Gibberellic Acid-induced synthesis of protease by isolated aleurone layers of barley

The production of protease by isolated aleurone layers of barley in response to gibberellic acid has been examined. The protease arises in the aleurone layer and is mostly released from the aleurone cells. The courses of release of amylase and protease from aleurone layers, the dose responses to gibberellic acid and the effects of inhibitors on the production of both enzymes are parallel. As is the case for amylase, protease is made de novo in response to the hormone. These data give some credence to the hypothesis that the effect of gibberellic acid is to promote the simultaneous synthesis and secretion of a group of hydrolases.     

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.     

Gibberellic Acid-enhanced Release of beta-1,3-Glucanase from Barley Aleurone Cells

A β-1, 3-glucanase of barley (Hordeum vulgare) aleurone cells accumulates when half-seeds are imbibed on water, and accumulation continues when the aleurone layers are incubated in buffer solution. The release of the enzyme is a gibberellic acid-dependent process, however. Although gibberellic acid stimulates glucanase release, it does not markedly affect the total amount of glucanase obtained from these cells when compared with water controls. β-1, 3-Glucanase release from aleurone cells is a function of gibberellic acid concentration and commences after a 4-hour lag period. Processes occurring during this lag period are also dependent upon gibberellic acid concentration. Removal of gibberellic acid from the incubation medium at the end of the lag period, however, does not affect subsequent release of glucanase. The release of glucanase from aleurone cells is an active process with a Q₁₀ greater than 3. Inhibitors of respiration and protein and RNA synthesis effectively inhibit the formation and release of glucanase. It is concluded that gibberellic acid functions primarily to enhance glucanase release rather than its formation.     

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.     

Hormonal control of enzyme synthesis: on the mode of action of gibberellic Acid and abscisin in aleurone layers of barley

Gibberellic acid (GA) enhances the synthesis of α-amylase and ribonuclease in isolated aleurone layers and this process is inhibited by abscisin. Removal of gibberellic acid in mid-course of α-amylase production results in a slowing down of α-amylase synthesis, suggesting a continued requirement of GA for enzyme synthesis. This is paralleled by a continuous requirement for RNA synthesis. Addition of 6-methylpurine or 8-azaguanine in mid-course results in an inhibition of α-amylase synthesis within 3 to 4 hours. However, actinomycin D added in mid-course is almost without effect. This is not due to its failure to enter the cells, because it does inhibit ¹⁴C-uridine incorporation at this stage. Addition of abscisin to aleurone layers which are synthesizing α-amylase results in an inhibition of this synthesis within 2 to 3 hours. Cycloheximide on the other hand inhibits enzyme synthesis immediately upon its addition. These data are consistent with the hypothesis that the expression of the GA effect requires the synthesis of enzyme-specific RNA molecules. The similarity in the kinetics of inhibition between abscisin on the one hand and 8-azaguanine or 6-methylpurine on the other suggests that abscisin may exert its action by inhibiting the synthesis of these enzyme-specific RNA molecules or by preventing their incorporation into an active enzyme-synthesising unit.     

An early response to gibberellic Acid not requiring protein synthesis

Cell-free extracts from gibberellic acid-treated barley (Hordeum vulgare L. cv. Himalaya) aleurone layers show phosphorylcholine glyceride transferase activity greater than that from control layers. The increase in activity is not prevented by a mixture of amino acid analogs nor by cordycepin under conditions in which it is demonstrated that the analogs and the cordycepin are entering the cells in effective concentrations. We conclude therefore that the GA₃-dependent increase in phosphorylcholine glyceride transferase activity (which occurs within the first 4 hours of GA₃ treatment) does not require RNA synthesis or protein synthesis.     

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.     

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.     

The role of ethylene in the induction of amylase activity in wheat aleurone tissue

When wheat aleurone layers ( Triticum aestivum L. var. Potam S-70) incubated in medium containing gibberellic acid were exposed to ethylene, the synthesis and release of amylase were enhanced relative to layers incubated in the presence of mercuric perchlorate. Exogenous ethylene stimulated gibberellic acid-induced amylase synthesis by approximately 2.2-fold. The ethylene-mediated stimulation of amylase formation was dependent upon the tissue being exposed to the gas during the lag phase of gibberellic acid action. Ethylene appeared to promote only quantitative changes in amylase synthesis and release, since the isoelectric focusing patterns of amylase is enzymes were not significantly altered by ethylene. Ethylene had no effect on the incorporation of [methyl-¹⁴C]choline into aleurone phospholipids, but stimulated the accumulation of [U-¹⁴C]adenine into poly(A) RNA of gibberellic acid-treated tissue by about 80%.     

