Localization of phytase and acid phosphatase isoenzymes in aleurone layers of barley

The localization of acid phosphatase (EC 3.1.3.2) in aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) grains was studied. Phosphatase (EC 3.1.3.26) activity, assayed with phytic acid as the substrate, is present in the dry grain at low leveis and increases during incubation in H₂O at 25°C for three days. When aleurone layers are isolated from imbibed grain and incubated for 18 h in buffer with or without 50 μM gibberellic acid (GA₃), the level of extractable phosphatase activity increases two- to threefold, and phosphatase is released into the medium. GA, promotes the release of phosphatase activity: aleurone layers incubated in GA, release twice as much phosphatase as layers incubated in buffer. Nine isoenzymes of phosphatase are found in aleurone layers of barley by non-denaturing polyacrvlamide gel electropho-resis. Six of these forms, isoenzymes 1,2,3,5,6 and 8, can be extracted from dry tissue, and after three days of imbibition in H₂O an additional isoenzyme, isoenzyme 9, is found in aleurone extracts. When isolated aleurone layers are incubated for a further 22 h in buffer with or without GA₃, isoenzyme 7 is found and yet another form, isoenzyme 4, is found in layers incubated in GA₃. Eight isoenzymes are released from aleurone layers into the incubation medium. Isoenzymes 5 and 6 are released in buffer both with and without GA₃, even when cycloheximide is present; cycloheximide inhibits the release of the other isoenzymes. Isoenzymes 1-4, 7 and 8, on the other hand, are secreted into the incubation medium only when GA₃, is present. Isoenzyme 9 is not released into the incubation medium. Acid phosphatase activity was localized in aleurone tissue using cytochemical, cell fractionation, and enzymatic methods. Cytochemical localization of ATPase (EC 3.6.1.8) in aleurone tissue showed the presence of enzyme activity in cell wall, protein bodies, endoplasmic reticulum, Golgi apparatus, and mitochondria. Analysis of organelle fractions isolated by density gradient centrifugation showed that the activity of acid phosphatase isoenzymes 1, 2 and 3 was prominently associated with the phytin globoid of protein bodies, and analysis of the activity released from the cell wall by enzymatic digestion showed that it was almost exclusively isoenzymes 5 and 6.     

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     

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.     

Hormonal regulation of the development of protease and carboxypeptidase activities in barley aleurone layers

Carboxypeptidase and protease activities of hormone-treated barley (Hordeum vulgare cv Himalaya) aleurone layers were investigated using the substrates N-carbobenzoxy-Ala-Phe and hemoglobin. A differential effect of gibberellic acid (GA₃) on these activities was observed. The carboxypeptidase activity develops in the aleurone layers during imbibition without the addition of hormone, while the release of this enzyme to the incubation medium is enhanced by GA₃. In contrast, GA₃ is required for both the production of protease activity in the aleurone layer and its secretion. The time course for development of protease activity in response to GA₃ is similar to that observed for α-amylase. Treating aleurone layers with both GA₃ and abscisic acid prevents all the GA₃ effects described above. Carboxypeptidase activity is maximal between pH 5 and 6, and is inhibited by diisopropylfluorophosphate and p-hydroxymercuribenzoate. We have observed three protease activities against hemoglobin which differ in charge but are all 37 kilodaltons in size on sodium dodecyl sulfate polyacrylamide gels. The activity of the proteases can be inhibited by sulfhydryl protease inhibitors, such as bromate and leupeptin, yet is enhanced by 2-fold with 2-mercaptoethanol. In addition, these enzymes appear to be active against the wheat and barley storage proteins, gliadin and hordein, respectively. On the basis of these characteristics and the time course of GA₃ response, it is concluded that the proteases represent the GA₃-induced, de novo synthesized proteases that are mainly responsible for the degradation of endosperm storage proteins.     

