RESULTS
Isolation, sequence analysis, and localization of mitochondrial ATP synthase β-subunit
The mitochondrial ATP synthase β- subunit (ATPB) is encoded by three genes (AT5G08670, AT5G08680, AT5G08690 ) with highly similar amino acid sequences (98% sequence identity). The mature protein consists of 556 amino acids and has a molecular mass of 59’630 Da and an isoelectric point of 6.53. Multiple sequence alignment analysis and the phylogenetic tree show that mitochondrial and chloroplast ATPB genes cluster in two branches (Figure 1a). We further examined the expression of these genes and found that AT5G08680 is a pseudogene that is not expressed. Thus, in this study, we only focus on theAT5G08670 and AT5G08690 genes. They are spaced approximately 4,400 bp apart, and their precursor proteins consist of a total of 566 amino acids (Figure 1b, c). The location and presequence size of these proteins were determined using several online protein-peptide prediction programs which predicted a mitochondrial presequence with a cleavage site localized specifically between amino acids numbers 51 and 52 (Figure S1). A comparison of the precursor proteins for AT5G08690 (NP 568204) and AT5G08670 (NP 568203) revealed that there are only two residues that differ between the two proteins (Figure 1c) within the presequence. To analyze the expression patterns of AT5G08670 and AT5G08690 , we generated transgenic plants expressingAT5G08670 p:β -glucuronidase (GUS) andAT5G08690 p:GUS. These two lines showed a similar spatiotemporal expression pattern of AT5G08670 and AT5G08690 in the early developmental stages of seedlings. However, at the later developmental stages, AT5G08670 expression declined, and by 21-days, it was no longer expressed (Figure 1d). These findings indicate that these two proteins are highly similar although their spatiotemporal expression patterns are different, which might contribute to functional differences in subsequent developmental stages.