Gibberellic Acid-induced synthesis of protease by isolated aleurone layers of barley

The production of protease by isolated aleurone layers of barley in response to gibberellic acid has been examined. The protease arises in the aleurone layer and is mostly released from the aleurone cells. The courses of release of amylase and protease from aleurone layers, the dose responses to gibberellic acid and the effects of inhibitors on the production of both enzymes are parallel. As is the case for amylase, protease is made de novo in response to the hormone. These data give some credence to the hypothesis that the effect of gibberellic acid is to promote the simultaneous synthesis and secretion of a group of hydrolases.     

Effect of ethylene on the gibberellic Acid-enhanced synthesis and release of amylase by isolated barley aleurone layers

Methods were developed and extended to enable the incubation of isolated barley (Hordeum vulgare cv. Himalaya) aleurone layers under carefully controlled conditions for studies on effects of ethylene on amylase synthesis and release. When layers in medium containing gibberellic acid were exposed to ethylene, the synthesis and release of amylase were altered relative to layers maintained in an ethylene-free environment. These ethylene effects were detected at the smallest concentration used, 0.041 nl/ml, indicating a very low threshold value. During the initial 24 h, ethylene accelerated both the appearance of total amylase activity, and the release of this activity from the aleurone layers. On the other hand, ethylene reduced the total amount of amylase activity that was recovered from samples after 48 and 72 h.     

Modes of ethylene action in the release of amylase from barley aleurone layers

The development of xylanase activity by isolated barley (Hordeum vulgare cv. Himalaya) aleurone layers exposed to gibberellic acid was enhanced by ethylene, whereas the rate of glucanase synthesis was unaffected by ethylene. The elevated xylanase activity expressed in ethylene-treated tissue may be responsible for enhanced release of amylase in response to ethylene.     

Selective expression of a probable amylase/protease inhibitor in barley aleurone cells: Comparison to the barley amylase/subtilisin inhibitor

We have cloned and sequenced a 650-nucleotide cDNA from barley (Hordeum vulgare L.) aleurone layers encoding a protein that is closely related to a known -amylase inhibitor from Indian finger millet (Eleusine coracana Gaertn.), and that has homologies to certain plant trypsin inhibitors. mRNA for this probable amylase/protease inhibitor (PAPI) is expressed primarily in aleurone tissue during late development of the grain, as compared to that for the amylase/subtilisin inhibitor, which is expressed in endosperm during the peak of storage-protein synthesis. PAPI mRNA is present at high levels in aleurone tissue of desiccated, mature grain, and in incubated aleurone layers prepared from rehydrated mature seeds. Its expression in those layers is not affected by either abscisic acid or gibberellic acid, hormones that, respectively, increase and decrease the abundance of mRNA for the amylase/subtilisin inhibitor. PAPI mRNA is almost as abundant in gibberellic acid-treated aleurone layers as that for -amylase, and PAPI protein is synthesized in that tissue at levels that are comparable to -amylase. PAPI protein is secreted from aleurone layers into the incubation medium.Abbreviations ABA abscisic acid - ASI barley amylase/subtilisin inhibitor - bp nucleotide base pairs - Da dalton - dpa days post anthesis - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor - poly(A)RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Hormonal regulation of the development of protease and carboxypeptidase activities in barley aleurone layers

