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1.
R. D. Firn 《Planta》1975,125(3):227-233
Summary Gel filtration and centrifugation studies were used to study the distribution of -amylase activity in homogenates of barley (Hordeum vulgare L.) aleurone layers. The results obtained were consistent with the hypothesis that -amylase is secreted via membrane-bound vesicles. The -amylase activity in an homogenate of barley aleurone layers was derived not only from the enzyme retained in the aleurone cells but also from enzyme previously secreted from the cells but apparently retained by the cell walls. The amount of -amylase retained by the cell wall was influenced by factors such as the buffer in which the layers were incubated or the presence of Actinomycin D in the incubation medium.Abbreviations GA3 gibberellic acid - RER rough endoplasmic reticulum - Act. D Actinomycin D  相似文献   

2.
Summary The treatment of barley aleurone layers with gibberellic acid (GA3) results in the synthesis of two groups of -amylase isozymes. Addition of abscisic acid (ABA) at the same time as GA3 inhibited the synthesis of both groups of isozymes. However, midcourse ABA addition (12 h or later after GA3) had a more inhibitory effect on the high pI -amylase group than on the low pI -amylase group. This midcourse inhibition was detectable within 2 h of ABA addition. Northern analysis results using cDNA probes for the high pI and low pI -amylase groups paralleled the protein synthesis results for both isozyme groups. High pI -amylase mRNA levels began to decrease within 2 h of midcourse ABA treatment and were less than 10% of the original level by 4 h. The levels of low pI -amylase mRNA were decreased less by midcourse ABA addition than were high pI mRNA levels. Cordycepin and cycloheximide blocked the effects of midcourse ABA addition on -amylase mRNA. These observations indicate that ABA inhibits -amylase expression at the pretranslational level and that protein and RNA synthesis are required for midcourse ABA action to occur. Our results also show that -amylase mRNA, which has been thought to be very stable, is degraded after midcourse ABA treatment.  相似文献   

3.
4.
The effects of GA3 and/or ABA on the α-amylase activity and the ultrastructure of aleurone cells in halves of seeds without embryos (embryo-less half seeds) of oats (Avena sativa L.) were studied. α-Amylase activity was detected by the starch-agar gel method in the aleurone layers of embryo-less half seeds soaked in 1 μM GA3 solution or 100 μM GA3+10 μM ABA solution but not in those of seeds soaked in distilled water, 10 μM ABA solution, or 1 μM GA3+10 μM ABA solution. Ultrastructural examinations of aleurone cells with α-amylase activity showed a decrease in the number of sphaerosomes, the appearance of flattened saccules pressed to the surface of aleurone grains, and the development and transformations of the rER from a slender form to the one with wide inner spaces. In the aleurone cells in which the enzyme activity was not detected, components of the rER showed only slender profiles. The number of sphaerosomes did not decrease, and no flattened saccules appeared in the aleurone cells treated with 10 μM ABA or 1 μM GA3+10 μM ABA.  相似文献   

5.
Robert Locy  Hans Kende 《Planta》1978,143(1):89-99
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  相似文献   

6.
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  相似文献   

7.
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  相似文献   

8.
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  相似文献   

9.
J. V. Jacobsen  R. B. Knox 《Planta》1973,112(3):213-224
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.  相似文献   

10.
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  相似文献   

11.
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.  相似文献   

12.
Summary Using two step labeling with rhodamine-labeled secondary antibodies, -amylase (EC 3.2.1.1.) was detected in the scutellum, germ aleurone (monolayer partially encircling the scutellum) and normal aleurone (trilayer partially encircling the starchy endosperm; see Fulcher et al. 1972) in sections of Lowicryl-embedded barley (Hordeum vulgare L. cv. Himalaya) after imbibition of whole grains for 24, 48, and 72 h, but not after 2 h. Staining occurred over the protoplasts, cell walls or intercellular spaces of each tissue indicating that all three tissues had secreted as well as produced -amylase. The immunofluorescence in the scutellum was predominantly in the epithelium. Normal aleurone near the aleurone/ scutellum junction showed structural changes indicative of secretory activity by 24 h, and the pattern of cell erosion in the sub-aleurone and starchy endosperm at this and later stages supported this conclusion. The data show that normal aleurone is a major source of -amylase even at early stages of germination, but there is clear evidence also of production and secretion of some -amylase by both the scutellum and the germ aleurone, indicating that these tissues could also contribute to starch hydrolysis.Abbrevations GA3 gibberellic acid - SDS sodium dodecyl sulphate - TBS 10 mM Tris, pH 7.4, containing 0.15 M NaCl - Tris 2-amino-2-(hydroxymethyl)-1, 3-propanediol  相似文献   

