Title LOCALIZATION AND TIME OF SYNTHESIS OF WHEAT GERM AGGLUTININ IN

approved Redacted for Privacy Ralk S. Quatrano The activity of a highly purified plant lectin, wheat germ agglutinin (WGA), has been detected in wheat embryos as young as 25 days post-anthesis. The amount of hemagglutinating activity present in young embryos increases to a maximum level at 45 days post-anthesis. No lectin activity was found in the endosperm, roots, leaves or in a wheatembryo tissue culture line. In order to test for lectin activity during development, a procedure for isolating large quantities of mature and developing wheat embryos has been devised. Grain is ground in a Waring Blendor, collected on a sieve (12 mesh/inch), floated on 2M sucrose and purified at the interface of 1M-2M sucrose step gradient. From 100 grams of mature wheat , one to two grams of embryos can be purified by this procedure. There is no contamination of the embryos with other wheat tissues. Developing embryos of T. aestivum, cultivar Twin were labelled by placing the cut stems of 28 day post-anthesis flowers in a solution containing H 2 35 SO 4 for 10 days. The embryos were extracted with 0.05 M HC1 and the resulting extract was chromatographed on a chitin affinity column (Bloch, R. and M. Burger, 1974. Biochem. Biophys. Res. Commun. 58: 113). Approximately 0.12% of the labelled material extracted by 0.05 M HC1 eluted in a peak coincident with authentic WGA. The identity of the 35 S protein eluting from the chitin column in 0.05 M HC1 was further substantiated by its immunological cross-reaction with purified rabbit anti-WGA IgG followdd by autoradiography of the precipitation arc. Wheat germ agglutinin appears to be synthesized de novo in developing wheat embryos. Antibodies directed against WGA were raised in rabbits by intramuscular injection of 3.0 mg of purified WGA. Ammonium sulfate fractionation of the antiserum followed by DEAE-cellulose chromatography yielded a population of gamma immunoglobulins active against wheat germ agglutinin. The purified antibody was used in immunodiffusion and immunoelectrophoresis tests to detect WGA in embryos, endosperm, roots and leaves of 3 day-old seedlings, and in an embryo tissue culture line. Only wheat embryos contained a protein that would cross-react with the purified rabbit anti-WGA IgG. A radioimmune assay has been devised to increase the sensitivity of detecting wheat germ agglutinin in tissue extracts. Using constant amounts of 14 C-wheat germ agglutinin and rabbit anti-WGA IgG, 0.2 pg of unlabelled wheat germ agglutinin can be detected by this method. Localization and Time of Synthesis of Wheat Germ Agglutinin in Developing Wheat (Triticum aestivum, L).