cGMP Is Required for Gibberellic Acid-Induced Gene Expression in Barley Aleurone.

The occurrence and roles of cGMP were investigated in aleurone layers and protoplasts isolated from barley (cv Himalaya) grain. Levels of cGMP in freshly isolated barley aleurone layers ranged from 0.065 to 0.08 pmol/g fresh weight of tissue, and cGMP levels increased transiently after incubation in gibberellic acid (GA). Abscisic acid (ABA) did not increase cGMP levels in aleurone layers. LY 83583 (LY), an inhibitor of guanylyl cyclase, prevented the GA-induced increase in cGMP and inhibited GA-induced [alpha]-amylase synthesis and secretion. The inhibitory effects of LY could be overcome by membrane-permeant analogs of cGMP. LY also prevented GA-induced accumulation of [alpha]-amylase and GAMYB mRNAs. cGMP alone was not sufficient to induce the accumulation of [alpha]-amylase or GAMYB mRNA. LY had a less dramatic effect on the accumulation of mRNAs encoding the ABA-responsive gene Rab21. We conclude that cGMP plays an important role in GA, but not ABA, signaling in the barley aleurone cell.

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

Gibberellic acid-induced alpha-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.

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

Relative levels of different alpha-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 alpha-amylase groups allowed the levels of different alpha-amylase mRNAs to be assessed both within and between the two groups. In all aleurone systems the same set of alpha-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 alpha-amylase mRNAs. In particular, the regulation of alpha-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.

Control of transient expression of chimaeric genes by gibberellic acid and abscisic acid in protoplasts prepared from mature barley aleurone layers.

Gibberellic acid (GA3) and abscisic acid (ABA) control the transcription of alpha-amylase genes in barley aleurone cells. This control is likely to be exerted through cis-acting hormone-responsive elements in the promoter region of the gene. In order to further define these elements, we have developed procedures for obtaining transient expression of chimaeric genes in protoplasts prepared from mature barley aleurone layers. Constructs with heterologous constitutive promoters and with heterologous and homologous GA3- and ABA-regulated promoters were expressed specifically by these cells. This system would appear to offer great potential in gene regulation studies especially for hormonally regulated homologous genes. Functional analysis of a barley alpha-amylase gene has been performed using this system. A 2050 bp fragment from a high-pI alpha-amylase gene was fused to a reporter gene (GUS) and control of its expression was examined. Deletion analysis of this promoter fragment showed that major GA- and ABA-responsive elements occurred between 174 and 41 bp upstream from the transcription initiation site.

A structural study of germination in celery (Apium graveolens L.) seed with emphasis on endosperm breakdown.

Germination of celery seed occurred after 6 d of imbibition in light. During this time the embryo enlarged at the expense of the adjacent endosperm cells and at the time of germination was 2-3 times as long as in the dry seed. Breakdown of the endosperm cells near the root cap preceeded radicle emergence. None of these changes occurred in darkness.Endosperm digestion began adjacent to the embryo and spread radially. In degrading cells, the aleurone grains often became larger and fewer in number. The cell walls were modified and appeared to undergo partial degradation. Ultimately the cells seemed to lose their contents. In cells adjacent to the root cap, similar changes occurred except there was a transient appearance of starch grains. Radial progression of endosperm breakdown also occurred in isolated endosperm treated with gibberellin A4+7.The results indicate that (1) the stimulus for breakdown of celery endosperm emanates from the embryo in response to light; (2) the stimulus may be a gibberellin because changes in endosperm cells and the sequence of endosperm digestion during germination resemble the responses of isolated endosperm to gibberellin; and (3) the radial progression of endosperm breakdown during germination may be the result of a sequential response of cells to a uniformly applied stimulus rather than the result of gradual embryo expansion.

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.

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 alpha-amylase synthesis: (a) synthesis of alpha-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 alpha-amylase synthesis to occur but that alpha-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 alpha-amylase and other hydrolytic enzymes whose synthesis is under gibberellic acid control.

Cytochemical localization of phosphatase in barley aleurone cells: The pathway of gibberellic-acid-induced enzyme release.

