Transcript analysis of Pa_3_10440 (PaCtr3), Pa_2_4460 (PaSod2) and Pa_3_1710 (PaAox).

<p>Upper row: RNA of the three biological replicates (individuals) was isolated, pooled and analyzed by SuperSAGE. Gene expression was quantified as “tags per million” (tpm). Lower row: The same RNA was used for qRT-PCR analysis. RNA samples from each individual were individually analyzed for relative gene expression. Error bars represent the standard error. In both analyses, x-axes indicate the age of the individuals at which total RNA was isolated.</p

Comparison of down-regulated genes in the grisea mutant and the wild type.

<p>(a) Common down-regulated genes within the age-dependent transcriptome of the wild-type ‘s’ and the long-lived mutant grisea of <i>P. anserina</i>. 556 genes of the grisea transcriptome were identified to be down-regulated with a factor of at least 3 and a p-value of ≤0.01 for differential expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083109#pone.0083109-Servos1" target="_blank">[22]</a>. The 1,202 genes, identified as being down-regulated during aging in this study, have 89 genes in common. (b) 10,000 simulated experiments with two sets of randomly picked gene names, each set of the same size as the two sets (wild type, grisea), were applied. The box plot shows that the 89 common genes were extreme outliers. (c) The results of the random experiments are normally distributed with a mean of μ≈66.05 and a standard deviation of σ≈7.39. Our determined value of 89 is greater than three times sigma, indicating that the corresponding genes do not occur randomly. (d) All enriched GO terms in the set of the 89 common genes with a p-value ≤0.01 are depicted. Copper associated terms can be found on the very top. (e) Four genes already identified as putative target genes of transcription factor GRISEA are found within these 89 common genes. Due to its irregular expression profile, the gene, encoding the putative copper transporter PaCTR1, is not among the 89 common genes.</p

GO term enrichment map for the “autophagy” category in the up-regulated gene set.

<p>Analogous to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083109#pone-0083109-g004" target="_blank">Figure 4</a>, each node represents one GO term and node colors correspond to the group and the degree of significance. Edge thickness indicates the amount of genes shared by two terms, and node sizes are proportional to the number of genes assigned to the corresponding term.</p

A Genome-Wide Longitudinal Transcriptome Analysis of the Aging Model <i>Podospora anserine</i>

<div><p>Aging of biological systems is controlled by various processes which have a potential impact on gene expression. Here we report a genome-wide transcriptome analysis of the fungal aging model <i>Podospora anserina</i>. Total RNA of three individuals of defined age were pooled and analyzed by SuperSAGE (serial analysis of gene expression). A bioinformatics analysis identified different molecular pathways to be affected during aging. While the abundance of transcripts linked to ribosomes and to the proteasome quality control system were found to decrease during aging, those associated with autophagy increase, suggesting that autophagy may act as a compensatory quality control pathway. Transcript profiles associated with the energy metabolism including mitochondrial functions were identified to fluctuate during aging. Comparison of wild-type transcripts, which are continuously down-regulated during aging, with those down-regulated in the long-lived, copper-uptake mutant grisea, validated the relevance of age-related changes in cellular copper metabolism. Overall, we (i) present a unique age-related data set of a longitudinal study of the experimental aging model <i>P. anserina</i> which represents a reference resource for future investigations in a variety of organisms, (ii) suggest autophagy to be a key quality control pathway that becomes active once other pathways fail, and (iii) present testable predictions for subsequent experimental investigations.</p></div

GO term enrichment map for the library of down- and up-regulated genes.

<p>Each node represents one GO term. The thickness of the edges represents the number of genes shared by two terms. The node color corresponds to the group and the degree of significance for enrichment (see color bar). Node sizes indicate the number of genes within the corresponding GO term. GO terms are grouped by their similarity degree defined by the number of common genes or related terminology. Striking groups were manually circled and labelled. Map regions with a high density of thick-sized nodes (center of figure) represent GO terms, describing very general processes with rather low information.</p

Transcriptome comparison of young (day 6) and old individuals (day 14).

<p>The figure depicts the enrichment map for the corresponding GO enrichment analysis. Analogous to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083109#pone-0083109-g004" target="_blank">Figure 4</a>, each node represents one GO term and node colors correspond to the group and the degree of significance. Edge thickness indicates the amount of genes shared by two terms and node sizes are proportional to the number of genes assigned to the corresponding term. Striking groups were subsequently circled and labelled.</p

GO term enrichment map for the “ribosome” and the “proteasome” categories in the set of down-regulated genes.

<p>As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083109#pone-0083109-g004" target="_blank">Figure 4</a>, each node represents one GO term and node colors correspond to the group and the degree of significance. Edge thickness indicates the amount of genes shared by two terms and the node sizes are proportional to the number of genes assigned to the corresponding term.</p

Fuzzy clustering analysis.

<p>All of the 7,467 “smoothed” expression profiles of the “cluster profile library” are distributed to eight fuzzy clusters. The x-axis represents the seven time points at which RNA was isolated from the three investigated <i>P. anserina</i> individuals. The y-axis indicates the relative expression strength. The color gradient corresponds to the membership value (see color bar on the right). A membership value of 1 means a perfect fitting to the corresponding cluster core (deep red). Arrows indicate the general tendency of the corresponding cluster. Below each cluster, a selection of categories to which significantly enriched GO terms (p-value ≤0.01) for particular functions were associated are listed (see description in the text).</p

Mitochondrial content.

<p><b>A</b> Mycelia were stained with Mitotracker Green and analysed by fluorescence microscopy. Representative hyphae are shown. Scale bar: 2 µm. <b>B</b> Protoplasts prepared from mutants grisea and <i>PaCox17</i>::ble contain significantly more mitochondria than the WT as revealed by NAO staining. Mitochondrial content in the WT was set to 100%. <b>C</b> Determination of the mtDNA/nuclear DNA ratio as a marker for mitochondrial quantity by PCR. <i>left</i>: representative 1% agarose gel showing separated <i>PaGpd</i> (nuclear DNA) and <i>PaLsu</i> (mtDNA) amplification products stained with ethidiumbromide, NC: negative control, pd: primer dimers. <i>right</i>: densitometric analysis of band intensities. The mtDNA/nuclear DNA ratio in the WT was set to 1. <b>D</b> Western blot analysis to detect PaPORIN levels in total protein extracts from the wild type strain and the two mutants. As a loading control the Coomassie-stained transfer membrane is shown. Data represent mean ± standard error. *: p<0.05; ***: p<0.001, Student's <i>t</i> test, two-tailed.</p

Western blot analysis of mitochondrial PaLON protease and the molecular chaperone HSP60.

<p><b>A</b> Mitochondrial proteins in WT and mutants grisea and <i>PaCox17</i>::ble were analysed with antibodies against HSP60 after transfer to a PVDF membrane. HSP60 levels are increased in the two mutants. <b>B</b> Protein levels of LON protease (PaLON) are moderately increased in the mutants compared to the WT. Below each immunodetection a densitometric analysis of signal intensities (x-fold level compared to the WT) is shown. Intensities of the PaPORIN signals were used for normalisation. UniProt accession numbers: PaLON: B2AZ54; PaHSP60: B2B270 and PaPORIN: B2B736.</p