Fig. 3.

Analysis of GS polypeptide, holoenzyme and GS activity in the leaves of RUB-Gmglnβ₁ and CaMV35S-Gmglnβ₁ alfalfa transformants. (A) Five µg of protein from leaf extracts of alfalfa control plants and independent transformants with the RUB-Gmglnβ₁ and CaMV35SGmglnβ₁ gene constructs was subjected to SDS PAGE followed by western blot analysis using anti-GS antibodies. Immunoreactive bands corresponding to GS₁ (39–40 kd) and GS₂ (42–43 kd) polypeptides are shown. (B) Band intensities of the GS₁ band from panel A is plotted. (C) Twenty five µg of of leaf extracts from the same plants as in panel A were subjected to native gel electrophoresis followed by western blot analysis using anti-GS antibodies. Immunoreactive bands corresponding to the endogenous GS₂ (MsGS₂) and GS₁ (MsGS₁) and the transgene product (GmGS₁) are designated. (D) Close-up of a immunoreactive native gel profile of leaf protein from control, RUB-Gmglnβ₁ (#1) and CaMV35S-Gmglnβ₁ (#12) transformants showing the bands corresponding to the endogenous MsGS₂ and MsGS₁, and the transgene product GmGS₁. Asterisk represents the broad immunoreactive band probably representing a mixture of heteromers made of the endogenous GS₁ and the transgene product. Bands a and b represent the turnover products of the MsGS and GmGS₁ holoproteins respectively. (E) GS activity was measured in the leaf protein samples used for western analysis in panel A. Average values ±SE of three experiments were plotted as µmol γ-glutamyl hydroxamate produced per minute per mg of protein at 30° C.