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1.
The major metabolite produced during incubation of [ 3H]gibberellin A 1 ([ 3H]GA 1) with barley aleurone layers is an amphoteric, water-soluble compound tentatively called [ 3H]ampho GA 1. Formation of [ 3H]ampho GA 1 in barley aleurones begins after a period of 2.5 hours. As judged by degradation studies as well as Sephadex column chromatography, GA 1 appears to be linked to a peptide; positions C-3 and C-7 were ruled out as conjugation sites. 相似文献
2.
Tritiated GA 1 and four of its synthetic derivatives were studiedin relation to their biological activity, uptake and metabolismby barley aleurone layers. Incubation was done in the presenceand absence of ABA. Tentative identification of some of themetabolites was made by TLC and GLC radiocounting of the metaboliteand its acid hydrolyzed derivative. Only GA 1 promoted -amylase synthesis. Uptake ranged from 20to 42%, varying with the derivative. ABA enhanced uptake of[ 3H]GA 1 and [ 3H]pseudoGA 1 and inhibited uptake of [ 3H]ketoGA 1the Wagner-Meerwein rearrangement product of [ 3H]GA 1 Uptakeof [ 3H]GA 1 methyl ester ([ 3H]GA 1-Me) and [ 3H]dihydroGA 1 wasunaffected by ABA. [ 3H]GA 1 was converted to an amphoteric GA 1 derivative ([ 3H]amphoGA 1)and [ 3H]GA 1-glycosyl ester. GA 1-Me was metabolized to four products,all of them GA 1 derivatives, including an apparent amphotericGA 1 derivative. DihydroGA 1 was quite stable; only one metabolitewas produced in sufficient yield to analyze. This product didnot cochromatograph with either of the expected acid hydrolyzedepimers of [ 3H]dihydroGA 1. [ 3H]ketoGA 1 was readily metabolizedto one product, probably the glycoside. [ 3H]pseudoGA 1 remainedessentially unmetabolized. Metabolism of all compounds testedwas not dramatically affected by ABA. Surprisingly, no metabolitesfrom hydroxylation at the 2-position were found.
1 Present address: Monsanto Agricultural Co., 800 N. LindberghBlvd., St. Louis, MO 63166, U.S.A. (Received January 31, 1977; ) 相似文献
3.
Summary When aleurone layers were treated with labeled gibberellin A 1 ( 3H-GA 1), gibberellin A 5 ( 3H-GA 5) and the methyl ester of 3H-GA 5 ( 3H-GA 5-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 (GA 5-ME>GA 5>GA 1). 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-GA 1 in aleurone tissue did not reach saturation when unlabeled GA 3 up to 10 -2 M was added to the incubation medium.Abbreviations GA
gibberellin
- GA 5
ME, gibberellin A 5 methyl ester
- RNase
ribonuclease 相似文献
5.
Summary When barley aleurone layers were incubated with 3H-Gibberellin A 1 ( 3H-GA 1), the hormone was converted to 3H-GA-X (not identified), 3H-GA 8 and two other compounds tentatively identified as 3H-GA 1-glucoside, and 3H-GA 8-glucoside. Uptake and metabolism of the 3H-GA 1 were markedly enhanced by simultaneous treatment with abscisic acid (ABA). Uptake of 3H-GA 1 from the medium containing ABA was linear over a 24-h period, whereas in the absence of ABA, uptake of 3H-GA 1 leveled off after 5 h. After 24 h, aleurones treated with 3H-GA 1 and 3H-GA 1 plus ABA, had taken up 9 and 24%, respectively, of the original 3H-GA 1 provided. Metabolism of 3H-GA 1 proceeded at a linear rate in the presence of ABA. The amount of 3H-GA 1-metabolites which had accumulated by the end of a 24-h incubation appeared to be linearly correlated to the logarithm of the ABA concentration. Gibberellins A 8 and-A 8-glucoside did not reverse GA 1-enhanced synthesis of -amylase. 相似文献
6.
The influence of an interstock of the dwarfing cultivar M9 and the nondwarfing cultivar MM115 on the distribution and metabolism of labeled gibberellic acid A 4 ([ 3H]GA 4) of high specific radioactivity (5.18 × 10 10 becquerel per millimole) applied to the xylem of the rootstock in grafted apple ( Malus × domestica Borkh.) trees was compared. Free [ 3H] GA-like metabolites of [ 3H]GA 4, including putative GA 1, GA 2, GA 3, and GA 34, as well as various 3H-putative GA glucosyl conjugates were detected in stem segments from both cultivars. M9 interstocks reduced the total uptake of [ 3H]GA 4 and decreased the proportion of 3H metabolites transported to the shoots and leaves of scions. The M9 interstock tissue and adjacent rootstock and scion tissue retained a much greater amount and a higher proportion of the label than did comparable tissue of the nondwarfing MM115 interstock. In addition, the amount and proportion of free [ 3H]GAs was higher, and the proportion of putative [ 3H]GA glucosyl conjugates lower, in M9 interstocks compared to MM115. These effects of the dwarfing interstock on GA distribution and metabolism indicate a significant role for GAs in any satisfactory explanation of the dwarfing mechanism in apple. 相似文献
8.
