A cell-free system for the study of alpha-amylase synthesis in barley aleurone layers.
Ontology highlight
ABSTRACT:
SUBMITTER: Duffus JH
PROVIDER: S-EPMC1270387 | biostudies-other | 1967 Apr
REPOSITORIES: biostudies-other
Similar Datasets
Project description:H2S is a signaling molecule in plants and animals. Here we investigated the effects of H2S on programmed cell death (PCD) in barley (Hordeum vulgare L.) aleurone layers. The H2S donor NaHS significantly delayed PCD in aleurone layers isolated from imbibed embryoless barley grain. NaHS at 0.25 mM effectively reduced the accumulation of superoxide anion (·O2 (-)), hydrogen peroxide (H2O2), and malondialdehyde (MDA), promoted the activity of superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), and decreased those of lipoxygenase (LOX) in isolated aleurone layers. Quantitative-PCR showed that NaHS treatment of aleurone tissue led to enhanced transcript levels of the antioxidant genes HvSOD1, HvAPX, HvCAT1, and HvCAT2 and repressed transcript levels of HvLOX (lipoxygenase gene) and of two cysteine protease genes HvEPA and HvCP3-31. NaHS treatment in gibberellic acid- (GA-) treated aleurone layers also delayed the PCD process, reduced the content of ·O2 (-), and increased POD activity while decreasing LOX activity. Furthermore, α-amylase secretion in barley aleurone layers was enhanced by NaHS treatment regardless of the presence or absence of GA. These data imply that H2S acted as an antioxidant in delaying PCD and enhances α-amylase secretion regardless of the presence of GA in barley aleurone layers.
| S-EPMC4442411 | biostudies-literature
Project description:Among natural populations, there are different colours of barley (Hordeum vulgare L.). The colour of barley grains is directly related to the accumulation of different pigments in the aleurone layer, pericarp and lemma. Blue grain colour is due to the accumulation of anthocyanins in the aleurone layer, which is dependent on the presence of five Blx genes that are not sequenced yet (Blx1, Blx3 and Blx4 genes clustering on chromosome 4HL and Blx2 and Blx5 on 7HL). Due to the health benefits of anthocyanins, blue-grained barley can be considered as a source of dietary food. The goal of the current study was to identify and characterize components of the anthocyanin synthesis regulatory network for the aleurone layer in barley.The candidate genes for components of the regulatory complex MBW (consisting of transcription factors MYB, bHLH/MYC and WD40) for anthocyanin synthesis in barley aleurone were identified. These genes were designated HvMyc2 (4HL), HvMpc2 (4HL), and HvWD40 (6HL). HvMyc2 was expressed in aleurone cells only. A loss-of-function (frame shift) mutation in HvMyc2 of non-coloured compared to blue-grained barley was revealed. Unlike aleurone-specific HvMyc2, the HvMpc2 gene was expressed in different tissues; however, its activity was not detected in non-coloured aleurone in contrast to a coloured aleurone, and allele-specific mutations in its promoter region were found. The single-copy gene HvWD40, which encodes the required component of the regulatory MBW complex, was expressed constantly in coloured and non-coloured tissues and had no allelic differences. HvMyc2 and HvMpc2 were genetically mapped using allele-specific developed CAPS markers developed. HvMyc2 was mapped in position between SSR loci XGBS0875-4H (3.4 cM distal) and XGBM1048-4H (3.4 cM proximal) matching the region chromosome 4HL where the Blx-cluster was found. In this position, one of the anthocyanin biosynthesis structural genes (HvF3'5'H) was also mapped using an allele-specific CAPS-marker developed in the current study.The genes involved in anthocyanin synthesis in the barley aleurone layer were identified and characterized, including components of the regulatory complex MBW, from which the MYC-encoding gene (HvMyc2) appeared to be the main factor underlying variation of barley by aleurone colour.
| S-EPMC5688479 | biostudies-literature
Project description:1. Turnovers of [14C]glycerol-labelled phospholipids in wheat aleurone tissue have been measured by using a pulse-decay technique. The most metabolically active phospholipids were phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylglycerol. 2. Gibberellic acid action on the tissue led to breakdown of phosphatidylcholine and stimulated turnover of the other phosphatides concomitant with the secretion of alpha-amylase from the tissue. After pulse-labelling of the aleurone tissue with [14C]glycerol, radioactivity was lost from the phospholipids and appeared quantitatively in triacylglycerols, suggesting a stoichiometric metabolism of the former into the latter. Although 1,2-diacylglycerol is an expected intermediate in such a conversion, the patterns of radioactivity in diacylglycerols gave no indication of this. 3. Several aspects of the response of aleurone tissue to gibberellic acid resemble the responses of exocytotic animal tissues to external agonists. In particular, our results and previous reports in the literature suggest that endomembrane flow, exocytosis, phosphatidylinositol turnover and a requirement of Ca2+ for enzyme secretion are common to both plant and animal systems. Although considerable differences also exist between the two, the similarities are sufficient to warrant further consideration that plants and animals might have conserved a similar hormone response-secretion mechanism.
