Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers

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.     

Hormonal regulation of gene expression in barley aleurone layers

As part of our investigation of the mode of action of plant hormones in barley (Hordeum vulgare L.) aleurone layers, we have studied the expression of five identified and three unidentified mRNA species in the presence of exogenous gibberellic acid (GA₃) and abscisic acid. Three of the mRNAs are GA₃-inducible, three are suppressed by GA₃, and two are constitutive. The extent of the GA₃ effect differs considerably for both inducible and suppressible mRNAs. For example, a ten-fold higher concentration of GA₃ (10^-8 M) is required for full induction of the high-pl group -amylase mRNA than is required for the low-pI -amylase mRNA (10^-9 M). Temporal regulation of mRNA abundance also varies between the two -amylase isoenzyme groups. The three GA₃-suppressible mRNA species studied, alcohol dehydrogenase (ADH1), a probable amylase and protease inhibitor, and an unidentified barley mRNA species also varied in response to GA₃. The ADH1 mRNA decreased drastically within 8 h of GA₃ treatment, whereas the other two began to decrease in abundance only after 12–16 h of GA₃ treatment. Abscisic-acid treatment counteracted the GA₃ effects for both the inducible and suppressible mRNA species. Comparison of -amylase-mRNA levels and -amylase-synthesis rates showed a strong correlation between the two parameters, the only exception being a lack of -amylase synthesis in the presence of -amylase mRNA at low GA₃ concentrations. Therefore, the expression of -amylase seems to be regulated primarily by its mRNA levels.Abbreviations ABA abscisic acid - ADH1 alcohol dehydrogenase 1 - cDNA copy DNA - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor     

The responsiveness of Avena fatua aleurone protoplasts to gibberellic acid

The sensitivity to gibberellic acid (GA₃) of aleurone protoplasts isolated from a single harvest of an inbred line of Avena fatua seed that had been after-ripened over anhydrous CaCl₂ at 25±2°C and 4±2°C for three years was assessed. Protoplasts isolated from aleurones of seed stored at 25°C produced substantially more -amylase in response to 10^–7 M GA₃ than those isolated from aleurones of seed stored at 4°C. The apparent difference in responsiveness does not appear to be due to a change in the duration of the lag phase between addition of GA₃ and the production of -amylase. The dose response of aleurone protoplasts to GA₃, measured as -amylase production, is complex and appears to have three phases. Protoplasts from seed stored at both temperatures respond appreciably to 10^–14 M GA₃. With increasing concentrations of GA₃, up to 10^–9 M, -amylase production increases similarly in protoplasts from both lots of seed, reaching a level approximately 2.7–3.8 times greater than when no GA₃ is applied. GA₃-induced -amylase production increases markedly as the concentration is raised from 10^–9 M to 10^–6 M, and the response then appears to be saturated. Over this part of the response curve protoplasts from the two seed lots differ markedly in their responsiveness to GA₃. Those from seed stored at 25°C produce considerably more -amylase, >130-fold higher than the minus GA₃ control, than those from seed stored at 4°C, <35-fold higher than the minus GA₃ control. This apparent difference in the responsiveness of aleurone protoplasts to GA₃ could be correlated with the loss of embryo dormancy in seed stored at 25°C. Seed stored at 4°C retained the dormancy characteristics present immediately after harvesting.     

Barley aleurone layer cell protoplasts as a transient expression system

Protoplasts were prepared from barley aleurone layers using Onozuka cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of -amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley -amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormoneresponsive elements in hydrolase genes.     

Isolation and transformation of rice aleurone protoplasts

Summary Protoplasts isolated from the aleurone have been used extensively in molecular studies focusing on hormone-mediated regulation of gene expression in barley seed. To extend the use of aleurone protoplasts to other species, we have determined the conditions necessary for the isolation of protoplasts from rice aleurone layers of germinated seed. Many of the common cell wall degrading enzymes used in making protoplasts were tested for their ability to release protoplasts from rice aleurone layers. Cellulysin was found to be the most effective. Transformation of these aleurone protoplasts was accomplished using polyethylene glycol and DNA constructs containing the firefly luciferase reporter gene under the control of two different promoters were tested. Luciferase expression was 24-fold greater when the reporter gene was under the control of the CaMV 35S promoter than when the promoter from the alcohol dehydrogenase 1 gene was used. With the isolation and transformation of aleurone protoplasts from rice, it is now possible to investigate molecular events occurring in this tissue during germination.     