Acid phosphatase has been localized by cytochemical techniques in aleurone layers of dry barley (Hordeum vulgare L.) grains, in imbibed half-grains and in isolated layers treated with and without gibberellic acid (GA3). A major fraction of the enzyme activity is located in the cell walls. During imbibition and incubation of layers without GA3 a steady increase of enzyme activity in the inner wall region indicates a continued release of enzyme into the walls, but there is no essential change in the distribution of wall-enzyme sites. On the other hand, when GA3 is present enzyme activity is found for the first time in regions of the wall that become digested during GA3 treatment. These results indicate that the digested wall channels act as preferential routes through which acid phosphatase is released from the aleurone layer. No digested wall channels are formed in the absence of GA3 and, there being no route for release of the enzyme, it accumulates in the inner regions of the wall around aleurone cells. Assays of enzyme activity in vitro support the conclusions based on the histochemical data. They indicate that release of acid phosphatase from aleurone layers is under strict GA3 control, but that some of the increase in acid phosphatase activity in the isolated during incubation is not GA3 dependent.Acid phosphatase is present in the protein matrix of aleurone grains in all stages except the dry grain. Enzyme activity persists in aleurone grains throughout GA3 treatment when enlargement of the grains and mobilization of reserves takes place. It is suggested that this phosphatase hydrolyses phosphate reserves within the aleurone grains.

The proteins released by isolated barley aleurone layers before and after gibberellic-acid treatment.

Isolated barley (Hordeum vulgare L.) aleurone layers released a number of proteins into an aqueous medium in the absence of gibberellic acid (GA3). Evidence from molecular weight determinations and a number of immunological tests indicated that these proteins were water-soluble endosperm proteins which apparently arose from endosperm cells which adhered to the layers during isolation. They were not aleurone-cell proteins. By means of immunofluorescence, the water-soluble endosperm proteins were found to be concentrated around starch grains in the starchy endosperm. These proteins were resistant to hydrolysis by GA3-induced hydrolases released from aleurone tissue.Isolated aleurone layers could be washed free of soluble endosperm proteins. After treatment with GA3, such layers released another group of proteins which were shown by immunological and electrophoretic methods to be uncontaminated by soluble endosperm proteins. The pure GA3-induced proteins were separated, using SDS-acrylamide gel disc electrophoresis, into 12 components which had molecular weights (monomer) from 15500 to 81000. Ten of these protein bands became radioactive if GA3-treatment of layers was carried out in the presence of radioactive amino acids, and therefore probably contained de novo synthesized proteins. The two protein bands which were not labelled contributed about 40% of the protein released by washed aleurone layers after GA3 treatment, and their production appeared to be dependent on proteolysis.

Cytochemical localization and antigenicity of α-amylase in barley aleurone tissue.

Gibberellic-acid(GA3)-induced α-amylase has been localised in barley aleurone layers using cytochemical methods and light microscopy. Evidence obtained from the use of a starch substrate film method as well as immunofluorescence indicated that the first amylase to appear in the cell was associated with aleurone grains, apparently with the outer membrane, and also with the peripheral cytoplasm. In GA3-treated tissue, the amylase distribution was much more diffuse, although patchy, throughout the cytoplasm and it tended to accumulate in the endosperm side of the cell. The possibility that the aleurone grain membrane is the site of gibberellin-induced enzyme synthesis and that it proliferates to become rough endoplasmic reticulum is considered. Immunological information was obtained which supports earlier indications that induced α-amylase consists of two different proteins, each with molecular heterogeneity.

Characterization of the alpha-Amylases Synthesized by Aleurone Layers of Himalaya Barley in Response to Gibberellic Acid.

The gibberellic acid (GA(3))-induced alpha-amylases from the aleurone layers of Himalaya barley (Hordeum vulgare L. cv Himalaya) have been purified by cycloheptaamylose-Sepharose affinity chromatography and fractionated by DEAE-cellulose chromatography. Four fractions (alpha-amylases 1-4) were obtained which fell into two groups (A and B) on the basis of a number of characteristics. Major differences in serological characteristics and in proteolytic fingerprints were found between group A (alpha-amylases 1 and 2) and group B (alpha-amylases 3 and 4). Also, the lag time for appearance of group B enzyme activity was longer than for group A, and the appearance of group B required higher GA(3) levels than group A. The components of each group behaved similarly, although differences in proteolytic fingerprints were detected.These results together with those from other studies indicate that GA(3) differentially controls the expression of two alpha-amylase genes or groups of genes giving rise to two groups of alpha-amylases with many different properties.

The role of the endoplasmic reticulum in the synthesis and transport of α-amylase in barley aleurone layers.

The subcellular site of α-amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA3). Using [(35)S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA3 was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker α-amylase. This protein was identified as authentic α-amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized α-amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled α-amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of α-amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA3 were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA3-treated aleurone layers, five peaks of α-amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA3 only three peaks of activity were found. In incubation media, four isoenzymes were found after GA3 treatment and two were found after incubation without GA3. We conclude that at least five α-amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.

