Fig. 3.

Confocal laser scanning microscopy of starch granules from the endosperm of developing grains at the late grain filling stage (30 DAP) of transgenic barley plants expressing HvGBSSIa^WT-eGFP (B, G, H, K) or HvGBSSIa^CDC_Alamo-eGFP (A, E, F). (A), (B), (E) and (F) are recorded with same sensitivity, whereas (D), (G) and (H) were recorded with lower sensitivity to avoid oversaturation. (C) The relative fluorescence intensities were much stronger in HvGBSSIa^WT-eGFP starch granules than in HvGBSSIa^CDC_Alamo-eGFP starch granules (n=10). Bars indicate standard errors. (D) Magnified subcellular body from HvGBSSIa^CDC_Alamo-eGFP endosperm cells from panel A. (E) The subcellular bodies in panel (A) disappeared after treatment with proteinase K (1 mg ml^–1 in H₂O) for 10 min. (G, H) Both HvGBSSIa^WT-eGFP and HvGBSSIa^CDC_Alamo-eGFP are localized into concentric spheres (indicates by arrows) within starch granules. (H) These concentric spheres became blurred (indicated by the arrow) upon treatment with α-amylase. (I, J) Transmission light was used to visualize subcellular structures in thin sections of endosperm cells simultaneously with localization of HvGBSSIa^CDC_Alamo-eGFP recorded as overlaid eGFP fluorescence. (K, L) HvGBSSIa^WT-eGFP (green; K) was located in granules inside chloroplasts (red fluorescence) in leaves whereas HvGBSSIa^CDC_Alamo-eGFP (L) was mostly localized in subcellular bodies (green, indicated by arrows) outside of the red chloroplasts. The scale bars in (A, B, E) are 25 µm; the scale bar in (I) is 20 µm; the scale bars in (F, H) are 10 µm; the scale bar in (G) is 7.5 µm; the scale bars in (J, K, L) are 5 µm; and the scale bar in (D) is 1 µm.