Expression of the R1-homologous region in mtDNA of cms-S sterile, restored and cytoplasmically revertant plants

--Gracia Zabala, Susan Gabay-Laughnan and John R. Laughnan

The molecular lesion responsible for the S-type of cytoplasmic male sterility (cms-S) in maize appears to involve sequences derived from mitochondrial episomes. All cms-S cytoplasmic revertants analyzed show mitochondrial DNA (mtDNA) rearrangements involving S1-, S2- and R1-homologous sequences. The type of rearrangement depends on the nuclear background. However, all cytoplasmic revertants analyzed to date contain rearrangements at the termini of the mitochondrial genome linearized by recombination between S episome terminal inverted repeats (IRs) and IR-homologous sequences present in the main mitochondrial genome. The termini of these linear molecules that rearrange contain a sequence, R, homologous to the unique end of the R1 episome present in RU cytoplasm. Plants with RU cytoplasm are fully fertile, however.

We have analyzed the expression of this R-homologous sequence in mitochondria of cytoplasmic revertants that arose in different nuclear backgrounds and their respective sterile progenitors. The complex transcriptional profile of the R region changes upon reversion in the seven revertants analyzed, representing four nuclear backgrounds. Although the changes in transcriptional profile vary among the different nuclear backgrounds, all seven revertants lack a 1.39kb transcript present in all cms-S mitochondria.

The nuclear restorer gene Rf3 restores fertility to cms-S plants. Our laboratory has identified newly arisen Rf genes, not allelic to Rf3, that also restore fertility to cms-S plants. We have examined the expression of the R sequence in cms-S lines with and without a nuclear Rf gene in two different nuclear backgrounds, WF9 and B37. The Rf3 gene affects expression from the R region while two of the new restorers do not. The Rf3 gene reduces the steady-state abundance of the transcripts and alters the transcriptional profile of the R region. The 1.39kb transcript is among those transcripts of which the steady-state abundance is reduced. This effect is more pronounced in Rf3 Rf3 homozygotes than in Rf3 rf3 heterozygotes although transcription is not eliminated entirely. Even though the newly arisen restorer genes do not affect the R transcriptional profile, we can not rule out the possibility that they may regulate the expression of these sequences at the post-transcriptional level.

We have analyzed mitochondrial RNA from strains containing RU cytoplasm and have determined that the R1 episome, that has homology to the R sequence of cms-S, is not transcribed in the RU cytoplasm in a manner similar to the R sequence in cms-S.

Normal (N) cytoplasm mitochondria carry the R sequence (R1 homology) integrated next to one copy of the 5.27kb repeat. Houchins et al. (EMBO J. 5:27-2788, 1986) have reported a 2.85kb transcript hybridizing to a 4.13kb clone containing the R sequence in normal cytoplasm. This transcript is thought to arise within R with transcription proceeding towards and into the 5.27kb repeat. We do not detect this or any other transcript with our R probe. It is possible that transcription in cms-S takes place in the region of the R sequence not adjacent to the 5.27kb repeat in N. These results suggest that the transcriptional pattern we have observed for the R region in cms-S mitochondria is specific to the particular arrangement of the R sequence in the cms-S genome and not to the R sequence per se.

We now have three compelling results supporting the involvement of mtDNA regions harboring the R sequence in the cms-S defect. These regions undergo rearrangements in all cytoplasmic revertants analyzed, regardless of nuclear background. The transcriptional profile of the R region changes upon reversion in all revertants analyzed. The Rf3 nuclear restorer gene reduces and alters the transcriptional activity of the R region in both nuclear backgrounds studied.

We will extend our analysis of gene expression of the R region into the developmental stage when pollen dysfunction first becomes evident. Such a developmental focus is essential since cms-S restoration is gametophytic, not sporophytic as in cms-T and cms-C.

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