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.     

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.     

Alpha-amylase secretion by single barley aleurone layers

The secretion of α-amylase from single isolated (Hordeum vulgare L. cv Himalaya) aleurone layers was studied in an automated flow-through apparatus. The apparatus, consisting of a modified sample analyzer linked to a chart recorder, automatically samples the flow-through medium at 1 minute intervals and assays for the presence of α-amylase. The release of α-amylase from aleurone layers begins after 5 to 6 hours of exposure to gibberellic acid and reaches a maximum rate after 10 to 12 hours. The release of α-amylase shows a marked dependence on Ca²⁺, and in the absence of Ca²⁺ it is only 20% of that in the presence of 10 millimolar Ca²⁺. Withdrawal of Ca²⁺ from the flow-through medium results in the immediate cessation of enzyme release and addition of Ca²⁺ causes immediate resumption of the release process. The effect of Ca²⁺ is concentration-dependent, being half-maximal at 1 millimolar Ca²⁺ and saturated at 10 millimolar Ca²⁺. Ruthenium red, which blocks Ca²⁺ but not Mg²⁺ efflux from barley aleurone layers, renders α-amylase release insensitive to Ca²⁺ withdrawal. Inhibitors of respiratory metabolism cause a burst of α-amylase release which lasts for 0.5 to 5 hours. Following this phase of enhanced α-amylase release, the rate of release declines to zero. Pretreatment of aleurone layers with HCl prior to incubation in HCN also causes a burst of α-amylase release, indicating that the inhibitor is affecting the secretion of α-amylase and not its movement through the cell wall. The rapid inhibition of α-amylase release upon incubation of aleurone layers at low temperature (5°C) or in 0.5 molar mannitol also indicates that enzyme release is dependent on a metabolically linked process and is not diffusion-limited. This conclusion is supported by cytochemical observations which show that, although the cell wall matrix of aleurone layers undergoes extensive digestion after gibberellin treatment, the innermost part of the cell wall is not degraded and could influence enzyme release.     

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.     

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.     

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.

     

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.     

Effect of Ethylene on the Release of alpha-Amylase through Cell Walls of Barley Aleurone Layers

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.     

Characterization of the increased lysophospholipase activity in gibberellic Acid-treated barley aleurone layers

A lysophospholipase (LPL) activity appears in the aleurone of barley (Hordeum vulgare L. cv Himalaya) half seeds during imbibition on moist agar. Secretion of LPL by half seeds is promoted by GA₃; the increase in secretory rate is almost linear from 10⁻¹⁰ to 10⁻⁶ molar GA₃. LPL activity is likewise promoted in isolated aleurone layers by GA₃. Its secretion into the incubation medium requires the continued presence of GA₃ and commences after a 10 to 14 hour lag period when 10 millimolar Ca²⁺ is present. In the absence of Ca²⁺, the lag period remains unchanged but attainment of the maximum secretory rate is delayed. Ca²⁺ alone has very little effect either on LPL activity accumulated in the aleurone layer or in the surrounding medium. However, 50 millimolar Ca²⁺ together with GA₃ dramatically increase the level of secreted activity and of total (accumulated and secreted) activity.

The metabolic inhibitors cycloheximide and actinomycin D inhibit the accumulation of LPL activity in the aleurone and also the secreted activity. Actinomycin D added after the lag period results in a much lower inhibition. The increase in LPL activity in response to GA₃ occurs as a result of de novo synthesis; LPL activity from barley half seeds incubated in 80% D₂O in the presence of GA₃ undergoes a shift to higher density compared with the activity from similar controls incubated in H₂O. The characteristics of the GA₃ enhancement of LPL activity are compared specifically with α-amylase and generally with other GA₃-controlled hydrolases.

     

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.     

Dormancy of the barley grain is correlated with gibberellic Acid responsiveness of the isolated aleurone layer

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.     

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.     

Control of lactate dehydrogenase, lactate glycolysis, and alpha-amylase by o(2) deficit in barley aleurone layers

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.     

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.     

Embryoless Wheat Grain: A Natural System for the Study of Gibberellin-induced Enzyme Formation

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.     

Quantitative and qualitative changes in the endoplasmic reticulum of barley aleurone layers

Changes in the level of the endoplasmicreticulum (ER) marker enzyme cytochrome-c reductase (EC 1.6.2.1) were followed with time of imbibition of de-embryonated half-seeds of barley (Hordeum vulgare L.) and the subsequent incubation of their aleurone layers in gibberellic acid (GA₃) and H₂O. During imbibition there is an increase in the level of cytochrome-c-reductase activity and in the amount of 280-nm absorbance associated with this enzyme. When aleurone layers are incubated for a further 42 h in water, there is a doubling of the cytochrome-c-reductase activity. In GA₃, the activity of cytochrome-c reductase reaches a maximum at 24 h of incubation and thereafter falls to below 70% of its level at the beginning of the incubation period. Changes in the cytochrome-c-reductase activity correlate with changes in the fine structure of the aleurone cell. The ER isolated in low Mg²⁺ from aleurone layers incubated in buffer for up to 18 h has buoyant density of 1.13–1.14 g cc^-1 while that from layers incubated in GA₃ for 7.5–18 h has a density of 1.11–1.12 g cc^-1. The -amylase (EC3.2.1.1) isolated with the organelle fraction by Sepharose gel filtration is associated with the ER on isopycnic and rate-zonal density gradients, and its activity can be enhanced by Triton X-100. The soluble -amylase fraction from Separose-4B columns, on the other hand, is not Triton-activated but is acid-labile. Acid phosphatase (EC3.1.3.2) is distributed in at least three peaks on isopycnic gradients. In low Mg²⁺ the second peak of activity has a density of 1.12 g cc^-1 in GA₃-treated tissue and 1.13–1.14 g cc^-1 in H₂O-treated tissue. With high-Mg²⁺ buffers, this peak of phosphatase activity disappears. Acid-phosphatase activity is not enhanced by Triton X-100 nor is it acid-labile.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid     

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.