The isolation of endoplasmic reticulum from barley aleurone layers

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.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid - Trizma tris(hydroxymethyl)aminomethane     

Isolation of a raw starch-binding fragment from barley alpha-amylase

Barley -amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with -cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel -sheets in domain C and the ₇-and ₈-helices of the (/)₈ domain.     

Primer extension studies on α-amylase mRNAs in barley aleurone. II. Hormonal regulation of expression

Relative levels of different -amylase mRNAs were assessed by primer extension experiments using RNA prepared from aleurone of barley (Hordeum vulgare L. cv. Himalaya). Three different aleurone systems were studied: protoplasts prepared from aleurone layers, isolated aleurone layers, and aleurone from germinated grain. Oligonucleotide primers specific for the low-pI and high-pI -amylase groups allowed the levels of different -amylase mRNAs to be assessed both within and between the two groups.In all aleurone systems the same set of -amylase mRNAs was produced in response to either applied gibberellic acid (aleurone protoplasts, isolated aleurone layers) or, presumably, native gibberellin(s) (germinated grain). This result indicates that the same set of genes is being expressed in each case. Differences were observed between the different aleurone systems in regulation of levels of -amylase mRNAs. In particular, the regulation of -amylase mRNA levels in aleurone of germinated grain has unique features which are not adequately explained by the response of isolated aleurone layers to gibberellic acid.     

Characterization of active barley alpha-amylase 1 expressed and secreted by Saccharomyces cerevisiae

Recombinant barley -amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified -amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The k _cat/K _m was 2.7 × 10² mM^–1.s^–1, consistent with those of -amylases from plants and other sources.     

Structural organization of ultrarapidly frozen barley aleurone cells actively involved in protein secretion

The ultrastructural organization of actively secreting barley (Hordeum vulgare L. cv. Himalaya) aleurone cells was examined using ultrarapid-freezing (<-10 000°C s^-1) followed by freeze-fracture and freeze-substitution. Our analysis indicates that much of the evidence supporting a direct pathway from the endoplasmic reticulum (ER) to the plasma membrane (i.e. bypassing the Golgi apparatus) for the secretion of -amylase (EC 3.2.1.1) may not be valid. Cryofixed ER cisternae show no sign of vesiculation during active -amylase secretion in gibberellic acid (GA₃)-treated cells. At the same time, Golgi complexes are abundant and numerous small vesicles are associated with the edges of the cisternae. Vesicles appear to be involved in the delivery of secretory products to the plasma membrane since depressions containing excess membrane material appear there. Treatment with GA₃ also induces changes in the composition of Golgi membranes; most notably, the density of intramembrane particles increases from 2700 m^-2 to 3800 m^-2 because of an increase of particles in the 3–8.5-nm size range. A slight decrease in 9–11-nm particles also occurs. These changes in membrane structure appear to occur as the Golgi complex becomes committed to the processing and packaging of secretory proteins. We suggest that secretory proteins in this tissue are synthesized in the abundant rough ER, packaged in the Golgi apparatus, and transported to the plasma membrane via Golgi-derived secretory vesicles. Mobilization of reserves is also accompanied by dynamic membrane events. Our micrographs show that the surface monolayer of the lipid bodies fuses with the outer leaflet of the bilayer of protein-body membranes during the mobilization of lipid reserves. Following the breakdown of the protein reserves, the protein bodies assume a variety of configurations.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - P protoplasmic - E exoplasmic     

Quantitative and qualitative changes in the endoplasmic reticulum of barley aleurone layers

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 -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 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.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid     

Messenger RNA from isolated aleurone cells directs the synthesis of an alpha-galactosidase found in the endosperm during germination of guar (Cyamopsis tetragonaloba) seed

In cereals and some legumes the aleurone layer is a site of synthesis of enzymes which mobilize endosperm reserves. It has been established whether or not the aleurone cells of the seed endosperm of Cyamopsis tetragonaloba are a site of synthesis of -galactosidase. The isolation and cultivation of aleurone cells demonstrated that they contain mRNA which directs the synthesis and secretion of -galactosidase into the endosperm where along with a -mannanase it is responsible for the degradation of the galactomannan storage polymer. A method was developed to purify the mRNA from the aleurone cells of germinating seeds. This mRNA was analysed by: i) Northern blot hybridization using oligo-nucleotide mixed probes derived from the protein's NH₂-terminal amino acid sequence and ii) in vitro translation in a wheat germ system and detection of the -galactosidase protein using antibodies. The molecular mass of the protein synthesized in vitro is slightly larger (44 kDa) than that of the mature -galactosidase (40.5 kDa) which is as expected for the precursor of a secreted protein.     

