Sugar sensing and α-amylase gene repression in rice embryos

We used a transient expression system to study the mechanism by which carbohydrates repress a rice (Oryza sativa L.) α-amylase (EC 3.2.1.1) gene. Exogenously fed metabolizable carbohydrates are able to elicit repression of the α-amylase gene RAmy3D in the rice embryo, and our results indicate that repression is also triggered efficiently by endogenous carbohydrates. Glucose analogs that are taken up by plant cells but not phosphorylated by hexokinase are unable to repress the α-amylase gene studied, while 2-deoxyglucose, which is phosphorylable but not further metabolized, down-regulates RAmy3D promoter activity, indicating a role for hexokinase in the sugar-sensing mechanism triggering repression of the RAmy3D gene. We tested two different hexokinase inhibitors, mannoheptulose and glucosamine, but only the latter was able to relieve RAmy3D promoter activity from repression by endogenous carbohydrates. This correlates with the higher ability of glucosamine to inhibit the activity of rice hexokinases in vitro. The glucosamine-mediated relief of RAmy3D promoter activity from repression by endogenous carbohydrates does not correlate with a reduced rate of carbohydrate utilization. Received: 22 April 1997 / Accepted: 9 September 1997     

Glucose and glycerol repression of α-amylase in Streptomyces kanamyceticus and isolation of deregulated mutants

Summary Glucose and glycerol at concentrations of 2 % negatively affected amylase synthesis in plate and submerged Streptomyces kanamyceticus cultures. This microorganism was insensitive to growth inhibition by glucose analogs and deregulated mutants were identified by a clearing zone around colonies grown on starch and glycerol or glucose, and selected. Three kinds of mutants were obtained: one insensitive to glucose (Mutant 41), another insensitive to glycerol repression (Mutant E) and the last (Mutant 29) an amylase-hyperproducing mutant, albeit regulated by glucose or glycerol like the wild type. The levels of glucokinase, an enzyme involved in catabolite regulation of Enterobacteria, were determined and results showed no differences between the parental strain and the mutants.     

The mode of secretion of α-amylase in barley aleurone layers

The involvement of the endomenbrane system of barley (Hordeum vulgare L.) aleurone cells in the secretion of gibberellin-induced hydrolases has been investigated at the biochemical level. Our results show that at least 40–60% of the -amylase activity in homogenates of aleurone layers occurs in a membrane-bound, latent form. The latent -amylase can be assayed quantitatively following disruption of membranes by treatment with Triton X-100, ethanol, sonication, or osmotic shock and shear. The association of -amylase with the membrane is not an artifact arising from homogenization of the tissue, and acid protease is also enriched in the same subcellular fraction as the -amylase. The membrane fraction with which the -amylase is associated has many properties of the endoplasmic reticulum (ER). When membranebound -amylase is prepared in buffers containing 3 mM MgCl₂ two fractions from a sucrose step gradient contain most of the -amylase activity. These fractions are enriched in the ER marker enzyme, NADH-dependent cytochrome-c reductase, and show densities characteristic of smooth and rough ER during subsequent purification on continuous gradients. In step gradients prepared with ethylenediaminete-traacetic-acid-treated membranes, -amylase activity is contained primarily in one fraction having the density of smooth ER. Electron microscopy of the purified fractions is consistent with -amylase being associated with smooth and rough ER. However, it has not been ruled out that the enzyme is also associated with plasma membrane, Golgi membranes, or tonoplast. Examination of the isoenzyme patterns of secreted, of total-homogenate and of membrane-associated -amylases, as well as the results from pulsechase experiments using L-[³H]leucine for labeling of -amylase, are all consistent with the hypothesis that membrane-associated -amylase is an intermediate in the secretory process.Abbreviations CNTPE N-carbobenzoxy-L-tyrosine p-nitrophenylester - Cyt oxidase Cytochrome oxidase - ER endoplasmic reticulum - EDTA ethylenediaminetetraacetic acid - GA₃ gibberellic acid - IDPase inosine diphosphatase - K⁺-ATPase pH 6.5 K⁺-stimulated adenosine triphosphatase - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - NADH: Cyt c reductase cyanide-insensitive NADH-linked cytochrome-c reductase - RER rough endoplasmic reticulum - Tris tris-(hydroxymethyl)-aminomethane     

