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     

Hormonal regulation of gene expression in barley aleurone layers

As part of our investigation of the mode of action of plant hormones in barley (Hordeum vulgare L.) aleurone layers, we have studied the expression of five identified and three unidentified mRNA species in the presence of exogenous gibberellic acid (GA₃) and abscisic acid. Three of the mRNAs are GA₃-inducible, three are suppressed by GA₃, and two are constitutive. The extent of the GA₃ effect differs considerably for both inducible and suppressible mRNAs. For example, a ten-fold higher concentration of GA₃ (10^-8 M) is required for full induction of the high-pl group -amylase mRNA than is required for the low-pI -amylase mRNA (10^-9 M). Temporal regulation of mRNA abundance also varies between the two -amylase isoenzyme groups. The three GA₃-suppressible mRNA species studied, alcohol dehydrogenase (ADH1), a probable amylase and protease inhibitor, and an unidentified barley mRNA species also varied in response to GA₃. The ADH1 mRNA decreased drastically within 8 h of GA₃ treatment, whereas the other two began to decrease in abundance only after 12–16 h of GA₃ treatment. Abscisic-acid treatment counteracted the GA₃ effects for both the inducible and suppressible mRNA species. Comparison of -amylase-mRNA levels and -amylase-synthesis rates showed a strong correlation between the two parameters, the only exception being a lack of -amylase synthesis in the presence of -amylase mRNA at low GA₃ concentrations. Therefore, the expression of -amylase seems to be regulated primarily by its mRNA levels.Abbreviations ABA abscisic acid - ADH1 alcohol dehydrogenase 1 - cDNA copy DNA - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor     

Regulation of α-amylase activity in Amaranthus caudatus seeds by methyl jasmonate, gibberellin A3, benzyladenine and ethylene

Methyl jasmonate (JA-Me) at 10^–3 Minhibited Amaranthus caudatus seed germination anddecreased -amylase activity. Exogenous gibberellin A₃(GA₃) and ethylene, but not benzyladenine (BA), increased activity ofthe enzyme in the presence of JA-Me, with ethylene being the most effective. Theinhibitor of ethylene action, 2,5-norbornadiene (NBD) inhibited seed germinationand decreased -amylase activity. The inhibitory action of JA-Me onAmaranthus caudatus seed germination is associated with theinhibition of -amylase activity. Exogenous GA₃ and ethylenecontrol both -amylase activity and seed germination in the presence of JA-Me.     

Expression of α-amylase and other gibberellin-regulated genes in aleurone tissue of developing wheat grains

Aleurone tissue from freshly harvested immature wheat grains (Triticum aestivum L. cv. Sappo) which is normally unresponsive to gibberellic acid can be made responsive by subjecting the tissue to a pre-incubation treatment in a simple buffered medium prior to the addition of the growth substance. The effectiveness of this treatment is dependent on grain age, with grains less than 15–20 days post anthesis failing to become converted to a responsive state whilst tissue from grains older than this become increasingly susceptible. Tissue from grains of a certain age (approx. 25–28 days post anthesis) produce small amounts of -amylase following this treatment even in the absence of exogenously applied growth substance. Using different ³²-labelled complementary-DNA probes for -amylase in wheat it was demonstrated that the failure of freshly harvested tissue to produce -amylase was correlated with the absence of the appropriate mRNA species. Inability to accumulate -amylase mRNA in response to gibberellic acid was removed by the pre-iccubation treatment and also by enforced drying. The gibberellin-regulated expression of other unidentified genes also responds to pre-incubation or drying. Induction of gibberellin-responsiveness in immature aleurone cells did not extend to the secretion of acid phosphatase, protease and ribonuclease.Abbreviations cDNA complementary DNA - dpa days post anthesis - GA gibberellin - GA₃ gibberellic acid     

The effects of gibberellic acid and abscisic acid on α-amylase mRNA levels in barley aleurone layers studies using an α-amylase cDNA clone

