共查询到20条相似文献,搜索用时 31 毫秒
1.
Polyamine metabolism and its relation to the induction of α-amylase formation in the aleurone layers of barley seeds ( Hordeum vulgare cv Himalaya) in response to gibberellic acid (GA 3) has been investigated. A high-performance liquid chromatographic system has been employed for qualitative and quantitative analyses of putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), and agmatine (Agm). Active polyamine metabolism occurs in the aleurone cells of deembryonate barley half seeds during imbibition. The aleurone layers isolated from fully imbibed half seeds contain about 880 nanomoles of Put, 920 nanomoles of Spd, and 610 nanomoles of Spm as free form per gram tissue dry weight while the levels of Cad and Agm are relatively low. The polyamine levels do not change significantly in the aleurone layers in response to added GA3 (1.5 micromolar) during the 8-hour lag period of the growth substance-induced formation of α-amylase. Also, the polyamine levels are not altered by the presence of abscisic acid (3 micromolar) which inhibits the enzyme induction by GA3. Kinetic studies show that both applied [U-14C]ornithine and [U-14C]arginine are primarily incorporated into Put during 2 hours of incubation, but the incorporation is not significantly affected by added GA3. Additionally, added GA3 does not affect the uptake and turnover of [1,4-14C]Put, nor does it affect the conversion of Put → Spd or Spd → Spm. Treatment of the aleurone layers with GA3 for 2 hours results in no significant changes in the total activities or the specific activities of ornithine decarboxylase and arginine decarboxylase. Experiments with polyamine synthesis inhibitors demonstrate that the level of Spd in the aleurone layers could be substantially reduced by the presence of methylglyoxal-bis(guanylhydrazone) (MGBG) during imbibition. MGBG treatment does not affect in vivo incorporation of [8-14C] adenosine into ATP. The lower the level of Spd the less α-amylase formation is induced by added GA3. The reduction of GA3-induced α-amylase formation by MGBG treatment can be either completely or partially overcome by added Spd, depending upon the concentration of MGBG used in the imbibition medium. The results indicate that the early action of GA3, with respect to induction of α-amylase formation in barley aleurone layers, appears to be not on polyamine metabolism. However, polyamines, particularly Spd, may be involved in regulation of the growth substance-dependent enzyme induction. 相似文献
2.
Barley (c.v. Himalaya) aleurone layers were incubated in [ 3H]gibberellin A 1 (GA 1) at low temperatures. At 3 and 4 C, 3H-activity was steadily accumulated in aleurone layers, and this accumulation was correlated with significant [ 3H]GA 1 metabolism. At 1 and 1.5 C, metabolism could not be detected, and at these temperatures aleurone layers equilibrated with the [ 3H]GA 1 concentration in the incubation medium. At equilibrium, the total amount of 3H-activity per unit volume in the aleurone layers was higher than in the incubation medium. Aleurone layers incubated at 0.5 C for 72 hours with [ 3H]GA 1 in the presence of saturating levels of carrier GA 1 consistently retained lower levels of 3H-activity than when incubated in [ 3H]GA 1 alone. The retention of [ 3H]GA 1 was unaffected by saturating levels of carrier GA 8. GA 1 retained by barley aleurone layers that were incubated at 0.5 C for 72 hours was able to induce α-amylase synthesis when aleurone layers were subsequently washed and transferred to a gibberellin-free medium at 25 C. 相似文献
3.
The role of gibberellic acid (GA 3) in controlling the secretion(across the plasma membrane) and release (through the cell wall)of acid phosphatase (E.C. 3.1.3.2
[EC]
.) from Avena aleurone layershas been investigated. Evidence from this comparative studywith intact aleurone layers and isolated aleurone protoplastsreveals that the secretion of acid phosphatase is under GA 3control. The mechanism underlying secretion and release of theenzyme from aleurone cells is discussed. Key words: Avena fatua, Acid phosphatase, Aleurone protoplasts, Gibberellic acid, Secretion 相似文献
4.
