Effect of gibberellic Acid and actinomycin d on the formation and distribution of rough endoplasmic reticulum in barley aleurone cells

Analysis of structural changes in barley aleurone cells during germination or following incubation of isolated layers in gibberellic acid with or without actinomycin D revealed extensive development of rough endoplasmic reticulum. Following the assembly of stacked rough endoplasmic reticulum, vesiculation occurred mainly in basal regions of the cell, resulting in a polar distribution of rough endoplasmic reticulum vesicles. It is postulated that these vesicles are involved in protein secretion, because smooth vesicles, derived from the rough endoplasmic reticulum, apparently become appressed to the plasma membrane. The increased α-amylase in the ambient medium and in cell homogenates correlated directly with formation and subsequent vesiculation of the rough endoplasmic reticulum. Furthermore, when cells were treated with actinomycin D and gibberellic acid, α-amylase synthesis was inhibited by 45% and secretion by 63%. These cells were characterized cytologically by large areas of disarrayed segments of fragmented rough endoplasmic reticulum, corresponding to a high intracellular level of α-amylase. In addition, small lipid bodies common to the segmented regions of rough endoplasmic reticulum were surrounded by fine fibrous material, short segments of rough endoplasmic reticulum, and free ribosomes, suggesting that actinomycin D had interfered with development and organization of rough endoplasmic reticulum.     

Gibberellin modulation of phosphatidyl-choline turnover in wheat aleurone tissue

Phosphatidyl choline (PC) is synthesised in wheat (Triticum aestivum L. cv. Flanders) aleurone tissue during early germination when new endomembranes are being formed. Although gibberellic acid does not ostensibly affect PC levels, it inhibits the incorporation of choline and differentially and specifically modulates the turnover of the N-methyl and methylene carbons of the choline headgroup of PC. Gibberellic acid has no effect on turnover of the phosphate moiety of either PC or the other major phosphatides. The possible biological importance of the findings is discussed.Abbreviations ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid - PA phosphatidic acid - PC phosphatidyl choline - PE phosphatidyl ethanolamine - PG phosphatidyl glycerol - PI phosphatidyl inositol - t_1/2 half-life     

Localization of gibberellic acid-induced acid phosphatase activity in the endoplasmic reticulum of barley aleurone cells with the electron microscope

A metal-salt precipitation method with p-nitrophenyl phosphate as substrate has been used to localize in the electron microscope acid phosphatase activity in isolated aleurone layers of barley (Hordeum vulgare L.), treated for 16 h in the presence or absence of gibberellic acid (GA₃). The paper confirms results obtained earlier with an azo-dye precipitation method of enzyme localization. In addition the results show for the first time that in GA₃-treated tissue enzyme activity is associated with the endoplasmic reticulum (ER), there being reaction product deposited in the ER cisternae. It is suggested that this activity represents new enzyme synthesized on ER in response to GA₃ and probably destined for secretion.Abbreviation ER endoplasmic reticulum     

Amino acids and quaternary nitrogen compounds in the germinating wheat grain

The tissues of the quiescent wheat grain contained free amino acids and quaternary nitrogen compounds. During germination the amino acid levels increased several fold. In the aleurone tissue and starchy endosperm glutamine was the predominant amino acid. Asparagine was predominant in the seedling tissues. Choline and glycine betaine were the principal quaternary nitrogen compounds present. The aleurone tissue and the embryo/seedling contained large quantities of glycine betaine. The increase in free amino acid levels in the aleurone tissue during the first 2 days of germination occurred independently of the embryo. After the second day, the further increase in levels was dependent upon the presence of the embryo and of gibberellic acid (GA). Estimation of the individual amino acids and quaternary nitrogen compounds released from incubating aleurone layers into aqueous media revealed a selective release of some compounds and retention of others. The process was regulated by GA. Possible mechanisms for the release of amino acid and its control by GA are discussed.     

