Dormancy of the barley grain is correlated with gibberellic Acid responsiveness of the isolated aleurone layer

The relationship between barley grain dormancy and gibberellic acid (GA₃) responsiveness of aleurone layers has been investigated. Barley (Hordeum distichum L. cvs Triumph and Kristina) grains were matured under defined conditions in a phytotron. Grains of Triumph plants grown under long-day/warm conditions had lower dormancy levels than grains of plants grown under short-day/cool conditions. Aleurone layers isolated from grains of long-day Triumph plants secreted more α-amylase and had a higher responsiveness to GA₃ as measured by α-amylase secretion. Storage of the grains increased both the percentage of germination and the responsiveness of the aleurone to GA₃. Use of different sterilization methods to break dormancy confirmed the correlation between germination percentage and aleurone layer GA₃ responsiveness. The response of embryoless Triumph grains to GA₃ was lower than that of the isolated aleurone layers, suggesting a role of the starchy endosperm in regulating the GA₃ response of the aleurone layer. Grains of the cultivar Kristina harvested from short day- and long day-grown plants lacked dormancy, and their isolated aleurone layers had a similar responsiveness to GA₃ as measured by α-amylase secretion. The data indicate that the physiological state of the aleurone layers contributes to the percentage germination of the grains.     

Amylases in developing barley seeds

The amylases of developing barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated by colorimetric and electrophoretic methods. Maxima of amylolytic activity appeared in the aleurone layers and starchy endosperm at 5 and 20 days after anthesis. Amylase from 5-day-old aleurone layers could be separated into four rapidly moving bands with α-amylase activity. By 20 days the four bands had been replaced by seven bands of medium mobility. These seven bands of amylase were electrophoretically identical to those observed when mature aleurone layers are treated with gibberellic acid. Immature aleurone layers failed to respond to exogenous gibberellic acid. In the starchy endosperm the seven bands of medium mobility were also present. Calcium-dependent alterations in the electrophoretic mobility and activity of particular bands occurred during the maturation of the starchy endosperm. Treatment of the immature starchy endosperm with papain yielded four forms of β-amylase.     

Characteristics of alpha-Amylase during Germination of Two High-Sugar Sweet Corn Cultivars of Zea mays L

The role of the scutellum and the aleurone in α-amylase production in the high-sugar sweet corn cultivars Illini X-tra Sweet (shrunken-2, sh2) and Illinois 677a (sugary, sugary enhancer; su se) was compared to that in the starchy (Su) hybrid Funks G4646 with the use of α-amylase enzyme assays, isoelectric focusing, electron microscopy, and laser scanning confocal microscopy. The scutellum of Illinois 677a had low levels of α-amylase activity compared to that of Funks G4646 through 10 days after imbibition, and the aleurone of Illini X-tra Sweet had negligible activity. On the isoelectric focusing gels, the Illinois 677a scutellum had fewer α-amylase isozymes at 7 days compared to the Funks G4646 scutellum. The Illini X-tra Sweet aleurone had no α-amylase isozymes. Funks G4646 scutellar epithelial and aleurone cells contained abundant rough endoplasmic reticulum, polysomes, and dictyosomes at 5 and 7 days, respectively. The scutellar epithelial cells of Illinois 677a contained fewer of these structures by 5 days, and the Illini X-tra Sweet aleurone contained mostly lipid bodies through 7 days. Few cytoplasmic membranes and little RNA were detected with laser scanning confocal microscopy in the Illini X-tra Sweet aleurone compared to Funks G4646 at 7 days. These data suggest that the scutellum of Illinois 677a and the aleurone of Illini X-tra Sweet have impaired abilities to produce α-amylase.     

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

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 α-amylase components by isoelectric focusing and rocket-line immunoelectrophoresis. Although aleurone and scutellar tissues appeared to synthesize the same α-amylase components, enzyme was detected first in the scutellum. A larger proportion of scutellar α-amylase was excreted into the endosperm compared to aleurone synthesized α-amylase. Aleurone cells appeared to synthesize appreciably more α-amylase than did scutellar tissue.     

