Abstract
Localization of the product of a bacterium-originated reporter gene (β-glucuronidase: GUS) was studied using histochemical and electron-immunocytochemical methods in transgenic tobacco plants introduced by GUS reporter gene with no targeting signals. In leaf and stem cells the insoluble blue dye associated with GUS gene product was found without exception on the subcellular organelles and in the moiety around them in the cytoplasm. However, in the cells with higher GUS activity, uniform blue color was often observed all over the cell as if blue ink had dissolved. When such cells, which have high GUS activity, were treated with 1 M KNO3 solution, the closed area with blue uniform color began to shrink and could be distinguished from cytoplasm and the cell wall as a result of plasmolysis. The blue round compartment was very similar to a vacuole which could also be detected as a red closed area after neutral red-staining and plasmolysis. GUS localization was further studied electron microscopically by the method of protein A-immunogold. Many gold particles were found in the electron-dense small bodies in vacuoles, however they were sparsely found in the other areas. These results indicated that GUS was produced in the cytoplasm, and was finally secreted into the vacuole in these cells.
