RNA editing and mutagenesis of the soybean (Glycine max) mitochondrial atp9 gene

A plethora of information has been amassed in attempts to understand the basic principles governing living systems. Since the birth of the modern scientific method, many compelling theories have been devised as scientists attempt to understand the mysteries of life. The endo-symbiont theory for the biogenesis of mitochondria and chloroplasts is one such theory that has recently gained increased in acceptance with the maturity of molecular and biochemical techniques. These two organelles are unique in that they contain DNA which codes for some of the proteins involved in organellar function. Maternal inheritance of some traits such as cytoplasmic male sterility (cms) in plants can be linked to mitochondrial DNA. However, further understanding of mitochondrial gene expression, regulation and DNA rearrangements has been hindered by the lack of a transformation system for these organelles. A longterm goal in the field is to develop a transformation and expression system for soybean mitochondria.

Despite recent transformations of yeast mitochondria, no successful transformations of higher plant mitochondria have been reported to date. Mitochondrial transformation occurs at low frequencies, and therefore requires a method of selection, i.e. a means by which to differentiate cells containing a transformed mitochondrion from the background of largely untransformed cells. The work presented in this paper is the foundation for the future development of a selectable marker for plant mitochondrial transformation. The antibiotic oligomycin is an inhibitor of the mitochondrial ATP synthase complex, and therefore has potential for the isolation of a marker gene. Two approaches, soybean tissue culture mutagenesis and site-directed mutagenesis of the soybean [Glycine max (L.) Merr.] atp9 gene, have been explored in the hopes of isolating an oligomycin-resistant marker. In yeast (Saccharomyces cerevisiae), atp9 mutants have been shown to contain nucleotide mutations resulting in single amino acid substitutions that confer resistance to oligomycin. Sites for mutagenesis of the soybean atp9 gene were chosen based on DNA sequence homology between the yeast and soybean genes. Before site-directed mutagenesis, atp9 cDNA clones were cloned and sequenced, revealing RNA editing sites.

Among these cDNA clones, we have also sequenced atp9 from an oligomycin-resistant soybean suspension culture for possible mutations in the gene. Suspension cultures were mutagenized with EMS and selected by growth inhibition with oligomycin. Inhibition curves showed an increased tolerance for oligomycin for EMS-treated cultures compared to wild-type.

For future testing of the mutagenized atp9 gene we have constructed a particle inflow gun to introduce the gene(s) of interest into the nucleus of soybean suspension culture cells. A mitochondrial target sequence from β-atp has been fused in frame to the atp9 gene to direct the mutagenized gene product, now the product of a nuclear gene, correctly into the mitochodrion.