RNA complementary to α-amylase mRNA in barley

Two experimental approaches demonstrate that different types of RNA complementary to -amylase mRNA are present in barley. S1 nuclease assays identify an RNA that is complementary to essentially the full length of both the type A and type B -amylase mRNAs. Complementarity, however, is imperfect: the S1 nuclease-resistant products can only be identified if they are electrophoresed as RNA-DNA hybrids. This RNA is present in developing endosperm + aleurone tissue and in mature aleurone tissue cultured in the absence of hormonal treatment or in the presence of abscisic acid, but not in shoot or root tissue. In mature aleurone tissue treated with abscisic acid, its steady-state abundance is similar to that of -amylase mRNA. Northern blot analysis indicated the presence of a second type of antisense RNA. Under conditions of moderate stringency, antisense-specific probes detect discrete hybridizing species of 1.6, 1.4, and 1.0 kilobases in mature aleurone and shoot tissues that do not represent spurious hybridization to rRNA, -amylase mRNA, or the abundant, G+C-rich mRNA for a probable amylase/protease inhibitor. The different results are consistent with the fact that the hybridization assay can tolerate relatively short regions of complementarity separated by large, nonhomologous sequences, while the nuclease protection assay cannot.     

Cytochemical localization and antigenicity of α-amylase in barley aleurone tissue

Summary Gibberellic-acid(GA₃)-induced -amylase has been localised in barley aleurone layers using cytochemical methods and light microscopy. Evidence obtained from the use of a starch substrate film method as well as immunofluorescence indicated that the first amylase to appear in the cell was associated with aleurone grains, apparently with the outer membrane, and also with the peripheral cytoplasm. In GA₃-treated tissue, the amylase distribution was much more diffuse, although patchy, throughout the cytoplasm and it tended to accumulate in the endosperm side of the cell. The possibility that the aleurone grain membrane is the site of gibberellin-induced enzyme synthesis and that it proliferates to become rough endoplasmic reticulum is considered. Immunological information was obtained which supports earlier indications that induced -amylase consists of two different proteins, each with molecular heterogeneity.     

Transglycosylation by barley α-amylase 1

The transglycosylation activity of barley α-amylase 1 (AMY1) and active site AMY1 subsite mutant enzymes was investigated. We report here the transferase ability of the V47A, V47F, V47D and S48Y single mutants and V47K/S48G and V47G/S48D double mutant AMY1 enzymes in which the replaced amino acids play important role in substrate binding at subsites at −3 through −5. Although mutation increases the transglycosylation activity of enzymes, in the presence of acceptors the difference between wild type and mutants is not so significant. Oligomer transfer reactions of AMY1 wild type and its mutants were studied using maltoheptaose and maltopentaose donors and different chromophore containing acceptors. The conditions for the chemoenzymatic synthesis of 4-methylumbelliferyl-α-d-maltooligosaccharides (MU-α-d-MOSs) were optimized using 4-methylumbelliferyl-β-d-glucoside as acceptor and maltoheptaose as donor. 4-Methylumbelliferyl-α-d-maltoside, -maltotrioside, -maltotetraoside and -maltopentaoside have been synthesized. Products were identified by MALDI-TOF MS. ¹H and ¹³C NMR analyses showed that AMY1 V47F preserved the stereo- and regioselectivity. The produced MU-α-d-MOSs of degree of polymerization DP 2, DP 3 and DP 5 were successfully applied to detect activity of Bacillus stearothermophilus maltogenic α-amylase, human salivary α-amylase and Bacillus licheniformis α-amylase, respectively in a fast and simple fluorometric assay.     

Differential expression of α-amylase genes in germinating rice and barley seeds

Steady-state levels of mRNA from individual -amylase genes were measured in the embryo and aleurone tissues of rice (Oryza sativa) and two varieties of barley (Hordeum vulgare L. cv. Himalaya and cv. Klages) during germination. Each member of the -amylase multigene families of rice and barley was differentially expressed in each tissue. In rice, -amylase genes displayed tissue-specific expression in which genes RAmy3B, RAmy3C, and RAmy3E were preferentially expressed in the aleurone layer, genes RAmy1A, RAmy1B and RAmy3D were expressed in both the embryo and aleurone, and genes RAmy3A and RAmy2A were not expressed in either tissue. Whenver two or more genes were expressed in any tissue, the rate of mRNA accumulation from each gene was unique. In contrast to rice, barley -amylase gene expression was not tissue-specific. Messenger RNAs encoding low- and high-pI -amylase isozymes were detectable in both the embryo and aleurone and accumulated at different rates in each tissue. In particular, peak levels of mRNA encoding high-pI -amylases always preceded those encoding low-pI -amylases. Two distinct differences in -amylase gene expression were observed between the two barley varieties. levels of high-pI -amylase mRNA peaked two days earlier in Klages embryos than in Himalaya embryos. Throughout six days of germination, Klages produced three times as much high-pI -amylase mRNA and nearly four times as much low-pI -amylase mRNA than the slower-germinating Himalaya variety.     

