A Competitive Enzyme-Linked Immunosorbent Assay to Quantify Acetaldehyde-Protein Adducts That Accumulate in Dry Seeds during Aging

Abstract

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) ...

Induction of mitochondrial aldehyde dehydrogenase by submergence facilitates oxidation of acetaldehyde during re‐aeration in rice

“…Acetaldehyde is harmful to cells because of its tendency to form adducts with protein and DNA [15,16]. Cells possess mechanisms to metabolize acetaldehyde.…”

Rice Aldehyde Dehydrogenase7 Is Needed for Seed Maturation and Viability

“…Lipid peroxidation and respiration result in the formation of reactive aldehydes such as malondialdehyde (MDA) and acetaldehyde, which tend to react with proteins and amino acids (Mueller, 1998;Almeras et al, 2003;Weber et al, 2004). Those reactions cause aging and seed damage (Zhang et al, 1995(Zhang et al, , 1997. Until recently, a physiological approach has been taken in research on seed aging, and molecular and genetic studies have been seldom reported (Clerkx et al, 2004a).…”

Metabolite Profiling of adh1 Mutant Response to Cold Stress in Arabidopsis

“…In the response to cold stress in soybeans, the accumulation of ADH1 and other typical cold response genes was not significant, which was considered to be the reason for poor acclimation capability and cold intolerance (Yamasaki and Randall, 2016). Environmental stresses such as hypoxia (Johnson et al, 1994), drought (Senthil-Kumar et al, 2010), and abscisic acid phytohormone (Zhang et al, 1997) induce a high expression of ADH1 . ADH gene expression, enzyme activity, and related metabolites were analyzed under different temperatures, and the results revealed that the levels of acetaldehyde, ethanol, acetic acid, and ethyl acetate fluctuated at 10°, 20°, and 30°C (Cirilli et al, 2012), suggesting temperature affecting ADH activity and the accumulation of related metabolites.…”

“…Metabolomic studies revealed ADH to play a central role in the biosynthesis of an important group of aroma volatiles (C6-derivative compounds) including different aldehydes, alcohols, and esters (Bicsak et al, 1982; Molina et al, 1986; Longhurst et al, 1990; Millán et al, 1990; Speirs et al, 1998). These metabolites are related to fruit flavors, ripening of fruits, antiviral resistance, environmental stresses responses and the ABA phytohormone (Dolferus et al, 1994; Zhang et al, 1997). The ADH1 gene mutant of Chlamydomonas reinhardtii elicits metabolic restructuring during dark anoxic growth (Catalanotti et al, 2012; Magneschi et al, 2012).…”