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  • 1
    ISSN: 1432-2048
    Keywords: Conjugates ; Gibberellin ; Lactuca ; Metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The properties of the water-soluble metabolites of [3H]gibberellin A1 ([3H]GA1) from lettuce (Lactuca sativa L.) hypocotyls were compared with those of authentic samples of gibberellin (GA) glucosyl esters and ethers. Partitioning against l-butanol at high and low pH was not an efficient method of differentiating between ester and ether conjugates of GA1 or GA3. Extraction into l-butanol at pH 2.5 was, however, useful as a group purification step. Gel-filtration on acrylamide indicated a mean molecular weight of ca. 600 for the polar material and high-voltage electrophoresis separated two compounds (LH 1 and LH 2) with differing charge properties. Both metabolites incorporated 14C from glucose and 3H from GA1. Subsequent enzymatic hydrolysis of LH 1 released material with identical properties to [14C]glucose together with a second uncharacterised component. Feeding with [3H]GA1 methyl ester greatly reduced the formation of LH 1 but not LH 2. The metabolites were provisionally identified as GA1-glucosyl ester (LH 1) and GA1-glucosyl ether (LH 2).
    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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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Planta 152 (1981), S. 442-449 
    ISSN: 1432-2048
    Keywords: Gibberellin ; Growth kinetics ; Hypocotyl, growth ; Lactuca ; pH and growth
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The short-term kinetics of growth of the excised lettuce (Lactuca sativa L.) hypocotyl were characterized with respect to the effects of gibberellic acid (GA3), indole-3-acetic acid (IAA), KCl and pH. A Hall-device-based, miniaturized, linear displacement transducer was developed to measure the growth of 2-mm hypocotyl sections with 1-μm resolution. Following treatment with GA3, a lag time of less than 10 min was typically followed by an increase in growth rate with two acceleration phases, reaching a final elevated rate within about 1 h. The kinetics of the response to GA1, a mixture of GA4 and GA7, and GA9 were similar to the response to GA3. There was no response to IAA treatment either in the presence or absence of GA3. KCl alone had no effect on the growth rate, but caused an increase in rate when added after GA3, with a lag time of usually less than 1 h. Responses to pH changes had lag times of a few minutes in all cases. A shift from H2O to pH 6 buffer inhibited growth, while a shift from H2O to pH 4 buffer resulted in a transient increase to a rate comparable to that induced by GA3. A shift from pH 6 to pH 5 caused an increase in growth rate, followed by a gradual decline to an H2O control rate after more than an hour. The responses to GA3 at pH 4 and pH 5 were similar to that found for addition of GA3 to water controls.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Planta 152 (1981), S. 450-456 
    ISSN: 1432-2048
    Keywords: Calcium ; Cell elongation ; Gibberellin ; Lactuca ; pH and growth
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The relationship between calcium ions and gibberellic acid (GA3)-induced growth in the excised hypocotyl of lettuce (Lactuca sativa L.) was investigated. The short-term kinetics of growth responses were measured using a linear displacement transducer. Test solutions were added either as drops to the filter paper on which the hypocotyl stood (“non-flow-past”) or by switching solution flowing past the base of hypocotyl (“flow-past”), resulting in differences in growth behavior. Drops of CaCl2 added at a high concentration (10 mM) inhibited growth within a few minutes. This inhibition was reversed by ethylenediaminetetraacetic acid (EDTA). Drops of EDTA or ethyleneglycol-bis(2-aminoethylether)-tetraacetic acid caused a rapid increase in growth rate. Growth induced by EDTA was not further promoted by GA3. A continuous H2O flow resulted in growth rates comparable to those in response to GA3. Addition of CaCl2 to the flow-past medium inhibited growth and this inhibition was reversed by a decrease in CaCl2 concentration. The growth rate was found to be a function of CaCl2 concentration. When a constant CaCl2 concentration was maintained by the flow-past medium, a shift in pH from 5.5 to 4.25 had no obvious effect on hypocotyl elongation. Gibberellic acid was found to reverse the inhibitory effect of CaCl2, causing an increase in growth rate similar to that found previously when GA3 was added to hypocotyls grown in H2O under non-flow-past conditions. We propose that gibberellin controls extension growth in lettuce hypocotyl sections by regulating the uptake of Ca2+ by the hypocotyl cells.
