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.     

Response of barley aleurone layers to abscisic Acid

Cordycepin, an inhibitor of RNA synthesis in barley (Hordeum vulgare L.) aleurone cells, does not inhibit the gibberellic acid-enhanced α-amylase (EC 3.2.1.1.) synthesis in barley aleurone layers if it is added 12 hours or more after the addition of the hormone. However, the accumulation of α-amylase activity after 12 hours of gibberellic acid can be decreased by abscisic acid. The accumulation of α-amylase activity is sustained or quickly restored when cordycepin is added simultaneously or some time after abscisic acid, indicating that the response of aleurone layers to abscisic acid depends on the continuous synthesis of a short lived RNA. By analysis of the newly synthesized proteins by gel electrophoresis with sodium dodecylsulfate, we observed that the synthesis of α-amylase is decreased in the presence of abscisic acid while the synthesis of most of the other proteins remains unchanged. From the rate of resumption of α-amylase production in the presence of cordycepin and abscisic acid, it appears that abscisic acid does not have a measurable effect on the stability of α-amylase mRNA.     

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.     

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.     

Heat Shock Inhibits alpha-Amylase Synthesis in Barley Aleurone without Inhibiting the Activity of Endoplasmic Reticulum Marker Enzymes

The effects of heat shock on the synthesis of α-amylase and on the membranes of the endoplasmic reticulum (ER) of barley (Hordeum vulgare) aleurone were studied. Heat shock, imposed by raising the temperature of incubation from 25°C to 40°C for 3 hours, inhibits the accumulation of α-amylase and other proteins in the incubation medium of barley aleurone layers treated with gibberellic acid and Ca²⁺. When ER is isolated from heat-shocked aleurone layers, less newly synthesized α-amylase is found associated with this membrane system. ER membranes, as indicated by the activities of NADH cytochrome c reductase and ATP-dependent Ca²⁺ transport, are not destroyed by heat stress, however. Although heat shock did not reduce the activity of ER membrane marker enzymes, it altered the buoyant density of these membranes. Whereas ER from control tissue showed a peak of marker enzyme activity at 27% to 28% sucrose (1.113-1.120 grams per cubic centimeter), ER from heat-shocked tissue peaked at 30% to 32% sucrose (1.127-1.137 grams per cubic centimeter). The synthesis of a group of proteins designated as heat-shock proteins (HSPs) was stimulated by heat shock. These HSPs were localized to different compartments of the aleurone cell. Several proteins ranging from 15 to 30 kilodaltons were found in the ER and the mitochondrial/plasma membrane fractions of heat-shocked cells, but none of the HSPs accumulated in the incubation medium of heat-shocked aleurone layers.     

Cyclic Purine Mononucleotides: Induction of Gibberellin Biosynthesis in Barley Endosperm

The de novo synthesis of α-amylase in barley endosperm and isolated aleurone layers is induced by 3′,5′-cyclic purine mononucleotides and gibberellic acid. The induction of α-amylase by cyclic purine mononucleotides is prevented by 2,4-DNP, inhibitors of RNA and protein syntheses, CCC, AMO-1618 and phosfon. The induction of α-amylase formation by 3′,5′-cyclic purine mononucleotides, but not by gibberellic acid, is also blocked by inhibitors of DNA synthesis. Extracts from cyclic AMP-treated endosperm halves exhibit a characteristic gibberellin-like activity which is detectable within 12 hours from the addition of the cyclic AMP. On paper chromatograms this gibberellin-like activity is located at the Rf typical for GA₃. Its formation is prevented by inhibitors of DNA synthesis, CCC and AMO-1618. Glucose inhibits the formation of α-amylase induced by gibberellic acid. Glucose has no effect on the cAMP-induced gibberellin biosynthesis. The evidence shows that the cyclic purine mononucleotides induce DNA synthesis, which results in gibberellin biosynthesis, which in turn activates the synthesis of α-amylase.     

Control of alpha-amylase mRNA accumulation by gibberellic Acid and calcium in barley aleurone layers

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.     

Regulation of the accumulation of mRNA for alpha-amylase in barley aleurone

The effect of gibberellic acid and Ca²⁺ on the accumulation of α-amylase mRNAs in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) was studied using cDNA clones containing sequences of mRNAs for the high and low isoelectric point (pI) α-amylases. There is no significant hybridization between the two α-amylase cDNA clones under the hybridization and washing conditions employed. These clones were therefore used to monitor levels of mRNAs for high and low pI α-amylases. It is shown that although the synthesis of the high pI α-amylase proteins depends on the presence of Ca²⁺ in the incubation medium, the accumulation of mRNA for this group occurs to the same degree in the presence or the absence of Ca²⁺. The accumulation of low pI α-amylase mRNA is also not affected by the presence or absence of Ca²⁺ in the incubation medium. These results establish gibberellic acid, not Ca²⁺, as the principal regulator of α-amylase mRNA accumulation in barley aleurone, while Ca²⁺ controls high pI α-amylase synthesis at a later step in the biosynthetic pathway.     