Summary Two cDNA clones were characterized which correspond to different RNA species whose level is increased by gibberellic acid (GA₃) in barley (Hordeum vulgare L.) aleurone layers. On the criteria of amino terminal sequencing, amino acid composition and DNA sequencing it is likely that one of these clones (pHV19) corresponds to the mRNA for -amylase (1,4--D-glucan glucanohydrolase, EC 3.2.1.1.), in particular for the B family of -amylase isozymes (Jacobsen JV, Higgins TJV: Plant Physiol 70:1647–1653, 1982). Sequence analysis of PHV19 revealed a probable 23 amino acid signal peptide. Southern hybridization of this clone to barley DNA digested with restriction endonucleases indicated approximately eight gene-equivalents per haploid genome.The identity of the other clone (pHV14) is unknown, but from hybridization studies and sequence analysis it is apparently unrelated to the -amylase clone.Both clones hybridize to RNAs that are similar in size (1500b), but which accumulate to different extents following GA₃ treatment: -amylase mRNA increases approximately 50-fold in abundance over control levels, whereas the RNA hybridizing to pHV14 increases approximately 10-fold. In the presence of abscisic acid (ABA) the response to GA₃ is largely, but not entirely, abolished. These results suggest that GA₃ and ABA regulate synthesis of -amylase in barley aleurone layers primarily through the accumulation of -amylase mRNA.Abbreviations ABA abscisic acid - CHA cyclohepta-amylose - CMC carboxymethyl cellulose - GA₃ gibberellic acid     

α-Amylase production and leaf protein synthesis in a gibberellin-responsive dwarf mutant of ‘Himalaya’ barley (Hordeum vulgare L.)

A dwarf mutant, M117, was isolated following sodium-azide mutagenesis of barley (Hordeum vulgare L. Himalaya). Treatment of the mutant with gibberellic acid (GA₃) restored growth to levels of the tall parent, -Amylase production was examined in germinated grains of the dwarf mutant and in Himalaya plants treated with gibberellin (GA) biosynthesis inhibitors. The mutant showed reduced -amylase activity relative to the parent when grains were germinated on water, but activities were equivalent to the parent following germination on GA₃ solution. Germination of normal or mutant grains in the presence of GA biosynthesis inhibitors led to reduced -amylase activity levels, but normal levels were restored if GA₃ was included in the inhibitor solution. These data are consistent with a model in which -amylase production in the germinated grain is regulated by the supply of active GAs. Treatment of M117 with GA₃ increased the length, fresh weight, dry weight, volume, cell number, and protein content of the first leaf. Proteins being synthesized in the first leaf were labelled with [³⁵S]methionine and fractionated by two-dimensional electrophoresis. No reproducible qualitative or quantitative differences in protein profiles were detected in response to GA₃ treatment. In contrast, first leaves from seedlings exposed to dehydration stress had profiles clearly distinguishable from those of control seedlings. Stem sections from dwarf plants maintained on 10 M GA₃ in the presence of sucrose elongated significantly more than controls without GA₃, but two-dimensional analysis of the [³⁵S]methionine-labelled radioactive polypeptides again revealed no GA₃-induced differences. It was concluded that enhanced elongation rates of leaves or stem segments were not associated with major changes in gene expression.Abbreviations 2D two-dimensional - GA gibberellin - GA₃ gibberellic acid - PB paclobutrazol We would like to thank Dr Barbara Read (Agricultural Research Institute, Wagga Wagga, Australia) for assistance with growth of barley plants, and Tony Carter, Alison McInnes, and Mark Cmiel for skilled technical assistance.     

Gibberellin perception at the plasma membrane of Avena fatua aleurone protoplasts