13.
Summary A glucanase from barley aleurone layers can be assayed using the algal polysaccharide laminarin as substrate. Gibberellic acid (GA3) enhances the release of this enzyme from isolated aleurone layers but has no significant effect on its synthesis. Concentrations of GA3 effective in stimulating this release are in the range of 3×10-11-3×10-7M. The time course of glucanase release was found to be significantly different from that of -amylase, glucanase release being completed before that of -amylase. Evidence based on using various histochemical stains suggests that barley aleurone cell walls contain a -1,3-linked polymer. Following treatment of aleurone layers with GA3, digestion of these walls is seen to occur. These observations strongly suggest that the -1,3-glucanase produced by aleurone cells is resposible for the observed cell-wall digestion.Supported by National Science Foundation Grant GB-8332. The skillful technical assistance of Mrs. Janet Price is gratefully acknowledged.  相似文献   

14.
15.
The effect of gibberellic acid (GA3) on gene expression in wheat aleurone cells has been characterised. In-vitro translation of polyadenylated RNA indicated that α-amylase and other messenger-RNA (mRNA) species increase in relative concentration in GA3-treated tissue. At least one mRNA species declines in relative level in response to GA3. There is also a GA3-dependent, four-fold increase in the level of polyadenylated RNA. This effect is largely the result of increased levels of many mRNA species which are also present in untreated tissue. Seven GA3-induced polyadenylated RNA species including the Amyl α-amylase gene product have been cloned as complementary DNA in the plasmid pBR322. These cloned DNAs have been used as hybridisation probes to show that the GA3-induced increase in α-amylase mRNA is more prolonged than the accumulation of the other GA3-regulated mRNA species. A polyadenylated-RNA sequence showing reduced concentration in GA3-treated tissue has also been cloned.  相似文献   

16.
(p-Chlorophenoxy)isobutyric acid (PCIB) inhibited indole-3-acetic acid (IAA)-induced ethylene production in etiolated mung bean hypocotyl sections. The endogenous level of 1-aminocyclopropane-1-carboxylic acid (ACC) was not significantly affected by PCIB, indicating that PCIB exerted its effect primarily by inhibiting the activity of the ethylene-forming enzyme (EFE). This conclusion was supported by the observations that PCIB inhibited the conversion of exogenously applied ACC to ethylene. The inhibitory effect of PCIB was already evident with 0.05 mM PCIB, and it increased with time after application of the inhibitor. PCIB also significantly inhibited ethylene production in apple fruit tissues, but it only slightly reduced the level of endogenous ACC. Similar to mung bean, EFE activity in apple tissue was significantly inhibited by PCIB. The possibility that PCIB also inhibits auxin-induced ACC synthase activity is discussed.  相似文献   

17.
Using sensitive and selective immunological assays we have shown that in germinating caryopses of Hordeum vulgare L. cv. Himalaya, the level of gibberellin A4 (GA4) rises approximately 18-to 20-fold shortly (2–4 h) before -amylase activity increases. Gibberellin A4 is the predominant immunoreactive gibberelin during these developmental stages and reaches a peak amount of approximately 9 pmol per caryopsis about 48 h after imbibition. Isolated aleurone layers produce GA4 in the presence of an exogenous gibberellin, such as GA1, which is not a biosynthetic precursor for GA4. Experiments with inhibitors of gibberellin biosynthesis indicate that gibberellin synthesis is required in this tissue for the induction of -amylase. The inductive effect of exogenously applied GA1 is indirect and appears to be mediated by GA4. Embryos form predominantly GA1; however, very little of this material is released by isolated embryos into the incubation medium. The results presented make it unlikely that the role of the embryo in the process of -amylase induction in aleurone layers is to provide gibberellins or gibberellin precursors.Abbreviations ABA abscisic acid - GA gibberellin - GA3 gibberellic acid - RIA radioimmunoassay - TLC thin-layer chromatography  相似文献   

18.
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  相似文献   

19.
Fitness cost is usually associated with insecticide resistance and may be mitigated by increased energy accumulation and mobilization. Preliminary evidence in the maize weevil (Coleoptera: Curculionidae) suggested possible involvement of amylases in such phenomenon. Therefore, α-amylases were purified from an insecticide-susceptible and two insecticide-resistant strains (one with fitness cost [resistant cost strain], and the other without it [resistant no-cost strain]). The main α-amylase of each strain was purified by glycogen precipitation and ion-exchange chromatography (≥70-fold purification, ≤19% yield). Single α-amylase bands with the same molecular mass (53.7 kDa) were revealed for each insect strain. Higher activity was obtained at 35-40 °C and at pH 5.0-7.0 for all of the strains. The α-amylase from the resistant no-cost strain exhibited higher activity towards starch and lower inhibition by acarbose and wheat amylase inhibitors. Opposite results were observed for the α-amylase from the resistant cost strain. Although the α-amylase from the resistant cost strain exhibited higher affinity to starch (i.e., lower Km), its Vmax-value was the lowest among the strains, particularly the resistant no-cost strain. Such results provide support for the hypothesis that enhanced α-amylase activity may be playing a major role in mitigating fitness costs associated with insecticide resistance.  相似文献   

20.
D. Melroy  R. L. Jones 《Planta》1986,167(2):252-259
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  相似文献   

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