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1.
Grandinol, an inhibitor of seed germination and photosynthesis in Eucalyptus sp., inhibits transpiration and stomatal opening. The acylphloroglucinol structure in grandinol seemed to be essential for these activities. Enhancement of activity was achieved by the introduction of a formyl group into the molecule. Therefore, structural requirements for these activities were very similar to that for the inhibition of seed germination and photosynthesis. Other grandinol-related compounds having two electron-withdrawing groups on the phloroglucinol nuclei were also active in these assays.  相似文献   

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The barley aleurone layer is a terminally differentiated secretory tissue whose activity is hormonally controlled. The plant hormone gibberellic acid (GA) stimulates the secretion of hydrolytic enzymes and triggers the onset of programmed cell death (PCD). Abscisic acid (ABA) antagonizes the effects of GA and inhibits enzyme secretion and PCD. Reactive oxygen species (ROS) are key players in many types of PCD, and data presented here implicate ROS in hormonally regulated death of barley aleurone cells. Incubation of aleurone layers or protoplasts in H(2)O(2)-containing media results in death of GA-treated but not ABA-treated aleurone cells. Cells that are programmed to die are therefore less able to withstand ROS than cells that are programmed to remain alive. Illumination of barley aleurone protoplasts with blue or UV-A light results in a rapid increase in intracellular H(2)O(2) production. GA-treated protoplasts die rapidly in response to this increase in intracellular H(2)O(2) production, but ABA-treated protoplasts do not die. The rate of light-induced death could be slowed by antioxidants, and incubating protoplasts in the dark with the antioxidant butylated hydroxy toluene reduces the rate of hormonally induced death. Taken together, these data demonstrate that GA-treated aleurone protoplasts are less able than ABA-treated protoplasts to tolerate internally generated or exogenously applied H(2)O(2), and strongly suggest that ROS are components of the hormonally regulated cell death pathway in barley aleurone cells.  相似文献   

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Using in vivo pulse labeling, changes in the pattern of protein synthesis were detected in isolated barley aleurone layers treated with fibberellic acid (GA3). GA3 greatly altered the relative rates of synthesis of many polypeptides, increasing some, notably -amylase, and decreasing others. -Amylase synthesis increased until it was the major product (over 60%) of protein synthesis after 24h. The pulse-labeled pattern of secreted polypeptides was also changed by GA3. There was the expected increase in -amylase together with a number of other polypeptides but there was reduced secretion of several polypeptides also.Cell-free translation of RNA isolated from control and hormone-treated tissues was used to measure changes in mRNA levels. GA3 caused many changes, particularly in the level of mRNA for -amylase. In vitro synthesized -amylase, identified by immunoaffinity chromatography, had an Mr of 46 000. This polypeptide was partially processed to a polypeptide with Mr 44 000 by the addition of dog pancreas membranes to the in vivo translation mixture. The level of mRNA for -amylase began to increase 2–4 h after GA3 was added and reached a maximum level of about 20% of total mRNA after 16 h. Thus after 16 h, the synthesis of -amylase as a proportion of total protein synthesis, continued to increase while the level of its mRNA as a proportion of total mRNA remained constant. These results indicate that protein synthesis was modified more extensively than we can account for by changes in mRNA.Abscisic acid (ABA) reversed all of the effects of GA3 on protein synthesis and mRNA levels. It also promoted synthesis of a small number of new polypeptides and increased the level of some mRNAs. GA3 reversed the accumulation of ABA-promoted mRNAs. Although, ABA strongly suppressed the increase in the level of translatable mRNA for -amylase, there was an even stronger inhibition of enzyme synthesis and accumulation.We conclude that both GA3 and ABA regulate protein synthesis both positively and negatively in aleurone cells largely by regulating levels of mRNA and in the case of -amylase, possibly also by changing the efficiency of translation of its mRNA.  相似文献   

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Exposure of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers to 40°C for a period of 3 h results in the selective suppression of the synthesis and secretion of hydrolytic enzymes; other normal cellular protein synthesis continues during heat shock. This suppression is correlated with secretory protein mRNA destabilization and the dissociation of stacked ER lamellae during heat shock (Belanger et al. 1986, Proceedings of the National Academy of Sciences USA 83, pp. 1354–1358). In this report we examined the effect of exposure to extended periods of heat shock. If exposure to 40°C was continued for a period of 18 h, the synthesis of α-amylase, the predominant secreted hydrolase, resumed. This was accompanied by increased α-amylase mRNA levels and the reformation of ER lamellae. Though initial exposure (3 h) to 40°C reduced protein secretion to ~10% of that observed in aleurone cells maintained at 25°C, exposure for prolonged periods (16–20 h) permitted the resumption of protein secretion to ~66% of non-heat-shocked control levels. The resumption of normal secretory protein synthesis during prolonged exposure to 40°C was correlated with an increase in the incorporation of [14C]glycerol into phosphatidylcholine and an increase in the ratio of saturated to unsaturated fatty acids in lipids isolated from ER membrane preparations. Increased fatty acid saturation has been demonstrated to enhance thermostability in biological membranes, and such changes in membrane composition may be important to the recovery of secretory protein synthesis at the ER.  相似文献   

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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.  相似文献   