Localization of carboxypeptidase I in germinating barley grain

Activity measurements and Northern blot hybridizations were used to study the temporal and spatial expression of carboxypeptidase I in germinating grains of barley (Hordeum vulgare L. cv Himalaya). In the resting grain no carboxypeptidase I activity was found in the aleurone layer, scutellum, or starchy endosperm. During germination high levels of enzyme activity appeared in the scutellum and in the starchy endosperm but only low activity was found in the aleurone layer. No mRNA for carboxypeptidase I was observed in the resting grain. By day 1 of germination the mRNA appeared in the scutellum where its level remained high for several days. In contrast, little mRNA was observed in the aleurone layer. These results indicate that the scutellum plays an important role in the production of carboxypeptidase I in germinating barley grain.     

Evidence for osmotic regulation of hydrolytic enzyme production in germinating barley seeds

α-Amylase levels in intact seeds of barley (Hordeum vulgare L. cv. Himalaya) reach a maximum at 3 to 4 days of germination while gibberellin levels continue to increase beyond 6 days of germination. In contrast to its effect on half seeds, gibberellic acid does not increase the total amount of α-amylase produced in germinating seeds. The inability of gibberellic acid to stimulate α-amylase production is not related to its availability; rather, evidence suggests that a factor(s) in whole seeds prevents further enhancement of α-amylase formation and accumulation. Hydrolysis products accumulate in the subaleurone space of the endosperm of germinating seeds up to concentrations of 570 milliosmolar. Chromatography of these hydrolysis products indicate the presence of maltose and glucose. Calculations based on reducing sugar determinations show that glucose accounts for as much as 57% of the solutes present in the endosperm fluid. Both maltose and glucose in the range of 0.2 to 0.4 M effectively inhibit the production of α-amylase by isolated barley aleurone layers. This inhibition is quantitatively similar to that brought about by solutions of polyethylene glycol and mannitol. On the basis of these data we propose that hydrolysis products which accumulate in the starchy endosperm of germinating seeds function to regulate the production of hydrolytic enzymes by the aleurone layer.     

Amylases from aleurone layers and starchy endosperm of barley seeds

Amylases from incubated aleurone layers or from starchy endosperm of barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated using acrylamide gel electrophoresis and analytical gel filtration with Sephadex G-200. Electrophoresis of amylase from aleurone layers yields seven visually distinct isozymes with an estimated molecular weight of 43,000. Because each isozyme hydrolyzes β-limit dextrin azure and incorporates calcium-45, they are α-amylases. On Sephadex G-200, amylase from the aleurone layers is separated into seven fractions ranging in estimated molecular weights from 45,000 to 3,000. Little or no activity is observed when six fractions are subjected to electrophoresis. Electrophoresis of only the fraction with the estimated molecular weight of 45,000 gave the seven isozymes. The amylases are heat labile and cannot be stabilized by the presence of substrate or by the protease inhibitor, phenylmethylsulfonylfluoride. Electrophoresis of amylase from the starchy endosperm yields nine β-amylases. Four of these β-amylases are isozymes with an estimated molecular weight of 43,000. The other five forms of β-amylase represent molecular aggregates of the four basic β-amylase monomers. A dimer, a tetramer, and an octamer of β-amylase can be identified with estimated molecular weights of about 86,000, 180,000 and 400,000, respectively. These estimated molecular weights were confirmed on Sephadex G-200. There are five additional fractions of β-amylase with estimated molecular weights ranging from 30,000 to 4,000. These fractions are not observed electrophoretically.     

Secretion of basic and acidic chitinases from salt-adapted and -unadapted winged bean cells

Northern hybridizations were used to study the site of synthesis of three carboxypeptidases (Cpases I-III) which occur in the starchy endosperm of germinating barley grain ( Hordeum vulgare L.). Further evidence was obtained by studying secretion of these enzymes from scutella or aleurone layers separated from germinating grains. Messenger RNA for Cpase II was detected only in developing grain, and the bulk of the mRNA was localized in the starchy endosperm. This suggests that Cpase II is synthesized at the site of its accumulation, the starchy endosperm. In contrast, Cpase I is expressed during germination and the predominant site of synthesis is the scutellum, from which it is secreted into the starchy endosperm. Cpase III is also synthesized during germination, but the bulk of it is synthesized in and secreted from the aleurone layer. Thus, the three carboxypeptidases, all of which seem to play a role in hydrolysis of the reserve proteins in the starchy endosperm during germination, have different sites of synthesis.     