Carboxypeptidase and protease activities of hormone-treated barley (Hordeum vulgare cv Himalaya) aleurone layers were investigated using the substrates N-carbobenzoxy-Ala-Phe and hemoglobin. A differential effect of gibberellic acid (GA₃) on these activities was observed. The carboxypeptidase activity develops in the aleurone layers during imbibition without the addition of hormone, while the release of this enzyme to the incubation medium is enhanced by GA₃. In contrast, GA₃ is required for both the production of protease activity in the aleurone layer and its secretion. The time course for development of protease activity in response to GA₃ is similar to that observed for α-amylase. Treating aleurone layers with both GA₃ and abscisic acid prevents all the GA₃ effects described above. Carboxypeptidase activity is maximal between pH 5 and 6, and is inhibited by diisopropylfluorophosphate and p-hydroxymercuribenzoate. We have observed three protease activities against hemoglobin which differ in charge but are all 37 kilodaltons in size on sodium dodecyl sulfate polyacrylamide gels. The activity of the proteases can be inhibited by sulfhydryl protease inhibitors, such as bromate and leupeptin, yet is enhanced by 2-fold with 2-mercaptoethanol. In addition, these enzymes appear to be active against the wheat and barley storage proteins, gliadin and hordein, respectively. On the basis of these characteristics and the time course of GA₃ response, it is concluded that the proteases represent the GA₃-induced, de novo synthesized proteases that are mainly responsible for the degradation of endosperm storage proteins.     

Interactions between Gibberellic Acid, Ethylene, and Abscisic Acid in Control of Amylase Synthesis in Barley Aleurone Layers

Gibberellic acid-induced α-amylase synthesis in barley (Hordeum vulgare L.) aleurone layers was inhibited by abscisic acid, and the inhibition was partly removed by additional gibberellic acid alone and by ethylene alone. Together additional gibberellic acid and ethylene almost eliminated abscisic acid inhibition of amylase synthesis. Time course studies of these phenomena showed that the effect of abscisic acid, ethylene, and varying concentrations of gibberellic acid on the course of amylase synthesis were either to speed up or slow down the whole process and not to affect the lag phase or the linear phase separately. The data are discussed in relation to previous studies of abscisic acid-gibberellic acid interaction.     

Hydrolysis of storage proteins in barley endosperms : analysis of soluble products

Soluble products, released by the hydrolysis of hordeins into the media of barley (Hordeum vulgare cv. Perth) half-seeds were analyzed. Large polypeptide fragments (methanol-insoluble) were identified using the Western immunoblot technique with the antibodies prepared against B and C polypeptides of hordein. A number of hordein IgG-reacting bands were noted in the samples from dry kernels. In samples incubated in the absence of gibberellic acid, polypeptide fragments in the size range of 25 to 30 kilodaltons appeared within 24 hours, and those in the size range of 40 kilodaltons became more prominent. In samples incubated in the presence of gibberellic acid, polypeptide fragments in the size range of 45 to 67 kilodaltons were less apparent and those in the size range of less than 15 kilodaltons were more pronounced. The hordein-related polypeptide fragments were present in low amounts after 72 hours in the presence of gibberellic acid. Methanol-soluble peptides were fractionated, on the basis of size, into two broad peaks. In the absence of gibberellic acid, there was no significant change in their profile over a 72 hour incubation period. In the presence of this growth substance, however, there was a decrease in the proportion of large size peptides (50-70 amino acid residues in length), and an increase in the levels of small peptides (15-35 amino acid residues in length) and amino acids. Our interpretation of the results is that the release of the initial large polypeptide fragments from hordein proteins is mediated by a protease(s) whose appearance is not dependent on the exogenously added gibberellic acid. Further hydrolysis is, however, mediated by proteases induced in the presence of this growth substance.     

The role of ethylene in the induction of amylase activity in wheat aleurone tissue

When wheat aleurone layers ( Triticum aestivum L. var. Potam S-70) incubated in medium containing gibberellic acid were exposed to ethylene, the synthesis and release of amylase were enhanced relative to layers incubated in the presence of mercuric perchlorate. Exogenous ethylene stimulated gibberellic acid-induced amylase synthesis by approximately 2.2-fold. The ethylene-mediated stimulation of amylase formation was dependent upon the tissue being exposed to the gas during the lag phase of gibberellic acid action. Ethylene appeared to promote only quantitative changes in amylase synthesis and release, since the isoelectric focusing patterns of amylase is enzymes were not significantly altered by ethylene. Ethylene had no effect on the incorporation of [methyl-¹⁴C]choline into aleurone phospholipids, but stimulated the accumulation of [U-¹⁴C]adenine into poly(A) RNA of gibberellic acid-treated tissue by about 80%.     

