SummarySeveral lines of evidence have implicated reactive oxygen species (ROS) in the pathogenesis of various degenerative diseases and in organismal ageing. Furthermore, it has been shown recently that the alternative pathway respiration present in plants lowers ROS mitochondrial production. An alternative oxidase (AOXp) also occurs in the filamentous fungus Podospora anserina. We show here that overexpression of this oxidase does not decrease ROS production and has no effect on longevity, mitochondrial stability or ageing in this fungus. In the same way, inactivation of the gene has no effect on these parameters. In contrast, overexpression of the alternative oxidase in the long-lived cox5::BLE mutant, deficient in cytochrome c oxidase, considerably increases ROS production of the mutant. It rescues slow growth rate and female sterility, indicating an improved energy level. This overexpression also restores senescence and mitochondrial DNA instability, demonstrating that these parameters are controlled by the energy level and not by the expression level of the alternative oxidase. We also suggest that expression of this oxidase in organisms naturally devoid of it could rescue respiratory defects resulting from cytochrome pathway dysfunctions.
In the filamentous fungus Podospora anserina, the association of two nuclear genes inevitably leads to a "premature death" phenotype consisting of an early end of vegetative growth a few days after ascospore germination. Mycelia showing this phenotype contain a mitochondrial chromosome that always bears the same deletion. One of the break points is exactly at the 5' splice site of a particular mitochondrial intron, suggesting that the deletion event could result from molecular mechanisms also involved in intron mobility.One of the nuclear genes involved in triggering this site-specific event belongs to the mating-type minus haplotype; the other is a mutant allele of a gene encoding a cytosolic ribosomal protein.In obligate aerobes, rearrangements of mitochondrial DNA (mtDNA) are responsible for various deleterious symptoms, such as maternally inherited male sterility in plants (1), several mycelial degenerative phenomena in fungi (2), and, as described more recently in humans, numerous neuromuscular (3) or hematological (4) diseases. In some cases, nuclear genes have been shown to control the mtDNA rearrangements (5, 6). The molecular mechanisms producing these rearrangements and the role played by nuclear-encoded proteins remain unclear in all cases.In the filamentous fungus Podospora anserina, vegetative growth is limited by a maternally inherited syndrome, called senescence, ending in cessation of mycelial elongation and apical cell death (7,8). In this species, senescence is clearly associated with mtDNA rearrangements and most probably is caused by them. More precisely, it has been shown that short mtDNA sequences are amplified as circular multimeric DNA molecules in senescent cultures (9)(10)(11)(12)(13)(14). It was shown that the most frequently amplified sequence corresponds exactly to a mitochondrial intron (15), intron a, and that most of the mutations allowing mycelia to escape senescence are rearrangements in intron a (12,(16)(17)(18). It has been known for a long time (19) that the nuclear genome controls the life-span of the fungus. For instance, the life-span of our reference strains differs according to their mating type: the process of senescence is delayed in mat+ strains compared with matones. However the differences, although significant, are not striking (see Table 1). Moreover the functions encoded by these genes are still unknown.The work presented here follows the observation that, in Podospora, mutations in several genes involved in the control of translational accuracy have an effect on the life-span (A. Raynal, personal communication; Table 1). As a first step, we focused our attention on the AS1-4 mutation. This particular allele confers a very long life-span to mat+ mycelia, while, when associated with mat-, it leads systematically to the premature death phenotype, consisting of an early end of mycelial growth a few days after ascospore germination.We showed that mycelia presenting this phenotype were in all cases heteroplasmic and that they systematically contained the same ...
Podospora anserina is a filamentous fungus with a limited life span. It expresses a degenerative syndrome called senescence, which is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. A mobile group II intron (alpha) has been thought to play a prominent role in this syndrome. Intron alpha is the first intron of the cytochrome c oxidase subunit I gene (COX1). Mitochondrial mutants that escape the senescence process are missing this intron, as well as the first exon of the COX1 gene. We describe here the first mutant of P. anserina that has the alpha sequence precisely deleted and whose cytochrome c oxidase activity is identical to that of wild-type cells. The integration site of the intron is slightly modified, and this change prevents efficient homing of intron alpha. We show here that this mutant displays a senescence syndrome similar to that of the wild type and that its life span is increased about twofold. The introduction of a related group II intron into the mitochondrial genome of the mutant does not restore the wild-type life span. These data clearly demonstrate that intron alpha is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron alpha plays in the instability of the mitochondrial chromosome and the link between this instability and longevity. Our results strongly support the idea that in Podospora, "immortality" can be acquired not by the absence of intron alpha but rather by the lack of active cytochrome c oxidase.
Senescence in Podospora anserina has long been shown to be under cytoplasmic control. Comparison of DNAs isolated from young and senescent cultures made it possible to detect the presence, in senescent cultures only, of a specific DNA (SEN-DNA). This DNA consists of repeated sequences arranged in a multimeric set of circular molecules. In this study we have examined one particular SEN-DNA whose monomer unit is 6300 bp long. Using the Southern hybridization technique, we have demonstrated that this SEN-DNA results from the amplication of a sequence of the mitochondrial chromosome. This amplification, which resembles the process leading to rho- ("petite") mutations in yeast, is discussed in relation to the determinism of senescence.
A fifth cytoplasmic mutation (capr 1) obtained in Podospora anserina is described. In addition to chloramphenicol resistance it confers a strong deficiency in cytochrome aa3 and impairs the germination of ascospores. Genetic analysis shows: 1) strict maternal inheritance of (capr 1) allele; 2) selection against the (capr 1) allele as well in sexual crosses as during vegetative growth; 3) complete reversion of this selection by even low concentration of CAP. On the basis of their cytoplasmic inheritance and altered cytochrome spectra the five cytoplasmic mutations are assumed to be mitochondrial. Analysis of crosses between them allows to class them in 3 loci, 2 of which being closely linked.
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