Alpha-amylase secretion by single barley aleurone layers

The secretion of α-amylase from single isolated (Hordeum vulgare L. cv Himalaya) aleurone layers was studied in an automated flow-through apparatus. The apparatus, consisting of a modified sample analyzer linked to a chart recorder, automatically samples the flow-through medium at 1 minute intervals and assays for the presence of α-amylase. The release of α-amylase from aleurone layers begins after 5 to 6 hours of exposure to gibberellic acid and reaches a maximum rate after 10 to 12 hours. The release of α-amylase shows a marked dependence on Ca²⁺, and in the absence of Ca²⁺ it is only 20% of that in the presence of 10 millimolar Ca²⁺. Withdrawal of Ca²⁺ from the flow-through medium results in the immediate cessation of enzyme release and addition of Ca²⁺ causes immediate resumption of the release process. The effect of Ca²⁺ is concentration-dependent, being half-maximal at 1 millimolar Ca²⁺ and saturated at 10 millimolar Ca²⁺. Ruthenium red, which blocks Ca²⁺ but not Mg²⁺ efflux from barley aleurone layers, renders α-amylase release insensitive to Ca²⁺ withdrawal. Inhibitors of respiratory metabolism cause a burst of α-amylase release which lasts for 0.5 to 5 hours. Following this phase of enhanced α-amylase release, the rate of release declines to zero. Pretreatment of aleurone layers with HCl prior to incubation in HCN also causes a burst of α-amylase release, indicating that the inhibitor is affecting the secretion of α-amylase and not its movement through the cell wall. The rapid inhibition of α-amylase release upon incubation of aleurone layers at low temperature (5°C) or in 0.5 molar mannitol also indicates that enzyme release is dependent on a metabolically linked process and is not diffusion-limited. This conclusion is supported by cytochemical observations which show that, although the cell wall matrix of aleurone layers undergoes extensive digestion after gibberellin treatment, the innermost part of the cell wall is not degraded and could influence enzyme release.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 9. DE NOVO SYNTHESIS OF beta-AMYLASE

Germinating rice seeds were fed with [³⁵S]methionine and the incorporation of ³⁵S into β-amylase demonstrated by quantitative immunoprecipitation using rabbit anti-β-amylase immunoglobulin G fraction. Separation of the antigen-antibody complex by Na-dodecylsulfate gel electrophoresis and subsequent radioautography clearly showed the radioactive labeling of the β-amylase molecule. The specific radioactivity of β-amylase derived from scutellum by immunoprecipitation was significantly greater than that of the endosperm. The results strongly indicate that at the onset of germination of rice seeds β-amylase is synthesized de novo in the scutellum and that in later stages there occurs activation of an inactive, latent form of the enzyme associated with starch granules in the endosperm. In later stages of germination this activated form of the enzyme becomes dominant.     

beta-Amylases in Cereals : A Study of the Maize beta-Amylase System

β-Amylase of maize (Zea mays L.) caryopses was studied during development and germination by means of enzymic, electrophoretic, and immunochemical techniques. β-Amylase activity increased during caryopsis development to a maximum value at the beginning of the water content plateau (at this stage the enzyme was located primarily within the pericarp) and then decreased. Almost no β-amylase (activity or antigen) was found in either free or bound forms in the mature maize caryopsis. The activity increased again during seedling growth and reached much higher values. Both the aleurone layer (to a major extent) and the scutellum produced and secreted β-amylase during germination, the secretion being stimulated by Ca²⁺. No posttranslational modification of the enzyme was detected during germination. The molecular specific activity of the enzyme remained unchanged during the observed periods, indicating that the regulation of the activity is based essentially on protein turnover. The enzyme from developing and germinating caryopses was found to be identical in terms of antigenicity, isoelectric point, and molecular mass to the β-amylases extracted from the roots and the leaves of the maize seedling. The maize β-amylase resembles in all respects the ubiquitous β-amylase described for rye and wheat, whereas the major β-amylase of those cereals appears to be lacking in the maize caryopsis.     

Differential synthesis in vitro of barley aleurone and starchy endosperm proteins

To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA₃. B and C hordein polypeptides and the salt-soluble proteins β-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the α-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, β-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of α-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for α-amylase, these hormones have the opposite effect on ASI mRNA levels.     