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  • 7
    ISSN: 1432-2048
    Keywords: Aleurone ; α-Amylase ; Calcium and enzyme secretion ; Enzyme secretion ; Gibberellin ; Hordeum (enzyme secretion)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effect of calcium on the secretion of α-amylase (EC 3.2.1.1) and other hydrolases from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was studied. Withdrawal of Ca2+ from the incubation medium of aleurone layers preincubated in 5 μM gibberellic acid (GA3) and 5 mM CaCl2 results in a 70–80% reduction in the secretion of α-amylase activity to the incubation medium. Agar-gel electrophoresis shows that the reduction in α-amylase activity following Ca2+ withdrawal is correlated with the disappearance of group B isoenzymes from the incubation medium. The secretion of isoenzymes of group A is unaffected by Ca2+. The addition of Ca2+ stimulates the secretion of group-B isoenzymes but has no measurable effect on either the α-amylase activity or the isoenzyme pattern of aleurone-layer extracts. Pulse-labelling experiments with [35S]methionine show that Ca2+ withdrawal results in a reduction in the secretion of labelled polypeptides into the incubation medium. Immunochemical studies also show that, in the absence of Ca2+, α-amylase isoenzymes of group B are not secreted into the incubation medium. In addition to its effect on α-amylase, Ca2+ influences the secretion of other proteins including several acid hydrolases. The secretion of these other proteins shows the same dependence on Ca2+ concentration as does that of α-amylase. Other cations can promote the secretion of α-amylase to less and varying extents. Strontium is 85% as effective as Ca2+ while Ba2+ is only 10% as effective. We conclude that Ca2+ regulates the secretion of enzymes and other proteins from the aleurone layer of barley.
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  • 8
    ISSN: 1432-2048
    Keywords: Cell elongation ; Gibberellins ; Lactuca
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Elongation growth and gibberellin (GA9) metabolism in excised hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) were investigated. Exogenously supplied GA9 stimulates elongation of hypocotyl sections and this response is intermediate between that elicited by GA1 or GA20 and GA4/7 mixture. Although uptake of radioactivity from [3H]GA9 increases with time, this gibberellin does not accumulate in the tissue but is rapidly converted to a compound with HPLC properties resembling those of [3H]GA20. After 2 h incubation in [3H]GA9, the presumptive GA20 represents 90% of the acidic ethyl acetate-soluble radioactivity in the tissue. Radioactivity is also associated with an acidic butanol-soluble fraction containing two components resolvable by HVE. The major component is similar in electrophoretic properties to a GA-glucosyl ether while the other compares to a GA-glucosyl ester. Conversion of [3H]GA9 to its [3H]GA20-like metabolite is reduced by addition of carrier GA9 or GA4/7 at concentrations as low as 1 μM, while GA1, GA3 and L-proline are without effect. Formation of the GA20-like compound can be blocked by the addition of 2,2′-dipyridyl, and this inhibitory effect of dipyridyl can be reversed by addition of Fe2+. At 200 μM dipyridyl, elongation growth as well as [3H]GA9 metabolism are reduced by 80%. The relationship of the metabolism of GA9 to the growth response is discussed.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1432-2048
    Keywords: Cell growth, orientation ; Colchicine ; Gibberellin and cell growth ; Hypocotyl (growth) ; Lactuca ; Microtubules
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effect of colchicine on growth and microtubules in H2O-and gibberellic acid (GA3)-treated hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) was examined. Hypocotyls of seedlings from γ-irradiated as well as non-irradiated seedlings were used in order to establish that the drug was affecting cell elongation and not cell division. Although colchicine inhibited elongation of GA3-treated hypocotyl sections at concentrations as low as 1 μM, it did not affect the growth of sections. Rather, colchicine treatment caused a reorientation of the plane of cell expansion. Lumicolchicine had no effect on elongation of hypocotyl sections at a concentration (100 μM) at which colchicine abolished GA3-induced elongation. Colchicine was only effective in reorienting the plane of growth in light; in darkness, it inhibited both radial and longitudinal cell expansion. Analogs of colchicine as well as other antimicrotubular agents were ineffective in changing the polarity of growth in lettuce. Electron microscopy showed that transversely oriented microtubules were numerous in the cortical cytoplasm adjacent to the longitudinal wall of freshly excised hypocotyl sections. Incubation in GA3 did not change the number or orientation of these microtubules, while incubation in H2O caused a reduction in their number and altered their distribution. Colchicine abolished microtubules under all conditions of incubation tested, but did not affect other characteristics of cell ultrastructure.
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  • 10
    ISSN: 1432-2048
    Keywords: Aleurone ; α-Amylase ; Endoplasmic reticulum ; Gibberellin and enzyme formation ; Hordeum (α-amylase in aleurone)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The subcellular site of α-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 α-amylase. This protein was identified as authentic α-amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized α-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 α-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 α-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 α-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 α-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.
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