Kernels of Klages barley (Hordeum vulgare L.) were germinated for 1 to 4 days on moist sand at 18°C. Representative kernels from each time period were dissected to give the following fractions: scutellum, subscutellar endosperm, aleurone-scutellum interface, remaining aleurone, subaleurone endosperm, and core endosperm. These tissues were analyzed for a-amylase components by isoelectric focusing and rocket-line immunoelectrophoresis. Although aleurone and scutellar tissues appeared to synthesize the same a-amylase components, enzyme was detected first in the scutellum. A larger proportion of scutellar a-amylase was excreted into the endosperm compared to aleurone synthesized a-amylase. Aleurone cells appeared to synthesize appreciably more a-amylase than did scutellar tissue.There has been much discussion recently on the relative importance of aleurone and scutellar tissues in the production of hydrolytic enzymes during germination and early seedling growth of cereal grains (3,4,11,23,24). a-Amylase synthesis in barley has received particular attention because ofthe technological importance of this enzyme in malting and brewing and also because synthesis of the enzyme in aleurone cells from barley kernels has been used as a model system to study hormonal action on protein synthesis (7,13). Estimates on the contribution made by the scutellum to total a-amylase synthesis in barley seedlings have varied from little or none (24), to a relatively small proportion of 14% (4) direct tissue line immunoelectrophoresis (9) to determine quantitatively changes in the levels of a-amylases I and II in these different sections. These results provide information on the sites of synthesis of the two groups of a-amylase and the rates at which the enzymes move into the endosperm. MATERIALS AND METHODSGermination Conditions. The two-rowed barley (Hordeum vulgare L. cv Klages) used in this study was grown in test plots in Brandon, Manitoba in 1976. Barley kernels were dehusked by a 3-h steep in 50% (v/v) H2SO4 (8) followed by thorough washing with sterile, distilled H20. Petri dishes, each containing 25 dehusked kernels, 50 g sterile sand, and 10 ml sterile H20, were incubated in a germination chamber at 1 8°C and high humidity. After 24, 48, 72, and 96 h, Petri dishes were removed and frozen at -20°C until analyzed.Dissection. Frozen kernels were dissected on a cold plate using an Olympus dissecting microscope. Tissues were frozen immediately after they were separated from the kernel. Ten kernels from each sample were completely dissected to give the following six fractions (Fig. 1): center portion of the scutellum, crushed cell layer and subscutellar starch, strip of aleurone and adhering scutellum along the aleurone-scutellum interface (Fig. 1A), residual aleurone, sub-aleurone starch, and core starch. Care was taken to ensure that each fraction was clean and not contaminated by material from another fraction. Frozen tissues were freeze-dried, weighed, and ground to a fine powder. a-Amylase levels did not appear to be a...