Gibberellic acid causes increased synthesis of RNA which contains poly(A) in barley aleurone tissue.

Incubation of isolated barley aleurone layers with gibberellic acid for 16 hr caused a 50% increase in the synthesis of RNA that contains poly(A) sequences [poly(A)-RNA], but had no measurable effect on the syntheses of the major RNA species. The syntheses of both the poly(A) and the heteropolymeric fractions of the poly(A)-RNA were increased. The poly(A) sequences were separated into two classes by size, one containing an average of 250 nucleotides and the other about 70 nucleotides. The two classes occurred in a molar ratio of about 1:1. Gibberellic acid increased the syntheses of both sequences to the same extent. We interpret these results to mean that gibberellic acid increases specifically the synthesis of mRNA in this tissue.

Heat-stable proteins and abscisic Acid action in barley aleurone cells.

[(35)S]Methionine labeling experiments showed that abscisic acid (ABA) induced the synthesis of at least 25 polypeptides in mature barley (Hordeum vulgare) aleurone cells. The polypeptides were not secreted. Whereas most of the proteins extracted from aleurone cells were coagulated by heating to 100 degrees C for 10 minutes, most of the ABA-induced polypeptides remained in solution (heat-stable). ABA had little effect on the spectrum of polypeptides that were synthesized and secreted by aleurone cells, and most of these secreted polypeptides were also heatstable. Coomassie blue staining of sodium dodecyl sulfate polyacrylamide gels indicated that ABA-induced polypeptides already occurred in high amounts in mature aleurone layers having accumulated during grain development. About 60% of the total protein extracted from mature aleurone was heat stable. Amino acid analyses of total preparations of heat-stable and heat-labile proteins showed that, compared to heat-labile proteins, heat-stable intracellular proteins were characterized by higher glutamic acid/glutamine (Glx) and glycine levels and lower levels of neutral amino acids. Secreted heat-stable proteins were rich in Glx and proline. The possibilities that the accumulation of the heat-stable polypeptides during grain development is controlled by ABA and that the function of these polypeptides is related to their abundance and extraordinary heat stability are considered.

Gibberellin-responsive elements in the promoter of a barley high-pI alpha-amylase gene.

Deletion analysis has previously shown that the major gibberellic acid (GA)- and abscisic acid (ABA)-responsive elements in the promoter of a high-pI alpha-amylase gene of barley are located downstream of -174 (Jacobsen and Close, 1991). We have used transient expression assays in barley aleurone protoplasts to identify sequences between -174 and +53 that confer GA and ABA responsiveness on expression of a beta-glucuronidase reporter gene. Using alpha-amylase promoter fragments and synthetic oligonucleotides fused to minimal promoters, we have shown that the hormone-responsive region is located between -174 and -108. A single copy of this region fused to a minimal alpha-amylase promoter (-41) conferred both GA- and ABA-responsive expression on the reporter gene comparable to the positive control, Am(-174)IGN. Multiple copies of this region were able to activate even greater levels of expression. Site-directed mutagenesis was used to determine the functional importance of the conserved motifs (-169pyrimidine box, -143TAACAAA box, and -124TATCCAC box) and nonconserved intervening sequences within the region between -174 and -108. Our results showed that both the TAACAAA and TATCCAC boxes play an important role in GA-regulated expression. We propose that the TAACAAA box is a gibberellin response element, that the TATCCAC box acts cooperatively with the TAACAAA box to give a high level of GA-regulated expression, and that together these motifs form important components of a gibberellin response complex in high-pI alpha-amylase genes. The TAACAAA box also appears to be the site of action of ABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Endosperm acidification and related metabolic changes in the developing barley grain.

The starchy endosperm (SE) of the developing grain (caryopsis) of barley (Hordeum vulgare L.) cv Himalaya, as well as that of other barley cultivars examined, acidifies during maturation. The major decrease in pH begins with the attainment of maximum grain dry weight, onset of dehydration, and completion of chlorophyll loss. Acidification is correlated with the accumulation of malate and lesser amounts of citrate and lactate, produced and probably secreted by the pericarp/testa/aleurone (PTA). It is accompanied by large concurrent rises in phosphoeno/pyruvate carboxylase and alcohol dehydrogenase (ADH) activity in the PTA. The activity of seven other enzymes of oxaloacetate and pyruvate metabolism was found to fall or rise only slightly during acidification. Sequential changes in relative amount of ADH isozymes were found in both PTA and SE. The PTA maintained a high respiration rate and adenylate energy charge (AEC) throughout acidification, whereas the SE showed a low respiration rate and rising AEC. The data are consistent with the occurrence of hypoxia in the SE. It is suggested that the above enzyme changes are required for the development of a malate/ethanol fermentation (i.e. a mixed metabolism) in the aleurone layer during maturation.

