The complex genetic system of higher plant mitochondria could not be studied by transgenic approaches because conventional methods do not permit genetic transformation of these organelles. An alternative approach has been developed in the laboratory, thanks to the existence of a natural process of transfer RNA (tRNA) import from the cytosol into mitochondria. It was shown that a tRNA mimic can be used in vivo as a shuttle for importing into plant mitochondria passenger RNAsexpressed from nuclear transgenes. Taking a trans-cleaving ribozyme as a passenger sequence allowed to obtain the specific knockdown of a major messenger RNA (mRNA) in the mitochondria of transformed plant cells. We used this strategy to develop mitochondrial regulation studies. Five mitochondrial mRNAs (nad9, sdh3, cob, cox3 and atp9) were chosen as targets for specific transcleaving hammerhead ribozymes. After validating the in vitro activity of these ribozymes, the corresponding expression constructs served to transform Nicotiana tabacum cells, Arabidopsis thaliana plants (for nad9, cob, cox3 and atp9) and N. tabacum plants (for sdh3). Specific in vivo ribozyme-mediated knockdown of the targeted mitochondrial RNAs was established. The regulation response to the knockdown of the individual targets was analyzed at the whole transcriptome level.Whereas it has been generally considered so far that mitochondrial regulation processes in plants essentially occur at the post-transcriptional stage, our results strongly support mRNA coordination mechanisms within the organelles and between the organelles and the nucleus.
