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1.
Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA3) with or without 5 millimolar CaCl2 shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca2+. 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 Ca2+. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA3 with 5 millimolar CaCl2 and layers incubated in GA3 alone. RNA isolated from layers incubated for 12 hours in GA3 with and without Ca2+ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca2+ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca2+-deprived aleurone layers was translatable. Ca2+ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.  相似文献   

2.
3.
The effects of gibberellic acid (GA3) and Ca2+ on the synthesis and secretion of α-amylase from protoplasts of barley (Hordeum vulgare L. cv Himalaya) aleurone were studied. Protoplasts undergo dramatic morphological changes whether or not the incubation medium contains GA3, CaCl2, or both. Incubation of protoplasts in medium containing both GA3 and Ca2+, however, causes an increase in the α-amylase activity of both incubation medium and tissue extract relative to controls incubated in GA3 or Ca2+ alone. Isoelectric focusing shows that adding Ca2+ to incubation media containing GA3 increases the levels of α-amylase isozymes having high isoelectric points (pI). In the presence of GA3 alone, only isozymes with low pIs accumulate. The increase in α-amylase activity in the incubation medium begins after 36 hours of incubation, and secretion is complete after about 72 hours. Protoplasts require continuous exposure to Ca2+ to maintain elevated levels of α-amylase release. Immunoelectrophoresis shows that Ca2+ stimulates the release of low-pI α-amylase isozymes by 3-fold and high-pI isozymes by 30-fold over controls incubated in GA3 alone. Immunochemical data also show that the half-maximum concentration for this response is between 5 and 10 millimolar CaCl2. The response is not specific for Ca2+ since Sr2+ can substitute, although less effectively than Ca2+. Pulse-labeling experiments show that α-amylase isozymes produced by aleurone protoplasts in response to GA3 and Ca2+ are newly synthesized. The effects of Ca2+ on the process of enzyme synthesis and secretion is not mediated via an effect of this ion on α-amylase stability or on protoplast viability. We conclude that Ca2+ directly affects the process of enzyme synthesis and transport. Experiments with protoplasts also argue against the direct involvement of the cell wall in Ca2+-stimulated enzyme release.  相似文献   

4.
5.
To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA3. B and C hordein polypeptides and the salt-soluble proteins β-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the α-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, β-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of α-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for α-amylase, these hormones have the opposite effect on ASI mRNA levels.  相似文献   

6.
The effects of gibberellic acid (GA3) and calcium ions on the production of α-amylase and acid phosphatase by isolated aleurone layers of barley (Hordeum vulgare L. cv Himalaya) were studied. Aleurone layers not previously exposed to GA3 or Ca2+ show qualitative and quantitative changes in hydrolase production following incubation in either GA3 or Ca2+ or both. Incubation in H2O or Ca2+ results in the production of low levels of α-amylase or acid phosphatase. The addition of GA3 to the incubation medium causes a 10- to 20-fold increase in the amounts of these enzymes released from the tissue, and addition of Ca2+ at 10 millimolar causes a further 8- to 9-fold increase in α-amylase release and a 75% increase in phosphatase release. Production of α-amylase isoenzymes is also modified by the levels of GA3 and Ca2+ in the incubation medium. α-Amylase 2 is produced under all conditions of incubation, while α-amylase 1 appears only when layers are incubated in GA3 or GA3 plus Ca2+. The synthesis of α-amylases 3 and 4 requires the presence of both GA3 and Ca2+ in the incubation medium. Laurell rocket immuno-electrophoresis shows that two distinct groups of α-amylase antigens are present in incubation media of aleurone layers incubated with both GA3 and Ca2+, while only one group of antigens is found in media of layers incubated in GA3 alone. Strontium ions can be substituted for Ca2+ in increasing hydrolase production, although higher concentrations of Sr2+ are required for maximal response. We conclude that GA3 is required for the production of α-amylase 1 and that both GA3 and either Ca2+ or Sr2+ are required for the production of isoenzymes 3 and 4 of barley aleurone α-amylase.  相似文献   

