Changes in Levels of α-Amylase Components in Barley Tissues during Germination and Early Seedling Growth

Abstract

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, subscu-tellar endosperm, aleurone-scutellum interface, remaining aleurone, sub-aleurone endosperm, and core endosperm. These tissues were analyzed for a-amylase components by isoelectric focusing and rocket-line immu-noelectrophoresis. 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. Aleu-rone 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) and, finally, to a high value of 50% (12). Some studies were based on enzyme histological (23) or im-munohistological (1 1, 12) techniques that were not quantitative although they gave convincing qualitative data. In others (24), isolated, incubated barley embryos were shown to contain little or no a-amylase but possible secretion of the enzyme was not investigated nor was the effect of barley cultivar. Little quantitative information is available on the relative amounts of a-amylase isoenzymes in tissues of barley kernels at different stages of germination and early seedling growth. In the present study an attempt has been made to obtain such data. Barley kernels, germinated for different periods of time, were dissected into a number of sections. Immune sera specific for each a-amylase group (I and II) were used in conjunction with the sensitive technique of line immunoelectrophoresis (16) and 'Supported by the Union Generale de la Brasserie Fransaise. Paper No. 531 ofthe Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Canada R3C 3G8. 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 METHODS Germination Conditions. The two-rowed barley (Hordeum vul-gare 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 de-husked 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.