Plant Physiol, July 2002, Vol. 129, pp. 1045-1053

Immunohistochemistry of Active Gibberellins and Gibberellin-Inducible -Amylase in Developing Seeds of Morning Glory¹

Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan (A.N., S.P., X.Z.-J., M.N., I.Y.); and Bio-oriented Technology Research Advancement Institution (BRAIN), Minato-ku, Tokyo 105-0001, Japan (X.Z.-J.)

Gibberellins (GAs) in developing seeds of morning glory (Pharbitis nil) were quantified and localized by immunostaining. The starch grains began to be digested after the GA contents had increased and reached a plateau. Immunohistochemical staining with the antigibberellin A₁-methyl ester-antiserum, which has high affinity to biologically active GAs, showed that GA₁ and/or GA₃ were localized around starch grains in the integument of developing young seeds, suggesting the participation of GA-inducible -amylase in this digestion. We isolated an -amylase cDNA (PnAmy1) that was expressed in the immature seeds, and using an antibody raised against recombinant protein, it was shown that PnAmy1 was expressed in the immature seeds. GA responsiveness of PnAmy1 was shown by treating the young fruits 9 d after anthesis with GA₃. RNA-blot and immunoblot analyses showed that PnAmy1 emerged soon after the rapid increase of GA_1/3. An immunohistochemical analysis of PnAmy1 showed that it, like the seed GA_1/3, was also localized around starch grains in the integument of developing young seeds. The localization of GA_1/3 in the integument coincident with the expression of PnAmy1 suggests that both function as part of a process to release sugars for translocation or for the further development of the seeds.