An E. coli expression system for the extracellular secretion of barley alpha-amylase

Libraries of modified genes are often screened during the process of genetically engineering enzymes with specifically tailored activities. It is important, therefore, to create expression systems which allow for the rapid screening of many clones. We developed an Escherichia coli expression system which will secrete enzymes into the growth medium. We describe the first reported expression of barley -amylase in E. coli. The enzyme is secreted onto solid media containing starch to produce easily visualized halos. In addition, the enzyme is secreted into liquid media in an intact, active form.     

Isolation and characterization of aleurone protoplasts from a malting variety of barley (Hordeum vulgare L.)

Summary A procedure has been developed to isolate protoplasts from mature aleurone layers of the malting variety Alexis and four other barley genotypes. It combines induction of endogenous cell wall degrading enzymes together with use of Onuzuka cellulase R 10 and driselase and results in better yields for two varieties than can be obtained with the huskless variety Himalaya. The viability of the freshly isolated protoplasts is greater than 90% and in spite of the presence of gibberellic acid during isolation procedures, most of the protoplasts are at an early developmental stage, as judged by ultrastructure. Gibberellic acid-induced changes in protoplast structure resemble those reported for Himalaya protoplasts. The protoplasts secrete both -amylase (EC 3.2.1.1) and (1-3, 1-4)--glucanase (EC 3.2.1.73) into the surrounding medium. Transfection studies using a low pI -amylase promoter to direct chloramphenicol acetyltransferase expression in aleurone protoplasts from Alexis and Himalaya revealed significant differences in their hormone responsiveness. In the absence of hormones, low levels of expression of the reporter enzyme were obtained in Alexis protoplasts, while high levels were characteristic for Himalaya protoplasts. An 8-fold increase in the expression of the reporter gene was induced by supplying the transfected Alexis protoplasts with gibberellin A₃, whereas expression in Himalaya protoplasts remained unchanged. When Himalaya protoplasts were isolated from aleurone layers that had not been incubated with GA₃ during the initial stages of protoplasting (the classical procedure), the hormone response of the promoter was 2.5-fold. It is thus possible to optimize the aleurone protoplast isolation procedure for different barley genotypes and mutants of interest in studies of transgenic gene expression and hormone induced secretion of proteins from this unique secretory plant tissue.Abbreviations ABA abscisic acid - APIM aleurone protoplast isolation medium - CAT chloramphenicol acetyltransferase - EDTA ethylenediamine tetraacetic acid - ER endoplasmic reticulum - GA₃ gibberellin A₃ - IgG immunoglobulin G - MES 2-(N-morpholino)-ethanesulfonic acid - PAGE polyacrylamide gel electrophoresis - PEG polyethylene glycol - pI isoelectric point - PIPES piperazine N,N-bis-(diethanesulfonic acid) - SDS sodium dodecyl sulfate     

Tnf-α and IL-1α inhibit both pyruvate dehydrogenase activity and mitochondrial function in cardiomyocytes: Evidence for primary impairment of mitochondrial function

Cytokines such as tumor necrosis factor (TNF) and Interleukin-1 (IL1) are known to influence energy metabolism and mitochondrial function in tumor and vascular smooth muscle cells. The aim of the present study was to investigate whether in cardiomyocytes mitochondrial function and PDH activity may also be impaired by TNF and IL1. Pyruvate dehydrogenase (PDH) activity and mitochondrial oxygen consumption of cultured cardiomyocytes were determined after subchronic exposure (24 h) to TNF (1, 10, 100, 1000 I.U./ml) and IL1 (0.1, 1, 10, 100 I.U./ ml).TNF- and IL1- exposure of the cardiomyocytes resulted in a concentration dependent decrease of PDH activity up to 38%. In parallel, selective oxygen consumption of the respiratory chain complexes I (NADH:ubiquinone oxidoreductase) and II (succinate:ubiquinone oxidoreductase) decreased by up to 45%. Addition of the PDH activator dichloracetate (0.01 M) resulted in complete restoration of PDH activity but not of mitochondrial function. The results suggest a primary inhibition of the mitochondrial respiratory chain by TNF and IL1 and a subsequent down regulation of PDH activity.     