After 4 days in an atmosphere of N 2, aleurone layers of barley ( Hordeum vulgare L. cv Himalaya) remained viable as judged by their ability to produce near normal amounts of α-amylases when incubated with gibberellic acid (GA 3) in air. However, layers did not produce α-amylase when GA 3 was supplied under N 2, apparently because α-amylase mRNA failed to accumulate. 相似文献
9.
The gibberellin (GA) binding properties of a cytosol fraction from hypocotyls of cucumber ( Cucumis sativus L. cv National Pickling) were examined using a DEAE filter paper assay, [ 3H]GA 4, and over 20 GAs, GA derivatives and other growth regulators. The results demonstrate structural specificity of the binding protein for γ-lactonic C-19 GAs with a 3 β-hydroxyl and a C-6 carboxyl group. Additional hydroxylations of the A, C, or D ring of the ent-gibberellane skeleton and methylation of the C-6 carboxyl impede or abolish binding affinity. Bioassay data are generally supported by the in vitro results but significantly GA 9 and GA 36, both considered to be precursors of GA 4 in cucumber, show no affinity for the binding protein. The results are discussed in relation to the active site of the putative GA 4 receptor in cucumber. 相似文献
10.
The expression of the Adh1 gene (alcohol dehydrogenase, EC 1.1.1.1) was studied in the aleurone layer of barley ( Hordeum vulgare cv. Himalaya). Expression increased markedly during grain development at the levels of activity, enzyme protein and mRNA. mRNA content, but not enzyme activity, could be increased further by exogenous abscisic acid (ABA) when isolated, de-embryonated developing grains were pre-treated with gibberellic acid (GA 3) or fluridone. In isolated mature aleurone layers incubated with exogenous hormones, ADH mRNA was strongly up-regulated by ABA and down-regulated by GA 3 within 6 h. With ABA, this increase in mRNA was followed by an increase in ADH protein and activity, peaking at 18 h. With GA 3, the decrease in mRNA was accompanied by simultaneous decreases in protein and activity. In general, GA 3 counteracted the effect of ABA and vice versa. In the aleurone of germinating grain, ADH activity decayed in a distal direction from the embryo, consistent with down-regulation by gibberellin(s) diffusing from it. It was concluded that ADH gene expression in the aleurone of the intact grain is regulated by an ABA/gibberellin interaction. 相似文献
11.
Heat shock in barley aleurone layers induces heat shock protein synthesis and suppresses secretory protein synthesis by selectively destabilizing their mRNAs. In addition, the endoplasmic reticulum (ER) membranes upon which secretory protein mRNAs are translated become vesiculated during heat shock, leading to the hypothesis that ER dissociation and targeted mRNA destabilization are linked mechanistically. Supporting this, ER can be heat adapted, and heat-adapted ER has higher levels of fatty acid saturation in membrane phospholipids which do not vesiculate upon heat shock. Secretory protein mRNAs are also more stable in heat-adapted cells. To understand better heat shock-induced changes in ER membranes, we examined ER membrane proteins and enzymes involved in phosphatidylcholine biosynthesis and phospholipid turnover in heat-shocked aleurone cells. Heat shock significantly increased the activity of phospholipases A2 and D, and shortly thereafter significant but gradual increases in choline kinase and phosphocholine glyceride transferase activities and a sharp increase in phosphorylcholine citidyl transferase activity were observed. Only minor changes were observed in SDS-PAGE analyses of proteins from sonicated ER membranes fractionated on continuous sucrose gradients. Overall, heat shock reduced total lipid in ER membranes relative to protein, and in intact, ultracentrifuged aleurone cells examined by light and electron microscopy the ER band appeared to increase in density. The changes in phospholipid metabolism coupled with the suppression of secretory protein synthesis indicate that in addition to inducing a classic heat shock response, high temperature also induces a classic unfolded protein response in the ER of this secretory cell. 相似文献
12.
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 (GA 3) 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. 相似文献
15.
When aleurone layers of barley ( Hordeum vulgare L.) are incubated with gibberellic acid (GA 3) xylose and arabinose—both as free sugars and bound to larger molecules—are released into the medium. Release begins 10–12h after the start of incubation and continues for at least 60h. At the same time there is a GA 3-induced breakdown of the cell wall resulting in a loss of 2/3 of the cell-wall pentose during 60h of incubation. GA 3 causes the appearance in the medium of an enzyme (or enzymes) which hydrolyze larchwood xylan and aleurone-layer arabinoxylan. Release of the enzyme(s) into the medium begins 28–32h after the start of incubation. Enzyme activity does not accumulate to any large extent in the tissue prior to release into the medium, and is present in very low levels only in the absence of GA 3. Xylanase activity is associated with a protein (or proteins) with a molecular weight of 29,000. The hydrolysis of the xylans is largely caused by endoxylanase activity, indicating the importance of endoglycosidases in the GA 3-induced breakdown of the aleurone cell wall. 相似文献
16.
