiana sylvestris cultures (Aviv & Galun 1988; Durand 1987; Durand & Harada 1989). Studies with cytoplasmic hybrids indicated that resistance is inherited with cytoplasmic organelles, specifically the mitochondria (Aviv & Galun 1988; Durand & Harada 1989; Perl et al. 1991). Oligomycin resistance has been studied as a possible selectable marker for mitochondrial transformation studies. Both