A comparison of the effects of Ca2+ and gibberellic acid on enzyme synthesis and secretion in barley aleurone

The effects of the addition and withdrawal of gibberellic acid (GA₃) and Ca²⁺ on enzyme synthesis and secretion by barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. Incubation of layers in GA₃ plus Ca²⁺ affects the total amount of secreted α-amylase (EC 3.2.1.1) and acid phosphatase (EC 3.1.3.2) by promoting the appearance of different isoenzymic forms of these enzymes. The release of α-amylase isoenzymes 1–4 in response to GA₃ plus Ca²⁺ has a lag of 6 h. When layers are incubated in GA₃ alone for 6 h prior to the addition of Ca²⁺, isoenzymes 1–4 appear in the medium after only 30 min. When the addition of Ca²⁺ to layers pretreated in GA₃ is delayed beyond 12 h, its effectiveness in stimulating the synthesis and release of isoenzymes 3 and 4 is diminished. After 35 h of preincubation in GA₃, addition of Ca²⁺ will not stimulate synthesis of α-amylase isoenzymes 3 and 4. Aleurone layers preincubated for 6 h in GA₃ will respond to Ca²⁺ when the GA₃ is withdrawn from the incubation medium by producing α-amylase isoenzymes 1–4. The converse is not the case, however, since layers preincubated in Ca²⁺ for 6 h will not produce all isoenzymes of α-amylase when subsequently incubated in GA₃. The Ca²⁺-stimulated release of α-amylase from GA₃ pre-treated layers is dependent on the time of incubation in Ca²⁺ and the concentration of the ion. The response to Ca²⁺ is temperature-dependent, and other divalent cations such as Mg²⁺ cannot substitute for Ca²⁺. We conclude that Ca²⁺ influences α-amylase release by influencing events at the biochemical level.     

Externally applied gibberellic acid and α-amylase formation in grains of barley (Hordeum distichon)

During germination the aleurone layer of barley grains becomes progressively less able to form more α-amylase in response to a dose of gibberellic acid (GA₃). This decline appears to be linked to the presence of a growing embryo. In whole grains the embryo ‘modulates’ the response (α-amylase formation) to controlled external applications of GA₃ in a dose-dependent manner. Sugars, and some other metabolites, repress α-amylase formation in transected grains, apparently by reducing levels of endogenously produced gibberellins. This effect is partly, but not completely, reversed by additions of GA₃. External applications of GA₃ augment the levels of several gibberellin fractions within the grain. The nature of the gibberellin material remaining on the surface of the grains alters with time. Grains treated with GA₃ contain a conjugate of low biological activity, possibly a glycoside, that is hydrolysed by a mixed glycosidase preparation to release a biologically-active gibberell in resembling GA₃.     

Abscisic Acid Inhibition of Gibberellic Acid and Cyclic 3',5'-Adenosine Monophosphate Induced α-Amylase Synthesis

The induction of α-amylase synthesis in barley aleurone by cyclic 3′,5′-adenosine monophosphate or GA₃ was inhibited by abscisic acid. The concentration of ABA required to inhibit α-amylase induction by the cyclic nucleotide in the extract was one-fiftieth to one hundredth of that required for GA₃-induced α-amylase. It is concluded that the effects of ABA on GA₃ and cyclic nucleotide induced α-amylase synthesis in the aleurone are independent and indirect.     

Regulation of the synthesis of barley aleurone alpha-amylase by gibberellic Acid and calcium ions

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.     

Calcium regulation of the secretion of α-amylase isoenzymes and other proteins from barley aleurone layers

The effect of calcium on the secretion of α-amylase (EC 3.2.1.1) and other hydrolases from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was studied. Withdrawal of Ca²⁺ from the incubation medium of aleurone layers preincubated in 5 μM gibberellic acid (GA₃) and 5 mM CaCl₂ results in a 70–80% reduction in the secretion of α-amylase activity to the incubation medium. Agar-gel electrophoresis shows that the reduction in α-amylase activity following Ca²⁺ withdrawal is correlated with the disappearance of group B isoenzymes from the incubation medium. The secretion of isoenzymes of group A is unaffected by Ca²⁺. The addition of Ca²⁺ stimulates the secretion of group-B isoenzymes but has no measurable effect on either the α-amylase activity or the isoenzyme pattern of aleurone-layer extracts. Pulse-labelling experiments with [³⁵S]methionine show that Ca²⁺ withdrawal results in a reduction in the secretion of labelled polypeptides into the incubation medium. Immunochemical studies also show that, in the absence of Ca²⁺, α-amylase isoenzymes of group B are not secreted into the incubation medium. In addition to its effect on α-amylase, Ca²⁺ influences the secretion of other proteins including several acid hydrolases. The secretion of these other proteins shows the same dependence on Ca²⁺ concentration as does that of α-amylase. Other cations can promote the secretion of α-amylase to less and varying extents. Strontium is 85% as effective as Ca²⁺ while Ba²⁺ is only 10% as effective. We conclude that Ca²⁺ regulates the secretion of enzymes and other proteins from the aleurone layer of barley.     

