Two Pentatricopeptide Repeat Proteins Are Required for the Splicing ofnad5Introns in Maize

Mitochondrial genes in flowering plants contain predominantly group II introns that require precise splicing before translation into functional proteins. Splicing of these introns is facilitated by various nucleus-encoded splicing factors. Due to lethality of mutants, functions of many splicing factors have not been revealed. Here, we report the function of two P-type PPR proteins PPR101 and PPR231, and their role in maize seed development. PPR101 and PPR231 are targeted to mitochondria. Null mutation ofPPR101andPPR231arrests embryo and endosperm development, generatingempty pericarpandsmall kernelphenotype, respectively, in maize. Loss-of-function in PPR101 abolishes the splicing ofnad5intron 2, and reduces the splicing ofnad5intron 1. Loss-of-function in PPR231 reduces the splicing ofnad5introns 1, 2, 3 andnad2intron 3. The absence of Nad5 protein eliminates assembly of complex I, and activates the expression of alternative oxidase AOX2. These results indicate that both PPR101 and PPR231 are required for mitochondrialnad5introns 1 and 2 splicing, while PPR231 is also required fornad5intron 3 andnad2intron 3. Both genes are essential to complex I assembly, mitochondrial function, and maize seed development. This work reveals that the splicing of a single intron involves multiple PPRs.