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  • 1
    ISSN: 1432-2048
    Keywords: Aleurone ; Calcium uptake ; Calmodulin ; Endoplasmic reticulum ; Gibberellic acid ; Hordeum ; (Ca2+ uptake)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-2048
    Keywords: Acid growth ; Cell elongation ; Gibberellin ; Fusicoccin ; Lactuca ; Hordeum ; Potasium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The roles of gibberellic acid (GA3) and fusicoccin (FC) in the elongation growth and acidification of the medium by excised hypocotyl sections of lettuce (Lactuca sativa L.) were investigated. Hypocotyl sections incubated in buffer without GA3 elongate optimally at pH 4.0–4.25 while sections incubated with GA3 show the same growth between pH 4.25 and 6.0. Preincubation of sections at pH 6.0 for 6 h does not affect the subsequent elongation response to acidic medium (pH 4.25); however, the sections become refractory to further acid treatment after their initial burst of growth in response to pH 4.25. Sections made refractory to acid are responsive to GA3 application, however, and the rate of growth in response to GA3 of sections pretreated for 6 h at pH 4.25 is 85% of that of sections pretreated at pH 6.0. Although preincubation of sections for 48 h in medium at pH 6.0 abolishes the GA3 response, it does not affect the response to buffer at pH 4.25. FC stimulates elongation growth in letuce hypocotyls at an optimal concentration of 1 μM, and pretreatment of sections at pH 4.25 does not affect this elongation response. Although both GA3 and FC increase elongation of the section, neither causes appreciable acidification of the medium. Addition of KCl or NaCl to FC-treated sections causes rapid medium acidification but addition of salts to GA3-treated tissue does not cause acidification. Abrasion of the hypocotyl to remove the cuticle does not enhance acidification of the medium by the sections nor deos it affect elongation of the sections in response to GA3 or FC. Medium acidification by the sections is not a passive process since it is abolished both by low temperature (2° C) and metabolic inhibitors (carbonyl cyanide-m-chlorophenyl-hydrazone, azide). The acidification of the medium by barley (Hordeum vulgare L.) roots in response to FC is also dependent on the presence of KCl. We conclude that the acid-growth hypothesis does not explain GA3- or FC-induced elongation in lettuce hypocotyls.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Planta 150 (1980), S. 58-69 
    ISSN: 1432-2048
    Keywords: Aleurone ; α-Amylase ; Endoplasmic reticulum ; Hordeum ; Organelle isolation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Planta 150 (1980), S. 70-81 
    ISSN: 1432-2048
    Keywords: Aleurone ; α-Amylase ; Endoplasmic reticulum ; Gibberellin ; Hordeum ; Organelle isolation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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 α-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 α-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.
    Type of Medium: Electronic Resource
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