A functional assay for gibberellin (GA) receptors is described based on the induction of -amylase gene expression in isolated aleurone protoplasts of Avena fatua L. by GA₄ immobilised to Sepharose beads. A 17-thiol derivative of GA₄, shown to be biologically active with aleurone protoplasts, has been coupled to epoxy-activated Sepharose 6B. This GA₄-17-Sepharose induces high levels of -amylase when incubated with isolated aleurone protoplasts, while cells of the intact aleurone layer do not respond appreciably to the immobilised GA₄. In order to eliminate the possibility that GA₄ may be released from the Sepharose when incubated with protoplasts, aleurone layers and isolated aleurone protoplasts have been co-incubated, and their responses to GA₄, GA₄-17-Sepharose and control Sepharose estimated by determining the relative amounts of -amylase mRNA induced in each tissue. Evidence from these experiments is consistent with the view that GA₄17-Sepharose induces -amylase gene expression in aleurone protoplasts by interacting with the protoplast surface. This indicates that GA receptors may be located at, or near, the external face of the aleurone plasma membrane.Abbreviation GA(n) gibberellin A(n) We thank Professor Jake MacMillan and Drs. Peter M. Chandler (CSIRO, Division of Plant Industry, Canberra, Australia), Peter Hedden and Johnathan Weir (Unilever, Port Sunlight, UK) for helpful discussions and suggestions. Computer graphics were performed by the University of Bristol Molecular Recognition Centre.     

Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers

Using in vivo pulse labeling, changes in the pattern of protein synthesis were detected in isolated barley aleurone layers treated with fibberellic acid (GA₃). GA₃ greatly altered the relative rates of synthesis of many polypeptides, increasing some, notably -amylase, and decreasing others. -Amylase synthesis increased until it was the major product (over 60%) of protein synthesis after 24h. The pulse-labeled pattern of secreted polypeptides was also changed by GA₃. There was the expected increase in -amylase together with a number of other polypeptides but there was reduced secretion of several polypeptides also.Cell-free translation of RNA isolated from control and hormone-treated tissues was used to measure changes in mRNA levels. GA₃ caused many changes, particularly in the level of mRNA for -amylase. In vitro synthesized -amylase, identified by immunoaffinity chromatography, had an Mr of 46 000. This polypeptide was partially processed to a polypeptide with Mr 44 000 by the addition of dog pancreas membranes to the in vivo translation mixture. The level of mRNA for -amylase began to increase 2–4 h after GA₃ was added and reached a maximum level of about 20% of total mRNA after 16 h. Thus after 16 h, the synthesis of -amylase as a proportion of total protein synthesis, continued to increase while the level of its mRNA as a proportion of total mRNA remained constant. These results indicate that protein synthesis was modified more extensively than we can account for by changes in mRNA.Abscisic acid (ABA) reversed all of the effects of GA₃ on protein synthesis and mRNA levels. It also promoted synthesis of a small number of new polypeptides and increased the level of some mRNAs. GA₃ reversed the accumulation of ABA-promoted mRNAs. Although, ABA strongly suppressed the increase in the level of translatable mRNA for -amylase, there was an even stronger inhibition of enzyme synthesis and accumulation.We conclude that both GA₃ and ABA regulate protein synthesis both positively and negatively in aleurone cells largely by regulating levels of mRNA and in the case of -amylase, possibly also by changing the efficiency of translation of its mRNA.     

Primer extension studies on α-amylase mRNAs in barley aleurone. II. Hormonal regulation of expression

Relative levels of different -amylase mRNAs were assessed by primer extension experiments using RNA prepared from aleurone of barley (Hordeum vulgare L. cv. Himalaya). Three different aleurone systems were studied: protoplasts prepared from aleurone layers, isolated aleurone layers, and aleurone from germinated grain. Oligonucleotide primers specific for the low-pI and high-pI -amylase groups allowed the levels of different -amylase mRNAs to be assessed both within and between the two groups.In all aleurone systems the same set of -amylase mRNAs was produced in response to either applied gibberellic acid (aleurone protoplasts, isolated aleurone layers) or, presumably, native gibberellin(s) (germinated grain). This result indicates that the same set of genes is being expressed in each case. Differences were observed between the different aleurone systems in regulation of levels of -amylase mRNAs. In particular, the regulation of -amylase mRNA levels in aleurone of germinated grain has unique features which are not adequately explained by the response of isolated aleurone layers to gibberellic acid.     

