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.
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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