A gibberellin-induced nuclease is localized in the nucleus of wheat aleurone cells undergoing programmed cell death

The aleurone layer of cereal grains undergoes a gibberellin-regulated process of programmed cell death (PCD) following germination. We have applied a combination of ultrastructural and biochemical approaches to analyze aleurone PCD in intact wheat grains. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed that PCD was initiated in aleurone cells proximal to the embryo and then extended to distal cells. DNA fragmentation and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis revealed PCD of aleurone cells in maize grains, although the process was delayed as compared with wheat. Aleurone cells undergoing PCD showed a rapid vacuolation with high lytic activity in the cytoplasm, whereas the nucleus, which adopted an irregular shape, appeared essentially intact and showed symptoms of degradation at the end of the process. A nuclease activity was identified localized in the nucleus of aleurone cells undergoing PCD, just prior to the appearance of DNA laddering. This nuclease was induced by gibberellic acid treatment and was not detected when gibberellin synthesis was inhibited or in gibberellic acid-insensitive mutants. This nuclease was activated by Ca(2+) and Mg(2+), strongly inhibited by Zn(2+), and showed optimum activity at neutral pH, resembling nucleases involved in apoptosis of animal cells.     

Isolation and sequence analysis of a barley alpha-amylase cDNA clone

We have isolated a cDNA clone derived from poly(A+) RNA from barley aleurone cells stimulated with gibberellic acid. This cDNA clone contains one open reading frame coding for 438 amino acids. The cloned DNA hybridizes to a poly(A+) RNA species 1550 bases in size, the same size as the most abundant poly(A+) RNA molecules in stimulated cells. RNA complementary to this clone can be translated to make immunoprecipitable alpha-amylase in the wheat germ system and increases about 5-fold in quantity after gibberellic acid stimulation of aleurone cells. In contrast, hybridization experiments using a total cDNA probe demonstrate that the most abundant mRNA population, identical in size with our cloned sequence and presumably that for alpha-amylase, increases at least 17-fold after gibberellic acid stimulation. We therefore infer that there must be at least two populations of alpha-amylase mRNA molecules derived from separate structural genes differently influenced by gibberellic acid in aleurone cells.     

Synthesis and release of sucrose by the aleurone layer of barley: Regulation by gibberellic acid

Summary Aleurone layers of barley contain large amounts of a soluble oligosaccharide which was identified as sucrose (30–40 g/mg fresh weight). Treatment of the layers with gibberellic acid (GA₃) causes the release of sucrose from the cells. This release requires the participation of metabolic processes, including protein synthesis. When embryoless half-seeds are incubated sucrose accumulates in the aleurone layers, but when seeds are germinated the sucrose content of the aleurone layers declines. Labeling experiments with radioactive glucose and fructose show that aleurone layers continuously synthesize sucrose and that the release, but not the synthesis of sucrose is enhanced by GA₃.     

The Role of Membrane-Bound Enzymes in an Early Response of Aleurone Tissue to Gibberellic Acid

Treatment of aleurone layers of barley seed with gibberellicacid increases the observable phosphorylcholine glyceride transferaseactivity in a membrane fraction prepared from extracts of thealeurone cells. This gibberellic acid-dependent increase inglyceride transferase activity requires neither RNA synthesisnor protein synthesis. Membrane fractions prepared from mixturesof extracts of gibberellic acid-treated layers and control layershave a specific activity of glyceride transferase higher thanexpected on the basis of simple addition of the activities fromthe two sources. Therefore, some kind of activation is occurring.     

Selective expression of a probable amylase/protease inhibitor in barley aleurone cells: Comparison to the barley amylase/subtilisin inhibitor

We have cloned and sequenced a 650-nucleotide cDNA from barley (Hordeum vulgare L.) aleurone layers encoding a protein that is closely related to a known -amylase inhibitor from Indian finger millet (Eleusine coracana Gaertn.), and that has homologies to certain plant trypsin inhibitors. mRNA for this probable amylase/protease inhibitor (PAPI) is expressed primarily in aleurone tissue during late development of the grain, as compared to that for the amylase/subtilisin inhibitor, which is expressed in endosperm during the peak of storage-protein synthesis. PAPI mRNA is present at high levels in aleurone tissue of desiccated, mature grain, and in incubated aleurone layers prepared from rehydrated mature seeds. Its expression in those layers is not affected by either abscisic acid or gibberellic acid, hormones that, respectively, increase and decrease the abundance of mRNA for the amylase/subtilisin inhibitor. PAPI mRNA is almost as abundant in gibberellic acid-treated aleurone layers as that for -amylase, and PAPI protein is synthesized in that tissue at levels that are comparable to -amylase. PAPI protein is secreted from aleurone layers into the incubation medium.Abbreviations ABA abscisic acid - ASI barley amylase/subtilisin inhibitor - bp nucleotide base pairs - Da dalton - dpa days post anthesis - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor - poly(A)RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

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.     

Involvement of the Golgi apparatus in the secretion of α-amylase from gibberellin-treated barley aleurone cells

Localisation of -amylase (EC 3.2.1.1) in barley aleurone cells treated with gibberellic acid has been achieved using protein A-gold-labelled polyclonal antibodies. Gold particles were located almost exclusively over the lumen of the rough endoplasmic reticulum and cisternae of the Golgi apparatus. The label was most concentrated over the Golgi apparatus. This indicates that the Golgi is involved in the secretion of -amylase protein from aleurone cells.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - PBS phosphate-buffered saline