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     

Fine Structural Changes in Barley Aleurone Cells During Gibberellic Acid-induced Enzyme Secretion

The fine structure of barley aleurone cells was studied in theenzyme secretion phase. An ultrastructural feature of this phaseis the formation of stacked rough endoplasmic reticulum (rER),for such a structure was never found in cells during the enzymesynthesis phase. Other structural features frequently observedin the secretion phase were amoeboid-shaped nuclei, the stackedrER wound round the nucleus and mitochondria, and a stream ofthe stacked rER directed to the plasmamembrane. Hordeum vulgare L, barley, aleurone cells, enzyme secretion, gibberellic acid     

Free sterols and glycolipids in the aleurone tissue of germinating wheat

The action of gibberellic acid on wheat aleurone tissue led to reduced levels of free sterols and glycolipids compared with control tissue. Radio-labelled precursors were not incorporated into sterols or glycolipids, although mevalonate and glycerol were incorporated into polyisoprenoid hydrocarbon and phospholipid respectively. It is concluded that sterols and glycolipids are not synthesized in the tissue during germination; this is in contrast to earlier reports of the active, gibberellin-regulated metabolism of phospholipids.     

Subcellular localization of nuclease in barley aleurone

The intracellular localization of an endonuclease (nuclease I) in barley aleurone responding to gibberellic acid was investigated by subcellular fractionation and immunocytochemistry with monoclonal and polyclonal antibodies. Organelle separations were performed with aleurone layers and protoplasts; immunefixations were carried out on protoplasts only. Nuclease was detected in fractions from isopycnic sucrose density gradients which were enriched in either endoplasmic reticulum or Golgi apparatus membranes. These two organelles were also labelled by the indirect immunogold method on thin sections. Intensive labelling of protein and developing vacuoles was observed. Therefore, as noted in other plants nuclease in barley is essentially a vacuolar enzyme.     

The release of α-amylase through gibberellin-treated barley aleurone cell walls

Localisation of -amylase (EC 3.2.1.1.) in low-temperature-embedded isolated barley (Hordeum vulgare L.) aleurone has been achieved using rhodamine-labelled secondary antibodies and the protein A-gold technique. Treatment with gibberellic acid (GA₃) resulted in an increase of immunofluorescence in the cytoplasm of aleurone cells and also its appearance in specific regions of the cell walls. Cytoplasmic label was neither perinuclear nor associated specifically with aleurone grains as had been found in earlier work, but was present throughout the cytoplasm of all cells. A relatively high level of labelling occurred in hydrolysed wall regions. Label was also associated with plasmodesmata in both hydrolysed and unhydrolysed wall regions. The pattern of labelling indicates that -amylase is released from aleurone via digested wall channels and that, except for the inner wall layer, unhydrolysed regions are impermeable to the enzyme. It is suggested that the resistant wall tubes around plasmodesmata may facilitate enzyme release by providing a pathway for transfer, especially of wall hydrolases, into the more impermeable parts of the wall.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - RER rough endoplasmic reticulum     

Gibberellic acid controls specific acid-phosphatase isozymes in aleurone cells and protoplasts of Avena fatua L.

In the presence of gibberellic acid (GA₃) aleurone layers and isolated aleurone protoplasts of Avena fatua accumulate specific isozymes of acid phosphatase (EC 3.1.3.2). Some of these may be involved in mobilizing aleurone-grain phosphate reserves during germination. The hormone also controls secretion of other specific molecular forms of the enzyme that probably assist in endosperm hydrolysis. The accumulation and secretion of putative cell-wall-associated isozymes are stimulated by the action of GA₃ in isolated protoplasts. This effect however, is apparently over-ridden in the intact tissue, possibly by a cell-wall-based feedback mechanism.Abbreviations GA₃ gibberellic acid - pI isoelectric point(s)     

Proteomes of the barley aleurone layer: A model system for plant signalling and protein secretion

The cereal aleurone layer is of major importance due to its nutritional properties as well as its central role in seed germination and industrial malting. Cereal seed germination involves mobilisation of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellic acid produced by the embryo, the aleurone layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the aleurone layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted analysis of germination-related changes and the thioredoxin system. Here, analysis of intracellular and secreted proteomes reveals features of the aleurone layer system that makes it promising for investigations of plant protein secretion mechanisms.     