Density labeling of proteins with 13C-labeled amino acids: no accumulation of an inactive alpha-amylase precursor in barley aleurone cells.

Gibberellic acid enhances α-amylase (EC 3.2.1.1) production in isolated barley aleurone layers after a lag period of 4 to 8 h, and most of the enzyme is produced after 12 h of hormone treatment. Amino acids necessary for protein synthesis in barley aleurone layers are derived from the degradation of storage proteins in this tissue. Since bromate is an inhibitor of barley protease, in the presence of bromate the production of α-amylase in aleurone layers becomes dependent on exogenous amino acids. We have incubated aleurone layers with bromate plus ¹³C-labeled amino acids and [³H]leucine from 0 to 24, 0 to 12, and 12 to 24 h after the application of gibberellic acid. The chemical quantity of [³H]leucine was negligible in comparison to that of ¹³C-labeled amino acids. Therefore, any density shift of proteins observed must be due to the incorporation of ¹³C-labeled amino acids. The density shift of α-amylase and that of newly synthesized proteins (radioactivity profile) were determined by isopycnic centrifugation in CsCl density gradients. The density shift of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 12 to 24 h after the addition of hormone was much larger than that of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 0 to 12 h of hormone treatment. By comparing the density shift of α-amylase with that of newly synthesized proteins, it is apparent that essentially all the amylase molecules are de novo synthesized. We can conclude that there is little or no accumulation of an inactive α-amylase precursor in barley aleurone cells between the time of the application of gibberellic acid and the time of the rapid increase in α-amylase activity.     

Inhibition of Early Steps in the Gibberellin-Activated Synthesis of α-Amylase

Subsequent production of amylase is severely inhibited if barban [4-chloro-2-butynyl N-(3-chlorophenyl) carbamate] is added to embryo-free half seeds of barley within 4 to 5 hours after gibberellic acid treatment of these seeds. Thirty to 50 mg/L concentrations of barban are effective. Barban inhibition is non-competitive with respect to gibberellic acid. Addition of barban 7 hours or more after gibberellic acid treatment is almost without effect.The delay between gibberellic acid treatment and amylase formation tends to become shorter with more prolonged imbibition periods. Regardless of imbibition period, susceptibility to barban is lost within 7 hours after gibberellic acid treatment.Other herbicidally active phenylurethanes are also inhibitors, but none are as effective as barban. Phenethyl alcohol and 2 arylcarbamates can act as inhibitors.     

A Correlation between a Ribonucleic Acid Fraction Selectively Labeled in the Presence of Gibberellic Acid and Amylase Synthesis in Barley Aleurone Layers

The effects of gibberellic acid on the incorporation of radio-active uridine and adenosine into RNA of barley aleurone layers were investigated using a double labeling method combined with acrylamide gel electrophoresis. After 16 hours of incubation, gibberellic acid stimulated the incorporation of label into all species of RNA, but the effects were very small (0-10%) for ribosomal and transfer RNA and comparatively large (up to 300%) for RNA sedimenting between 5S and 14S. This result was obtained for both isolated aleurone layers and for layers still attached to the endosperm. A similar but less marked pattern occurred in layers incubated for 8 hours, but the effect was not observed after 4 hours. The gibberellic acid-enhanced RNA labeling was not due to micro-organisms. The following evidence was obtained for an association between the gibberellic acid-enhanced RNA synthesis and α-amylase synthesis: (a) synthesis of α-amylase took place in parallel with incorporation of label into gibberellic acid-RNA; (b) actinomycin D inhibited amylase synthesis and gibberellic acid-RNA by similar percentages; (c) 5-fluorouracil halved incorporation of label into ribosomal RNA but had no effect on amylase synthesis and gibberellic acid-RNA; and (d) abscisic acid had little effect on synthesis of RNA in the absence of gibberellic acid, but when it was included with gibberellic acid the synthesis of both enzyme and gibberellic acid-RNA was eliminated. We conclude that large changes in the synthesis of the major RNA species are not necessary for α-amylase synthesis to occur but that α-amylase synthesis does not occur without the production of gibberrellic acid-RNA. Gibberellic acid-RNA is probably less than 1% of the total tissue RNA, is polydisperse on acrylamide gels, and could be messenger species for α-amylase and other hydrolytic enzymes whose synthesis is under gibberellic acid control.     