Protoplasts isolated from aleurone layers of wild oat (Avena fatua L.) exhibit the classic response to gibberellic acid

Viable, long-lived, gibberellic acid (GA₃)-responsive protoplasts have, for the first time, been isolated from aleurone layers of mature wild oat (Avena fatua L.) grain. More than 90% of the cells of aleurone layers are recovered as protoplasts, and these respond to treatment with GA₃ in essentially the same manner as the tissue from which they were derived. Protoplasts become vacuolate during incubation in vitro and, although not dependent upon GA₃, vacuolation is markedly stimulated by the hormone. Amylase and ribonuclease (RNase) are produced and secreted only in the presence of GA₃ and only after lag periods of 3 d and 4 d respectively. The amounts of amylase produced and secreted are proportional to GA₃ concentrations as low as 1.61·10^-13 M. With increasing concentrations of mannitol in the culture medium both vacuolation and the GA₃-induced production and secretion of enzymes are inhibited progressively, the latter being precluded by 0.6 M to 0.7 M mannitol.Abbreviations GA₃ gibberellic acid₃ - RNase ribonuclease     

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.     

Tannins as gibberellin antagonists in the synthesis of alpha-amylase and Acid phosphatase by barley seeds

The tannins chebulinic acid or tara tannin were added to an incubation system in which GA₃ induces enzyme synthesis in endosperm half seeds of barley (Hordeum vulgare L.). The activity of amylase and acid phosphatase in the incubation medium was reduced compared to the activity in the medium after incubation with GA₃ alone. When embryo half seeds of barley were incubated with chebulinic acid or tara tannin in the absence of added GA₃, the enzyme activity of the incubation medium was also reduced. The activity of preformed enzymes obtained from endosperm half seeds previously induced with GA₃ was not reduced by the addition of tannin. Comparisons were made of the amount of enzyme activity from breis of aleurone layers incubated with GA₃ in the presence and absence of tannins. The amounts of activity were relatively small and approximately equal in both cases, indicating that secretion from the aleurone was not blocked by the tannins. The reduction of enzyme activity caused by tannins in both endosperm and embryo half seeds could be completely reversed by the addition of GA₃.     

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.

     

Resistance of wheat aleurone cell walls to acid and xylanase action

Isolated wheat (Triticum aestivum var. Potam) aleurone layers have a high capacity to acidify their environment, and secrete hydrolytic enzymes (endoxylanase, glucanase, α-amylase, proteases, etc.) under the control of GA₃. Acidic pH and xylanases are found to be essential for cell wall relaxation in growing tissues, but aleurone is a non-growing, non-dividing tissue. In this tissue, we studied the effect of these loosening factors on aleurone cell walls.Exposure to pH 3.0 caused the release of carbohydrates and calcium ions from the pericarp, and a small amount of carbohydrates, mainly polysaccharides, from aleurone layers from which pericarp tissue had been removed. 50 percnt; of the total sugars released into the incubation medium by these isolated aleurone tissue was arabinose, but no xylose, calcium ions, or phenolic compounds were found. Acid preincubation decreased by 30 percnt; the susceptibility of aleurone cell walls to degradation by exogenously-applied endoxylanase, and also modified the architecture of cell wall as observed by autofluorescence of phenolic groups. These findings suggest that acid treatment and endoxylanase action, rather than having a loosening effect on aleurone cell wall, can have an opposite effect, increasing the resistance of aleurone cell walls to loosening.     