Identification and characterization of gibberellin-insensitive mutants selected from among dwarf mutants of rice

In rice, many dwarf mutants have been isolated and characterized. We have investigated the relationship between dwarfism and the gibberellin (GA)-mediated control of physiological processes. Twenty-three rice cultivars and mutants (9 normal, 3 semi-dwarf, 11 dwarf) were analyzed in terms of two GA-mediated processes, namely, elongation of shoots and production of -amylase activity in the endosperm. As a result, we identified four different groups (groups N, T, D and E). Two-dimensional plotting of the extent of induction of -amylase in the endosperm versus the extent of enhancement of shoot elongation upon treatment with exogenous gibberellic acid (GA₃) provided a useful method for the rapid allocation of large numbers of dwarf mutants of rice to the various groups. Members of group N (normal type), which included all normal cultivars and semi-dwarf mutants, showed a slight increase in elongation of shoots and a remarkable increase in production of -amylase with the application of GA₃ during germination. All of the dwarf mutants were classified as being members of the other three groups. Members of group T (Tan-ginbozu type), including three dwarf mutants, were highly responsive to exogenous GA₃ in terms of elongation of shoots and production of -amylase, with associated lower levels of endogenous GA. In contrast, members of the other three groups, including group N, had normal levels of endogenous GAs. Members of group D (Daikoku type) were only slightly responsive to exogenous GA₃, an indication that they are GA-insensitive mutants. Members of group E (Ebisu type) had responses to GA₃ similar to those of group N, not only in terms of elongation of shoots but also in terms of -amylase production, an indication that they are dwarf mutants that can be considered as neither GA-deficient nor GA-insensitive mutants. We also examined a GA-insensitive mutant selected from among 19 near-isogenic dwarf lines of Shiokari, and we concluded that the d-1 gene is associated with the phenotype of GA-insensitive dwarf mutants.     

Hormonal regulation of gene expression in the “slender” mutant of barley (Hordeum vulgare L.)

The slender mutant of barley resembles a normal barley plant treated with high doses of gibberellic acid (GA₃). Expression of GA₃-regulated and abscisic acid (ABA)-regulated mRNAs was studied in the endosperm and roots of mutant and wild-type (WT) plants.Production of -amylase (EC 3.2.1.1) by WT embryoless half-grains was dependent on the presence of GA₃, and was prevented by ABA. In contrast, -amylase was produced by half-grains of the slender mutant in the absence of added GA₃, although it was still reduced by ABA. The spectrum of -amylase mRNAs in slender embryoless half-grains incubated in the absence of added GA₃ was the same as in WT endosperm half-grains incubated in the presence of GA₃. These results indicate that the endosperm of the slender mutant exhibits similar properties to WT endosperm treated with GA₃.In roots the expression of an ABA-inducible mRNA was similar in slender and WT seedlings either treated with exogenous ABA or exposed to dehydration. This result, and the effect of ABA on -amylase production by the endosperm, indicate that the slender plants retain sensitivity to ABA.Abbreviations ABA abscisic acid - AMV avian myeloblastosis virus - GA gibberellin - GA₁ gibberellin A₁ - GA₃ gibberellic acid - WT wild-type     

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     

Endogenous gibberellin A1 level and α-amylase activity in germinating rice seeds

The role of endogenous gibberellin A₁ (GA₁) in the induction of -amylase activity was investigated during germination of rice (Oryza sativa L.) seeds. The level of endogenous GA₁ and the -amylase activity in the seeds of normal rice, cv. Nipponbare, increased simultaneously from 3 days after the imbibition of water. The -amylase activities in the dwarf rice, cv. Waito-C and Tan-ginbozu, were less than that in the normal rice. The level of endogenous GA₁ and -amylase activity were decreased in proportion to the concentration of a growth retardant, uniconazole. The retardation in -amylase activity caused by the treatment of uniconazole was recovered by the application of exogenous GA₁. These results indicate that the endogenous GA₁ biosynthesized de novo regulates -amylase production in germinating rice seeds.Abbreviations GA(s) gibberellin(s) - ABA abscisic acid - AE fraction acidic ethyl acetate-soluble fraction - HPLC high performance liquid chromatography - R _t retention time - GC-SIM gas chromatography-selected ion monitoring     

Characterization of active barley alpha-amylase 1 expressed and secreted by Saccharomyces cerevisiae

Recombinant barley -amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified -amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The k _cat/K _m was 2.7 × 10² mM^–1.s^–1, consistent with those of -amylases from plants and other sources.     

