A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantify endogenous acetaldehyde-protein adducts (APAs) produced in plant seeds at low acetaldehyde concentrations without exogenous reducing agents. The key point of this technique is the use of a gelatin-acetaldehyde adduct, which is synthesized under 1 mM acetaldehyde and 10 mM NaCNBH,, to pre-coat plate wells to obtain the proper binding parameters for the quantification of APA in seed proteins. Compared with the traditional, direct ELISA method, the competitive one has higher sensitivity and less background. Using competitive ELISA, we determined the accumulation of endogenous APAs in seeds in relation to the loss of seed viability. Lettuce seeds were exposed to 2 mM gaseous acetaldehyde during storage for 30 or 45 d; the relative humidity and temperature of storage were studied independently. Viability decreased only in acetaldehyde-treated seeds, as either the temperature or the relative humidity increased. A loss in viability was accompanied by an increase in the accumulation of APA. The APA content also increased as viability decreased in five species of seeds, which were aged naturally without exposure to acetaldehyde. It is suggested that the modification of functional seed proteins with endogenously evolved acetaldehyde may be an important cause of seed aging.Acetaldehyde is an intermediate of alcoholic fermentation in plants. The toxic effects of acetaldehyde may arise from its nonenzymatic binding with proteins (Gaines et al., 1977;Stevens et al., 1981;Donohue et al., 1983) and DNA (Restow and Obe, 1978;Fraenkel-Conrat and Singer, 1988). Israel et al. (1986) found that the immunization of mice with acetaldehyde conjugated to human plasma proteins results in the production of a polyclonal antibody, which reacts with various APAs. The production of acetaldehyde in tissue culture is thought to cause cell damage (Perata et al., 1988;Perata and Alpi, 1991). Perata et al. (1992) tried to detect APA from suspension-cultured carrot cells using a direct ELISA method. However, APAs were detected only when both ethanol and a reducing agent (NaCNBH,) were supplied in the culture medium, and only trace amounts of endogenous APA were detected without the presence of a reducing agent. Among the many kinds of volatile compounds that have evolved from dry seeds, acetaldehyde and ethanol are common components in the various seed species (Zhang et al., 1993). Acetaldehyde is effective in causing the rapid deterioration of seeds when it is applied as a gas to seeds during storage periods (Zhang et al., 1994). We proposed a hypothesis that seed aging may be caused by a modification of seed proteins by endogenously produced acetaldehyde (Zhang et al., 1995). In this paper we report the detection of endogenous APA from five species of dry seeds using a sensitive, competitive ELISA technique and demonstrate the increased accumulation of APA in aged seeds. MATERIALS AND METHODS Seed Materials and Storage MethodsSeeds with high viability (90% or more) ...

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