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University of Illinois

A second mitochondrial cox2 mutation associated with an NCS phenotype --M. Lauer, K. Newton, C. Knudsen, S. Gabay-Laughnan and J. Laughnan
The maternally inherited nonchromosomal stripe (NCS) mutants are characterized by variable poor growth, abnormal morphologies and leaf striping. We have suggested that the NCS phenotypes are due to lesions involving essential mitochondrial genes, that NCS plants carry both mutant and "normal" mtDNAs and that somatic segregation of the mixed organelles leads to sectors of defective and normal growth (Newton and Coe, PNAS 83:7363). Last year we reported that NCS5 plants carried a partially deleted version of the mitochondrial cytochrome oxidase subunit 2 gene. We have now found that a second NCS mutant, NCS6, which has a phenotype indistinguishable from that of NCS5, also carries a partially deleted cox2 gene. The cox2 rearrangement in NCS6 occurs at a site (within the first exon) different from that in NCS5 (within the intron)

The NCS6 mutant was found among plants descended from a fertile revertant of the RD (=R) cytoplasm, a member of the cms-S group. Like NCS5, the NCS6 leaf and plant stripes are yellow, and clonal sectors with aborted kernels are seen on ears from striped plants.

Mitochondrial DNA was isolated from NCS6 striped and from non-striped control plants (RD-revertant relatives). DNA blots were hybridized with several cloned gene probes. The only consistent striped/nonstriped difference involved the cytochrome oxidase subunit 2 gene (cox2). A 2.4 kb EcoRI fragment carrying the whole cox2 gene (pZmE1; Fox and Leaver, Cell 26:315) hybridized to a 5.5 kb XhoI fragment in mtDNAs from control, nonstriped plants. However, in mtDNAs from the NCS6 plants, the amount of 5.5 kb XhoI hybridizing fragment was reduced and an additional 9.2 kb restriction fragment hybridized strongly. By restriction endonuclease analyses, we determined that the restriction map of a 9.2 kb XhoI mutant fragment diverged from that of the 5.5 kb fragment within the first exon of the cox2 gene. The proximal third of the first exon and the 5' flanking region of the cox2 gene is not carried on the 9.2 kb XhoI fragment. A 2.65 kb XhoI/BamHI restriction fragment from the 9.2 kb XhoI insert, outside of the region of homology with the cox2 gene, was used as a probe onto DNA blots. This probe identified the 9.2 kb XhoI fragment in NCS6 mtDNA and an additional 4.6 kb XhoI band in both NCS6 and control DNA.

A comparison of the restriction maps of the three cloned XhoI fragments suggested that the molecular origin of this mutation was a recombination event. The two "parental" (4.6 kb and 5.5 kb) restriction fragments do not show detectable homology to each other under stringent hybridization conditions. However, subsequent sequencing of portions of the three clones showed that they all contain the same 31 base-pair sequence at the site of the rearrangement.

The partially deleted cox2 gene in NCS6 plants does not appear to be expressed. Transcripts corresponding to the cox2 gene are specifically reduced in NCS6 plants. The cox2 protein is also present at reduced levels in mitochondrial preparations from NCS6 plants. This was shown by Western blot analyses using antibodies directed to a petunia cox2 peptide sequence (Nivison and Hanson, The Plant Cell 1:1121).

Studies with NCS mutations suggest that they result from homologous recombination occurring between small repeats. The sizes of the repeats vary: 6 bp in NCS5 (Newton et al., The Plant Cell, in press, 1990), 12 bp in NCS3 (Hunt and Newton, MNL 63:70, 1989) and 31 bp in NCS6. The repeats associated with different NCS mutations do not appear to be related to one another.

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