Circular and linear mitochondrial genomes in cytoplasmic male sterile maize [abstract]

Abstract only availableCytoplasmic male sterility (CMS) is a maternally inherited condition in which a plant has an inability to produce viable pollen. It is usually due to the production of a toxic chimeric protein within the mitochondria during the maturation of pollen grains. In maize (Zea mays), there are three types of CMS: CMS-T, CMS-C and CMS-S. The S-type of cytoplasmic male sterility (CMS-S) in maize is associated with the expression of a rearranged mitochondrial DNA region. This CMS-S-specific region includes two co-transcribed chimeric open reading frames, orf355 and orf77. The nuclear restorer-of-fertility gene, Rf3, cleaves all transcripts containing both orfs, including the CMS-S-specific linear 1.6 kb mRNA; this results in male fertility. The Lancaster Surecrop-derived inbred line A619 carries a different and weaker restorer called Rf9. Fertility restoration by Rf3 and Rf9 was compared for their effects upon the CMS-associated region of mitochondrial DNA. Unlike Rf3, Rf9 affects the organization of the CMS-S-specific region. It appears to do this by affecting recombination between linear "S" plasmids and the CMS-S-specific region of the main mitochondrial genome, which produces a linear end from which transcripts for the 1.6 kb mRNA are initiated. By reducing the amount of recombination, Rf9 reduces the amount of linear template available for transcribing the S-associated 1.6 kb RNA. A reduction in this transcript is associated with an increase in pollen survival. We have studied the effects of the two restorer-of-fertility genes from several different inbred lines on the amounts of integrated and linearized orf355/orf77 genes within CMS-S mtDNA.MU Monsanto Undergraduate Research Fellowshi

Unique Changes in Mitochondrial Genomes Associated with Reversions of S-Type Cytoplasmic Male Sterility in Maizemar

Cytoplasmic male sterility (CMS) in plants is usually associated with the expression of specific chimeric regions within rearranged mitochondrial genomes. Maize CMS-S plants express high amounts of a 1.6-kb mitochondrial RNA during microspore maturation, which is associated with the observed pollen abortion. This transcript carries two chimeric open reading frames, orf355 and orf77, both unique to CMS-S. CMS-S mitochondria also contain free linear DNA plasmids bearing terminal inverted repeats (TIRs). These TIRs recombine with TIR-homologous sequences that precede orf355/orf77 within the main mitochondrial genome to produce linear ends. Transcription of the 1.6-kb RNA is initiated from a promoter within the TIRs only when they are at linear ends. Reversions of CMS-S to fertility occur in certain nuclear backgrounds and are usually associated with loss of the S plasmids and/or the sterility-associated region. We describe an unusual set of independently recovered revertants from a single maternal lineage that retain both the S plasmids and an intact orf355/orf77 region but which do not produce the 1.6-kb RNA. A 7.3-kb inversion resulting from illegitmate recombination between 14-bp microrepeats has separated the genomic TIR sequences from the CMS-associated region. Although RNAs containing orf355/orf77 can still be detected in the revertants, they are not highly expressed during pollen development and they are no longer initiated from the TIR promoter at a protein-stabilized linear end. They appear instead to be co-transcribed with cytochrome oxidase subunit 2. The 7.3-kb inversion was not detected in CMS-S or in other fertile revertants. Therefore, this inversion appears to be a de novo mutation that has continued to sort out within a single maternal lineage, giving rise to fertile progeny in successive generations

Characteristics of cms-S reveversion to male fertility in maize : (cytoplasmic male sterility, male fertility restoration S-type cytoplasm, mitochondrial DNA)

The association of cytoplasmic reversion of cms-S male-sterile strains to male fertility with disappearance of the S1 and S2 mtDNA plasmids as discrete molecules has been established for all 23 cytoplasmic revertant strains that have been studied so far. This correspondence between mutational step and molecular event provides the first unequivocal evidence that the genetic determination of cms-S male-sterility; male-fertility expression is located in mtDNA. When cytoplasmic reversion of cms-S strains to the male-fertile condition occurs, at least some, perhaps all, of the S1 and S2 plasmid sequences are transposed and integrated into the main high molecular weight mitochondrial DNA. These findings are consistent with the model proposed earlier for cytoplasmic reversion that connected it with "fixation" at the cytoplasmic level of an episomal fertility element carried by reversion-prone cms-S strains. Whether a corresponding transposition and integration of S1 and/or S2 mtDNA sequences into nuclear chromosomes is involved in the nuclear reversions, as called for by the model, is uncertain. If such a correlation were established it would provide the first example in higher plants of inter-organellar transposition of a naturally occurring genetic element. The S1 and S2 mtDNA plasmids, like the IS elements of bacterial transposons, have terminal inverted repeat sequences that probably equip them for integration into high molecular weight genomes. The possibilities for use of S1 and S2, and other mtDNA plasmids that have been identified in maize, as vehicles in interorganellar gene transfer are discussed briefly.JOHN R. LAUGHNAN, SUSAN GABAY-LAUGHNAN and JOHN E. CARLSON, Department of Genetics and Development, University of Illinois Urbana, IL

Restorer-of-Fertility Mutations Recovered in Transposon-Active Lines of S Male-Sterile Maize

Mitochondria execute key pathways of central metabolism and serve as cellular sensing and signaling entities, functions that depend upon interactions between mitochondrial and nuclear genetic systems. This is exemplified in cytoplasmic male sterility type S (CMS-S) of Zea mays, where novel mitochondrial open reading frames are associated with a pollen collapse phenotype, but nuclear restorer-of-fertility (restorer) mutations rescue pollen function. To better understand these genetic interactions, we screened Activator-Dissociation (Ac-Ds), Enhancer/Suppressor-mutator (En/Spm), and Mutator (Mu) transposon-active CMS-S stocks to recover new restorer mutants. The frequency of restorer mutations increased in transposon-active stocks compared to transposon-inactive stocks, but most mutants recovered from Ac-Ds and En/Spm stocks were unstable, reverting upon backcrossing to CMS-S inbred lines. However, 10 independent restorer mutations recovered from CMS-S Mu transposon stocks were stable upon backcrossing. Many restorer mutations condition seed-lethal phenotypes that provide a convenient test for allelism. Eight such mutants recovered in this study included one pair of allelic mutations that were also allelic to the previously described rfl2-1 mutant. Targeted analysis of mitochondrial proteins by immunoblot identified two features that consistently distinguished restored CMS-S pollen from comparably staged, normal-cytoplasm, nonmutant pollen: increased abundance of nuclear-encoded alternative oxidase relative to mitochondria-encoded cytochrome oxidase and decreased abundance of mitochondria-encoded ATP synthase subunit 1 compared to nuclear-encoded ATP synthase subunit 2. CMS-S restorer mutants thus revealed a metabolic plasticity in maize pollen, and further study of these mutants will provide new insights into mitochondrial functions that are critical to pollen and seed development