| S-EPMC1153934 | biostudies-other
Project description:The phytohormone gibberellic acid (GA) is well known to promote seed germination in plants. One of its functions is to stimulate the production of hydrolytic enzymes in the aleurone and their secretion to the adjacent endosperm. The storage in the endosperm is thus degraded by these hydrolases into small molecules, which are utilized as nutrients for embryo growth to establish the young seedling. ABA in contrast plays antagonistic role to GA to keep seed in dormancy. Cereal aleurone has been established as a model system to investigate giberrellin (GA) and abscisic acid (ABA) responses. Using Barley 1 GeneChip, we examined the mRNA accumulation of over 22 000 genes in barley aleurone treated with GA, ABA, GA plus ABA, and sln1 mutant.
2010-01-01 | GSE18758 | GEO
Project description:Treatment of barley alpha-amylase/subtilisin inhibitor (BASI) with reagents specific for arginine, histidine, methionine and tyrosine residues and amino and carboxyl groups indicates that an arginine residue(s) is essential for its action on the target enzyme barley alpha-amylase 2. Phenylglyoxal modified eight out of 12 arginine residues in BASI. Kinetic analysis shows that the inactivation of BASI follows a pseudo-first-order reaction and is due to reaction with one molecule of phenylglyoxal; the second-order rate constant is determined to be 2.95 M-1.min-1. At pH 8.0, BASI and barley alpha-amylase 2 form an inactive 1:1 complex. The Ki value of this association is 2.2 x 10(-10) M. The alpha-amylase protects four arginine residues and also the alpha-amylase inhibitory activity of BASI against phenylglyoxal. When BASI from the phenylglyoxal-modified target enzyme-inhibitor complex is isolated and subjected to a second treatment with phenylglyoxal, four additional arginine residues are modified, with concomitant loss of the inhibitory activity. These results are discussed in relation to a three-dimensional model of BASI based on the known structure of the corresponding inhibitor from wheat.
| S-EPMC1134333 | biostudies-other
Project description:Arginine residues in barley alpha-amylase/subtilisin inhibitor (BASI) involved in binding to barely alpha-amylase 2 (AMY2) were differentially labelled using AMY2 as protectant and phenylglyoxal (PGO) and [14C]PGO as modifying agents. Chymotryptic fragments of labelled BASI were purified by reverse-phase HPLC, and we concluded that the radiolabelled Arg-27, Arg-155 and most likely Arg-127, identified by amino acid, sequence and 14C analyses, are protected by AMY2. While Arg-106 and Arg-107 showed intermediate reactivity and apparently were only partly accessible, Arg-15, Arg-41 and Arg-61 reacted with PGO and were thus exposed in the BASI-AMY2 complex. Patterns of arginine modification by [14C]PGO in free or in AMY2-complexed BASI were consistent with the results of differential labelling. The AMY2-protected arginines in BASI are at a distance from each other, as deduced from crystal structures of different beta-trefoil proteins (Erythrina caffra and soybean trypsin inhibitors, interleukin-1 alpha and -1 beta and WASI, the wheat homologue), suggesting that the BASI-AMY2 complex has multiple contacts at a larger interface. Accordingly, 11-16-residue-long BASI oligopeptides synthesized to include Arg-27, Arg-106/Arg-107 or Arg-127 were unable to suppress the formation of BASI-AMY2 or the effect of an inhibitory monoclonal antibody to BASI. Since Arg-27 is not conserved in rice and wheat ASIs, we further propose that Arg-155 in BASI is the kinetically identified PGO-sensitive group that is essential for inhibition [Abe, Sidenius and Svensson (1993) Biochem. J. 293, 151-155].