Summary When barley aleurone layers are treated with gibberellic acid (GA 3) in the presence of increasing concentrations (0.2–0.8 M) of mannitol, the rate of 32Pi incorporation into phospholipids becomes progressively inhibited. Mannitol does not affect this process in aleurone layers not treated with GA 3, nor does it appreciably inhibit GA 3-effected increases of 32Pi incorporation into organic phosphates or the activities of the particulate enzymes of the CDP-choline pathway. These results suggest that some of the early events controlled by GA 3 can be separated from later activities regulated by the hormone, including -amylase synthesis. 相似文献
5.
Water stress inhibits the gibberellic acid (GA 3)-induced synthesis of α-amylase in aleurone layers of barley ( Hordeum vulgare L.). Electron microscope evidence indicates that the effect of water stress induced by 0.6 M solutions of polyethylene glycol (PEG) is to reduce the binding of ribosomes to the endoplasmic reticulum. This was confirmed by sucrose density gradient centrifugation of polyribosome preparations from stressed cells. The reduction in polyribosome formation does not result from reduced ribosome activity as measured by [ 3H]peptidylpuromycin formation. Thus, calculation of percent active ribosomes shows that osmoticum has little effect on the ability of ribosomes to incorporate puromycin into nascent protein. Water stress does not cause a marked decrease in the total RNA level of aleurone cells. Estimates of total RNA in postmitochondrial supernatant fractions from stressed cells show only a reduction of 8–9% relative to the control. Membrane synthesis measured by [ 14C]choline incorporation is depressed by 15% in cells stressed with 0.6 M PEG for 2.5 hours. 相似文献
6.
Phospholipids of barley ( Hordeum vulgare L. cv Himalaya) aleurone layers were labeled with myo-[2- 3H]inositol or [ 32Pi], extracted, and analyzed by physical (chromatography) and chemical (deacylation) techniques. Three phospholipids were found to incorporate both myo-[2- 3H]inositol and [ 32Pi]—phosphatidylinositol, phosphatidylinositol-monophosphate, and phosphatidylinositol-bisphosphate. Stimulation of [ 3H]inositol prelabeled aleurone layers with GA 3 showed enhanced incorporation of label into phosphatidylinositol within 30 seconds and subsequent rapid breakdown. Stimulation of phosphatidylinositol labeling observed in these studies is the earliest response of aleurone cells to gibberellic acid reported. 相似文献
7.
Endosperms of quiescent barley grains contained, on average, 54.5 μg of neutral glyceride-glycerol, equivalent to ca 480 μg glyceride. Of this probably 90% was located in the aleurone layer. During germination the level of glyceride-glycerol declined. It also declined in degermed grains and aleurone layers incubated in vitro. The fall was accelerated by GA 3, but indoleacetic acid, kinetin and glutamine were without effect. Increases in the levels of malate synthase and isocitrate lyase from very low initial values, and the results of incorporation studies with [ 14C]-labelled substrates, indicate that the glyoxylate cycle functions to convert glycerides to sucrose in germinating grains and degermed grains incubated with GA 3, but not in degermed grains without the hormone. In the absence of GA 3 the glyceride could be a respiratory substrate in degermed grains. The aleurone layers converted exogenous glucose to sucrose. Little label from [ 14C]-amino acids appeared in sucrose but in some cases considerable incorporation occurred into glutamine. 相似文献
8.
Summary The cytochemical localization of adenosine triphosphatase (ATPase) was studied in the aleurone layer of barley ( Hordeum vulgare L. cv. Himalaya). Isolated barley aleurone layers secrete numerous enzymes having acid phosphatase activity, including ATPase. The secretion of these enzymes was stimulated by incubation of the aleurone layer in gibberellic acid (GA 3). ATPase was localized using the metal-salt method in tissue incubated in CaCl 2 with and without GA 3. In sections of tissue incubated without GA 3, cytochemical staining was confined to a narrow band of cytoplasm adjacent to the starchy endosperm and to the cell wall of the innermost tier of aleurone cells. Cytochemical staining was absent from the organelles of tissues not treated with GA 3. In tissue incubated in the presence of GA 3, cytochemical staining was evident throughout the cytoplasm and cell walls of the tissue. In the cell wall, electron-dense deposits were found only in digested channels. The cell-wall matrix of GA 3-treated aleurone did not stain, indicating that it does not permit diffusion of enzyme. In the cytoplasm of GA 3-treated aleurone, all organelles except microbodies, plastids, and spherosomes stained for ATPase activity; endoplasmic reticulum (ER), Golgi apparatus, and mitochondria showed intense deposits of stain. The ER of the aleurone is a complex system made up of flattened sheets of membrane, which may be associated with both the Golgi apparatus and the plasma membrane. The dictyosome did not stain uniformly for ATPase activity; rather there was a gradation in staining of the cisternae from the cis (lightly stained) to the trans (heavily stained) face. Vesicles associated with dictyosome cisternae also stained intensely as did the protein bodies of GA 3-treated aleurone cells. 相似文献
9.