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During germination of barley grains, DNA fragmentation was observed in the aleurone. The appearance of DNA fragmentation in the aleurone layer, observed by TUNEL staining in aleurone sections, started near the embryo and extended to the aleurone cells far from the embryo in a time dependent manner. The same spatial temporal activities of hydrolytic enzymes such as -amylase were observed in aleurone. DNA fragmentation could also be seen in vitro under osmotic stress, in isolated aleurone. During aleurone protoplast isolation, a very enhanced and strong DNA fragmentation occurred which was not seen in protoplast preparations of tobacco leaves. ABA was found to inhibit DNA fragmentation occurring in barley aleurone under osmotic stress condition and during protoplast isolation, while the plant growth regulator gibberellic acid counteracted the effect of ABA. Addition of auxin or cytokinin had no significant effect on DNA fragmentation in these cells. To study the role of phosphorylation in ABA signal transduction leading to control of DNA fragmentation (apoptosis), the effects of the phosphatase inhibitor okadaic acid and of phenylarisine oxide on apoptosis were studied. We hypothesize that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleurone activities during germination. The possible signal transduction pathway of ABA leading to the regulation of DNA fragmentation is discussed.  相似文献   

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The effects of the addition and withdrawal of gibberellic acid (GA3) and Ca2+ on enzyme synthesis and secretion by barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. Incubation of layers in GA3 plus Ca2+ affects the total amount of secreted α-amylase (EC 3.2.1.1) and acid phosphatase (EC 3.1.3.2) by promoting the appearance of different isoenzymic forms of these enzymes. The release of α-amylase isoenzymes 1–4 in response to GA3 plus Ca2+ has a lag of 6 h. When layers are incubated in GA3 alone for 6 h prior to the addition of Ca2+, isoenzymes 1–4 appear in the medium after only 30 min. When the addition of Ca2+ to layers pretreated in GA3 is delayed beyond 12 h, its effectiveness in stimulating the synthesis and release of isoenzymes 3 and 4 is diminished. After 35 h of preincubation in GA3, addition of Ca2+ will not stimulate synthesis of α-amylase isoenzymes 3 and 4. Aleurone layers preincubated for 6 h in GA3 will respond to Ca2+ when the GA3 is withdrawn from the incubation medium by producing α-amylase isoenzymes 1–4. The converse is not the case, however, since layers preincubated in Ca2+ for 6 h will not produce all isoenzymes of α-amylase when subsequently incubated in GA3. The Ca2+-stimulated release of α-amylase from GA3 pre-treated layers is dependent on the time of incubation in Ca2+ and the concentration of the ion. The response to Ca2+ is temperature-dependent, and other divalent cations such as Mg2+ cannot substitute for Ca2+. We conclude that Ca2+ influences α-amylase release by influencing events at the biochemical level.  相似文献   

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The effect of temperature on α-amylase synthesis and secretion from barley (c.v. Himalaya) half-seeds and aleurone layers is reported. Barley half-seeds incubated at 15 C in gibberellic acid (GA) concentrations of 0.5 and 5 micromolar for 16 hours do not release α-amylase. Similarly, isolated aleurone layers of barley do not release α-amylase when incubated for 2 or 4 hours at temperatures of 15 C or below following 12 hours incubation at 25 C at GA concentrations from 50 nanomolar to 50 micromolar. There is an interaction between temperature and GA concentration for the process of α-amylase release from aleurone layers; thus, with increasing GA concentration, there is an increase in the Q10 of this process. A thermal gradient bar was used to resolve the temperature at which the rate of α-amylase release changes; thermal discontinuity was observed between 19 and 21 C. The time course of the response of aleurone tissue to temperature was determined using a continuous monitoring apparatus. Results show that the effect of low temperature is detectable within minutes, whereas recovery from exposure to low temperature is also rapid. Although temperature has a marked effect on the amount of α-amylase released from isolated aleurone layers, it does not significantly affect the accumulation of α-amylase within the tissue. At all GA concentrations above 0.5 nanomolar, the level of extractable α-amylase is unaffected by temperatures between 10 and 28 C. It is concluded that the effect of temperature on α-amylase production from barley aleurone layers is primarily on the process of enzyme secretion.  相似文献   

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β-Amylase is involved in the starch degradation process and therefore influences grain quality. Starch degradation efficiency is dependent on the enzyme thermostability during malting and mashing. Four alleles resulting in different enzyme thermostability are known. These alleles are distinguished by coding single nucleotide polymorphism (cSNP). Pyrosequencing was used for cSNP genotyping of β-amylase alleles in 79 spring barley varieties by using analyser PSQ MA96 System (Pyrosequencing, Biotage). A new cSNP was revealed by means of Pyrosequencing analysis of sequence flanking cSNP698, thus recognizing a fifth β-amylase allele. Pyrosequencing is a high-throughput, fast, and precise system for barley SNP genotyping.  相似文献   

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The osmotic regulation of gibberellic acid-enhanced hydrolase synthesis in aleurone cells of barley is mediated via a general inhibition of protein synthesis. This inhibition of protein synthesis occurs both in the absence and in the presence of gibberellic acid. Osmotica do not specifically inhibit gibberellic acid elicited responses in aleurone cells as was thought in the past.  相似文献   

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cDNAs of barley α-amylase andA. niger glucoamylase were cloned in oneE. coli-yeast shuttle plasmid resulting in the construction of expression secretion vector pMAG15. pMAG15 was transformed intoS. cerevisiae GRF18 by protoplast transformation. The barley α-amylase andA. niger glucoamylase were efficiently expressed under the control of promoter and terminator of yeast PGK gene and their own signal sequence. Over 99% of the enzyme activity expressed was secreted to the medium. The recombinant yeast strain, S.cerevisiae GRF18 (pMAG15), hydrolyzes 99% of the starch in YPS medium containing 15% starch in 47 h. The glucose produced can be used for the production of ethanol. Project supported by the Guangdong Natural Science Foundation.  相似文献   

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