Messenger RNAs from the Scutellum and Aleurone of Germinating Barley Encode (1-->3,1-->4)-beta-d-Glucanase, alpha-Amylase and Carboxypeptidase

Polyclonal antibodies raised against barley (1→3,1→4)-β-d-glucanase, α-amylase and carboxypeptidase were used to detect precursor polypeptides of these hydrolytic enzymes among the in vitro translation products of mRNA isolated from the scutellum and aleurone of germinating barley. In the scutellum, mRNA encoding carboxypeptidase appeared to be relatively more abundant than that encoding α-amylase or (1→3,1→4)-β-d-glucanase, while in the aleurone α-amylase and (1→3,1→4)-β-d-glucanase mRNAs predominated. The apparent molecular weights of the precursors for (1→3,1→4)-β-d-glucanase, α-amylase, and carboxypeptidase were 33,000, 44,000, and 35,000, respectively. In each case these are slightly higher (1,500-5,000) than molecular weights of the mature enzymes. Molecular weights of precursors immunoprecipitated from aleurone and scutellum mRNA translation products were identical for each enzyme.     

Effect of temperature on the synthesis and secretion of alpha-amylase in barley aleurone layers

The effect of temperature on α-amylase synthesis and secretion from barley (c.v. Himalaya) half-seeds and aleurone layers is reported. Barley half-seeds incubated at 15 C in gibberellic acid (GA) concentrations of 0.5 and 5 micromolar for 16 hours do not release α-amylase. Similarly, isolated aleurone layers of barley do not release α-amylase when incubated for 2 or 4 hours at temperatures of 15 C or below following 12 hours incubation at 25 C at GA concentrations from 50 nanomolar to 50 micromolar. There is an interaction between temperature and GA concentration for the process of α-amylase release from aleurone layers; thus, with increasing GA concentration, there is an increase in the Q₁₀ of this process. A thermal gradient bar was used to resolve the temperature at which the rate of α-amylase release changes; thermal discontinuity was observed between 19 and 21 C. The time course of the response of aleurone tissue to temperature was determined using a continuous monitoring apparatus. Results show that the effect of low temperature is detectable within minutes, whereas recovery from exposure to low temperature is also rapid. Although temperature has a marked effect on the amount of α-amylase released from isolated aleurone layers, it does not significantly affect the accumulation of α-amylase within the tissue. At all GA concentrations above 0.5 nanomolar, the level of extractable α-amylase is unaffected by temperatures between 10 and 28 C. It is concluded that the effect of temperature on α-amylase production from barley aleurone layers is primarily on the process of enzyme secretion.     

Inhibition of Gibberellic Acid-induced alpha-Amylase Formation by Polyethylene Glycol and Mannitol

Both polyethylene glycol (PEG) and mannitol inhibit gibberellic acid-induced α-amylase production in barley aleurone layers. The effect of the osmotic solution is on enzyme synthesis rather than α-amylase secretion. The inhibition of α-amylase synthesis does not appear to be mediated via an indirect effect on respiration or protein synthesis. Rather it seems that the osmotic solutions reduce the extent of proteolysis of the stored aleurone grain protein thus making available less substrate for new protein synthesis.     

Enzymic mechanism of starch breakdown in germinating rice seeds : 15. Immunochemical study on multiple forms of amylase

The formation of multiple forms of amylases in germinating rice (Oryza sativa L. cv Kimmaze) grains was examined by means of isoelectric focusing, cross-immunoelectrophoresis, and rocket-line immunoelectrophoresis followed by a reaction of enzymic characterization by using β-limit dextrin or starch as substrate. The constituents detected by isoelectric focusing were identified as three electrophoretically heterogeneous antigens. The major α-amylase bands A and B corresponded to a same antigen, the main portion of which was produced within 2 days' germination. The bulk of α-amylase D appeared between 2 and 4 days' germination. Component E, a debranching enzyme according to its action on the β-limit dextrin, already exists in the ungerminated seeds; its amount decreases within the first 2 days of germination and increases again thereafter.

Evidence showing that β-amylase (band C) is produced by the scutellum at an early stage of germination was provided. The enzyme appeared in a suspension of the scutellum after a prolonged incubation.