Ca-stimulated secretion of alpha-amylase during development in barley aleurone protoplasts

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.     

Control of protein synthesis in barley aleurone layers by the plant hormones gibberellic acid and abscisic acid

The patterns of protein synthesis in barley aleurone layers treated with gibberellic acid (GA₃) 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, GA₃ and ABA both induce the formation of new translatable mRNAs and cause new proteins to be synthesized. The effects of GA₃ are more dramatic than those of ABA. In GA₃-treated tissues, overall protein synthesis is redirected to produce large quantities of α-amylase and a few other GA₃-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 GA₃ 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 GA₃-induced mRNAs does occur. This indicates that ABA does not interfere with GA₃ induction of translatable mRNAs but prevents the translation of these mRNAs in vivo. Thus ABA and potentially GA₃ regulate the translation of proteins in vivo in barley aleurone layers.     

Polyamine metabolism and its relation to response of the aleurone layers of barley seeds to gibberellic Acid

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.

     

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     

Tannins as gibberellin antagonists in the synthesis of alpha-amylase and Acid phosphatase by barley seeds

The tannins chebulinic acid or tara tannin were added to an incubation system in which GA₃ induces enzyme synthesis in endosperm half seeds of barley (Hordeum vulgare L.). The activity of amylase and acid phosphatase in the incubation medium was reduced compared to the activity in the medium after incubation with GA₃ alone. When embryo half seeds of barley were incubated with chebulinic acid or tara tannin in the absence of added GA₃, the enzyme activity of the incubation medium was also reduced. The activity of preformed enzymes obtained from endosperm half seeds previously induced with GA₃ was not reduced by the addition of tannin. Comparisons were made of the amount of enzyme activity from breis of aleurone layers incubated with GA₃ in the presence and absence of tannins. The amounts of activity were relatively small and approximately equal in both cases, indicating that secretion from the aleurone was not blocked by the tannins. The reduction of enzyme activity caused by tannins in both endosperm and embryo half seeds could be completely reversed by the addition of GA₃.     

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

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

Benzaldehyde O-Alkyloximes as New Plant Growth Regulators

Fourty-two kinds of benzaldehyde O-alkyloximes derived from benzaldehydes were prepared and their biological activities were investigated. Introduction of a fluorine or bromine atom to the benzene ring of the oximes enhanced their phytotoxic activity. The O-alkyloximes with a fluorine atom at the 3 or 4 position of the benzene ring were more active than the other oximes in the GA₃-induced α-amylase induction inhibition test. In the transpiration test, 4-bromobenzaldehyde O-carboxylmethyloxime was the most active. The O-alkyloximes exhibited weak abscisic acid-like activity by inhibiting not only the germination, root growth and transpiration of higher plants but also GA₃-induced α-amylase induction in embryoless barley seeds.     

On the uptake, metabolism and retention of [h] gibberellin a(1) by barley aleurone layers at low temperatures

Barley (c.v. Himalaya) aleurone layers were incubated in [³H]gibberellin A₁ (GA₁) at low temperatures. At 3 and 4 C, ³H-activity was steadily accumulated in aleurone layers, and this accumulation was correlated with significant [³H]GA₁ metabolism. At 1 and 1.5 C, metabolism could not be detected, and at these temperatures aleurone layers equilibrated with the [³H]GA₁ 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]GA₁ in the presence of saturating levels of carrier GA₁ consistently retained lower levels of ³H-activity than when incubated in [³H]GA₁ alone. The retention of [³H]GA₁ was unaffected by saturating levels of carrier GA₈. GA₁ 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.     

The induction of sensitivity to gibberellin in aleurone tissue of developing wheat grains

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 (GA₃) 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 GA₃ 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 GA₃ can be manipulated represents a useful step towards investigating the role of cell membranes in growth-regulator action.     

Screening for barley mutants with altered hormone sensitivity in their aleurone layers

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.     

