Enzyme formation and release by isolated barley aleurone layers

Aleurone layers, with testa attached, were prepared from degermed, decorticated barley with the aid of a fungal enzyme preparation. The preparations appeared intact under the scanning electron microscope. By using antibiotics only in an early stage preparations were obtained uncontaminated by micro-organisms and which, when incubated under optimal conditions with gibberellic acid, GA₃, produced near-maximal amounts of α-amylase. The enzyme accumulated in the tissue before it was released into the incubation medium. Daily replacement of the incubation medium, containing GA₃, depressed the quantity of α-amylase produced. α-Amylase was also produced in response to gibberellins GA₁, GA₄ and GA₇ and, to a much lesser extent, helminthosporol and helminthosporic acid. A range of other substances, reported elsewhere to induce α-amylase formation, failed to do so in these trials. At some concentrations, glutamine marginally enhanced the quantity of enzyme formed during prolonged incubations. It is confirmed that α-glucosidase occurs in the aleurone layer and embryo of ungerminated barley, and increases in amount during germination. GA₃ is shown to enhance this increase. When embryos arc burnt, to prevent gibberellin formation, no rise in α-glucosidase levels occurs unless GA₃ is supplied to the grains. As the activity of α-glucosidase and other enzymes have been determined as ‘α-amylase’ by some assay methods, their alterations in activity in response to GA₃ necessitates a re-evaluation of the evidence for de novo) synthesis of α-amylase in aleurone tissue.     

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

[³⁵S]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°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.     

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.     

Differential synthesis in vitro of barley aleurone and starchy endosperm proteins

To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA₃. B and C hordein polypeptides and the salt-soluble proteins β-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the α-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, β-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of α-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for α-amylase, these hormones have the opposite effect on ASI mRNA levels.     

Hormonally regulated programmed cell death in barley aleurone cells

Cell death was studied in barley (cv Himalaya) aleurone cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of alpha-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley aleurone. We conclude that cell death in aleurone cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.     

Synthesis and release of sucrose by the aleurone layer of barley: Regulation by gibberellic acid

Summary Aleurone layers of barley contain large amounts of a soluble oligosaccharide which was identified as sucrose (30–40 g/mg fresh weight). Treatment of the layers with gibberellic acid (GA₃) causes the release of sucrose from the cells. This release requires the participation of metabolic processes, including protein synthesis. When embryoless half-seeds are incubated sucrose accumulates in the aleurone layers, but when seeds are germinated the sucrose content of the aleurone layers declines. Labeling experiments with radioactive glucose and fructose show that aleurone layers continuously synthesize sucrose and that the release, but not the synthesis of sucrose is enhanced by GA₃.     

Identification of thioredoxin target disulfides in proteins released from barley aleurone layers

Thioredoxins are ubiquitous disulfide reductases involved in a wide range of cellular processes including DNA synthesis, oxidative stress response and apoptosis. In cereal seeds thioredoxins are proposed to facilitate the germination process by reducing disulfide bonds in storage proteins and other targets in the starchy endosperm. Here we have applied a thiol-specific labeling approach to identify specific disulfide targets of barley thioredoxin in proteins released from barley aleurone layers incubated in buffer containing gibberellic acid.     

Total protein release from barley aleurone layers as a bioassay for gibberellins

The effect of gibberellic acid on the secretion of proteins from barley (Hordeum vulgare L.) aleurone layers has been investigated for its suitability as a gibberellin bioassay. Concentrations from 10^–4 g/ml to 100 g/ml of GA₃ resulted in the release of proportionally increasing amounts of total protein. The release of proteins is not affected by indoleacetic acid and kinetin. This method has been applied and compared with the -amylase assay for the estimation of gibberellin in extracts of tomato fruits and maize seedlings.Abbreviations GA₃ gibberellic acid - IAA indoleactic acid - K kinetin     

Gibberellic-acid-induced synthesis and release of cell-wall-degrading endoxylanase by isolated aleurone layers of barley

When aleurone layers of barley (Hordeum vulgare L.) are incubated with gibberellic acid (GA₃) xylose and arabinose—both as free sugars and bound to larger molecules—are released into the medium. Release begins 10–12h after the start of incubation and continues for at least 60h. At the same time there is a GA₃-induced breakdown of the cell wall resulting in a loss of 2/3 of the cell-wall pentose during 60h of incubation. GA₃ causes the appearance in the medium of an enzyme (or enzymes) which hydrolyze larchwood xylan and aleurone-layer arabinoxylan. Release of the enzyme(s) into the medium begins 28–32h after the start of incubation. Enzyme activity does not accumulate to any large extent in the tissue prior to release into the medium, and is present in very low levels only in the absence of GA₃. Xylanase activity is associated with a protein (or proteins) with a molecular weight of 29,000. The hydrolysis of the xylans is largely caused by endoxylanase activity, indicating the importance of endoglycosidases in the GA₃-induced breakdown of the aleurone cell wall.     

