Seed longevity is a complex process of key ecological and agronomic importance. DNA damage is a significant factor affecting seed ageing. Likewise, epigenetic changes can control gene expression and, therefore, seed response to ageing. The aim of the present work was to investigate the effect of ageing on nucleic acid stability and to identify reliable molecular markers that might help to monitor epigenetic changes within plant genetic resources during conservation. DNA profiles, evaluated by RAPD (random amplified polymorphic DNA), and methylation patterns, obtained by MSAP (methylation-sensitive amplification polymorphism), were compared in non-aged and aged Mentha aquatica seeds and plants produced by them. Germination decreased to 50% by storing seeds at 35°C and 12% wc for 28 days. RAPD profiles were 99% similar in these aged seeds compared to non-aged seeds. However, seedlings produced from the aged seeds showed a 13% dissimilarity compared to seedlings produced from the non-aged seeds. About 8% difference in the MSAP epigenetic profile was detected in seeds after storage and 16% difference was detected in the seedlings produced from them. This indicates that stress from high temperature and humidity during storage induced changes on the methylation state of seeds, and that changes were also detectable in the regenerated plants. Our results suggest that DNA integrity was compromised in seeds during ageing, and on seedlings produced by aged seeds. Genotype screening techniques such as RAPD and MSAP have the potential as markers of nucleic acid stability during seed ageing.

The mouse mammary tumor virus (MMTV) gag-pro frameshifting pseudoknot is an H-type RNA pseudoknot that contains an unpaired adenosine (A14) at the junction of the two helical stems required for efficient frameshifting activity. The thermodynamics of folding of the MMTV vpk pseudoknot have been compared with a structurally homologous mutant RNA containing a G[bull ]U to G-C substitution at the helical junction (U13C RNA), and an A14 deletion mutation in that context (U13CΔA14 RNA). Dual wavelength optical melting and differential scanning calorimetry reveal that the unpaired adenosine contributes 0.7 (±0.2) kcal mol−1 at low salt and 1.4 (±0.2) kcal mol−1 to the stability (ΔG°37) at 1 M NaCl. This stability increment derives from a favorable enthalpy contribution to the stability ΔΔH = 6.6 (±2.1) kcal mol−1 with ΔΔG°37 comparable to that predicted for the stacking of a dangling 3′ unpaired adenosine on a G-C or G[bull ]U base pair. Group 1A monovalent ions, NH4+, Mg2+, and Co(NH3)63+ ions stabilize the A14 and ΔA14 pseudoknots to largely identical extents, revealing that the observed differences in stability in these molecules do not derive from a differential or specific accumulation of ions in the A14 versus ΔA14 pseudoknots. Knowledge of this free energy contribution may facilitate the prediction of RNA pseudoknot formation from primary nucleotide sequence (Gultyaev et al., 1999, RNA 5:609–617).