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.