Variation of seed α-amylase inhibitors in the common bean

Variation of seed -amylase inhibitors was investigated in 1 154 cultivated and 726 non-cultivated (wild and weedy) accessions of the common bean, Phaseolus vulgaris L. Four -amylase inhibitor types were recognized based on the inhibtion by seed extracts of the activities of porcine pancreatic -amylase and larval -amylase and larval -amylase of the Mexican bean weevil, Zabrotes subfasciatus Boheman. Of the 1 880 accessions examined most (1 734) were able to inhibit porcine pancreatic -amylase activity, but were inactive against the Z. subfasciatus larval -amylase; 41 inhibited only the larval -amylase activity, 52 inhibited the activities of the two -amylases, and 53 did not inhibit the activity of either of the -amylases. The four different inhibitor types were designated as AI-1, AI2, AI-3, and AI-0, respectively. These four inhibitor types were identified by the banding patterns of seed glycoproteins in the range of 14–20 kDa by using SDSpolyacrylamide gel electrophoresis. Additionally, four different banding patterns were recognized in accessions with AI-1, and were designated as AI-1a, 1b, 1c, and 1d. Two different patterns of the accessions lacking an -amylase inhibitory activity were identified and designated as AI-0a and AI-0b. The largest diversity for seed -amylase inhibitors was observed in non-cultivated accessions collected from Mexico where all eight inhibitor types were detected. The possible relationships between the variation of seed -amylase inhibitors and bruchid resistance are discussed.     

Properties and synthesis de novo of auxin-induced α-amylase in pea cotyledons

Analysis of starch-degrading enzymes in a crude extract of detached cotyledons of Pisum sativum L. by polyacrylamide gel electrophoresis (PAGE) demonstrated the presence of one band of -amylase (EC 3.2.1.1) activity. The activity of only this amylase was promoted in cotyledons incubated with 2,4-dichlorophenoxyacetic acid (2,4-D). The auxin-induced -amylase from pea cotyledons was purified to homogeneity, as judged by the criterion of a single band after PAGE. The relative molecular mass (M_r), estimated by gel filtration, was approx. 42 000 and the enzyme contained no carbohydrate moiety. Sodium dodecylsulfate-PAGE yielded a single band that corresponded to an M_r of 41 000. The isoelectric point was 5.85 and the aminoacid composition was similar to that of -amylase from other plants. When [³H]leucine was fed to detached dry cotyledons prior to incubation, the radioactivity in -amylase from cotyledons incubated in the presence of 2,4-D was found to be approx. 10-fold higher than that from cotyledons incubated in distilled water. When -amylase from cotyledons incubated with ²H₂O that contained 2,4-D and the tritiated amylase were centrifuged together in a CsCl density gradient, the peak of enzymatic activity of deuterated -amylase was shifted to a denser fraction than the peak of radioactivity of the tritiated enzyme. These results show that auxin-induced -amylase in pea cotyledons is synthesized de novo.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - pI isoelectric point - SDS sodium dodecyl sulfate We are very grateful to Mr. Kazuo Itoh and Mrs. Matsumi Doe for carrying out the analysis of amino-acid composition.     

Effect of inhibitors of gibberellin biosynthesis on the induction of α-amylase in embryoless caryopses of Hordeum vulgare cv. Himalaya

The induction of -amylase by exogenously supplied gibberellin A₁ (GA₁) and GA₄ in embryoless caryopses of Hordeum vulgare (cv. Himalaya) was determined indirectly by measuring reducing sugars released from the endosperm. The presence of the inhibitors of GA biosynthesis, 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (Amo 1618), Ancymidol, 2-chloroethyl trimethyl ammonium chloride (CCC) or (R,S)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,3-triazolyl)pentan-3-ol (PP333) did not inhibit -amylase production by either GA₁ or GA₄.Abbreviations Amo-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride - CCC 2-chloroethyl trimethyl ammonium chloride - cv. cultivar - GA gibberellin - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - PP333 (R,S)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,3-triazolyl) pentan-3-01     