7.
α-Amylase has been purified from de-embryonated seeds of barley (Hordeum vulgare L. cv. Betzes) which have been incubated on 10−6 m gibberellic acid (GA3) following 3 days of imbibition in buffer. Incubation of the half-seeds in up to 10−2 m 5-fluorouracil (5-FU) during the entire incubation period, including imbibition, had no effect on any of the following characteristics of purified α-amylase: thermal stability in the absence of calcium, molecular weight of the enzyme, isozyme composition, specific activity, or the amount of α-amylase synthesized by the aleurone tissue. The synthesis of rRNA and tRNA was strongly inhibited by 5-FU, indicating that the analog had entered the aleurone cells. These results are not in agreement with those of Carlson (Nature New Biology 237: 39-41 [1972]) who found that treatment of barley aleurone with 10−4 m 5-FU prior to the addition of GA3 resulted in decreased thermal stability of GA3-induced α-amylase and who interpreted this as evidence that the mRNA for α-amylase was synthesized during the imbibition of the aleurone tissue and independently of gibberellin action. Results of the present experiments indicate that the thermal stability of highly purified α-amylase is not altered by treatment of barley half-seeds with 5-FU, and that 5-FU cannot be used as a probe to examine the timing of α-amylase mRNA synthesis.  相似文献   

8.
Lin PP 《Plant physiology》1984,74(4):975-983
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 (GA3) 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   

9.
After 4 days in an atmosphere of N2, aleurone layers of barley (Hordeum vulgare L. cv Himalaya) remained viable as judged by their ability to produce near normal amounts of α-amylases when incubated with gibberellic acid (GA3) in air. However, layers did not produce α-amylase when GA3 was supplied under N2, apparently because α-amylase mRNA failed to accumulate.  相似文献   

10.
Ho TH  Shih SC 《Plant physiology》1980,66(1):153-157
A method, based on the diffusion assay of α-amylase on agar plates, was developed to screen for barley (Himalaya) mutants with altered sensitivity to gibberellic acid (GA3) or abscisic acid (ABA) in their aleurone layers. The seeds produced by sodium azide-mutagenized barley were screened for their ability to synthesize and secrete α-amylase when treated with different combinations of hormones. Various GA3-insensitive or supersensitive, ABA-insensitive, temperature-dependent GA3-insensitive, and constitutive mutants have been identified. Several stable mutants with altered GA3 sensitivity were recovered. Two of the homozygous GA3-insensitive mutants have been preliminarily characterized. The GA3-enhanced production of α-amylase and release of phosphatase are hampered in these mutants. However, they have normal stem height, and the uptake of GA3 by their aleurone layers appears to be the same as that of wild-type barley. They are most likely regulatory mutants affecting both α-amylase synthesis and phosphatase release.  相似文献   

11.
Ho TH  Abroms J  Varner JE 《Plant physiology》1982,69(5):1128-1131
A large portion of the gibberellic acid (GA3)-induced α-amylase in isolated aleurone layers is transported into the incubation medium. In the presence of GA3 and ethylene, an even larger portion of the enzyme is found in the medium. Employing an acid washing technique developed by Varner and Mense (Plant Physiol 1972 49:187-189), it was observed that ethylene significantly reduces the amount of α-amylase trapped by the thick cell walls of aleurone layers. However, the amount of enzyme remaining in the cell (within the boundary of plasma membrane) is not affected by ethylene. Ethylene has no observable effect on membrane formation as measured by the incorporation of [32P]orthophosphate into phospholipids. Because of these observations it is suggested that ethylene enhances the release of α-amylase, i.e. transport of α-amylase across cell walls, but not the secretion of α-amylase, i.e. transport of α-amylase past the barrier of plasma membrane. The possible mechanism of this ethylene effect is discussed.  相似文献   