Gibberellic acid and the fine structure of barley aleurone cells

Summary This paper describes changes in the fine structure of barley aleurone cells following treatment with gibberellic acid (GA₃). Within 2 hr of GA₃ treatment the aleurone grains lose the spherical appearance characteristic of aleurone cells incubated in water and buffer alone. This swelling increases with increased exposure of the cells to GA₃ and reaches a maximum at about 10 hr. Accompanying this increase in volume of the aleurone grains is an increase in the amount of rough endoplasmic reticulum. The relevance of these GA₃-stimulated changes in aleurone-cell fine-structure to GA₃-regulated -amylase production is discussed.Work supported by National Science Foundation grants GB-5863 and GB-8332. The skillful technical assistance of Mrs. Janet Price is gratefully acknowledged.     

Using a Semi-Aqueous Medium for the Subcellular Study of Acid Phosphatase in Wheat Aleurone Tissue

Subcellular fractionation of the aleurone tissue in ethan-1,2-diol:H₂O(80:20, v/v) provided a suitable means to study acid phosphataseactivity in the protein bodies (aleurone grains). Enzyme activityin the fraction increased 7-fold during 4 d germination. Theincrease was dependent upon the presence of the embryo, butit was not regulated by gibberellic acid, indole acetic acidor glutamine when these compounds were presented either singlyor in combination. The regulatory activities of these compoundswere restricted to an acid phosphatase located in the cell wall. Key words: Triticum aestivum, aleurone tissue, acid phosphatase, plant growth regulators     

The isolation of endoplasmic reticulum from barley aleurone layers

Techniques for the isolation and purification of endoplasmic reticulum (ER) from aleurone layers of barley (Hordeum vulgare L.) were assessed. Neither differential centrifugation nor density gradient centrifugation of a homogenate separate the ER or other organelles of this tissue from the lipidcontaining spherosomes. Isopycnic sucrose gradient centrifugation of organelles first purified by molecular sieve chromatography on Sepharose 4B, however, results in separation of the organelles based on their differing buoyant densities. Manipulation of the magnesium concentration of the isolation media and density-gradient solutions affords isolation of ER at a density of 1.13–1.14 g cc^-1 and 1.17–1.18 g cc^-1. Electron microscopy shows that the membranes sedimenting at 1.13–1.14 g cc^-1 are devoid of ribosomes and are characteristic of smooth ER, while those sedimenting at 1.17–1.18 g cc^-1 are studded with ribosomes and have the features of rough ER. Endoplasmic reticulum isolated by isopycnic density gradient centrifugation can be further purified by rate-zonal centrifugation.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid - Trizma tris(hydroxymethyl)aminomethane     

Gibberellic acid and the fine structure of barley aleurone cells

Summary Ultrastructural changes in barley aleurone, cells treated with gibberellic acid (GA₃) for 24–36 hr are described. Many large vacuoles are seen in the ground cytoplasm; the coalasce to form one large central vacuole. Evidence is presented indicating that the vacuoles are formed from the aleurone grains. The dictyosomes of aleurone cells treated with GA₃ for 24 hr or longer proliferate many vesicles. This proliferation of dictyosome vesicles is associated with the phase of rapid ribonuclease release from the aleurone cell. Estimates indicate that microbodies are considerably reduced in number with GA₃ treatment from 24–36, hr while the number of mitochondria is not substantially affected relative to controls. P-Protein-like material is seen in the cytoplasm of these cells often in close proximity to endoplasmic reticulum and spiny vesicles.Supported by National Science Foundation Grant No. GB8332.     