A tetrameric inhibitor of insect α-amylase from barley

A tetrameric inhibitor that is active against α-amylase from the larvae of the insect Tenebrio molitor, but inactive against the enzyme from human saliva and against the endogenous one, has been described in barley endosperm. The subunits of the inhibitor have been identified as the previously characterized proteins CMa, CMb and CMd, of which only CMa was inhibitory by itself.     

Hydrolysis of starch particles using immobilized barley α-amylase

Barley α-amylase has been immobilized on silica particles with diameters between 0.5 and 10 μm using a covalent binding method. Immobilization procedures were adjusted to optimize enzyme activity. The effects of product inhibition, thermal stability and operational stability have been determined. The feasibility of using the immobilized enzyme to hydrolyze wheat starch particles at temperatures below the gelatinization temperature (<55 °C) was proven. The optimal conditions for the hydrolysis were found to be: pH 4.5, 40 °C, calcium ion concentration 0.002 M and immobilized enzyme loading of 30 mg/ml. At these conditions, the immobilized enzyme was able to hydrolyze wheat starch particles at concentrations as high as 100 mg/ml with a final conversion of 90% after 24 h of operation. Maltose and glucose were found to inhibit the immobilized enzyme in a similar manner as reported previously using soluble enzyme. Although the thermostability of the immobilized enzyme was superior to the soluble enzyme, the immobilized enzyme degraded at the same rate as the soluble enzyme during cold wheat starch hydrolysis (operational stability unchanged). Model equations are presented for product inhibition, hydrolysis kinetics and enzyme degradation. Using best-fit parameters, the equations are shown to fit the experimental data well.     

Localization of α-amylase isozymes within the endomembrane system of barley aleurone

Summary The localization of -amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts was studied using electron microscope immunocytochemistry. Antibodies were raised against total barley -amylase, i.e., -amylase containing both highisoelectric point (high-pI) and low-pI isoforms, as well as against purified high- and low-pI isoforms. All antibodies localized -amylase to the endoplasmic reticulum (ER) and Golgi apparatus (GApp) of the aleurone cell, and various controls showed that the labeling was specific for -amylase. Labeling of protein bodies and spherosomes, which are the most abundant organelles in this cell, was very low. There was no evidence that -amylase isoforms were differentially distributed within different compartments of the endomembrane system. Rather, both high- and low-pI isoforms showed the same pattern of distribution in ER and in the cis, medial, and transregions of the GApp. We conclude that in the Himalaya cultivar of barley, all isoforms of -amylase are transported to the plasma membrane via the GApp.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - GApp Golgi apparatus - PBS phosphate buffered saline - PCR partially coated reticulum - PM plasma membrane - TBS Tris buffered saline - TGN trans-Golgi network     

Composition of α-amylase secreted by aleurone layers of grains of himalaya barley

α-Amylases secreted by the aleurone layer of whole barley grains were relatively rich in histidine and relatively poor in glutamate/glutamine and serine when compared to other eukaryotic proteins. The secreted α-amylases had an estimated 0.5 residues each of glucose, mannose and N-acetylglucosamine per molecule of protein (MW 41 400 daltons), and gave positive staining reactions for carbohydrate on sodium dodecylsulfate polyacrylamide gels. Because the average α-amylase molecule had less than one sugar residue per enzyme molecule, it was concluded that secreted α-amylases were heterogeneous with respect to glycosylation. A second protein co-purified with α-amylase, but the amino acid composition of this protein was different from that of barley or wheat α-amylase. This protein was composed of two 21 500 dalton polypeptides. No significant amounts of L-leucine (¹⁴C-U) were incorporated into this second protein in isolated aleurone tissue during incubation with gibberellic acid, perhaps because much of it was already present in the starchy endosperm at the time of hormone addition.     

A cell-free system for the study of α-amylase synthesis in barley aleurone layers

1. A cell-free system capable of alpha-amylase synthesis has been obtained from the aleurone layers of germinating barley. 2. This system requires potassium chloride, sucrose and an amino acid mixture in order to function. The crude preparation does not require calcium chloride. Chloramphenicol inhibits alpha-amylase synthesis as indicated both by increase in measurable enzyme activity and incorporation of l-[U-(14)C]glutamic acid.     

