Application of direct tissue rocket-line immunoelectrophoresis to the study of a-amylase production and its localization in wheat seeds during the early stages of germinationLine immunoelectrophoresis was adapted to the quantitative analysis of antigens directly from tissues without any previous extraction. The technique was used in a study on the production and the distribution of two a-amylase isozymes (I and 11) in tissues of wheat seeds that sprouted (radicle emergence) after different periods of germination. The a-amylase content was examined in the scutellum and in three tissue fractions dissected from a 0.5 mm slice parallel and next to the scutellum (aleurone layer, outer and inner part of starchy endosperm). At the stages of germination investigated, a-amylase I was not detected in these tissues. The results indicate that a-amylase I1 accumulated in the scutellum and also in the aleurone layer after one day's germination; after about two day's germination, the a-amylase I1 content in the aleurone layer was similar to or higher than that in the scutellum. Moreover, the production and distribution of a-amylase I1 do not seem to be correlated with the radicle emergence.
IntroductionRecent enzymic and immunohistological studies on aamylase production during the germination of whole cereal kernels have emphasized the role of the scutellum in its production [l-41. In these studies, the enzyme was clearly detected in the aleurone layer only after several day's germination. The difficulty of detecting the enzyme in the aleurone layer at the beginning of germination may be due to a reduced permeability of the tissue to the substrate and to the antibodies. Such a difficulty was reported in a study on the 1 1s globulin of pumpkin seeds for its immunohistological detection in the protein bodies [51. In order to overcome this difficulty and to get a quantitative insight into the productivity of both tissues at the beginning of germination, their aamylase contents were compared by rocket-line immunoelectrophoresis [61. The aleurone layer, the outer and inner parts of the starchy endosperm contained in a 0.5 mm thick slice cut next to the scutellum and in parallel direction to the scutellum, as well as the scutellum were analyzed. Rocket-line immunoelectrophoresis was adapted for quantitating the enzyme directly from the tissues because only minute amounts of tissues were collected and because their enzyme contents at this early stage of germination were small. Direct tissue analysis avoids the dilution involved in the extraction procedure. The tissues were dissected from three groups of germinating seeds which were distinguished by the emergence of the radicle after different periods of time. in order to eliminate dormancy (100 % germination capacity). The seeds were soaked for 10 min in 1 % sodium hypochlorite, then washed and germinated on filter paper moistened with deionized water at 20 "C in the dark. Three groups of seeds were gathered: 1) The first 10 seeds which had "just sprouted" (visible protusion of the ra...
Polymorphism of barley a-amylase was studied using immuno-electrophorests and immuno-absorption in a gel medium with an anti-barley malt a-amylase immune serum: a-amylase from germinated seeds is antigenically heterogeneous. The two antigens which were demonstrated evolved differently upon germin ation. The bulk of the enzyme activity extracted from the seeds at different stages of germination differed antigenically from a-amylases found in devel oping barley seeds.
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