12.
Schuurink RC  Sedee NJ  Wang M 《Plant physiology》1992,100(4):1834-1839
The relationship between barley grain dormancy and gibberellic acid (GA3) responsiveness of aleurone layers has been investigated. Barley (Hordeum distichum L. cvs Triumph and Kristina) grains were matured under defined conditions in a phytotron. Grains of Triumph plants grown under long-day/warm conditions had lower dormancy levels than grains of plants grown under short-day/cool conditions. Aleurone layers isolated from grains of long-day Triumph plants secreted more α-amylase and had a higher responsiveness to GA3 as measured by α-amylase secretion. Storage of the grains increased both the percentage of germination and the responsiveness of the aleurone to GA3. Use of different sterilization methods to break dormancy confirmed the correlation between germination percentage and aleurone layer GA3 responsiveness. The response of embryoless Triumph grains to GA3 was lower than that of the isolated aleurone layers, suggesting a role of the starchy endosperm in regulating the GA3 response of the aleurone layer. Grains of the cultivar Kristina harvested from short day- and long day-grown plants lacked dormancy, and their isolated aleurone layers had a similar responsiveness to GA3 as measured by α-amylase secretion. The data indicate that the physiological state of the aleurone layers contributes to the percentage germination of the grains.  相似文献   

13.
Russell L. Jones 《Plant physiology》1969,44(10):1428-1429,1431,1433,1435-1438
Ultracentrifugation of barley aleurone cells results in the stratification of organelles thus allowing for a quantitation of those organelles. Gibberellic acid (GA3)-stimulated α-amylase production in stratified cells is reduced by centrifugation at gravitational forces greater than 40,000g. Forces below 30,000g do not affect GA3-stimulated α-amylase production although stratification of organelles occurs at these forces. The ability of centrifuged cells to respond maximally to GA3 by producing α-amylase is related to the degree of redistribution of organelles within these cells. Thus, recovery of cells from centrifugation at forces below 30,000g is rapid, while recovery from forces above 40,000g is slow.  相似文献   

14.
Yorkstar wheat, grown in New York State, has a high percentage (10-11) of grains without embryos. The embryoless grains have viable aleurone layers and show no sign of injury. These grains are able to support α-amylase synthesis only in the presence of gibberellin A3 (GA3). In the absence of GA3 some protein synthesis occurs in embryoless grains during the early hours of soaking, indicating that such activity occurs prior to and independent of GA3 induction of α-amylase. The level of β-amylase on a dry weight basis is the same in embryoless and normal grains and decreases with time of soaking. In the presence of GA3, β-amylase decreases at a slower rate. Isoenzymes of α-amylase from GA3-treated embryoless and normal grains show quantitative as well as qualitative differences. Cycloheximide (60 μg/ml) completely inhibits the synthesis of α-amylase by embryoless grains. Of the RNA synthesis inhibitors, actinomycin D (60 μg/ml) was ineffective while 6-methylpurine (60 μg/ml) gave 65% inhibition without decreasing the number of isoenzymes.  相似文献   

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

16.
α-Amylase activities in extracts of different parts of barley grain (Hordeum vulgare L. cv Himalaya) were low after 1 day of germination at 20°C, but they began to increase afterwards. In the scutellum and the aleurone layer, the increases were small, but in the starchy endosperm a great increase took place between days 1 and 6.

When the aleurone layers were separated from germinating whole grains and incubated in 10 millimolar CaCl2, the α-amylase activity in the medium increased linearly for about 30 to 60 minutes, indicating secretion. The activity inside the aleurone layer decreased only slightly during the incubation, indicating that secretion of α-amylase was accompanied by synthesis. The rates of secretion in vitro by the aleurone layers separated at different stages of germination corresponded rather well to the rate of accumulation of α-amylase activity in the starchy endosperm in a whole grain.

Scutella separated after 1 day of germination released small amounts of α-amylase activity into 10 millimolar CaCl2. This release was linear for at least 1 hour and did not occur at 0°C; it is therefore likely to be due to secretion. At later stages of germination, the secretion by the scutella was slower than at day 1 and the total secretion accounted for only 5 to 10% of the increase of α-amylase activity in the starchy endosperm in a whole grain.

Since the times from the separation of the parts of the grain to the beginning of the secretion assay (10-40 minutes) as well as the duration of the assay itself (20-60 minutes) were short, the rates of secretion by the separated grain parts are likely to represent those in an intact grain. The results indicate therefore that at least in the conditions used the bulk of the total α-amylase in the starchy endosperm is secreted by the aleurone layer, the contribution by the scutellum being only 5 to 10% of the total activity.

