The S-adenosylmethionine dependent O-methyltransferase PaMTH1: a longevity assurance factor protecting Podospora anserina against oxidative stress

Abstract

PaMTH1 is an O-methyltransferase catalysing the methylation of vicinal hydroxyl groups of polyphenols. The protein accumulates during ageing of Podospora anserina in both the cytosol and in the mitochondrial matrix. The construction and characterisation of a PaMth1 deletion strain provided additional evidence about the function of the protein in the protection against metal induced oxidative stress. Deletion of PaMth1 was found to lead to a decreased resistance against exogenous oxidative stress and to a shortened lifespan suggesting a role of PaMTH1 as a longevity assurance factor in a new molecular pathway involved in lifespan control.

Keywords: O-methyltransferase; Podospora anserina; ageing; flavonoids; knock-out; reactive oxygen species.

Figure 1.

Construction and validation of a PaMth1 deletion strain of P. anserina. (A) Physical maps and sizes of the restriction products for the genomic region bearing PaMth1 and the recombined version with the hygromycin B resistance cassette. Reading frames of the two genes are indicated in grey. The genomic sequences flanking PaMth1 are indicated by punctuation. Restriction sites of EcoRV are indicated. (B) Southern blot analysis of Eco RV digested wild-type strain s genomic DNA and genomic DNA of two secondary transformants isolated from a primary deletion strain. The PaMth1 gene-specific probe (left panel) detects the 3.55 kbp fragment only in the sample of the wild-type s (wt) but not in the samples of the deletions strains (ΔPaMth1). The hygromycin B resistance gene-specific probe (right panel) detects the 3.26 kbp fragment only in the sample of the deletions strains. (C) Western blot analysis verifying the successful construction of a PaMth1 deletion strain using total- and mitochondrial protein samples of juvenile and senescent P. anserina wild-type strain s and the secondary transformants of the deletion strain, respectively. The PaMTH1 specific antibody detects PaMTH1 in the samples of the wild-type s strains but not in samples of the deletion strains. As loading controls an actin specific antibody for total proteins and a porin specific antibody for the mitochondrial proteins were used.

Figure 2.. Growth rates of wild-type strain s and of the PaMth1 deletion strain on synthetic PASM medium containing different amounts of CuSO4 and of hydrogen peroxide, respectively.

Pairs of strains (wild-type strain s and the PaMth1 knock-out strain) were grown on one plate and growth rates were recorded over 3 days. Cultures of the PaMth1 deletion strain (n = 20) were characterised by decreased growth rates compared to the wild-type strain s (n = 20) when incubated on medium containing 40 μM (p = 6.154e-08) or 80 μM (p = 1.921e-08) CuSO4 and 0.01% (p = 1.443e-08); 0.02% (p = 1.06e-07) or 0.04% (p = 0.99) hydrogen peroxide. Plates containing hydrogen peroxide were incubated in the dark.

Figure 3.

In comparison to wild-type strain s (n = 40), the mean lifespan of the PaMth1 deletion strain (n = 40) is decreased by 18%. Mean lifespan (wild-type strain s: 21 days and ΔPaMth1: 17 days) was determined on synthetic PASM medium.