We address here organellar genetic regulation and intercompartment genome coordination. We developed earlier a strategy relying on a tRNA-like shuttle to mediate import of nuclear transgene-encoded custom RNAs into mitochondria in plants. In the present work, we used this strategy to drive trans-cleaving hammerhead ribozymes into the organelles, to knock down specific mitochondrial RNAs and analyze the regulatory impact. In a similar approach, the tRNA mimic was used to import into mitochondria in Arabidopsis thaliana the orf77, an RNA associated with cytoplasmic male sterility in maize and possessing sequence identities with the atp9 mitochondrial RNA. In both cases, inducible expression of the transgenes allowed to characterise early regulation and signaling responses triggered by these respective manipulations of the organellar transcriptome. The results imply that the mitochondrial transcriptome is tightly controlled by a “buffering” mechanism at the early and intermediate stages of plant development, a control that is released at later stages. On the other hand, high throughput analyses showed that knocking down a specific mitochondrial mRNA triggered a retrograde signaling and an anterograde nuclear transcriptome response involving a series of transcription factor genes and small RNAs. Our results strongly support transcriptome coordination mechanisms within the organelles and between the organelles and the nucleus.

The maintenance of the plant organelle genomes involves factors that are mostly inherited from their bacterial symbiotic ancestors. In bacteria, a major player in genome maintenance is a 5'-3' exonuclease/flap-endonuclease required for multiple replication and repair functions. This activity is given by the 5'-3' exonuclease domain of DNA Polymerase I. Plant organellar DNA polymerases POL1A and POL1B are derived from DNA Pol I, but lack this domain. In Arabidopsis, proteins similar to this missing domain were identified, named OEX1 and OEX2 (Organellar Exonucleases 1 and 2), targeted to mitochondria and chloroplasts, respectively. An oex1 mutant shows severe developmental and fertility defects, which aggravate in subsequent generations. These defects correlate with mitochondrial genome (mtDNA) instability, resulting in differential segregation of subgenomes generated by recombination, suggesting that OEX1 processes replication and/or recombination intermediates whose accumulation compromises genome stability. OEX1 is expressed in two isoforms via alternative splicing, which can differentially interact with POL1A and POL1B and variably affect mtDNA repair. Recombinant OEX1 digests double-strand DNA in a 5'-3' direction and has flapendonuclease activity, a key function in replication and repair processes. In addition, OEX1 has a high affinity for RNA:DNA hybrid substrates, rapidly degrading RNA in Okazaki-like structures and in R-loops. Consistent with a role in suppressing R-loops, oex1 mutant plants accumulate R-loops in highly transcribed regions of the mtDNA. Overall, our results support that OEX1 fulfills multiple important roles in the processes required to maintain mtDNA stability.