Gibberellic Acid and Ion Release from Barley Aleurone Tissue: Evidence for Hormone-dependent Ion Transport Capacity

The release of potassium, magnesium, and phosphate ions from aleurone cells of barley (Hordeum vulgare L. cv. Himalaya) is a gibberellic acid-dependent process. The release of these ions is preceded by a lag period of 6 to 8 hours after gibberellic acid addition. The effect of gibberellic acid on the release of ions is not mediated through an effect on ion solubilization. Thus, gibberellic acid does not apreciably affect the sum of extracted and released ions relative to controls. Rather, the effect of the hormone is on the release process itself. Inhibitors of oxidative phosphorylation when added with gibberellic acid or at times up to 6 hours after gibberellic acid inhibition release. When these inhibitors are added after ion release has begun, however, rapid efflux of ions occurs. These results suggest a strong correlation between energy levels and ion transport capacity. Inhibitors of RNA and protein synthesis also inhibit gibberellic acid-stimulated ion release. Evidence suggests that RNA and protein synthesis are required to establish and maintain ion release capacity of aleurone cells.     

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.     

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.     

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.

     

Gibberellic Acid-enhanced synthesis and release of alpha-amylase and ribonuclease by isolated barley and aleurone layers

Gibberellic acid enhances the synthesis of α-amylase in isolated aleurone layers of barley-seeds (Hordeum vulgare var. Himalaya). In the presence of 20 mm calcium chloride the amount of enzyme obtained from isolated aleurone layers is quantitatively comparable to that of the half-seeds used in earlier studies. After a lag period of 6 to 8 hours enzyme is produced at a linear rate. Gibberellic acid does not merely trigger α-amylase synthesis, but it is continuously required during the period of enzyme formation. Enzyme synthesis is inhibited by inhibitors of protein and RNA synthesis. Small amounts of actinomycin D differentially inhibit enzyme release and enzyme synthesis suggesting 2 distinct processes. Gibberellic acid similarly enhances the formation of ribonuclease which increases linearly over a 48 hour period. During the first 24 hours the enzyme is retained by the aleurone cells and this is followed by a rapid release of ribonuclease during the next 24 hour period. The capacity to release the enzyme is generated between 20 and 28 hours after the addition of the hormone. Ribonuclease formation is inhibited by inhibitors of protein and RNA synthesis. These inhibitors also prevent the formation of the release mechanism if added at the appropriate moment.     

Interactions between Gibberellic Acid, Ethylene, and Abscisic Acid in Control of Amylase Synthesis in Barley Aleurone Layers

Gibberellic acid-induced α-amylase synthesis in barley (Hordeum vulgare L.) aleurone layers was inhibited by abscisic acid, and the inhibition was partly removed by additional gibberellic acid alone and by ethylene alone. Together additional gibberellic acid and ethylene almost eliminated abscisic acid inhibition of amylase synthesis. Time course studies of these phenomena showed that the effect of abscisic acid, ethylene, and varying concentrations of gibberellic acid on the course of amylase synthesis were either to speed up or slow down the whole process and not to affect the lag phase or the linear phase separately. The data are discussed in relation to previous studies of abscisic acid-gibberellic acid interaction.     

Substrate induction of nitrate reductase in barley aleurone layers

Nitrate induces the formation of nitrate reductase activity in barley (Hordeum vulgare L. cv. Himalaya) aleurone layers. Previous work has demonstrated de novo synthesis of α-amylase by gibberellic acid in the same tissue. The increase in nitrate reductase activity is inhibited by cycloheximide and 6-methylpurine, but not by actinomycin D. Nitrate does not induce α-amylase synthesis, and it has no effect on the gibberellic acid-induced synthesis of α-amylase. Also, there is little or no direct effect of gibberellic acid (during the first 6 hr of induction) or of abscisic acid on the nitrate-induced formation of nitrate reductase. Gibberellic acid does interfere with nitrate reductase activity during long-term experiments (greater than 6 hr). However, the time course of this inhibition suggests that the inhibition may be a secondary one. Barley aleurone layers therefore provide a convenient tissue for the study of both substrate- and hormone-induced enzyme formation.     

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.     

Production of cell wall hydrolyzing enzymes by barley aleurone layers in response to gibberellic Acid

The cell walls of barley (Hordeum vulgare var. Himalaya) aleurone layers undergo extensive degradation during the tissue's response to gibberellic acid. Previous work had shown that these cell walls consist almost entirely of arabinoxylan. In this study we show that gibberellic acid stimulates endo-β-1,4-xylanase activity in isolated aleurone layers. In addition, gibberellic acid enhances the activity of two glycosidases: β-xylopyranosidase and α-arabinofuranosidase. No gibberellic acid-stimulated cellulase activity was detected. Germination studies showed a similar pattern of enzyme development in intact seeds.     

