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Polyclonal and monoclonal antibodies that recognize the 35-, 36-, and 37-kDa alternative oxidase proteins of Sauromatum guttatum (Schott) were used to isolate a cDNA clone, pAOSG81, from an S. guttatum cDNA expression library. A fusion protein with an apparent molecular mass of 48 kDa was expressed from a pUell9 derivative of pAOSG81 (pAOSG81-119) in Escherichia coli cells and was recognized by the monoclonal antibodies. When the in vitro translated and immunoprecipitated products made from mRNA hybridselected by pAOSG81 were analyzed, a single band corresponding to a protein with an apparent molecular mass of42 kDa was observed. DNA sequence characterization showed that pAOSG81 contains the entire coding region of a protein with a calculated molecular mass of 38.9 kDa, a putative 63-amino acid transit peptide, and a 9-amino acid match to the authentic N-terminal sequence of the 36-kDa alternative oxidase protein.Analyses of the deduced amino acid sequence indicate: (i) that the transit peptide is predicted to form amphiphilic helices, and (ii) that three regions of the processed protein are likely to form transmembrane a-helices. We conclude from these data that pAOSG81 represents a nuclear gene, aoxl, encoding a precursor protein of one or more of the alternative oxidase proteins of S. guttatum.

Chemical inhibition of the mitochondrial electron transport chain (mtETC) by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA) causes increased expression of nucleus-encoded alternative oxidase (AOX) genes in plants. In order to better understand the mechanisms of this mitochondrial retrograde regulation (MRR) of gene expression, constructs containing deleted and mutated versions of a promoter region of the Arabidopsis thaliana AOX1a gene (AtAOX1a) controlling expression of the coding region of the enhanced firefly luciferase gene were employed to identify regions of the AtAOX1a promoter important for induction in response to mtETC or TCA cycle inhibition. Transient transformation coupled with in vitro and in vivo assays as well as plants containing transgenes with truncated promoter regions were used to identify a 93 base pair portion of the promoter, termed the MRR region, that was necessary for induction. Further mutational analyses showed that most of the 93 bp MRR region is important for both AA and MFA induction. Sub-regions within the MRR region that are especially important for strong induction by both AA or MFA were identified. Specific mutations in a W-box and Dof motifs in the MRR region indicate that these specific motifs are not important for induction. Recent evidence suggests that MRR of AOX genes following inhibition of the mtETC is via a separate signaling pathway from MRR resulting from metabolic shifts, such as those that result from MFA treatment. Our data suggest that these signaling pathways share regulatory regions in the AtAOX1a promoter. Arabidopsis proteins interacted specifically with a probe containing the MRR region, as shown by electrophoretic mobility shift assays and Southwestern blotting. These interactions were eliminated under reducing conditions.