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17.
The gibberellic acid (GA3)-induced α-amylases from the aleurone layers of Himalaya barley (Hordeum vulgare L. cv Himalaya) have been purified by cycloheptaamylose-Sepharose affinity chromatography and fractionated by DEAE-cellulose chromatography. Four fractions (α-amylases 1-4) were obtained which fell into two groups (A and B) on the basis of a number of characteristics. Major differences in serological characteristics and in proteolytic fingerprints were found between group A (α-amylases 1 and 2) and group B (α-amylases 3 and 4). Also, the lag time for appearance of group B enzyme activity was longer than for group A, and the appearance of group B required higher GA3 levels than group A. The components of each group behaved similarly, although differences in proteolytic fingerprints were detected.

These results together with those from other studies indicate that GA3 differentially controls the expression of two α-amylase genes or groups of genes giving rise to two groups of α-amylases with many different properties.

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18.
Kernels of Klages barley (Hordeum vulgare L.) were germinated for 1 to 4 days on moist sand at 18°C. Representative kernels from each time period were dissected to give the following fractions: scutellum, subscutellar endosperm, aleurone-scutellum interface, remaining aleurone, subaleurone endosperm, and core endosperm. These tissues were analyzed for α-amylase components by isoelectric focusing and rocket-line immunoelectrophoresis. Although aleurone and scutellar tissues appeared to synthesize the same α-amylase components, enzyme was detected first in the scutellum. A larger proportion of scutellar α-amylase was excreted into the endosperm compared to aleurone synthesized α-amylase. Aleurone cells appeared to synthesize appreciably more α-amylase than did scutellar tissue.  相似文献   

19.
Gibberellic acid (GA3) sensitivity (measured as α-amylase production) of the isolated aleurone tissue/deembryonated seed of two wheat (Triticum aestivum L. var Kite and var Aroona) varieties each containing either one of the dwarfing genes, Rht1 and Rht2, was increased significantly as a result of low temperature treatment. The magnitude of the low temperature-induced increase occurred without any change in the lag time of α-amylase production. This low temperature induction of GA3 sensitivity was found to be operative in aleurone tissue of only those varieties having at least one of the three Rht alleles. It is likely, therefore, that the low temperature treatment effect which `cures' or circumvents the genetic lesions manifest in the Rht1 and Rht2 genotypes is the same as that effective in the Rht3-containing genotype and probably involves an increase in hormone (GA3) receptor sites. Furthermore, this increase appears to be a quantitative temporal one.  相似文献   

20.
The sensitivity to gibberellic acid (GA3) of aleurone protoplasts isolated from a single harvest of an inbred line of Avena fatua seed that had been after-ripened over anhydrous CaCl2 at 25±2°C and 4±2°C for three years was assessed. Protoplasts isolated from aleurones of seed stored at 25°C produced substantially more -amylase in response to 10–7 M GA3 than those isolated from aleurones of seed stored at 4°C. The apparent difference in responsiveness does not appear to be due to a change in the duration of the lag phase between addition of GA3 and the production of -amylase. The dose response of aleurone protoplasts to GA3, measured as -amylase production, is complex and appears to have three phases. Protoplasts from seed stored at both temperatures respond appreciably to 10–14 M GA3. With increasing concentrations of GA3, up to 10–9 M, -amylase production increases similarly in protoplasts from both lots of seed, reaching a level approximately 2.7–3.8 times greater than when no GA3 is applied. GA3-induced -amylase production increases markedly as the concentration is raised from 10–9 M to 10–6 M, and the response then appears to be saturated. Over this part of the response curve protoplasts from the two seed lots differ markedly in their responsiveness to GA3. Those from seed stored at 25°C produce considerably more -amylase, >130-fold higher than the minus GA3 control, than those from seed stored at 4°C, <35-fold higher than the minus GA3 control. This apparent difference in the responsiveness of aleurone protoplasts to GA3 could be correlated with the loss of embryo dormancy in seed stored at 25°C. Seed stored at 4°C retained the dormancy characteristics present immediately after harvesting.  相似文献   

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