Effect of Ecdysterone on Morphological and Physiological Processes in Plants

In order to elucidate the physiological role of phytoecdysteroids in plants, we investigated the effects of exogenous ecdysterone (ECD) and phytohormones (IAA, GA₃, and 24-epibrassinolide (EBL)) on the growth of wheat coleoptiles and Arabidopsis thaliana seedlings (wild-type ecotype Columbia (Col) and its det2 mutant), on -amylase activity in the barley aleurone layer, and on the pigment content in the kidney bean senescent leaves. The range of effective ECD concentrations depended on the type of a reaction to be regulated. The regulation of growth processes was affected by a wide range of ECD concentrations (10^–13–10^–5 M), whereas some metabolic processes, such as the activation of -amylase and the retardation of leaf yellowing, by a narrow range, that is, 10^–9–10^–7 M and 10^–9–10^–8 M, respectively. We noted the synergetic effect of ECD and IAA on coleoptile elongation, the antagonistic effect of ECD and EBL on coleoptile elongation, as well as the antagonistic action of ECD and GA₃ on coleoptile elongation and -amylase activity. The data obtained demonstrate that ECD is a physiologically active compound. ECD might be supposed to act as a source of sterols or a regulator of IAA and protein synthesis. The effects of this regulator seems to be brought about by its interaction with the EBL and GA₃ receptors.     

Expression of α-amylase in response to wounding in mung bean

The activity of -amylase (EC 3.2.1.1) in mung bean (Vigna radiata (L.) Wilczek) cotyledons increased markedly in response to wounding. The changes in enzyme activity were in parallel with those in enzyme content. The level of -amylase mRNA also notably increased in wounded cotyledons and attained its maximum level during the period between 1 and 2 d after wounding. The level of mRNA for phenylalanine ammonia-lyase, which is one of the well-characterized stress-inducible proteins, also increased after wounding, but the increase in mRNA level was faster than that of -amylase mRNA. On the other hand, the content of mRNA for actin, a housekeeping protein, was almost the same in wounded and unwounded cotyledons. The increase in -amylase mRNA level in wounded cotyledons was severely inhibited by -amanitin and cordycepin. -Amylase expression in the first leaves of mung-bean seedlings was also induced by wounding.Abbreviations PAL phenylalanine ammonia-lyase - SSC standard saline citrate We greatly acknowledge Prof. Richard Meagher, Department of Genetics, University of Georgia, Athens, USA for the gift of soybean actin gene clone. We also thank Mr. Kaoru Ishiwata for technical assistance.     

Prejunctional α2-Adrenoceptors and Peroxide-Induced Potentiation of Norepinephrine Release from the Bovine Iris

Peroxides can enhance field-stimulated [³H]norepinephrine ([³H]NE) release in isolated irides from several mammalian species. In the present study, we investigated the role of prejunctional ₂-adrenoceptors in peroxide-induced potentiation of sympathetic neurotransmission in bovine isolated irides. Isolated hemi-irides were incubated in a Krebs buffered-solution containing [³H]NE and prepared for studies of neurotransmitter release using the superfusion method. ₂-Adrenoceptor agonists, oxymetazoline, UK-14304 and clonidine inhibited field-stimulated [³H]NE overflow without affecting basal tritium efflux. Pretreatment of tissues with H₂O₂ (300 M) had no effect on inhibition of evoked [³H]NE release caused by the ₂-adrenergic agonists. However, H₂O₂ (300 M) caused significant (P < 0.01) leftward shifts of excitatory concentration-response curves to yohimbine (10 nM–1 M). In contrast, yohimbine (1 M) did not prevent the enhancement of evoked [³H]NE overflow induced by H₂O₂ (300 M). In conclusion, excitatory effects of peroxides on sympathetic neurotransmission in bovine irides are not mediated by prejunctional ₂-adrenoceptors.