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 (GA 3) and H 2O. 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 GA 3, 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 Mg 2+ 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 GA 3 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 Mg 2+ the second peak of activity has a density of 1.12 g cc -1 in GA 3-treated tissue and 1.13–1.14 g cc -1 in H 2O-treated tissue. With high-Mg 2+ 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
- GA 3
gibberellic acid 相似文献
17.
The role of calmodulin (CaM) in gibberellic acid (GA 3)-stimulated Ca 2+ uptake was investigated in endomembranes isolated from aleurone cells of barley ( Hordeum vulgare L.). Unidirectional Ca 2+ -uptake activity of endoplasmic reticulum (ER) was higher in membranes isolated from aleurone layers treated for 16 h with GA 3 and Ca 2+ compared with those isolated from layers incubated in Ca 2+ alone. However, the level of uptake from Ca 2+-treated tissue could be stimulated to that of the GA 3-treated cells by applying exogenous CaM which increased the V max of the Ca 2+ transporter approximately threefold. Calcium uptake in ER from GA 3-treated tissue was inhibited by the CaM antagonist W7 in 50% of experiments, whereas the activity in membranes from non-GA 3-treated tissue was unaffected. Treatment with GA 3 also led to a twofold increase in CaM levels in aleurone layers within 4–6 h, paralleling the time course of the stimulation of Ca 2+ uptake and preceding the stimulation of α-amylase secretion. We propose that the elevation of Ca 2+ uptake into the ER induced by GA 3 may be coordinated and regulated by elevated levels of membrane-associated CaM and this may regulate Ca 2+-dependent α-amylase synthesis in the lumen of the ER. 相似文献
18.
Pulse-labeling of barley ( Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA 3) with or without 5 millimolar CaCl 2 shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca 2+. A cDNA clone for α-amylase was isolated and used to measure α-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca 2+. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA 3 with 5 millimolar CaCl 2 and layers incubated in GA 3 alone. RNA isolated from layers incubated for 12 hours in GA 3 with and without Ca 2+ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca 2+ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca 2+-deprived aleurone layers was translatable. Ca 2+ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing. 相似文献
19.
In G2 peas ( Pisum sativum L.) apical senescence occurs only in long days (LD), and indeterminate growth is associated with elevated gibberellin (GA) levels in the shoot in short days (SD). Metabolism of GA 12 aldehyde was investigated by feeding shoots grown in SD or LD with [ 14C]GA 12 aldehyde through the cut end of the stem for 0.5 to 6 hours in the light and analyzing the tissue extract by high performance liquid chromatography. More radioactive products were detected than can be accounted for by the two GA metabolic pathways previously known to be present in peas. Three of the major products appear to be GA conjugates, but an additional pathway(s) of GA metabolism may be present. The levels of putative C 20 GAs, [ 14C]GA 53, [ 14C]GA 44, [ 14C]GA 19, and/or [ 14C] GA 17, were all elevated in SD as compared to LD. Putative [ 14C]GA, was slightly higher in LD than in SD. Putative [ 14C]GA 53 was a major metabolite after 30 minutes of treatment in SD but had declined after longer treatment times to be replaced by elevated levels of putative [ 14C] GA 44 and [ 14C]GA 19/17. Metabolism of GA 20 was slow in both photoperiods. Although GA 20 and GA 19 are the major endogenous GAs as determined by gas chromatography-mass spectrometry, putative [ 14C]GA 20 and [ 14C]GA 19 were never major products of [ 14C]GA 12 aldehyde metabolism. Thus, photoperiod acts in G2 peas to change the rate of GA 53 production from GA 12 aldehyde, with the levels of the subsequent GAs on the 13-OH pathway being determined by the amount of GA 53 being produced. 相似文献
20.
Barley aleurone layers synthesize and secrete several proteases in response to gibberellic acid (GA3). Two major cysteine proteinases designated EP-A (37,000 M(r)) and EP-B (30,000 M(r)) have been described [Koehler and Ho (1988). Plant Physiol. 87, 95-103]. We now report the cDNA cloning of EP-B and describe the post-translational processing and hormonal regulation of both cysteine proteinases. Three cDNAs for cysteine proteinases were cloned from GA3-induced barley aleurone layers. Genomic DNA gel blot analysis indicated that these are members of a small gene family with no more than four to five different genes. The proteins encoded by two of these clones, pHVEP1 and 4, are 98% similar to each other and are isozymes of EP-B. The proteins contain large preprosequences followed by the amino acid sequence described as the mature N terminus of purified EP-B, and are antigenic to EP-B antiserum. The results of pulse-chase experiments indicated that the post-translational processing of large prosequences proceeds in a multistep fashion to produce the mature enzymes. Processing intermediates for EP-B are observed both in the aleurone layers and surrounding incubation medium, but only mature EP-A is secreted. The regulation of synthesis of EP-A, EP-B, and other aleurone cysteine proteinases was compared at the protein and mRNA levels. We conclude that barley aleurone cysteine proteinases are differentially regulated with respect to their temporal and hormonally induced expression. 相似文献
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