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Splicing of plant organellar group II introns is under accurate nuclear control that employs many nucleus-encoded protein cofactors from various families. For mitochondrial introns, only a few splicing factors have been characterized because disruption of their functions often causes embryo lethality. Here, we report the function of Empty Pericarp8 (Emp8) in the splicing of three group II introns in mitochondria, complex I biogenesis, and seed development in maize. Emp8 encodes a P subfamily pentatricopeptide repeat protein that localizes in mitochondria. The loss-of-function mutants of Emp8 are embryo lethal, showing severely arrested embryo and endosperm development in maize. The respiration rate in the emp8 mutants is reduced with substantially enhanced expression of alternative oxidases. Transcript analysis indicated that the trans-splicing of nad1 intron 4 and cis-splicing of nad4 intron 1 are abolished, and the cis-splicing of nad2 intron 1 is severely impaired in the emp8 mutants. These defects consequently lead to the disassembly of mitochondrial complex I and a dramatic reduction in its activity. Together, these results suggest that Emp8 is required for the trans-splicing of nad1 intron 4 and cis-splicing of nad4 intron 1 and nad2 intron 1, which is essential to mitochondrial complex I assembly and hence to embryogenesis and endosperm development in maize.

RNA editing, converting cytidines (C) to uridines (U) at specific sites in the transcripts of mitochondria and plastids, plays a critical role in organelle gene expression in land plants. Recently pentatricopeptide repeat (PPR) proteins were identified as site-specific recognition factors for RNA editing. In this study, we characterized an empty pericarp7 mutant (emp7) in Zea mays (maize), which confers an embryo-lethal phenotype. In emp7 mutants, mitochondrial functions are seriously perturbed, resulting in a strikingly reduced respiration rate. Emp7 encodes an E-subgroup PPR protein that is localized exclusively in the mitochondrion. Null mutation of Emp7 abolishes the C → U editing of ccmF(N) transcript solely at position 1553. CcmF(N) is coding for a subunit of heme lyase complex in the cytochrome c maturation pathway. The resulting Phe → Ser substitution in CcmF(N) leads to the loss of CcmF(N) protein and a strikingly reduced c-type cytochrome. Consequently, the mitochondrial cytochrome-linked respiratory chain is impaired as a result of the disassembly of complex III in the emp7 mutant. These results indicate that the PPR-E subgroup protein EMP7 is required for C → U editing of ccmF(N) -1553 at a position essential for cytochrome c maturation and mitochondrial oxidative phosphorylation, and hence is essential to embryo and endosperm development in maize.

The sfr6-1 mutant of Arabidopsis thaliana was identified previously on the basis of its failure to undergo acclimation to freezing temperatures following exposure to low positive temperatures. This failure is attributed to a defect in the pathway leading to cold on-regulated (COR) gene expression via CBF (C-box binding factor) transcription factors. We identified a region of chromosome 4 containing SFR6 by positional mapping. Fine mapping of the sfr6-1 mutation proved impossible as the locus resides very close to the centromere. Therefore, we screened 380 T-DNA lines with insertions in genes within the large region to which sfr6-1 mapped. This resulted in the identification of two further mutant alleles of SFR6 (sfr6-2 and sfr6-3); like the original sfr6-1 mutation, these disrupt freezing tolerance and COR gene expression. To determine the protein sequence, we cloned an SFR6 cDNA based on the predicted coding sequence, but this offered no indication as to the mechanism by which SFR6 acts. The SFR6 gene itself is not strongly regulated by cold, thus discounting regulation of SFR6 activity at the transcriptional level. We show that over-expression of CBF1 or CBF2 transcription factors, which constitutively activate COR genes in the wild-type, cannot do so in sfr6-1. We demonstrate that CBF protein accumulates to wild-type levels in response to cold in sfr6-1. These results indicate a role for the SFR6 protein in the CBF pathway -downstream of CBF translation. The fact that the SFR6 protein is targeted to the nucleus may suggest a direct role in modulating gene expression.