ORIGINAL RESEARCH Inter-nucleosomal DNA fragmentation and loss of RNA integrity during seed ageing Ilse Kranner • Hongying Chen • Hugh W. Pritchard • Stephen R. Pearce • Simona Birtic ´ Received: 13 May 2010 / Accepted: 29 July 2010 / Published online: 15 August 2010 Ó Springer Science+Business Media B.V. 2010 Abstract The germination of viable seeds is the basis for new plant growth and development. Seeds lose viability during storage, but the biochemical mechanisms of seed death are not fully understood. This study aimed to investi- gate degradation patterns of nucleic acids during seed ageing and subsequent water uptake. Seeds of Pisum sativum L. were artificially aged at 50°C and 12% seed water content (WC). Nucleic acids degradation was studied during ageing and during imbibition of four seed lots with differential viability from highly viable to dead. As seeds lost viability during ageing, DNA was gradually degraded into internu- cleosomal fragments, resulting in ‘DNA laddering’, in con- junction with disintegration of 18S and 28S rRNA bands. During imbibition, non-aged controls had high levels of DNA and RNA integrity through to radicle protrusion. In an aged seed lot with 85% total germination (TG) DNA frag- mentation decreased upon imbibition probably due to nucleosome degradation, while rRNA integrity did not improve. In an aged seed lot with 44% TG, neither DNA nor rRNA integrity improved upon imbibition. Dead seeds showed DNA degradation as laddering throughout imbibi- tion along with extensive degradation of rRNA. We present a model in which interlinked programmed and non- programmed events contribute to seed ageing, and suggest that protection of nucleic acids during ageing is key to seed longevity. Keywords Ageing Á DNA Á Pisum sativum Á Programmed cell death Á RNA Á Seed Introduction The ability of desiccation tolerant ‘orthodox’ (Roberts 1973) seeds to survive at very low intracellular water content (WC) is the basis for their longevity, maintaining plant genetic resources over centuries (Daws et al. 2007) or even millennia (Sallon et al. 2008; Shen-Miller et al. 1995). In spite of this remarkable longevity, all seeds age and die eventually. The underlying mechanisms of seed ageing and death are less understood than the empirical description of seed longevity as a function of seed WC and temperature (Ellis and Roberts 1980; Pritchard and Dickie 2003; Roberts 1973; Walters 1998; Walters et al. 2005). Ortho- dox seeds can be dried to extremely low WC (e.g., \ 5%), inducing ‘vitrification’, which is the transition of the cytoplasm to the ‘glassy’ state (Sun and Leopold 1993; Williams and Leopold 1989). They can then be stored in gene banks at low temperatures (e.g., at -20°C, or in or above liquid nitrogen) where their DNA may be preserved long-term (Walters et al. 2004; 2006). Taken together, the glassy state and low temperatures will restrict molecular mobility, delaying degenerative processes (Walters 1998). Under such circumstances, seed death through a process that potentially involves gene expression, is less likely than cell death through random damage. For agricultural purposes, air dried seeds are stored at higher relative I. Kranner (&) Á H. Chen Á H. W. Pritchard Á S. Birtic ´ Seed Conservation Department, Royal Botanic Gardens, Kew, Wakehurst Place, West Sussex RH17 6TN, UK e-mail: i.kranner@kew.org H. Chen Kunming Institute of Botany, Chinese Academy of Sciences, 661100 Kunming, People’s Republic of China S. R. Pearce Á S. Birtic ´ School of Life-Sciences, University of Sussex, Brighton, East Sussex BN1 9QG, UK 123 Plant Growth Regul (2011) 63:63–72 DOI 10.1007/s10725-010-9512-7