A Correlation between a Ribonucleic Acid Fraction Selectively Labeled in the Presence of Gibberellic Acid and Amylase Synthesis in Barley Aleurone Layers

The effects of gibberellic acid on the incorporation of radio-active uridine and adenosine into RNA of barley aleurone layers were investigated using a double labeling method combined with acrylamide gel electrophoresis. After 16 hours of incubation, gibberellic acid stimulated the incorporation of label into all species of RNA, but the effects were very small (0-10%) for ribosomal and transfer RNA and comparatively large (up to 300%) for RNA sedimenting between 5S and 14S. This result was obtained for both isolated aleurone layers and for layers still attached to the endosperm. A similar but less marked pattern occurred in layers incubated for 8 hours, but the effect was not observed after 4 hours. The gibberellic acid-enhanced RNA labeling was not due to micro-organisms. The following evidence was obtained for an association between the gibberellic acid-enhanced RNA synthesis and α-amylase synthesis: (a) synthesis of α-amylase took place in parallel with incorporation of label into gibberellic acid-RNA; (b) actinomycin D inhibited amylase synthesis and gibberellic acid-RNA by similar percentages; (c) 5-fluorouracil halved incorporation of label into ribosomal RNA but had no effect on amylase synthesis and gibberellic acid-RNA; and (d) abscisic acid had little effect on synthesis of RNA in the absence of gibberellic acid, but when it was included with gibberellic acid the synthesis of both enzyme and gibberellic acid-RNA was eliminated. We conclude that large changes in the synthesis of the major RNA species are not necessary for α-amylase synthesis to occur but that α-amylase synthesis does not occur without the production of gibberrellic acid-RNA. Gibberellic acid-RNA is probably less than 1% of the total tissue RNA, is polydisperse on acrylamide gels, and could be messenger species for α-amylase and other hydrolytic enzymes whose synthesis is under gibberellic acid control.     

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.     

Development of (1-->3,1-->4)-beta-d-Glucan Endohydrolase Isoenzymes in Isolated Scutella and Aleurone Layers of Barley (Hordeum vulgare)

An immunological assay has been used to investigate the synthesis of (1→3,1→4)-β-glucanase (EC 3.2.1.73) isoenzymes from isolated barley aleurone layers and scutella. Enzyme release from both tissues is enhanced by 1 micromolar gibberellic acid and 10 millimolar Ca²⁺, although increases induced by gibberellic acid are observed only in the presence of Ca²⁺. Isoenzyme I is synthesized predominantly in the scutellum, while isoenzyme II is synthesized exclusively in the aleurone. A third, putative isoenzyme III has been detected in significant proportions in scutellar secretions and may also be secreted from aleurone layers. Both gibberellic acid and Ca²⁺ appear to preferentially enhance isoenzyme II secretion from the aleurone and isoenzyme III secretion from scutella. The patterns of isoenzyme secretion are suggestive of tissue-specific differences in expression of the genes which code for (1→3,1→4)-β-glucanase isoenzymes. Qualitatively similar results were obtained with barley cultivars harvested in Australia and North America.     

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.     

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.     

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.     

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.     

Multiple Forms of Amylase Induced by Gibberellic Acid in Isolated Barley Aleurone Layers

The addition of gibberellic acid to isolated aleurone layers of barley (Hordeum vulgare L.) causes the production and secretion of four α-amylases. Two of these are stable at pH 3.7 and are not inactivated by ethylenediaminetetraacetate. The other two represent the classical barley α-amylases; i.e., they are inactivated at pH 3.7 and by reagents which from complexes with divalent metal ions. All four forms are synthesized de novo in response to the addition of gibberellic acid.     

Inhibition of Gibberellic Acid-induced alpha-Amylase Formation by Polyethylene Glycol and Mannitol

Both polyethylene glycol (PEG) and mannitol inhibit gibberellic acid-induced α-amylase production in barley aleurone layers. The effect of the osmotic solution is on enzyme synthesis rather than α-amylase secretion. The inhibition of α-amylase synthesis does not appear to be mediated via an indirect effect on respiration or protein synthesis. Rather it seems that the osmotic solutions reduce the extent of proteolysis of the stored aleurone grain protein thus making available less substrate for new protein synthesis.