The release of α-amylase through gibberellin-treated barley aleurone cell walls

Localisation of -amylase (EC 3.2.1.1.) in low-temperature-embedded isolated barley (Hordeum vulgare L.) aleurone has been achieved using rhodamine-labelled secondary antibodies and the protein A-gold technique. Treatment with gibberellic acid (GA₃) resulted in an increase of immunofluorescence in the cytoplasm of aleurone cells and also its appearance in specific regions of the cell walls. Cytoplasmic label was neither perinuclear nor associated specifically with aleurone grains as had been found in earlier work, but was present throughout the cytoplasm of all cells. A relatively high level of labelling occurred in hydrolysed wall regions. Label was also associated with plasmodesmata in both hydrolysed and unhydrolysed wall regions. The pattern of labelling indicates that -amylase is released from aleurone via digested wall channels and that, except for the inner wall layer, unhydrolysed regions are impermeable to the enzyme. It is suggested that the resistant wall tubes around plasmodesmata may facilitate enzyme release by providing a pathway for transfer, especially of wall hydrolases, into the more impermeable parts of the wall.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - RER rough endoplasmic reticulum     

Subcellular distribution of β-amylase in developing barley endosperm

Protein bodies were isolated from maturing barley (Hordeum vulgare L. cv. Menuet) endosperm using sucrose density gradient centrifugation. Isolated protein bodies were not contaminated by other organelles such as mitochondria and microbodies, and contained a large amount of the barley storage protein, hordein, indicating that the protein bodies were prepared in high purity and were intact. Enzymatic and immunochemical analysis showed that β-amylase, which was previously thought to be localized in protein bodies, was not detected in the protein body fraction, but was found only in the cytosol fraction. The results show that β-amylase is synthesized as a cytosolic protein in maturing barley endosperm.     

The role of the endoplasmic reticulum in the synthesis and transport of α-amylase in barley aleurone layers

The subcellular site of -amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA₃). Using [³⁵S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA₃ was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker -amylase. This protein was identified as authentic -amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized -amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled -amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of -amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA₃ were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA₃-treated aleurone layers, five peaks of -amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA₃ only three peaks of activity were found. In incubation media, four isoenzymes were found after GA₃ treatment and two were found after incubation without GA₃. We conclude that at least five -amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.Abbreviations CHA cyclohepataamylose - DEAE-cellulose diethylaminoethyl-cellulose - ER endoplasmic reticulum - GA₃ gibberellic acid - SDS-PAGE sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis     

Involvement of the Golgi apparatus in the secretion of α-amylase from gibberellin-treated barley aleurone cells

Localisation of -amylase (EC 3.2.1.1) in barley aleurone cells treated with gibberellic acid has been achieved using protein A-gold-labelled polyclonal antibodies. Gold particles were located almost exclusively over the lumen of the rough endoplasmic reticulum and cisternae of the Golgi apparatus. The label was most concentrated over the Golgi apparatus. This indicates that the Golgi is involved in the secretion of -amylase protein from aleurone cells.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - PBS phosphate-buffered saline     

The effect of monensin on intracellular transport and secretion of α-amylase isoenzymes in barley aleurone

The effect of monensin on the secretion of -amylase and other enzymes from the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya) was studied by electrophoresis followed by fluorography and by pulse-chase and organelle-isolation experiments. Monensin markedly inhibits the secretion, but not the synthesis, of -amylase, acid phosphatase, and at least four other proteins from the aleurone layer. Monensin treatment causes -amylase to accumulate within the protoplast, but its effect on the different -amylase isoenzymes is not equal. The accumulation of isoenzyme 2 is not influenced by monensin while isoenzymes 1, 3 and 4 are not secreted but rather accumulate in the cell when monensin is included in the incubation medium. The -amylase and acid-phosphatase activities which accumulate within the aleurone cells following treatment with monensin are localized in an organelle having a buoyant density greater than that of endoplasmic reticulum and less than that of mitochondria. In pulse-chase experiments with [³⁵S]methionine, labelled proteins accumulate in this organelle in the presence of monensin and do not appear in the incubation medium. We conclude that monensin inhibits the secretion of proteins from the barley aleurone layer by influencing their intracellular transport.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - SDS-PAGE sodium dodecyl-sulfate polyacrylamide-gel electrophoresis