| S-EPMC1135726 | biostudies-other
Project description:BACKGROUND:Vacuolar processing enzymes (VPEs) have been identified as the enzymes that regulate vacuole-mediated programmed cell death (PCD) in plants. The mechanism that VPE regulates the PCD in rice aleurone layers remains unknown. RESULTS:The aleurone layers treated with distilled water exerted caspase-1 and VPE activity, both of which were inhibited by the caspase-1 specific inhibitor Ac-YVAD-CMK but not by the caspase-3 specific inhibitor Ac-DEVD-CHO. However, the caspase-1 and caspase-3 inhibitors weakened the activity of caspase-3. Combined with the effects of endogenous gibberellin (GA) on the induction of OsVPEs, we suggest that the OsVPE3 in the aleurone layers, which exhibits caspase-1-like activity, is a key molecule in GA-induced PCD via regulating the protease with caspase-3-like activity. Many studies have confirmed that vacuolar fusion is an important feature of vacuole-mediated PCD in plants. In this experiment, the process of vacuole fusion was accompanied by changes in the structure of actin filaments (AFs), specifically, their depolymerization and polymerization. The process of vacuolar fusion was accelerated or delayed by the promotion or inhibition of the depolymerization of AFs, respectively. Here, the inhibition of OsVPE3 blocked the depolymerization of AFs and delayed the fusion of vacuoles, indicating that OsVPE3 can regulate the fusion of vacuoles in rice aleurone layers via mediating AFs. Furthermore, the depolymerization of AFs contributed to the up-regulation of OsVPE3 gene expression and VPE activity, resulting in accelerated PCD in rice aleurone layers. However, the inhibitor of VPE reversed the effects of AF depolymerization on the activity of VPE, then postponing the process of PCD, implying that AF can involve in GA-induced PCD of rice aleurone layers by mediating OsVPE3. CONCLUSIONS:Together, activation of OsVPE3 and depolymerization of AFs shortened the process of vacuolation and PCD in rice aleurone layers, and OsVPE3 interacted with AFs during regulation.
| S-EPMC7105518 | biostudies-literature
Project description:Haem oxygenase-1 (HO-1) confers protection against a variety of oxidant-induced cell and tissue injury in animals and plants. In this report, it is confirmed that programmed cell death (PCD) in wheat aleurone layers is stimulated by GA and prevented by ABA. Meanwhile, HO activity and HO-1 protein expression exhibited lower levels in GA-treated layers, whereas the hydrogen peroxide (H(2)O(2)) content was apparently increased. The pharmacology approach illustrated that scavenging or accumulating H(2)O(2) either delayed or accelerated GA-induced PCD. Furthermore, pretreatment with the HO-1 specific inhibitor, zinc protoporphyrin IX (ZnPPIX), before exposure to GA, not only decreased HO activity but also accelerated GA-induced PCD significantly. The application of the HO-1 inducer, haematin, and the enzymatic reaction product of HO, carbon monoxide (CO) aqueous solution, both of which brought about a noticeable induction of HO expression, substantially prevented GA-induced PCD. These effects were reversed when ZnPPIX was added, suggesting that HO in vivo played a role in delaying PCD. Meanwhile, catalase (CAT) and ascorbate peroxidase (APX) activities or transcripts were enhanced by haematin, CO, or bilirubin (BR), the catalytic by-product of HO. This enhancement resulted in a decrease in H(2)O(2) production and a delay in PCD. In addition, the antioxidants butylated hydroxytoluene (BHT), dithiothreitol (DTT), and ascorbic acid (AsA) were able not only to delay PCD but also to mimic the effects of haematin and CO on HO up-regulation. Overall, the above results suggested that up-regulation of HO expression delays PCD through the down-regulation of H(2)O(2) production.
| S-EPMC2993913 | biostudies-literature
Project description:The phytohormone gibberellic acid (GA) is well known to promote seed germination in plants. One of its functions is to stimulate the production of hydrolytic enzymes in the aleurone and their secretion to the adjacent endosperm. The storage in the endosperm is thus degraded by these hydrolases into small molecules, which are utilized as nutrients for embryo growth to establish the young seedling. ABA in contrast plays antagonistic role to GA to keep seed in dormancy. Cereal aleurone has been established as a model system to investigate giberrellin (GA) and abscisic acid (ABA) responses. Using Barley 1 GeneChip, we examined the mRNA accumulation of over 22 000 genes in barley aleurone treated with GA, ABA, GA plus ABA, and sln1 mutant. Barley aleurone tissues were separated from half-seed without embryo. Three independent RNA samples for each treatment including the control without any hormone were extracted and hybridized onto Affymetrix microarrays. We also did microarray in three replications for sln1 mutant aleurone without hormone treatment.
2010-01-01 | E-GEOD-18758 | biostudies-arrayexpress
Project description:Cell-free RNA and protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. Advances in reconstituted CFPS systems such as the Protein synthesis Using Recombinant Elements (PURE) system offer new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, protein microarrays, isotopic labeling, and incorporating unnatural amino acids. In this study, using firefly luciferase synthesis as a reporter system, we improved PURE system productivity up to 5 fold by adding or adjusting a variety of factors that affect transcription and translation, including Elongation factors (EF-Ts, EF-Tu, EF-G, and EF4), ribosome recycling factor (RRF), release factors (RF1, RF2, RF3), chaperones (GroEL/ES), BSA and tRNAs. The work provides a more efficient defined in vitro transcription and translation system and a deeper understanding of the factors that limit the whole system efficiency.
| S-EPMC4152126 | biostudies-literature