Proteins of the embryo and endosperm are readily labeled with 35S-methionine following the introduction of the label directly into kernels on developing ears. For the labeling experiments, immature ears (20-24 days post-pollination) were brought into the lab from the field, and a portion (about the distal third) of the ear broken off. A ring of kernels near the point of breakage were removed, and the label was introduced into selected kernels on the ear segment by injection of the solution directly into the base of the kernel, using a Hamilton syringe. The amount of 35S-methionine introduced into each kernel was about 80 uci (1.76 x 108 dpm), in a volume of 7.5 - 10 ul. Injected kernels were labeled with a marking pen for later identification. The ear segment was incubated at 30 C for 2-4 days in a water-saturated atmosphere. After the appropriate incubation period, injected kernels were removed from the ear, and each embryo and endosperm individually macerated in 1 ml of SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2.3% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 10% glycerol). These samples were boiled for 5 minutes and centrifuged in a Brinkmann tabletop centrifuge for 2-5 minutes. Aliquots (1-5 ul) of the supernatant were spotted onto filter paper disks, and placed in cold 10% trichloroacetic acid (TCA) to precipitate macromolecules. The filters were boiled in the same solution for ten minutes and washed with 5% TCA, followed by a wash with 95% ethanol. The filters were dried and counted in a scintillation counter. Incorporation of label was on the order of 104 dpm/ul sample, for both embryo and endosperm, for an incorporation efficiency of 5-10%. The samples were electrophoresed in polyacrylamide gels, stained with Coomassie Brilliant Blue, and dried down. The dried gels were used to expose Kodak X-OMAT AR film. The bands on the exposed autoradiograms corresponded to the stained bands in the gel.
The efficiency of labeling in this manner is variable, and the embryo and endosperm of the same kernel may show a significant difference in the amount of label incorporated. It should be noted that the site of injection of the label is critical: attempts to introduce the label by injection into the crown were not successful, as most of the label solution was extruded. Kernels injected at the crown show very little, if any, incorporation of label by the TCA precipitation assay, and no bands were detectable on autoradiograms in lanes in which such kernels were run. Studies involving the introduction of labelled amino acids into kernels with the ear remaining on the plant are in progress at the present time.
The 35S-methionine used in these preliminary studies was of the same quality as that used routinely in our laboratory as a tracer in an in vitro protein synthesizing system (Amersham SJ.204, 1385 ci/mmol).
Alan L. Kriz
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