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, 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...

Localization of Carboxypeptidase I in Germinating Barley Grain

“…This conclusion is supported by so si s2 s3 s5 a2 to be secreted by the scutellum (22). Scutellum also secretes little a-amylase (8,16). In addition, some RNAases and proteinases may originate in the scutellum (15).…”

“…Because lane a2 contains very low levels of carboxypeptidase mRNA compared to lane s2, the scutellum specific expression of carboxypeptidase I is apparent. As an additional control, the filter was hybridized with a probe for high pI a-amylase, which in germinating grain is predominantly expressed in the aleurone layer (7,8). As expected, the bulk ofthe a-amylase label of predicted size (17) was localized in the aleurone sample.…”

Development of (1→3,1→4)-β-d-Glucan Endohydrolase Isoenzymes in Isolated Scutella and Aleurone Layers of Barley (Hordeum vulgare)

“…Similarly, aamylase is now known to be secreted from both tissues (6,8,13,16) although, in contrast to the (1--3,1-+4).q3-glucanases, no major differences in a-amylase isoenzyme patterns are apparent (8). The preferential disappearance of (1-+3,1-4)-f3-glucanase isoenzymes II and III from aleurone and scutellar secretions when Ca2" is withdrawn from the medium may be compared with the disappearance of a-amylase group B isoenzymes following Ca2+ withdrawal from isolated aleurone layers (7).…”

“…Cells of the scutellar epithelial layer also appear to secrete hydrolytic enzymes which participate in the degradation of barley endosperm (4,6,8,13,16), although this has been disputed on the basis that the aleurone cells which adhere tightly to the periphery of the scutellum complicate interpretation of experimental data (2,14). Two (1--3, 1 -4)-f3-glucan 4-glucanohydrolases (EC 3.2.1.73), which specifically depolymerize (1--3,1-4)-#-glucans of endosperm cell walls, have been purified from germinating barley and characterized (20,21).…”

Messenger RNAs from the Scutellum and Aleurone of Germinating Barley Encode (1→3,1→4)-β-d-Glucanase, α-Amylase and Carboxypeptidase

“…Many of the hydrolases which participate in endosperm mobilization are secreted from the aleurone layer in response to the phytohormone gibberellic acid (7,21). Despite suggestions that the scutellum plays no part in endosperm modification (22), there is increasing evidence that cells ofthe scutellar epithelial layer are also capable of secreting hydrolytic enzymes, in particular a-amylase and cell wall-degrading enzymes, and that these contribute to the degradation of the endosperm in germinating barley (3,11,16,24). Furthermore, the initial site of protease activity in germinating barley is also adjacent to the epithelial cells ofthe scutellum (19).…”