Enhanced Expression of BiP Is Associated with Treatments that Extend Storage Longevity of Primed Tomato Seeds

While seed priming (hydration in water or osmotic solutions followed by drying) enhances seed germination performance, the longevity of primed seeds in storage often is reduced. Postpriming treatments including a reduction in seed water content followed by incubation at 37 or 40 °C for 2 to 4 h can substantially restore potential longevity in tomato (Lycopersicon esculentum Mill.) seeds. These conditions might induce heat-shock proteins (hsp) that could be involved in the extension of seed longevity. The abundance of BiP (78 kD Binding Protein), hsp70 and class I small hsp in primed seeds subjected to postpriming treatments was examined to assess this possibility. BiP mRNA and protein amounts increased during postpriming heat treatments that extended longevity of tomato seeds. Treatment of primed seeds with the calcium ionophore calcimycin (A21387) enhanced BiP protein accumulation in the absence of heat treatment and also extended potential seed longevity. Changes in the abundance of hsp70 and class I small hsps were not consistently associated with potential seed longevity. Thus, enhanced BiP expression may contribute to the improved longevity of primed seeds following postpriming treatments. small heat-shock proteins (shsps) cooperate with the large heatshock proteins such as hsp70 to reactivate heat-denatured proteins (Forreiter and Nover, 1998; Lee and Vierling, 2000). Plant hsps are expressed not only in response to heat stress, but also developmentally during embryogenesis and other stages of the life cycle (Schöffl et al., 1998). For example, several hsps accumulate to high amounts during seed development and have been implicated in the acquisition of desiccation tolerance during seed maturation (Helm and Abernethy, 1990; Reddy et al., 1998; Wehmeyer and Vierling, 2000). Among the heat-shock family of proteins are specific chaperones that are capable of facilitating refolding of abnormally folded proteins, preventing protein aggregation, and binding to damaged proteins to aid entry into proteolytic pathways (Parsell and Lindquist, 1993; reviewed by Nover and Scharf, 1997). Among these, the immunoglobulin binding protein (termed BiP or GRP78) (Haas and Wable, 1983; Munro and Pelham, 1986) is an endoplasmic reticulum (ER)-resident homolog of cytoplasmic hsp70. Evidence indicates that BiP, in addition to being expressed constitutively in normal development (Dupont et al., 1998), is involved in protein repair processes in plants in response to heat shock and other stresses (Hurkman et al., 1998; Leborgne-Castel et al., 1999; Lee and Vierling, 2000). The maize floury-2 endosperm mutant overproduces BiP during abnormal protein accumulation associated with seed maturation, possibly reflecting a protein repair function (Fontes et al., 1991). BiP is also upregulated in response to ER stresses such as loss of calcium due to the ionophore calcimycin (also known as A21387) (Jones and Bush, 1991; Wooden et al., 1991; Li et al., 1993) or to the inhibition of glycosylation by tunicamycin, resulting in misfolded proteins (Fontes et al., 1991). BiP and other members of the hsp70 family have been reported to promote cellular survival when they are up-regulated following stress (Bolliger et al., 1994; LeborgneCastel et al., 1999; Kawana et al., 2000). In particular, expression of elevated levels of BiP in transgenic tobacco plants conferred tolerance to water deficit during growth and to inhibition by tunicamycin during seed germination (Alvim et al., 2001). Induction of BiP by partial or slow drying of primed seeds could Received for publication 8 Jan. 2002. Accepted for publication 15 Apr. 2002. The authors thank Alan Bennett, University of California, Davis, for providing the tomato BiP cDNA and antibody, Choo Bong Hong, Institute of Molecular Biology and Genetics, Seoul National University, Korea for antibody to class I small heat shock proteins, Hiroyuki Nonogaki for assistance with protein immunoblotting, and Russell Wrobel for his constructive review of the manuscript. This study was supported by the Western Regional Seed Physiology Research Group and Regional Research Project W-168. Corresponding author; e-mail [email protected]. Seed priming is a process in which seeds are imbibed in water or osmotic solutions followed by drying before radicle emergence (McDonald, 2000). Priming can increase the speed and uniformity of germination when the seeds are subsequently planted (Taylor et al., 1998). However, primed seeds often exhibit reduced longevity in storage, particularly under adverse storage conditions (Alvarado and Bradford, 1988; Tarquis and Bradford, 1992). In some seeds, even relatively short hydration periods that do not advance germination can dramatically reduce storage longevity (Gurusinghe and Bradford, 2001; Tarquis and Bradford, 1992). Various treatments imposed after priming but before dehydration have been reported to improve seed longevity in storage (Bruggink et al., 1999). The most effective treatments included both a moderate reduction in seed moisture content and short periods of incubation at elevated temperature (Bruggink et al., 1999; Gurusinghe and Bradford, 2001; Schipper et al., 2001). This suggested that while seed longevity in storage was reduced by rapid dehydration following a period of hydration, a reduction in seed moisture content or exposure to elevated temperatures (heat shock) after priming might induce physiological or molecular changes that would render the seeds more resistant to deterioration during storage. As exposure to high temperatures for only a few hours was effective in restoring longevity to primed seeds, induction of the heat-shock response is implicated. Cellular processes triggered by brief heat shock, such as the expression of heat-shock proteins (hsps), can increase tolerance to subsequent stresses (Wisniewski et al., 1996; Vierling, 1997). Some hsps function as molecular chaperones that assist in protein folding (DeRocher and Vierling, 1994; Lee et al., 1995; Vierling, 1997) and evidence suggests that