     

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

An immunological assay has been used to investigate the synthesis of (1→3,1→4)-β-glucanase (EC 3.2.1.73) isoenzymes from isolated barley aleurone layers and scutella. Enzyme release from both tissues is enhanced by 1 micromolar gibberellic acid and 10 millimolar Ca²⁺, although increases induced by gibberellic acid are observed only in the presence of Ca²⁺. Isoenzyme I is synthesized predominantly in the scutellum, while isoenzyme II is synthesized exclusively in the aleurone. A third, putative isoenzyme III has been detected in significant proportions in scutellar secretions and may also be secreted from aleurone layers. Both gibberellic acid and Ca²⁺ appear to preferentially enhance isoenzyme II secretion from the aleurone and isoenzyme III secretion from scutella. The patterns of isoenzyme secretion are suggestive of tissue-specific differences in expression of the genes which code for (1→3,1→4)-β-glucanase isoenzymes. Qualitatively similar results were obtained with barley cultivars harvested in Australia and North America.     

Water stress enhances expression of an alpha-amylase gene in barley leaves

The amylases of the second leaves of barley seedlings (Hordeum vulgare L. cv Betzes) were resolved into eight isozymes by isoelectric focusing, seven of which were β-amylase and the other, α-amylase. The α-amylase had the same isoelectric point as one of the gibberellin-induced α-amylase isozymes in the aleurone layer. This and other enzyme characteristics indicated that the leaf isozyme corresponded to the type A aleurone α-amylase (low pI group). Crossing experiments indicated that leaf and type A aleurone isozymes resulted from expression of the same genes.

In unwatered seedlings, leaf α-amylase increased as leaf water potential decreased and ABA increased. Water stress had no effect on β-amylase. α-Amylase occurred uniformly along the length of the leaf but β-amylase was concentrated in the basal half of the leaf. Cell fractionation studies indicated that none of the leaf α-amylase occurred inside chloroplasts.

Leaf radiolabeling experiments followed by extraction of α-amylase by affinity chromatography and immunoprecipitation showed that increase of α-amylase activity involved synthesis of the enzyme. However, water stress caused no major change in total protein synthesis. Hybridization of a radiolabeled α-amylase-related cDNA clone to size fractionated RNA showed that water-stressed leaves contained much more α-amylase mRNA than unstressed plants. The results of these and other studies indicate that regulation of gene expression may be a component in water-stress induced metabolic changes.

     

Enzymic mechanism of starch breakdown in germinating rice seeds : 12. Biosynthesis of alpha-amylase in relation to protein glycosylation

The biosynthetic mechanism of α-amylase synthesis in germinating rice (Oryza sativa L. cv. Kimmazé) seeds has been studied both in vitro and in vivo. Special attention has been focused on the glycosylation of the enzyme molecule. Tunicamycin was found to inhibit glycosylation of α-amylase by 98% without significant inhibition of enzyme secretion. The inhibitory effect exerted by the antibiotic on glycosylation did not significantly alter enzyme activity.

In an in vitro system using poly-(A) RNA isolated from rice scutellum and the reticulocyte lysate translation system, a precursor form of α-amylase (precursor I) is formed. Inhibition of glycosylation by Tunicamycin allowed detection of a nonglycosylated precursor (II) of α-amylase. The molecular weight of the nonglycosylated precursor II produced in the presence of Tunicamycin was 2,900 daltons less than that of the mature form of α-amylase (44,000) produced in the absence of Tunicamycin, and 1,800 daltons less than the in vitro synthesized molecule.

The inhibition of glycosylation by Tunicamycin as well as in vitro translation helped clarify the heterogeneity of α-amylase isozymes. Isoelectrofocusing (pH 4-6) of the products, zymograms, and fluorography were employed on the separated isozyme components. The mature and Tunicamycin-treated nonglycosylated forms of α-amylase were found to consist of three isozymes. The in vitro translated precursor forms of α-amylase consisted of four multiple components. These results indicate that heterogeneity of α-amylase isozymes is not due to glycosylation of the enzyme protein but likely to differences in the primary structure of the protein moiety, which altogether support that rice α-amylase isozymes are encoded by multiple genes.