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.     

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.     

Differential rates of α-amylase and acid phosphatase induction by Ga3 in embryoless half-seeds and isolated aleurones of wheat

The induction of α-amylase and acid phosphatase by gibberellic acid (GA₃) was significantly higher (2–4-fold) in embryoless half-seeds of wheat than that observed in the excised aleurones. Addition of endosperm extract to excised aleurones enhanced the stimulatory effect of GA₃ on amylase activity by approximately 2-fold. Substitution of endosperm extract by 19 amino acids in GA₃-treated aleurones also brought about a 2–2.5-fold stimulation of α-amylase activity. Subsequent studies revealed that the addition of seven non-polar amino acids (0.5 mM each) was sufficient for the enhanced induction of α-amylase (1.8–2.5-fold) in GA₃-treated aleurones. A similatory effect of endosperm extract and amino acids on acid phosphatase activity was observed in GA₃-treated wheat aleurones. These observations are of physiological significance since an increased pool of free amino acids (5-fold) was also witnessed in the incubation medium of GA₃-treated half-seeds in comparison to the hormone-treated aleurones. The relative abundance of free amino acids in half-seed seems vital for the maximal induction of α-amylase and acid phosphatase. Thus, the presence of endosperm tissue associated with the aleurone layers is crucial for enhanced rate of production of GA₃-induced α-amylase and acid phosphatase in the wheat system.     

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.     

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

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

Calmodulin stimulation of unidirectional calcium uptake by the endoplasmic reticulum of barley aleurone

The role of calmodulin (CaM) in gibberellic acid (GA₃)-stimulated Ca²⁺ uptake was investigated in endomembranes isolated from aleurone cells of barley (Hordeum vulgare L.). Unidirectional Ca²⁺ -uptake activity of endoplasmic reticulum (ER) was higher in membranes isolated from aleurone layers treated for 16 h with GA₃ and Ca²⁺ compared with those isolated from layers incubated in Ca²⁺ alone. However, the level of uptake from Ca²⁺-treated tissue could be stimulated to that of the GA₃-treated cells by applying exogenous CaM which increased the V _max of the Ca²⁺ transporter approximately threefold. Calcium uptake in ER from GA₃-treated tissue was inhibited by the CaM antagonist W7 in 50% of experiments, whereas the activity in membranes from non-GA₃-treated tissue was unaffected. Treatment with GA₃ also led to a twofold increase in CaM levels in aleurone layers within 4–6 h, paralleling the time course of the stimulation of Ca²⁺ uptake and preceding the stimulation of α-amylase secretion. We propose that the elevation of Ca²⁺ uptake into the ER induced by GA₃ may be coordinated and regulated by elevated levels of membrane-associated CaM and this may regulate Ca²⁺-dependent α-amylase synthesis in the lumen of the ER.     

Differential effects of mannitol on gibberellin-regulated phospholipid synthesis and enzyme activities of the CDP-choline pathway in barley aleurone cells

Summary When barley aleurone layers are treated with gibberellic acid (GA₃) 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 GA₃, nor does it appreciably inhibit GA₃-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 GA₃ can be separated from later activities regulated by the hormone, including -amylase synthesis.     

Effect of temperature on the synthesis and secretion of alpha-amylase in barley aleurone layers

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.     

Amylases in developing barley seeds

The amylases of developing barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated by colorimetric and electrophoretic methods. Maxima of amylolytic activity appeared in the aleurone layers and starchy endosperm at 5 and 20 days after anthesis. Amylase from 5-day-old aleurone layers could be separated into four rapidly moving bands with α-amylase activity. By 20 days the four bands had been replaced by seven bands of medium mobility. These seven bands of amylase were electrophoretically identical to those observed when mature aleurone layers are treated with gibberellic acid. Immature aleurone layers failed to respond to exogenous gibberellic acid. In the starchy endosperm the seven bands of medium mobility were also present. Calcium-dependent alterations in the electrophoretic mobility and activity of particular bands occurred during the maturation of the starchy endosperm. Treatment of the immature starchy endosperm with papain yielded four forms of β-amylase.     

Hormonal control of polyribosome formation in barley aleurone layers

The addition of abscisic acid to barley (Hordeum vulgare L. cv. Himalaya) aleurone layers at the same time as gibberellic acid completely prevents the gibberellin-induced increases in the percentage of polysomes, the formation of polyribosomes, and the synthesis of α-amylase, even when the molar concentration of gibberellic acid is four times greater than the concentration of abscisic acid. The addition of abscisic acid to aleurone cells producing α-amylase (midcourse addition) inhibits the further synthesis of α-amylase and decreases the percentage of polysomes but does not change the number of ribosomes per cell.