Attenuation of changes in sarcoplasmic reticular gene expression in cardiac hypertrophy by propranolol and verapamil

The prothymosin a kinase (ProTK) is an apparently novel enzyme that is responsible for the phosphorylation of prothymosin (ProT), involved in the proliferation of mammalian cells. The present study investigated the properties of this enzyme. ProTK is more effectively activated by Mn²⁺ than by other divalent cations, and its activity is unaffected by RNA. Its principal substrate in proliferating cells appears to be ProTa. Both in vivo and in vitro, it is unable to phosphorylate the peptides thymosin ₁ and thymosin ₁₁, derived from the amino terminus of ProT, despite the fact that the sites of phosphorylation of ProT are contained within this part of its sequence. In trials in vivo, inhibition of gene expression abolished both phosphorylation of ProT and ProTK activity. ProTK is located in the cytosolic fractions throughout the cell cycle. Its activity, which is dependent on cell proliferation, increases markedly during S phase and begins to decline as the cell enters G2. Studies of the effects of activators and inhibitors of protein kinases involved in signal transduction pathways suggest that ProTK is activated by phosphorylation in a mitogen-initiated pathway that is dependent on PKC; however, PKC does not itself phosphorylate ProTK, which is therefore presumably phosphorylated by another kinase.     

Syntheses of 1-O-benzyl-α-glucoside and 1-O-benzyl-α-maltoside by transglycosylation of α- amylase from soluble starch in aqueous solution

Glycosides, 1-O-benzyl--glucoside (BG) and 1- O-benzyl--maltoside (BM), were synthesized from soluble starch and benzyl alcohol by transglycosylation with an -amylase in a water system. BG was mostly obtained in a reaction mixture of pH 5.0, while BM was synthesized in pH 8.0. The synthesized glycosides had -configuration linkage between sugar and benzyl alcohol. The BG was rapidly hydrolyzed to benzyl alcohol and glucose by -glucosidase. The BM was hydrolyzed to BG and glucose below pH 5.0 by the -amylase used for its synthesis but it was not hydrolyzed above pH 8.0.     

Promotion of murine antitumour activity by prothymosin α treatment: I. Induction of tumoricidal peritoneal cells producing high levels of tumour necrosis factor α

Summary The effect of prothymosin (ProT) on the survival of DBA/2 mice inoculated with syngeneic tumour cells was studied. DBA/2 mice inoculated intraperitoneally (i.p.) with 2×10⁵ syngeneic leukaemic L1210 cells developed ascites within 8–12 days and died 10–14 days later. Treatment with ProT consistently inhibited the development of ascites in 20% of the treated animals and prolonged the survival of 40%–60% of the animals up to 70 days. The most effective treatment schedule of ProT was 300 ng/mouse given i.p. at 2-day intervals for 3 weeks followed by a rest period of 7 days, prior to tumour cell inoculation. Peritoneal exudate (PE) cells collected from mice treated with the optimal dose of ProT produced, in the absence of exogenous stimulus, six- to eightfold higher levels of tumour necrosis factor (TNF) than PE cells from control mice. Furthermore these cells exhibited cytotoxic activity against several tumour cell lines including the syngeneic L1210, the TNF-insensitive P815 mastocytoma, the human MOLT-4 lymphoblastic leukaemia, as well as the murine TNF-sensitive L929 fibroblast cell line. Kinetic studies revealed that both production of TNF and tumoricidal activity peaked 7 days after the last injection of ProT and were maintained at high levels over a period of 1 month. Injections with 150 ng ProT slightly improved the survival of mice whereas higher (500 ng and 1000 ng) doses of ProT and a wide range of thymosin 1 doses remained without any effect. PE cells collected from these mice produced extremely low levels of TNF and exhibited negligible tumoricidal activity. Our data demonstrate that ProT has a protective effect in vivo against the growth of adoptively transfered tumour cells and suggest that this effect is, at least in part, mediated by ProT-activated PE cells. These cells were demonstrated to produce high levels of TNF in vitro and to exhibit activity against both TNF-sensitive and TNF-resistant cell lines.Supported by a CEC grant to Dr. M. Papamichail