Spr2 encodes a mitochondrion-localized PPR protein. A, Transgenic construct for overexpressing Spr2. LB: left border; RB: right border; 3 × HA: triple HA tag. B, Analysis of transcript levels of Spr2 in spr2 mutants by RT–PCR analysis using primer Spr2-F3 and Spr2-R3. RNAs were normalized against maize Actin gene (GRMZM2G126010). C, Analysis of protein abundance of SPR2 in overexpression lines (Spr2-OE) and complemented lines (Spr2-Com) by western blotting analysis. Maize ACTIN protein was used as a loading control. D, Subcellular localization of SPR2. Red fluorescence signals are from mitochondrial marker MitoTracker. Scale bar, 20 μm. E, Immunoblotting localization of SPR2 in total protein (TP), nuclear protein (Nu), mitochondrial protein (Mito), and chloroplast protein (Chl) extracts obtained from overexpression lines Spr2-OE1 probed with antibodies against H3 (Histone H3, nuclei marker), D2 (chloroplastic marker), Cox2 (mitochondrial marker), and HA.