Uptake and metabolism of radioactive gibberellins by barley aleurone layers

Summary When aleurone layers were treated with labeled gibberellin A₁ (³H-GA₁), gibberellin A₅ (³H-GA₅) and the methyl ester of ³H-GA₅ (³H-GA₅-ME), radioactivity was accumulated by the tissue for a period of 20–30 h. After this time, radioactivity was released into the medium. Concomitantly, ribonuclease was also liberated by the tissue. The radioactivity accumulated by aleurone layers was associated with polar metabolites of the respective GAs, and the extent of extent of accumulation was a function of the degree of GA metabolism (GA₅-ME>GA₅>GA₁). Accumulation of radioactivity was inhibited in the cold and by the metabolic poisons NaF and dinitrophenol. This was thought to be due to inbition of GA metabolism. The accumulation of ³H-GA₁ in aleurone tissue did not reach saturation when unlabeled GA₃ up to 10^-2 M was added to the incubation medium.Abbreviations GA gibberellin - GA₅ ME, gibberellin A₅ methyl ester - RNase ribonuclease     

Regulation of the synthesis of barley aleurone alpha-amylase by gibberellic Acid and calcium ions

The effects of gibberellic acid (GA₃) and calcium ions on the production of α-amylase and acid phosphatase by isolated aleurone layers of barley (Hordeum vulgare L. cv Himalaya) were studied. Aleurone layers not previously exposed to GA₃ or Ca²⁺ show qualitative and quantitative changes in hydrolase production following incubation in either GA₃ or Ca²⁺ or both. Incubation in H₂O or Ca²⁺ results in the production of low levels of α-amylase or acid phosphatase. The addition of GA₃ to the incubation medium causes a 10- to 20-fold increase in the amounts of these enzymes released from the tissue, and addition of Ca²⁺ at 10 millimolar causes a further 8- to 9-fold increase in α-amylase release and a 75% increase in phosphatase release. Production of α-amylase isoenzymes is also modified by the levels of GA₃ and Ca²⁺ in the incubation medium. α-Amylase 2 is produced under all conditions of incubation, while α-amylase 1 appears only when layers are incubated in GA₃ or GA₃ plus Ca²⁺. The synthesis of α-amylases 3 and 4 requires the presence of both GA₃ and Ca²⁺ in the incubation medium. Laurell rocket immuno-electrophoresis shows that two distinct groups of α-amylase antigens are present in incubation media of aleurone layers incubated with both GA₃ and Ca²⁺, while only one group of antigens is found in media of layers incubated in GA₃ alone. Strontium ions can be substituted for Ca²⁺ in increasing hydrolase production, although higher concentrations of Sr²⁺ are required for maximal response. We conclude that GA₃ is required for the production of α-amylase 1 and that both GA₃ and either Ca²⁺ or Sr²⁺ are required for the production of isoenzymes 3 and 4 of barley aleurone α-amylase.     

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.     

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.

     

The role of endogenous gibberellins in the formation of α-amylase by aleurone layers of germinating barley caryopses

Using sensitive and selective immunological assays we have shown that in germinating caryopses of Hordeum vulgare L. cv. Himalaya, the level of gibberellin A₄ (GA₄) rises approximately 18-to 20-fold shortly (2–4 h) before -amylase activity increases. Gibberellin A₄ is the predominant immunoreactive gibberelin during these developmental stages and reaches a peak amount of approximately 9 pmol per caryopsis about 48 h after imbibition. Isolated aleurone layers produce GA₄ in the presence of an exogenous gibberellin, such as GA₁, which is not a biosynthetic precursor for GA₄. Experiments with inhibitors of gibberellin biosynthesis indicate that gibberellin synthesis is required in this tissue for the induction of -amylase. The inductive effect of exogenously applied GA₁ is indirect and appears to be mediated by GA₄. Embryos form predominantly GA₁; however, very little of this material is released by isolated embryos into the incubation medium. The results presented make it unlikely that the role of the embryo in the process of -amylase induction in aleurone layers is to provide gibberellins or gibberellin precursors.Abbreviations ABA abscisic acid - GA gibberellin - GA₃ gibberellic acid - RIA radioimmunoassay - TLC thin-layer chromatography     

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.     

