University of Florida and USDA-ARS

Intracellular immunolocalization of Wx protein in endosperm cells Yen-Ching Chen and Prem S. Chourey The Wx locus on chromosome 9 codes for a starch-granule-bound nucleoside diphosphate (NDP) sugar-starch glucosyltransferase, which transfers glucose from UDP-glucose (or ADP-glucose) to starch (Nelson and Rines, Biochem. Biophys. Res. Commun. 9:297-300, 1962). Echt and Schwartz (Genetics 99:275-284, 1981) and Shure et al. (Cell 35:225-233, 1983) reported isolation of a 58 kD protein from starch grains of Wx endosperm. Both groups presented various lines of evidence to show that the 58 kD protein is encoded by the Wx locus. SDS Western blot analyses revealed a single polypeptide in Wx and none in some of the stable wx endosperm starch-bound protein extracts using the polyclonal antiserum raised against the Wx protein (Shure et al. 1983). We have used this mono-specific Wx antiserum (kindly provided to us by Dr. Nina Fedoroff) to localize the Wx protein in developing Wx endosperm cells through immuno-gold silver enhancement reactions.

Figure 1. Immunocytological localization of Wx protein in endosperm cells. Sections stained with (A) preimmune serum (B) Wx antiserum. sg = starch grain. cw = cell wall. bar = 2µ.

The kernels at 15 days after pollination were fixed in cacodylate buffer (pH 7.2) containing 3% glutaraldehyde, 2% paraformaldehyde, 2.5% dimethyl sulfoxide and 0.5% acrolein. The thin sections of maize endosperm at crown region were treated with 1:200 diluted Wx antiserum, incubated with secondary antibody conjugated with 15 nm colloidal gold, and stained with silver enhancement reagents. As expected, the labeling as evidenced by the black dots was predominantly limited to the starch grains; background labeling over cytoplasm, organelles, and cell walls was very low (Fig. 1B). Immunostaining with a preimmune serum (1:200 dilution) gave low to undetectable levels of nonspecific staining (Fig. 1A).

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