Abscisic acid (ABA) in lanolin, applied to the internode of decapitated runner bean plants enhances the outgrowth of lateral buds. The optimum concentration of the paste is 10 -5 M. The effect of ABA is counteracted by indoleacetic acid (IAA) but not by gibberellic acid (GA 3). There is no effect when ABA is applied to the apical bud or lateral buds of intact plants. However, 13.2 ng given to the lateral buds of decapitated plants stimulate their growth, whereas higher concentrations are inhibitory. Consequently, ABA enhances growth of lateral buds directly, but only when apical dominance is already weakened. The growth of the decapitated 2 nd internode was not affected by ABA. Radioactivity from [2- 14C] ABA, applied to nonelongating 2 nd internode stumps of decapitated runner bean plants moves to the lateral buds, whereas [1- 14C]IAA-and [ 3H]GA 1-translocation is much weaker. ABA transport is inhibited if IAA or [ 3H]GA 1 is applied simultaneously. In elongating internodes [ 14C]ABA is almost completely immobile. [ 14C]IAA-and [ 3H]GA 1-translocation is not affected by ABA. The amount of radioactivity from labelled ABA, translocated to the lateral buds, is highest during the early stages of bud outgrowth.Abbreviations ABA
2,4-cis, trans-(+)-abscisic acid
- GA
gibberellic acid
- IAA
indoleacetic acid
- p.l.
plain lanolin 相似文献
10.
Viable, long-lived, gibberellic acid (GA 3)-responsive protoplasts have, for the first time, been isolated from aleurone layers of mature wild oat ( Avena fatua L.) grain. More than 90% of the cells of aleurone layers are recovered as protoplasts, and these respond to treatment with GA 3 in essentially the same manner as the tissue from which they were derived. Protoplasts become vacuolate during incubation in vitro and, although not dependent upon GA 3, vacuolation is markedly stimulated by the hormone. Amylase and ribonuclease (RNase) are produced and secreted only in the presence of GA 3 and only after lag periods of 3 d and 4 d respectively. The amounts of amylase produced and secreted are proportional to GA 3 concentrations as low as 1.61·10 -13 M. With increasing concentrations of mannitol in the culture medium both vacuolation and the GA 3-induced production and secretion of enzymes are inhibited progressively, the latter being precluded by 0.6 M to 0.7 M mannitol.Abbreviations GA 3
gibberellic acid 3
- RNase
ribonuclease 相似文献
11.
When aleurone layers of barley ( Hordeum vulgare L.) are incubated with gibberellic acid (GA 3) xylose and arabinose—both as free sugars and bound to larger molecules—are released into the medium. Release begins 10–12h after the start of incubation and continues for at least 60h. At the same time there is a GA 3-induced breakdown of the cell wall resulting in a loss of 2/3 of the cell-wall pentose during 60h of incubation. GA 3 causes the appearance in the medium of an enzyme (or enzymes) which hydrolyze larchwood xylan and aleurone-layer arabinoxylan. Release of the enzyme(s) into the medium begins 28–32h after the start of incubation. Enzyme activity does not accumulate to any large extent in the tissue prior to release into the medium, and is present in very low levels only in the absence of GA 3. Xylanase activity is associated with a protein (or proteins) with a molecular weight of 29,000. The hydrolysis of the xylans is largely caused by endoxylanase activity, indicating the importance of endoglycosidases in the GA 3-induced breakdown of the aleurone cell wall. 相似文献
12.