Phosphate mobilization in grains of Hordeum distichon

When decorticated grains were germinated at 14·5°, inorganic substances moved from the endosperm, mainly the aleurone layer, to the embryo. The level of Pi rose in the embryo and endosperm, and the embryo appeared to accumulate Pi against a concentration gradient. The level of organic-P declined in the endosperm, particularly in the alcurone layer. Separated aleurone layers, incubated at 25°, released only small amounts of organic or inorganic phosphate. However, when incubated with gibberellic acid (GA₃), a massive release of Pi occurred at the expense of organic phosphates within the tissue. This release followed a sigmoid pattern with time following a lag and was virtually complete in 6 days. Allowing the aleurone layer to dry before incubating with GA₃ reduced or abolished the lag period of Pi release and only marginally depressed the total amount ultimately freed. In contrast, α-amylase production was depressed by the longer periods of drying. The major phosphate of the aleurone was phytate (meso-inositol hexaphosphate, IP₆), but traces of 1P₄, IP₃, IP₂, IP₁, Pi and unidentified phosphates were detected. During incubation with GA₃ the IP₆ content fell, and the lower esters of inositol rose slightly and then fell in a pattern indicating that the phosphate groups of each IP₆ molecule were being sequentially hydrolysed. After 6 days incubation, the tissue phosphates were reduced to a very low level. Attempts to isolate aleurone grains containing phytate were unsuccessful.     

Hormonal regulation of the development of protease and carboxypeptidase activities in barley aleurone layers

Carboxypeptidase and protease activities of hormone-treated barley (Hordeum vulgare cv Himalaya) aleurone layers were investigated using the substrates N-carbobenzoxy-Ala-Phe and hemoglobin. A differential effect of gibberellic acid (GA₃) on these activities was observed. The carboxypeptidase activity develops in the aleurone layers during imbibition without the addition of hormone, while the release of this enzyme to the incubation medium is enhanced by GA₃. In contrast, GA₃ is required for both the production of protease activity in the aleurone layer and its secretion. The time course for development of protease activity in response to GA₃ is similar to that observed for α-amylase. Treating aleurone layers with both GA₃ and abscisic acid prevents all the GA₃ effects described above. Carboxypeptidase activity is maximal between pH 5 and 6, and is inhibited by diisopropylfluorophosphate and p-hydroxymercuribenzoate. We have observed three protease activities against hemoglobin which differ in charge but are all 37 kilodaltons in size on sodium dodecyl sulfate polyacrylamide gels. The activity of the proteases can be inhibited by sulfhydryl protease inhibitors, such as bromate and leupeptin, yet is enhanced by 2-fold with 2-mercaptoethanol. In addition, these enzymes appear to be active against the wheat and barley storage proteins, gliadin and hordein, respectively. On the basis of these characteristics and the time course of GA₃ response, it is concluded that the proteases represent the GA₃-induced, de novo synthesized proteases that are mainly responsible for the degradation of endosperm storage proteins.     

No Effect of 5-Fluorouracil on the Properties of Purified alpha-Amylase from Barley Half-seeds

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.     

Density labeling of proteins with 13C-labeled amino acids: no accumulation of an inactive alpha-amylase precursor in barley aleurone cells.

Gibberellic acid enhances α-amylase (EC 3.2.1.1) production in isolated barley aleurone layers after a lag period of 4 to 8 h, and most of the enzyme is produced after 12 h of hormone treatment. Amino acids necessary for protein synthesis in barley aleurone layers are derived from the degradation of storage proteins in this tissue. Since bromate is an inhibitor of barley protease, in the presence of bromate the production of α-amylase in aleurone layers becomes dependent on exogenous amino acids. We have incubated aleurone layers with bromate plus ¹³C-labeled amino acids and [³H]leucine from 0 to 24, 0 to 12, and 12 to 24 h after the application of gibberellic acid. The chemical quantity of [³H]leucine was negligible in comparison to that of ¹³C-labeled amino acids. Therefore, any density shift of proteins observed must be due to the incorporation of ¹³C-labeled amino acids. The density shift of α-amylase and that of newly synthesized proteins (radioactivity profile) were determined by isopycnic centrifugation in CsCl density gradients. The density shift of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 12 to 24 h after the addition of hormone was much larger than that of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 0 to 12 h of hormone treatment. By comparing the density shift of α-amylase with that of newly synthesized proteins, it is apparent that essentially all the amylase molecules are de novo synthesized. We can conclude that there is little or no accumulation of an inactive α-amylase precursor in barley aleurone cells between the time of the application of gibberellic acid and the time of the rapid increase in α-amylase activity.     

On the secretion of α-amylase by barley aleurone layers after incubation in gibberellic acid

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