Physiological Effects of Gibberellic Acid. IX. Recovery of Gibberellic Acid Following Incubation with Endosperm

To determine the fate of gibberellic acid (GA₃), solutions were incubated for 24 hours with or without barley endosperm and were subsequently applied to dwarf maize seedlings. Hormone activity appeared to increase as a result of incubation with endosperm. This apparent increase in GA₃ concentration was probably due to a synergistic interaction between GA₃ and endosperm constituents, particularly carbohydrate, released during the incubation period. It is concluded that relatively little hormone is inactivated during the initiation of endosperm mobilization.     

Tannins as gibberellin antagonists in the synthesis of alpha-amylase and Acid phosphatase by barley seeds

The tannins chebulinic acid or tara tannin were added to an incubation system in which GA₃ induces enzyme synthesis in endosperm half seeds of barley (Hordeum vulgare L.). The activity of amylase and acid phosphatase in the incubation medium was reduced compared to the activity in the medium after incubation with GA₃ alone. When embryo half seeds of barley were incubated with chebulinic acid or tara tannin in the absence of added GA₃, the enzyme activity of the incubation medium was also reduced. The activity of preformed enzymes obtained from endosperm half seeds previously induced with GA₃ was not reduced by the addition of tannin. Comparisons were made of the amount of enzyme activity from breis of aleurone layers incubated with GA₃ in the presence and absence of tannins. The amounts of activity were relatively small and approximately equal in both cases, indicating that secretion from the aleurone was not blocked by the tannins. The reduction of enzyme activity caused by tannins in both endosperm and embryo half seeds could be completely reversed by the addition of GA₃.     

Detection and partial characterization of lymphoid cell surface proteases

Cultures of viable thymocytes and lymph node cells (LNC) were found to exhibit neutral protease activity toward radiolabeled protein substrates. Proteases were not actively secreted in serum-free culture. Thymocyte surface proteases were not affected by incubation of the cells in 1 mM ethylenediaminetetraacetic acid (EDTA) or 1 mM ethylene glycol bis(aminoethyl ether) N, N'-tetraacetic acid (EGTA); however, approximately 25% of lymph node cell surface protease activity was released from the cells by EDTA. It was concluded that the majority of protease activity displayed by both cell types was tightly associated with the cell surface. The inhibitor sensitivity of the cell surface proteases detected on hamster thymocytes and LNC and rat thymocytes was very similar. Cell surface protease activity was inhibited (85%) by the serine protease inhibitors diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF) and was partially inhibited by l-1-tosylamide-2-phenylethylchloromethyl ketone(TPCK) and soybean trypsin inhibitor (SBTI), but not by N-α-p-tosyl-l-lysine-chloromethyl ketone (TLCK) or ?-aminocaproic acid (EACA). The bacterial protease inhibitor antipain was strongly inhibitory whereas leupeptin was less effective and elastinal did not inhibit cell surface protease activity. Thymocyte surface proteases were also inhibited (65%) by ZnCl₂, but not be several other divalent cations. In LNC, both ZnCl₂ and NiCl₂ were inhibitory to a lesser extent (32% inhibition). At least one surface protease in both thymocytes and LNC could function as a plasminogen activator.     