The structure and composition of aleurone grains in the barley aleurone layer

Summary Cytochemical methods have been used in conjunction with light and electron microscopy to determine the nature of the inclusions in aleurone grains of barley aleurone layers. Two kinds of inclusions were found: (1) Globoids within globoid cavities which were not enclosed by a membrane: the globoids stained red with toluidin blue due to the presence of phytin, and with lipid stains; (2) Protein-carbohydrate bodies which stained green with toluidin blue. The characteristics of globoids and protein-carbohydrate bodies as seen in the electron microscope are described in detail using both glutaraldehyde- and permanganatefixed tissues. The protein-carbohydrate body was identified by silver-hexaminestaining; this was not caused by carbohydrate but by some component which stained green in toluidin blue and which also occurred in cell walls in a thin band adjacent to the cytoplasm. The characteristics of both bodies are discussed in relation to apparent confusion in their identities in previous electron-microscope studies.     

微胚乳玉米籽粒糊粉层细胞显微结构研究

研究了10类含油率不同玉米的糊粉层厚度、结构以及糊粉层厚度与籽粒含油量、去胚籽粒(即籽粒除胚以外的其它部分)含油量、籽粒重、去胚籽粒重、籽粒蛋白质含量、胚重及胚含油量的相关性。结果显示:(1)3类微胚乳玉米糊粉层厚度最大且显著高于第7类普通高油玉米和第10类普通硬粒玉米;(2)糊粉层厚度与籽粒含油率、去胚籽粒含油率均呈极显著正相关,与籽粒重和去胚籽粒重则呈极显著负相关;(3)有些微胚乳玉米的胚乳存在不同程度的解体。因此认为糊粉层厚度和籽粒重可以作为标记性状筛选超高油玉米;在有的微胚乳玉米中,高含油量可能依赖于淀粉降解。     

Fractionation of the enzymes of the barley aleurone layer: Evidence for a soluble mode of enzyme release

Summary Aleurone cells of barley (Hordeum vulgare L.) contain microbodies as determined by histochemical localization with diaminobenzidine. These microbodies can be isolated from both water and gibberellic acid (GA₃) treated cells and identified on sucrose density gradients as glyoxysomes on the basis of their buoyant densities (1.25 g cm^-3) and their enzyme complement. Fractionation of aleurone layer homogenates by differential centrifugation after varying times of exposure to GA₃, however, does not indicate the presence of a discrete secretory vesicle containing either -amylase or -1,3-glucanase. Cytological evidence also suggests that at least -1,3-glucanase is not released from these cells by means of a discrete secretory vesicle.Supported by National Science Foundation Grant GB-27468. The assistance of Dr. William Breidenbach in the assay of glyoxysomal enzymes is gratefully acknowledged.     

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.     

The fine structure of barley aleurone cells

Summary The ultrastructural morphology of both dry and water-imbibed barley aleurone cells is described. The aleurone cell is characterized by the presence of numerous aleurone grains and spherosomes. In addition, it contains organelles typical of other plant cells including structures similar to microbodies, and rough endoplasmic reticulum characterized by the presence of numerous polyribosomes. It is inferred that the morphological specialization of aleurone cells is related to their biochemical specialization.Work supported by National Science Foundation grant GB5863. The skillful technical assistance of Mrs. Janet Price is gratefully acknowledged.     

Subcellular localization of nuclease in barley aleurone

The intracellular localization of an endonuclease (nuclease I) in barley aleurone responding to gibberellic acid was investigated by subcellular fractionation and immunocytochemistry with monoclonal and polyclonal antibodies. Organelle separations were performed with aleurone layers and protoplasts; immunefixations were carried out on protoplasts only. Nuclease was detected in fractions from isopycnic sucrose density gradients which were enriched in either endoplasmic reticulum or Golgi apparatus membranes. These two organelles were also labelled by the indirect immunogold method on thin sections. Intensive labelling of protein and developing vacuoles was observed. Therefore, as noted in other plants nuclease in barley is essentially a vacuolar enzyme.