© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 791 Biotechnol. J. 2008, 3, 791–802 DOI 10.1002/biot.200800005 www.biotechnology-journal.com 1 Introduction Aging is a phenomenon occurring in most eukary- otic organisms and is generally (but not exclusive- ly) associated with a gradual dysfunctioning of the mitochondria. Different theories on the cause of senescence have been developed of which Har- man’s free radical theory of aging [1] is the most widely accepted: reactive oxygen species (ROS) produced as byproduct of the energy metabolism react with mitochondrial compounds like nucleic acids, lipids and proteins, and cause and enhance mitochondrial dysfunctioning.Also common mech- anisms exist that delay aging or extend lifespan in most organisms: calorie restriction (CR) and a de- crease in temperature are examples of such mech- anisms [2, 3]. Whereas the majority of mycelial fungi show lit- tle or no sign of aging, the ascomycete Podospora anserina has been a model organism for aging re- search for over 50 years ([4], for reviews see [5, 6]). In nature, P. anserina grows on an ephemeral and lifespan limiting substrate: the dung of herbivores. In general, with ample nutrients and at a tempera- ture of 27°C, P. anserina has a lifespan of 2–3 wk. During this period, P. anserina gradually acquires a senescent phenotype with reduced growth, sterili- ty, a change in pigmentation, and finally hyphal death at the growth tips. Several growth conditions Research Article Mitochondrial pAL2-1 plasmid homologs are senescence factors in Podospora anserina independent of intrinsic senescence Anne D. van Diepeningen, Alfons J. M. Debets, S. Marijke Slakhorst and Rolf F. Hoekstra Laboratory of Genetics, Plant Sciences, Wageningen University, Wageningen, the Netherlands Since the first description of a linear mitochondrial plasmid in Podospora anserina, pAL2-1, and ho- mologous plasmids have gone from being considered beneficial longevity plasmids, via neutral genetic elements, toward mutator plasmids causing senescence. The plasmid has an invertron structure, with terminal inverted repeats and encodes a DNA and a RNA polymerase. Here we test whether pAL2-1 homologs cause rapid aging independent of intrinsic and external conditions. We first analyzed a natural population of P. anserina and in 40% of the 112 isolates we detected pAL2- 1 homologous plasmids. Though the lifespan varied considerably among the strains, plasmid-in- fected wild-type strains are on average shorter lived than plasmid-free strains and typically show a reduced lifespan extending effect of calorie restriction (CR). However, interesting exceptions were found, inviting further study. To further investigate the effect of pAL2-1 homologs under various conditions, we constructed and analyzed isogenic lines with and without the plasmid. We found that the presence of pAL2-1 homologs did not significantly affect growth rate as suggested by the population analysis, but reduced lifespan under all conditions. This effect was particularly clear for the lifespan extending conditions tested (CR, low temperature, antibiotics) supporting the idea that pAL2-1 homologs are additional senescence factors independent of the intrinsic senescence determinants. Keywords: Aging · Calorie restriction · Longevity · Mitochondrial instability Correspondence: Dr. Anne D. van Diepeningen, Laboratory of Genetics, Plant Sciences, Wageningen University, Arboretumlaan 4, 6703 BD Wa- geningen, the Netherlands E-mail: anne.vandiepeningen@wur.nl Fax: +31-317-483146 Abbreviations: CR, calorie restriction; PASM, P. anserina synthetic medium; ROS, reactive oxygen species Received 10 January 2008 Revised 21 February 2008 Accepted 5 March 2008