Gibberellin induces endopeptidase activity in detached cotyledons of Pisum sativum

Endopeptidase activity in cotyledons of 5-day seedlings of Pisum sativum increased rapidly during germination. However, the increase of the activity in detached cotyledons was depressed. We examined whether a growth regulator can be substituted for the embryonic axis on the development of endopeptidase activity. As monitored by an assay with azoalbumin, the development of endopeptidase activity from crude extracts of detached cotyledons appeared to be slightly accelerated by incubation with 10^–5 M GA₃. However, the pattern after gelatin-polyacrylamide gel suggested that the activity induced in detached cotyledons during a 5-d incubation at 10^–7 M GA₃ was the same as that in attached ones during germination for 5 days and an even greater increase in activity was obtained with 10^–5 M GA₃. These results suggest that GA₃ from the embryonic axis induces endopeptidase activity in attached cotyledons at the first stage of germination.Abbreviations ABA abscisic acid - IAA indole-3-acetic acid - GA gibberellic acid     

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.     

Efficient GA3-assisted plant regeneration from cell suspensions of three grape genotypes via somatic embryogenesis

Petioles from in vitro grown plants of interspecific grapevine hybrids cvs `Bianca', `Podarok Magaracha' and `Intervitis Magaracha' were cultured on solid NN medium supplemented with 2,4-D and BA at various concentrations. The callus developed was cultured in liquid NN medium supplemented with 0.5 mg l<sup>–1</sup> BA to induce formation of somatic embryos. Somatic embryos of globular and heart-stage developed in suspensions of `Podarok Magaracha' and `Intervitis Magaracha'. In contrast, `Bianca' did not undergo embryogenesis beyond globular stage. This made it necessary to perform subculture of the suspensions to HTE liquid medium supplemented with 0.2 mg l^–1 BA for the development of globular embryos into heart stage. Heart-stage embryos developed into torpedo-stage after subculturing suspensions of all three cultivars to liquid HTE medium supplemented with 0.1 mg l^–1 IAA and 30 mg l^–1 sodium hummate. Torpedo-stage embryo suspensions were subcultured in liquid HTE medium supplemented with 0.5 mg l^–1 BA, 0.5 mg l^–1 GA₃ and 0.5 mg l^–1 GA₃ + 0.2 mg l^–1 BA. After 12 days of incubation, plantlets were cultured on solid M2MS medium: without growth regulators and with 0.5 mg l^–1 BA. Plantlets that developed in liquid HTE media with 0.5 mg l^–1 GA₃ or 0.5 mg l^–1 GA₃ + 0.2 mg l^–1 BA produced 82–90% shoots on solid M2MS medium with 0.5 mg l^–1 BA after 50 days of culture.     

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.     

Enzyme formation and release by isolated barley aleurone layers

Aleurone layers, with testa attached, were prepared from degermed, decorticated barley with the aid of a fungal enzyme preparation. The preparations appeared intact under the scanning electron microscope. By using antibiotics only in an early stage preparations were obtained uncontaminated by micro-organisms and which, when incubated under optimal conditions with gibberellic acid, GA₃, produced near-maximal amounts of α-amylase. The enzyme accumulated in the tissue before it was released into the incubation medium. Daily replacement of the incubation medium, containing GA₃, depressed the quantity of α-amylase produced. α-Amylase was also produced in response to gibberellins GA₁, GA₄ and GA₇ and, to a much lesser extent, helminthosporol and helminthosporic acid. A range of other substances, reported elsewhere to induce α-amylase formation, failed to do so in these trials. At some concentrations, glutamine marginally enhanced the quantity of enzyme formed during prolonged incubations. It is confirmed that α-glucosidase occurs in the aleurone layer and embryo of ungerminated barley, and increases in amount during germination. GA₃ is shown to enhance this increase. When embryos arc burnt, to prevent gibberellin formation, no rise in α-glucosidase levels occurs unless GA₃ is supplied to the grains. As the activity of α-glucosidase and other enzymes have been determined as ‘α-amylase’ by some assay methods, their alterations in activity in response to GA₃ necessitates a re-evaluation of the evidence for de novo) synthesis of α-amylase in aleurone tissue.