PLANT PHYSIOLOGY , Vol 113, Issue 2 397-402, Copyright © 1997 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
A Competitive Enzyme-Linked Immunosorbent Assay to Quantify Acetaldehyde-Protein Adducts That Accumulate in Dry Seeds during Aging
M. Zhang, S. Nagata, K. Miyazawa, H. Kikuchi and Y. Esashi
Laboratory of Environmental Biology, Botanical Garden, Faculty of Science, North Campus, Tohoku University, Kawauchi, Aobaku, Sendai 980-77, Japan (M.Z., K.M., H.K., Y.E.)
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 NaCNBH3, 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.