Fluorescent diphenylphosphonate-based probes for detection of serine protease activity during inflammation

Activity-based probes are small molecules that covalently bind to the active site of a protease in an activity-dependent manner. We synthesized and characterized two fluorescent activity-based probes that target serine proteases with trypsin-like or elastase-like activity. We assessed the selectivity and potency of these probes against recombinant enzymes and demonstrated that while they are efficacious at labeling active proteases in complex protein mixtures in vitro, they are less valuable for in vivo studies. We used these probes to evaluate serine protease activity in two mouse models of acute inflammation, including pancreatitis and colitis. As anticipated, the activity of trypsin-like proteases was increased during pancreatitis. Levels of elastase-like proteases were low in pancreatic lysates and colonic luminal fluids, whether healthy or inflamed. Exogenously added recombinant neutrophil elastase was inhibited upon incubation with these samples, an effect that was augmented in inflamed samples compared to controls. These data suggest that endogenous inhibitors and elastase-degrading proteases are upregulated during inflammation.     

Effects of Ga3 and Ca2+ on barley aleurone protoplasts: a freeze-fracture study

Summary Freeze-fracture electron microscopy was used to study changes in the endomembrane system of barley (Hordeum vulgare L. cv. Himalaya) aleurone protoplasts. Protoplasts were used for this study because their response to calcium and the plant hormone gibberellic acid (Ga₃) can be monitored prior to rapid freezing of cells for electron microscopy. Protoplasts incubated in Ga₃ plus Ca²⁺ secrete elevated levels of a-amylase relative to cells incubated in Ga₃ or Ca²⁺ alone. The endoplasmic reticulum (ER) and Golgi apparatus of protoplasts incubated in Ga₃ plus Ca²⁺ undergo changes that are well correlated with the synthesis and secretion of a-amylase. The ER, which appears as short, single sheets of membrane in Ca²⁺-and Ga₃-treated protoplasts, exists as a series of long fenestrated stacks of membranes following incubation in Ga₃ plus Ca²⁺. The Golgi apparatus is also more highly developed in protoplasts treated with Ga₃ plus Ca²⁺. This organelle is larger and has more vesicles associated with its periphery in protoplasts that actively secrete a-amylase. Evidence that the Golgi apparatus participates in a-amylase secretion is also provided by experiments with the ionophore monensin, which causes pronounced swelling of Golgi cisternae and inhibits the secretion of a-amylase. We interpret these observations as showing that the ER and Golgi apparatus of barley aleurone participate in the intracellular transport and secretion of a-amylase. The plasmalemma (PF face) of barley aleurone protoplasts shows a high density of intramembranous particles (IMPs) which, in general, are evenly distributed. Occasionally, ordered arrays of IMPs are observed, possibly resulting fro m osmotic stress. after 48 hours the plasmalemma of some Ga₃-treated protoplasts show particle-free areas considered to be indications of senescence.abbreviations ER endoplasmic reticulum - Ga₃ gibberellic acid - IEF isoelectric focusing - IMP intramembranous particle - PF protoplasmic fracture - PL plasmalemma     

Extracts of the Brown Seaweed <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> Induce Gibberellic Acid (GA<Subscript>3</Subscript>)-independent Amylase Activity in Barley

Extracts of the brown seaweed Ascophyllum nodosum have been used as a biostimulant to promote growth and productivity in a number of agricultural production systems. Although the extracts have been shown to improve seedling emergence and vigor in a variety of plants, including barley, the mechanism(s) of this growth-promoting effect is(are) largely unknown. In our study, A. nodosum extract induced amylase activity in barley seed-halves; a significant difference in amylase activity was observed in seeds without an embryo. The addition of activated charcoal to the treatment media negated the bioactivity of the extracts suggesting the organic nature of bioactive compounds in A. nodosum extracts. The extracts induced amylase activity in a gibberellic acid (GA)-deficient barley mutant (grd2). LC-MS-MS analysis failed to detect the presence of GA₃ in the extracts. ABA supplementation of the medium caused a significant reduction of amylase activity in GA-treated seeds compared with those treated with the A. nodosum extract. Taken together, our results suggest that the organic components of A. nodosum extract induce amylase activity independent of GA₃ and might act in concert with GA-dependent amylase production leading to enhanced germination and seedling vigor in barley. Being derived from a renewable resource, the bioactive compounds from A. nodosum could be used to improve crop productivity in sustainable agricultural systems.     

