Gibberellic acid and the fine structure of barley aleurone cells

Summary This paper describes the ultrastructural changes in barley aleurone cells following exposure to gibberollic acid (GA₃) for 10–12 hr and longer. These changes involve a further proliferation of the endoplasmic reticulum (ER), distention of the endoplasmic reticulum (ER) cisternae (12–16 hr of GA₃) and proliferation of vesicles from the ER and dictyosomes (14–22 hr). Accompanying these changes is a reduction in the size of the aleurone grains and a decrease in the number of spherosomes. Plastids and microbodies however appear to increase in number during this period of GA₃ treatment. The relevance of these ultrastructural changes to GA₃-stimulated synthesis of hydrolases is discussed.The skillful technical assistance of Mrs. Janet Price is gratefully acknowledged. Supported by National Science Foundation grant GB-8332.     

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.     

Glyceride metabolism and gluconeogenesis in barley endosperm

Endosperms of quiescent barley grains contained, on average, 54.5 μg of neutral glyceride-glycerol, equivalent to ca 480 μg glyceride. Of this probably 90% was located in the aleurone layer. During germination the level of glyceride-glycerol declined. It also declined in degermed grains and aleurone layers incubated in vitro. The fall was accelerated by GA₃, but indoleacetic acid, kinetin and glutamine were without effect. Increases in the levels of malate synthase and isocitrate lyase from very low initial values, and the results of incorporation studies with [¹⁴C]-labelled substrates, indicate that the glyoxylate cycle functions to convert glycerides to sucrose in germinating grains and degermed grains incubated with GA₃, but not in degermed grains without the hormone. In the absence of GA₃ the glyceride could be a respiratory substrate in degermed grains. The aleurone layers converted exogenous glucose to sucrose. Little label from [¹⁴C]-amino acids appeared in sucrose but in some cases considerable incorporation occurred into glutamine.     

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.     

Localization of ATPase in the endoplasmic reticulum and golgi apparatus of barley aleurone

Summary The cytochemical localization of adenosine triphosphatase (ATPase) was studied in the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya). Isolated barley aleurone layers secrete numerous enzymes having acid phosphatase activity, including ATPase. The secretion of these enzymes was stimulated by incubation of the aleurone layer in gibberellic acid (GA₃). ATPase was localized using the metal-salt method in tissue incubated in CaCl₂ with and without GA₃. In sections of tissue incubated without GA₃, cytochemical staining was confined to a narrow band of cytoplasm adjacent to the starchy endosperm and to the cell wall of the innermost tier of aleurone cells. Cytochemical staining was absent from the organelles of tissues not treated with GA₃. In tissue incubated in the presence of GA₃, cytochemical staining was evident throughout the cytoplasm and cell walls of the tissue. In the cell wall, electron-dense deposits were found only in digested channels. The cell-wall matrix of GA₃-treated aleurone did not stain, indicating that it does not permit diffusion of enzyme. In the cytoplasm of GA₃-treated aleurone, all organelles except microbodies, plastids, and spherosomes stained for ATPase activity; endoplasmic reticulum (ER), Golgi apparatus, and mitochondria showed intense deposits of stain. The ER of the aleurone is a complex system made up of flattened sheets of membrane, which may be associated with both the Golgi apparatus and the plasma membrane. The dictyosome did not stain uniformly for ATPase activity; rather there was a gradation in staining of the cisternae from thecis (lightly stained) to thetrans (heavily stained) face. Vesicles associated with dictyosome cisternae also stained intensely as did the protein bodies of GA₃-treated aleurone cells.     

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.     

The role of the endoplasmic reticulum in the synthesis and transport of α-amylase in barley aleurone layers

The subcellular site of -amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA₃). Using [³⁵S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA₃ was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker -amylase. This protein was identified as authentic -amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized -amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled -amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of -amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA₃ were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA₃-treated aleurone layers, five peaks of -amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA₃ only three peaks of activity were found. In incubation media, four isoenzymes were found after GA₃ treatment and two were found after incubation without GA₃. We conclude that at least five -amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.Abbreviations CHA cyclohepataamylose - DEAE-cellulose diethylaminoethyl-cellulose - ER endoplasmic reticulum - GA₃ gibberellic acid - SDS-PAGE sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis     

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     

Gibberellic acid (GA3)-induced enhancement of α-amylase activity in the aleurone of Shrunken-2 maize kernels

