University of Missouri


University of Illinois

Abnormal growth phenotype correlated with a mitochondrial gene (cox2) deletion --K. J. 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. NCS plants arise most frequently in inbred or hybrid WF9 or WF9-related lines; therefore, nuclear genotype plays a role in the generation or selection of these mutations. The previously described NCS2 and NCS3 defective phenotypes are each correlated with a specific mitochondrial DNA rearrangement (Newton and Coe, PNAS 82:6879, 1986; Feiler and Newton EMBO J. 6:1535, 1987). Both NCS2 and NCS3 arose in plants with cms-T cytoplasm. We are analyzing several other NCS mutants that have been found in plants carrying cms-S and S-revertant cytoplasms. These new NCS mutants also have altered mitochondrial DNAs and they arose in the WF9 nuclear background. We have suggested that the NCS phenotypes are due to lesions involving essential mitochondrial genes and that NCS plants carry both mutant and normal mtDNAs (Newton and Coe, 1986). Somatic segregation of the mixed organelles would lead to sectors of defective and normal growth.

In support of our hypothesis, we have found that one of the new NCS mutants carries a deleted form of the cytochrome oxidase subunit II gene, in addition to reduced levels of the normal form. This NCS mutant was found among plants descended from a fertile revertant of the ML cytoplasm, a member of the cms-S group. The phenotype is typical of NCS in that maternally inherited variable stunting and striping is seen. However, the stripes are yellow-green, distinct from the pale-green stripes observed for NCS2 and the necrotic striations of NCS3 plants. The mtDNA from the revertant striped plants was similar to the mtDNA of revertant, nonstriped relatives, as assayed by restriction enzyme analysis. Use of several cloned gene probes led to the finding that the only consistent striped/nonstriped difference involved the cytochrome oxidase subunit II gene (cox2). The whole cloned pZmE1 probe (Fox and Leaver, Cell 26:315, 1981) hybridized to a 5.5kb XhoI fragment in mtDNAs from both sterile and fertile-revertant ML nonstriped plants. However, in mtDNAs from the striped, ML-fertile plants, the amount of 5.5kb XhoI hybridizing fragment was reduced and an additional 8.3kb restriction fragment hybridized strongly. The maize cox2 gene consists of two exons separated by a 794bp intron (Fox and Leaver, 1981). We prepared probes that were specific for (a) exon1, (b) the intron or (c) exon2 plus 3' flanking regions. Hybridization of the 2nd exon probe (c) showed the same pattern of hybridization as did the whole cox2 probe. However, the intron probe hybridized much less strongly to the 8.3kb XhoI fragment and the exon1-specific probe did not hybridize at all to the 8.3kb restriction fragment. The 5'-flanking region, the first exon and a portion of the intron of the cox2 gene have apparently been deleted from this NCS mutant mtDNA. Cloning and detailed restriction mapping of the relevant clones suggest that the molecular origin of this mutation was a recombination event followed by a deletion. The two "parental" restriction fragments apparently lack long regions of homology to one another; however, the presence of very small repeats, such as the one seen at the site of an NCS3 rearrangement, has not yet been eliminated.

Transcripts corresponding to the cox2 gene are detected at reduced levels in the plants carrying the cox2 partially deleted gene. They are apparently specifically reduced because transcripts for cytochrome oxidase subunits I and III, as well as ATPase subunit 9, appear to be present at relatively normal levels. This finding of a quantitative reduction, rather than of aberrant transcripts, suggests that the partially deleted cox2 gene is not transcriptionally active. The cox2 transcripts in the striped plants would derive from the normal gene which is present in reduced amounts.

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