A method for isolating viable protoplasts in high yield from the aleurone layers of developing wheat grains is described, and the techniques for their subsequent culture outlined. Protoplasts from untreated tissue do not produce α-amylase in response to gibberellic acid (GA 3) if the incubation temperature is left at 25°C. However, pre-treatment of the protoplast preparation at temperatures above 27°C for at least 8 h followed by a short incubation at 25°C induces sensitivity to the growth regulator such that α-amylase is produced. The requirements of the sensitisation process are similar to those for intact aleurone tissue although additional adjustment to the calcium ion is beneficial. Pre-treatment of aleurone layers with the sensitising temperature regimes prior to protoplast isolation have the advantage of increasing protoplast viability. Once sensitised, the protoplasts respond to a GA 3 concentration as low as 10 -11 mol dm -3 with a maximal response at 10 -9 mol dm -3. The successful isolation of wheat aleurone protoplasts whose sensitivity to GA 3 can be manipulated represents a useful step towards investigating the role of cell membranes in growth-regulator action. 相似文献
13.
The role of calmodulin (CaM) in gibberellic acid (GA 3)-stimulated Ca 2+ uptake was investigated in endomembranes isolated from aleurone cells of barley ( Hordeum vulgare L.). Unidirectional Ca 2+ -uptake activity of endoplasmic reticulum (ER) was higher in membranes isolated from aleurone layers treated for 16 h with GA 3 and Ca 2+ compared with those isolated from layers incubated in Ca 2+ alone. However, the level of uptake from Ca 2+-treated tissue could be stimulated to that of the GA 3-treated cells by applying exogenous CaM which increased the V max of the Ca 2+ transporter approximately threefold. Calcium uptake in ER from GA 3-treated tissue was inhibited by the CaM antagonist W7 in 50% of experiments, whereas the activity in membranes from non-GA 3-treated tissue was unaffected. Treatment with GA 3 also led to a twofold increase in CaM levels in aleurone layers within 4–6 h, paralleling the time course of the stimulation of Ca 2+ uptake and preceding the stimulation of α-amylase secretion. We propose that the elevation of Ca 2+ uptake into the ER induced by GA 3 may be coordinated and regulated by elevated levels of membrane-associated CaM and this may regulate Ca 2+-dependent α-amylase synthesis in the lumen of the ER. 相似文献
14.
Phospholipid synthesis in wheat aleurone tissue was found tobe induced by imbibition. Radiotracer experiments using [Me- 14C]cholineprecursor showed that the synthesized phospholipid was locatedin the microsomal fraction of the tissue. The most active periodof phospholipid synthesis was during the first day of germinationbefore gibberellic acid (GA 3) responses were evident. GA 3 was,in fact, without effect upon the rate of phospholipid synthesis,but evidence is presented which indicates that the hormone increasedthe rate of phospholipid breakdown. Abscisic acid (ABA) alsoinduced phospholipid breakdown, and ABA did not inhibit theGA 3-induced breakdown. The relationship of phospholipid synthesisand membrane formation to the induction of hydrolase productionby GA 3 is discussed. 相似文献
15.
The patterns of protein synthesis in barley aleurone layers treated with gibberellic acid (GA 3) and abscisic acid (ABA) are compared with the patterns observed in wheat germ in vitro translation assays directed by RNA isolated from similarly treated layers. When used alone, GA 3 and ABA both induce the formation of new translatable mRNAs and cause new proteins to be synthesized. The effects of GA 3 are more dramatic than those of ABA. In GA 3-treated tissues, overall protein synthesis is redirected to produce large quantities of α-amylase and a few other GA 3-induced proteins, while other protein synthesis is reduced or stopped. Large amounts of new translatable mRNA for α-amylase are also induced such that the dominant in vitro translation product is α-amylase. These changes are blocked by the simultaneous addition of ABA to the tissue. In GA 3 plus ABA-treated layers, few changes in protein synthesis in vivo are observed when compared to protein synthesis in untreated tissue, although the induction of mRNA for α-amylase and the other GA 3-induced mRNAs does occur. This indicates that ABA does not interfere with GA 3 induction of translatable mRNAs but prevents the translation of these mRNAs in vivo. Thus ABA and potentially GA 3 regulate the translation of proteins in vivo in barley aleurone layers. 相似文献
16.
