Cytoplasmic reversions to fertility in cms-S maize have been correlated with the loss of freely-replicating S1 and S2 plasmid-like mitochondrial DNAs (plmtDNAs) (Laughnan, Gabay-Laughnan, and Carlson, 1981, Stadler Gen. Symp. 13: 93) and rearrangements of S1 and S2 sequences in high molecular weight mtDNA (Levings et al., 1980, Science 209:1021). These results were obtained with lines established from individual cytoplasmic revertants and propagated for two or more generations.

We have examined several tissues and developmental stages for the presence of S1 and S2 plmtDNAs during the expression of cytoplasmic reversion. Plants were identified in which revertant tassel sectors were large and included the ears within the subtending plant sector. From such sectored revertants, crude mitochondrial lysates were prepared using tissue of both sterile and fertile tassel from the same plant. The mitochondrial lysates were electrophoresed into agarose and the gels stained with ethidium bromide. S1 and S2 plmtDNAs were detected by fluorescence in samples from both sterile and revertant (fertile) tassel sectors, after electrophoresis. That is, cytoplasmically reverted tassel sectors still carried free S1 and S2 at relatively high copy number.

S1 and S2 plmtDNA levels were also assayed in seed from ears included within large cytoplasmic revertant sectors. These ears had been fertilized with pollen from maintainer plants, and seeds throughout the ear had been subsequently confirmed to carry fertile cytoplasm. Dry seeds were powdered, mitochondria extracted and crude lysates electrophoresed into agarose. S1 and S2 were visible by fluorescent staining in only 2 of 11 revertant seed samples. Remarkably, however, after blotting the gels to nitrocellulose and hybridizing with nick-translation labelled S2 probe, S2 plasmid was detected in all of the seed samples, albeit at various levels.

Whereas S1 and S2 plasmids in first generation revertant seed are contributed maternally, they were not found in male gametes of revertants. When mtDNA samples were assayed by Southern hybridization using an S2 probe, pollen from fertile cytoplasmic-revertant sectors showed no S2 hybrid band, just as with "N" pollen, while the S2 hybrid band was detected in mtDNA from cms-S nuclear restored pollen. This indicates a block in transmission of the S1 and S2 plmtDNAs through microsporogenesis in revertant tassels.

In the first and second generations of lines propagated from cytoplasmic reversion mutants, seedlings were assayed for S1 and S2 DNAs both by fluorescent staining and Southern hybridization. S1 and S2 were not detected in mitochondria of several assayed revertant lines following propagation.

It appears that a strict causal relationship cannot be drawn between the presence of autonomous S1 and S2 plmtDNAs and the cms-S phenotype in maize. The vegetative tassel tissues of cytoplasmic revertants -still carry the S1 and S2 plasmids at relatively high levels. Similarly, RI and R2 plmtDNAs have been observed in fertile South American maize races (Weissinger et al., 1982, PNAS USA 79:1). The basis for cytoplasmic reversions to fertility in cms-S maize is not a mutation affecting an all or none control over S1 and S2 plmtDNA levels. The continued presence of S1 and S2 in seed from revertant sectors suggests that, concomitant with the reversion event, replication of S1 and S2 ceases, but a finite number of cell divisions is required to dilute the remnant S1 and S2 levels. The stability of S1 and S2 after replication stops may be the result of covalently bound terminal proteins on these plasmids (Kemble and Thompson, 1983, in press). In microsporogenesis, blockage of transmission of non-replicating DNAs might explain the absence of S1 and S2 plasmids in pollen from revertant sectors. Further studies are to be attempted with isolated endosperm and embryo mtDNAs of cytoplasmic revertant seed to learn more about post reversion transmission of S1 and S2.

John E. Carlson,* Aubrey Miller, Susan Gabay-Laughnan, and John R. Laughnan

*JEC is currently at Kansas State University, Manhattan, Kansas

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