Senescence-dependent degradation of Lhcb3 is mediated by a thylakoid membrane-bound protease

A novel proteolytic activity integrally associated with barley thylakoid membranes has been discovered and characterized. This enzymatic activity mediates senescence-dependent degradation of Lhcb3, one of the apoproteins of the major light-harvesting chlorophyll a/b protein complex of photosystem II. Once senescence of barley leaves is initiated by detachment and dark incubation, the degradation of Lhcb3 can proceed and be followed in vitro in an experimental system composed of thylakoids isolated from senescing leaves incubated in darkness in suitable medium at 25 degrees C. The protease involved is present in its active form and Lhcb3 is susceptible for proteolytic attack already in fresh leaves, although Lhcb3 degradation does not take place unless undefined extrinsic membrane proteins protecting Lhcb3 are removed in a senescence-dependent manner. It is thus concluded that senescence-dependent Lhcb3 degradation is regulated at the substrate availability level. The protease involved is ATP stimulated, has an optimum activity at pH 7.8, and requires 3 mM added Mg2+ (replicable by micromolar doses of Zn2+) for its proper activity. Studies using typical inhibitors of various classes of proteases indicate that the enzyme is a metalloprotease with disulfide linkage essential for its activity. Micromolar doses of Zn2+ were demonstrated to restore the activity of Lhcb3-degrading enzymes abolished by an ethylenediaminetetraacetic acid pretreatment of the thylakoids and it is inferred that the protease involved is a zinc-binding metalloprotease. Mg2+ was shown to be able to partially replace zinc as the bound ion.     

A survey of the sequence of some effects of gibberellic Acid in the metabolism of cereal grains

The sequence in which a variety of enzymes and metabolites are affected by gibberellic acid after application of the hormone to aleurone layers of half seeds of barley (Hordeum vulgare var. Betzes) and half seeds of wheat (Triticum aestivum var. Gensee) was investigated. With barley aleurone layers the first hormonal effect observed was the increased secretion of soluble carbohydrate, some of which appears to be a glucan containing some β-1,3 linkages. This was followed by increased oxygen consumption and increased secretion of ATPase, GTPase, phytase, phosphomonoesterase, phosphodiesterase, inorganic phosphate, carbohydrates other than amylase, peroxidase and amylase. Similar sequential effects were seen in wheat half seeds. Increased activity of alcohol dehydrogenase in barley seeds was elicited by the hormone but there was no effect on glucose-6-phosphate isomerase.     

Involvement of ethylene in aphid infestation of barley

The recently arrived Russian wheat aphid (RWA) is a major pest of wheat and barley in the United States. RWA induced ethylene production in Morex, a barley variety susceptible to RWA, but induced little ethylene production in PI 366450, a barley resistant to RWA. Greenbugs, another aphid pest, induced ethylene production in PI 366450 and Morex, both of which are susceptible to greenbugs. RWA infestation results in pronounced symptoms on barley including leaf streaking, stunting of growth, and rolled leaves. Incubation of barley in ethylene (5 and 50 l/l) or other plant hormones (auxin, gibberellic acid, zeatin, kinetin, and abscisic acid at 10^-4 M) failed to produce streaking or rolling in uninfested plants or to alter the production of these symptoms in infested plants. Incubation of Morex and PI 366450 in ethylene caused some stunting of the leaves and internodes that emerged during or soon after ethylene incubation. However, the leaf and internode that emerged 9 days after incubation showed some compensatory, or increased, growth.Mention of specific product name by the United States Department of Agriculture does not constitute an endorsement and does not imply a recommendation over other suitable products.