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 (GA 3). Using [ 35S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA 3 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 GA 3 were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA 3-treated aleurone layers, five peaks of -amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA 3 only three peaks of activity were found. In incubation media, four isoenzymes were found after GA 3 treatment and two were found after incubation without GA 3. 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
- GA 3
gibberellic acid
- SDS-PAGE
sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis 相似文献
17.
Summary Aleurone layers of barley contain large amounts of a soluble oligosaccharide which was identified as sucrose (30–40 g/mg fresh weight). Treatment of the layers with gibberellic acid (GA 3) causes the release of sucrose from the cells. This release requires the participation of metabolic processes, including protein synthesis. When embryoless half-seeds are incubated sucrose accumulates in the aleurone layers, but when seeds are germinated the sucrose content of the aleurone layers declines. Labeling experiments with radioactive glucose and fructose show that aleurone layers continuously synthesize sucrose and that the release, but not the synthesis of sucrose is enhanced by GA 3. 相似文献
18.
In the presence of gibberellic acid (GA 3) aleurone layers and isolated aleurone protoplasts of Avena fatua accumulate specific isozymes of acid phosphatase (EC 3.1.3.2). Some of these may be involved in mobilizing aleurone-grain phosphate reserves during germination. The hormone also controls secretion of other specific molecular forms of the enzyme that probably assist in endosperm hydrolysis. The accumulation and secretion of putative cell-wall-associated isozymes are stimulated by the action of GA 3 in isolated protoplasts. This effect however, is apparently over-ridden in the intact tissue, possibly by a cell-wall-based feedback mechanism.Abbreviations GA 3
gibberellic acid
- pI
isoelectric point(s) 相似文献
19.
The localization of acid phosphatase (EC 3.1.3.2) in aleurone layers of barley ( Hordeum vulgare L. cv. Himalaya) grains was studied. Phosphatase (EC 3.1.3.26) activity, assayed with phytic acid as the substrate, is present in the dry grain at low leveis and increases during incubation in H 2O at 25°C for three days. When aleurone layers are isolated from imbibed grain and incubated for 18 h in buffer with or without 50 μM gibberellic acid (GA 3), the level of extractable phosphatase activity increases two- to threefold, and phosphatase is released into the medium. GA, promotes the release of phosphatase activity: aleurone layers incubated in GA, release twice as much phosphatase as layers incubated in buffer. Nine isoenzymes of phosphatase are found in aleurone layers of barley by non-denaturing polyacrvlamide gel electropho-resis. Six of these forms, isoenzymes 1,2,3,5,6 and 8, can be extracted from dry tissue, and after three days of imbibition in H 2O an additional isoenzyme, isoenzyme 9, is found in aleurone extracts. When isolated aleurone layers are incubated for a further 22 h in buffer with or without GA 3, isoenzyme 7 is found and yet another form, isoenzyme 4, is found in layers incubated in GA 3. Eight isoenzymes are released from aleurone layers into the incubation medium. Isoenzymes 5 and 6 are released in buffer both with and without GA 3, even when cycloheximide is present; cycloheximide inhibits the release of the other isoenzymes. Isoenzymes 1-4, 7 and 8, on the other hand, are secreted into the incubation medium only when GA 3, is present. Isoenzyme 9 is not released into the incubation medium. Acid phosphatase activity was localized in aleurone tissue using cytochemical, cell fractionation, and enzymatic methods. Cytochemical localization of ATPase (EC 3.6.1.8) in aleurone tissue showed the presence of enzyme activity in cell wall, protein bodies, endoplasmic reticulum, Golgi apparatus, and mitochondria. Analysis of organelle fractions isolated by density gradient centrifugation showed that the activity of acid phosphatase isoenzymes 1, 2 and 3 was prominently associated with the phytin globoid of protein bodies, and analysis of the activity released from the cell wall by enzymatic digestion showed that it was almost exclusively isoenzymes 5 and 6. 相似文献
20.
Pulse-labeling of barley ( Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA 3) with or without 5 millimolar CaCl 2 shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca 2+. A cDNA clone for α-amylase was isolated and used to measure α-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca 2+. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA 3 with 5 millimolar CaCl 2 and layers incubated in GA 3 alone. RNA isolated from layers incubated for 12 hours in GA 3 with and without Ca 2+ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca 2+ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca 2+-deprived aleurone layers was translatable. Ca 2+ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing. 相似文献
|