Calcium regulation of the secretion of α-amylase isoenzymes and other proteins from barley aleurone layers.

The effect of calcium on the secretion of α-amylase (EC 3.2.1.1) and other hydrolases from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was studied. Withdrawal of Ca(2+) from the incubation medium of aleurone layers preincubated in 5 µM gibberellic acid (GA3) and 5 mM CaCl2 results in a 70-80% reduction in the secretion of α-amylase activity to the incubation medium. Agar-gel electrophoresis shows that the reduction in α-amylase activity following Ca(2+) withdrawal is correlated with the disappearance of group B isoenzymes from the incubation medium. The secretion of isoenzymes of group A is unaffected by Ca(2+). The addition of Ca(2+) stimulates the secretion of group-B isoenzymes but has no measurable effect on either the α-amylase activity or the isoenzyme pattern of aleurone-layer extracts. Pulse-labelling experiments with [(35)S]methionine show that Ca(2+) withdrawal results in a reduction in the secretion of labelled polypeptides into the incubation medium. Immunochemical studies also show that, in the absence of Ca(2+), α-amylase isoenzymes of group B are not secreted into the incubation medium. In addition to its effect on α-amylase, Ca(2+) influences the secretion of other proteins including several acid hydrolases. The secretion of these other proteins shows the same dependence on Ca(2+) concentration as does that of α-amylase. Other cations can promote the secretion of α-amylase to less and varying extents. Strontium is 85% as effective as Ca(2+) while Ba(2+) is only 10% as effective. We conclude that Ca(2+) regulates the secretion of enzymes and other proteins from the aleurone layer of barley.

Control of lactate dehydrogenase, lactate glycolysis, and alpha-amylase by o(2) deficit in barley aleurone layers.

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 alpha-amylases when incubated with gibberellic acid (GA(3)) in air. However, layers did not produce alpha-amylase when GA(3) was supplied under N(2), apparently because alpha-amylase mRNA failed to accumulate.When an 8-hour pulse of [U-(14)C]glucose was supplied under N(2) to freshly prepared aleurone layers, both [(14)C]lactate and [(14)C]ethanol accumulated; the [(14)C]lactate/[(14)C]ethanol ratio was about 0.3. Prior incubation of layers for 1 day under N(2) changed this ratio to about 0.8, indicating an increase in the relative importance of the lactate branch of glycolysis.l(+)Lactate dehydrogenase (LDH) activity was low in freshly prepared aleurone layers and increased 10-fold during 2 days under N(2), whereas alcohol dehydrogenase activity (ADH) was high initially and rose by 60%. The responses of LDH and ADH activities to O(2) tension were dissimilar; when layers were incubated in various O(2)/N(2) mixtures, LDH activity peaked at 2 to 5% O(2) whereas ADH activity was highest at 0% O(2). The LDH activity was resolved into several enzymically active bands by native polyacrylamide gel electrophoresis.We conclude that barley aleurone layers are highly adapted to O(2) deficiency, that they possess an inducible LDH system as well as an ADH system, and we infer that the LDH and ADH systems are independently regulated.

Gibberellic-acid-responsive protoplasts from mature aleurone of Himalaya barley.

Gibberellic acid (GA3)-responsive protoplasts were prepared from mature aleurone layers of Himalaya barley. Protoplasts prepared in air (air-protoplasts) synthesized α-amylase (EC 3.2.1.1) in the presence of GA3 at a rate which was 4-5 times greater that in its absence. Protoplasts prepared in nitrogen (N2-protoplasts) took longer than air-protoplasts to respond to GA3 but α-amylase synthesis ultimately attained a rate which was similar to that for air-protoplasts and which was many times that occurring in the absence of the hormone. Many characteristics of the protoplast response were similar to those of intact aleurone layers. α-Amylase arose by new synthesis, its synthesis was inhibited by abscisic acid, it was isozymically similar to aleurone layer enzyme, most of it was secreted into the incubation medium and its synthesis was accompanied by accumulation of α-amylase mRNA. GA3-induced changes in protein synthesis and cell structure also resembled those of intact aleurone cells. We conclude that the response of the protoplasts to GA3 is normal and that they present a useful system for the study of GA3 action in barley aleurone.