The aleurone of RB-3 shrunken-2 (sh2) maize kernels is deficient in α-amylase activity during germination, but exogenous applications of gibberellic acid (GA₃) (0.001–10 μm ) induced low levels of activity. The highest activity was measured in the aleurone of kernels treated with 10 μm GA₃ (14,600 ± 945 units), but was lower than untreated Starchy (Su) aleurone tissues (35,280 ± 5,010 units). On isoelectric focusing gels, no α-amylase isozymes were detected in the untreated sh2 aleurone using starch zymograms or immunoblots, but the 1.0 and 10 μm mm GA₃ treatments induced nearly all the isozymes (eight to ten) present in the Su aleurone. There was a very low level of α-amylase mRNA in the untreated sh2 aleurone, an intermediate level in the 1.0 μm GA₃-treated sh2 aleurone, and the highest level in the untreated Su aleurone. On the confocal microscope, the 1.0 μm GA₃-treated aleurone cells had enhanced levels of cytoplasmic membranes and RNA compared to untreated sh2 aleurone cells. The 1.0 μm GA₃ treatment also induced shoot elongation in the sh2 seedlings. The data demonstrate that the sh2 aleurone is deficient in its function to produce α-amylases, and exogenous GA₃ can partially restore cell function in the sh2 kernels.     

Phosphoinositides in barley aleurone layers and gibberellic Acid-induced changes in metabolism

Phospholipids of barley (Hordeum vulgare L. cv Himalaya) aleurone layers were labeled with myo-[2-³H]inositol or [³²Pi], extracted, and analyzed by physical (chromatography) and chemical (deacylation) techniques. Three phospholipids were found to incorporate both myo-[2-³H]inositol and [³²Pi]—phosphatidylinositol, phosphatidylinositol-monophosphate, and phosphatidylinositol-bisphosphate. Stimulation of [³H]inositol prelabeled aleurone layers with GA₃ showed enhanced incorporation of label into phosphatidylinositol within 30 seconds and subsequent rapid breakdown. Stimulation of phosphatidylinositol labeling observed in these studies is the earliest response of aleurone cells to gibberellic acid reported.     

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.     

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     

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     

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.     

Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of α-ketobutyrate to l-isoleucine

Summary Corynebacterium glutamicum ATCC 13 032 produces 13 g/l l-isoleucine from 200 mM -ketobutyrate as a synthetic precursor. In fed batch cultures up to 19 g/l l-isoleucine is formed. For optimal conversion the addition of 0.3 mM l-valine plus 0.3 mM l-leucine to the fermentation medium is required. The affinity constants for the acetohydroxy acid synthase (AHAS) were determined. (This enzyme directs the flow of -ketobutyrate plus pyruvate towards l-isoleucine and that of two moles of pyruvate to l-valine and l-leucine, respectively.) For -ketobutyrate the K _m is 4.8×10^-3 M, and V _max 0.58 U/mg, for pyruvate the K _m is 8.4×10^-3 M, and V _max 0.37 U/mg. Due to these characteristics the presence of high -ketobutyrate concentrations apparently results in a l-valine, l-leucine deficiency. This in turn leads to a derepression of the AHAS synthesis from 0.03 U/mg to 0.29 U/mg and high l-isoleucine production is favoured. The derepression of the AHAS synthesis induced by the l-valine, l-leucine shortage was directly proven with a l-valine, l-leucine, l-isoleucine auxotrophic mutant where the starvation of each amino acid resulted in an increased AHAS level. This is in accordance with the fact that only one AHAS enzyme could be verified by chromatographic and electrophoretic separations as being responsible for the synthesis of all three branched-chain amino-acids.     

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.

     

Effects of gibberellic acid and abscisic acid on α-amylase activity and ultrastructure of aleurone cells ofAvena sativa L.

The effects of GA₃ and/or ABA on the α-amylase activity and the ultrastructure of aleurone cells in halves of seeds without embryos (embryo-less half seeds) of oats (Avena sativa L.) were studied. α-Amylase activity was detected by the starch-agar gel method in the aleurone layers of embryo-less half seeds soaked in 1 μM GA₃ solution or 100 μM GA₃+10 μM ABA solution but not in those of seeds soaked in distilled water, 10 μM ABA solution, or 1 μM GA₃+10 μM ABA solution. Ultrastructural examinations of aleurone cells with α-amylase activity showed a decrease in the number of sphaerosomes, the appearance of flattened saccules pressed to the surface of aleurone grains, and the development and transformations of the rER from a slender form to the one with wide inner spaces. In the aleurone cells in which the enzyme activity was not detected, components of the rER showed only slender profiles. The number of sphaerosomes did not decrease, and no flattened saccules appeared in the aleurone cells treated with 10 μM ABA or 1 μM GA₃+10 μM ABA.     

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.     

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.