Genes inhibiting senescence in the ascomycete Podospora anserina.

Summary Senescence occurs in all wild strains of Podospora anserina after continued growth. This syndrome can be inhibited by a synergistic interaction of two linked genes, incoloris and vivax. Whereas the wild strain starts to become senescent after 26 d and the mutants incoloris and vivax after 42 and 66 d respectively, the double mutant shows no signs of aging after culture for more than one year.     

The phenol oxidases of the ascomycete Podospora anserina

For the low molecular weight laccases II and III of Podospora anserina the kinetic parameters Michaelis constant (K _M) and maximum reaction velocity (V) were determined polarographically under pH optimum conditions for representative substrates of different substitution patterns.Laccase II showed two peaks in its pH optimum curve, each with a different substrate specificity, indicating structural differences to laccase III which exhibits only one broad peak.Under optimum conditions the affinities of various substrates are determined by their substitution patterns: high affinity for simple o-and p-diphenols, low affinity for m-phenols. The maximal velocity remains largely uninfluenced.This study of the effect of substitution on substrate utilization leads to the assumption that there is no specific reactive site for m-phenols in either laccase. Oxidation of m-phenols, however, takes only place at high pH values.     

What triggers senescence in Podospora anserina?

Senescence of Podospora anserina is triggered by a cytoplasmic and infectious factor (the determinant of senescence) and is always correlated with mitochondrial DNA modifications, especially with the accumulation of small circular subgenomic DNA molecules, the senDNAs. Several observations have suggested that the senDNAs could be the cytoplasmic and infectious determinant. However, we show here (1) that senDNA molecules can be transferred to a young culture without the cotransmission of the determinant of senescence and (2) that the determinant of senescence does not segregate as a mitochondrial DNA mutation. Overall, our data strongly argue that amplification of senDNA molecules in the mitochondria is not an intrinsic property of these small DNA molecules. They question the nature of the actual determinant of senescence.     

Genetic analysis of spore killing in the filamentous ascomycete Podospora anserina

In the present study, we analysed different Podospora anserina strains for their ability to induce spore killing and identified three new killer strains. Test crosses of killer strains with different sensitive strains revealed different second division segregation ratios suggesting an influence of the sensitive strain on the crossing-over frequency. In crosses of killer strain O with a sensitive strain, the frequency of two-spored asci was found to vary extremely from perithecium to perithecium. Furthermore, crosses of strain O with sensitive strain Us5 led to a significant proportion of asci containing an unexpected high number of surviving spores as the result of gene conversion. Finally, for the first time, we present data demonstrating that in a number of ascospores the killer and the corresponding sensitive allele is located in one individual nucleus. Mycelia derived from such ascospores display a "sensitive killer" phenotype. Crosses of these mycelia with a killer strain as well as with a sensitive strain result in spore killing. Strikingly, heterokaryotic spores containing the recombined "sensitive/killer" allele and a nucleus with a killer allele give rise to mycelia protected against spore killing during selfing.     

Stress-dependent opposing roles for mitophagy in aging of the ascomycete Podospora anserina

Mitochondrial dysfunction is causatively linked to organismal aging and the development of degenerative diseases. Here we describe stress-dependent opposing roles of mitophagy, the selective autophagic degradation of mitochondria, in aging and life-span control. We report that the ablation of the mitochondrial superoxide dismutase which is involved in reactive oxygen species (ROS) balancing, does not affect life span of the fungal aging model Podospora anserina, although superoxide levels are strongly increased and complex I-dependent respiration is impaired. This unexpected phenotype depends on functional autophagy, particularly mitophagy, which is upregulated during aging of this mutant. It identifies mitophagy as a prosurvival response involved in the control of mitohormesis, the well-known beneficial effect of mild mitochondrial oxidative stress. In contrast, excessive superoxide stress turns mitophagy to a prodeath pathway and leads to accelerated aging. Overall our data uncover mitophagy as a dynamic pathway that specifically responds to different levels of mitochondrial oxidative stress and thereby affects organismal aging.     

A Podospora anserina longevity mutant with a temperature-sensitive phenotype for senescence.

A Podospora anserina longevity mutant was identified with a temperature-sensitive phenotype for senescence. This mutant, termed TS1, grew for over 3 m at 27 degrees C, but when shifted to 34 degrees C, it underwent senescence between 10 and 18 cm. A previously described senescence-associated plasmid, alpha senDNA, derived from the mitochondrial genome, was not detected in TS1 at 27 degrees C but was present in senescent cultures at 34 degrees C. A similar result was observed in progeny strains obtained by crossing the TS1 mutant with a wild-type strain. Other mitochondrial excision-amplification DNAs in addition to alpha senDNA were also observed in the senescent cultures. Most were derived from a specific region of the mitochondrial genome. These results provide evidence that alpha senDNA is involved in TS1 senescence and suggest that this plasmid may play a role in the formation of other mitochondrial excision-amplification plasmids.     

The phenoloxidases of the ascomycete Podospora anserina. Structural differences between laccases of high and low molecular weight.

In order to investigate the extent of the relationship between the three copper-containing glycoproteins, laccases I, II and III (Mr70000, 80000 and 390000 respectively) of Podospora anserina, the following experiments were carried out on laccases II and III: (a) determination of amino acid composition; (b) determination of N-terminal and C-terminal amino acid; (c) determination of sugar composition; (d) dissociation studies on native and denatured laccases and also after removal of copper from the enzymes; (e) digestion of the carbohydrate moieties with the aid of glycosylhydrolases. A comparison between the results of these experiments and data previously obtained with laccase I allows the following conclusions to be drawn. 1. Laccases II and III are not identical. 2. Neither of these low molecular weight laccases are as complete molecules subunits of the oligomeric laccase I. 3. The possibility of partial identity of amino acid sequences of laccases I and III can not be excluded. 4. Laccase II possibly consists of subunits of Mr37000 whereas laccase III does not. 5. Digestion of 50% of the carbohydrate content leads to complete loss of serological specificity (serological reaction and cross reaction). This finding is discussed with regard to the possible role of the carbohydrate moiety as antigenic determinants and thus as the reason for the immunological relationship. As a consequence, at least three independent structural genes for laccases must be assumed.