Effect of Gibberellin and Heat Shock on the Lipid Composition of Endoplasmic Reticulum in Barley Aleurone Layers

The heat-shock responses of barley (Hordeum vulgare L. cv Hi- malaya) aleurone layers incubated with or without gibberellic acid (GA3) were compared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that heat shock blocked the synthesis and secretion of secretory proteins from GA3-treated layers but not untreated layers. This suppression of secretory protein synthesis has been correlated with changes in endoplasmic reticulum (ER) membranes (F.C. Belanger, M. R. Brodl, T.-h.D. Ho [1986] Proc Natl Acad Sci USA 83: 1354-1358; L. Sticher, A.K. Biswas, D.S. Bush, R.L. Jones [1990] Plant Physiol 92: 506-513). Our secretion data suggested that the ER membranes of aleurone layers incubated without GA3 may be more heat shock tolerant. To investigate this, the lipid profiles of membrane extracts in aleurone layers labeled with [14C]glycerol were examined. Heat shock markedly increased [14C]glycerol incorporation into phosphatidylcholine (PC), and gas chromatography revealed an increase in the amount of saturated fatty acids associated with thin layer chromatography-purified PC in GA3-treated layers. In contrast, aleurone layers incubated without GA3 at normal temperature contained PC-associated fatty acids with a greater degree of saturation than GA3-treated layers. Heat shock modestly increased the degree of fatty acid saturation in untreated aleurone layers. This same trend was noted in fatty acids isolated from ER membranes purified by continuous sucrose density centrifugation. We propose that increased fatty acid saturation may help sustain ER membrane function in heat-shocked aleurone layers incubated in the absence of GA3.     

Monitoring intra- and extracellular redox capacity of intact barley aleurone layers responding to phytohormones

Redox regulation is important for numerous processes in plant cells including abiotic stress, pathogen defence, tissue development, seed germination and programmed cell death. However, there are few methods allowing redox homeostasis to be addressed in whole plant cells, providing insight into the intact in vivo environment. An electrochemical redox assay that applies the menadione-ferricyanide double mediator is used to assess changes in the intracellular and extracellular redox environment in living aleurone layers of barley (Hordeum vulgare cv. Himalaya) grains, which respond to the phytohormones gibberellic acid and abscisic acid. Gibberellic acid is shown to elicit a mobilisation of electrons as detected by an increase in the reducing capacity of the aleurone layers. By taking advantage of the membrane-permeable menadione/menadiol redox pair to probe the membrane-impermeable ferricyanide/ferrocyanide redox pair, the mobilisation of electrons was dissected into an intracellular and an extracellular, plasma membrane-associated component. The intracellular and extracellular increases in reducing capacity were both suppressed when the aleurone layers were incubated with abscisic acid. By probing redox levels in intact plant tissue, the method provides a complementary approach to assays of reactive oxygen species and redox-related enzyme activities in tissue extracts.     

Study of barley endonucleases and alpha-amylase genes

We have identified an endonuclease(s) that preferentially cleaves the internucleosomal linker regions in the aleurone chromatin producing mono- and oligonucleosomes. This enzyme(s) has been designated as a "linker"-specific nuclease(s). This nuclease does not require divalent cations for activity, and therefore it is not the "Ca2+-Mg2+-DNase" found in mammalian cells. The linker-specific nuclease activity is not detectable in the dry aleurone tissue and in the tissue treated with 0.5 mM cordycepin. The endonuclease activity of the aleurone tissue incubated with gibberellic acid is higher than the level of this endonuclease in tissue treated with abscisic acid or water alone. Nuclei isolated from embryos have lower levels of endonuclease activities compared to those from aleurone tissue. Digestion of the nuclei from embryos with micrococcal nuclease revealed the subunit structure of chromatin. In Southern blots of the HindIII digests of DNA from embryos, five DNA bands hybridized to a nick-translated alpha-amylase cDNA clone. In similar autoradiograms with aleurone DNA, particular bands are less visible, notably in the DNA isolated from the tissue treated with gibberellic acid. This is the first report of the presence of a linker-specific nuclease activity in plant cells.     

Gibberellic Acid Regulates the Level of a BiP Cognate in the Endoplasmic Reticulum of Barley Aleurone Cells

The isolation of a 70-kilodalton protein from barley (Hordeum vulgare L.) aleurone layers that cross-reacts with an antibody against yeast binding protein (BiP) is reported. Endoplasmic reticulum isolated from aleurone layers treated with gibberellic acid contain much higher levels of the BiP cognate than do membranes isolated from layers treated with abscisic acid.     

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.