Effect of Gibberellic Acid on the Ultrastructure and {alpha}-Amylase Activity of Aleurone Cells of Avena sativa L.

The -amylase activity and ultrastructure of aleurone cells inseeds of Avena sativa L. were studied using seed halves withembryo (embryo seeds) which had imbibed water and seed halveswithout embryo (embryo-less seeds) which had imbibed water withor without GA₃. -Amylase activity was detected in the aleurone layers of embryoseeds that had imbibed water and embryo-less seeds that hadimbibed GA₃-water. The ultrastructure of aleurone cells withdetectable -amylase activity showed marked changes in the roughsurfaced endoplasmic reticulum (rER), in the flattened sacculesforming stacks and in the aleurone grains. The progressive changesin the rER were as follows: first, the number of slender rERincreased; then, the inner space became wider and showed roundor oval profile; and finally, the rER became slender again witha reduced number of adhering ribosomes. The flattened sacculesforming stacks were appressed to the surface of aleurone grains.With time, they decreased in number and finally disappeared.In parallel with the decrease of flattened saccules, digestionof proteinaceous material inside the aleurone grains proceeded. (Received February 24, 1987; Accepted September 3, 1987)     

The Effect of Calmodulin Antagonists on Gibberellic Acid-induced Enzyme Secretion in Barley Aleurone Layers

Calmodulin activity was detected and assayed in barley aleuronecells. The effect of calmodulin antagonists on GA₃-induced enzymesynthesis and secretion in barley aleurone layers was also investigated.These calmodulin antagonists (chlorpromazine, haloperidol) inhibitedonly GA₂-induced -amylase secretion. This inhibitory effectwas intensified after 6 h of GA₃-incubation. This leads us tosuggest that some calmodulin-controlled mechanism is involvedin GA₂-induced -amylase secretion. Hordeum vulgare L., barley aleurone cells, gibberellic acid, -amylase secretion, calmodulin, calmodulin antagonist     

Gibberellic acid-induced secretion of hydrolases in barley aleurone layers

Activities of phosphatases in the aleurone layers of a husklessbarley, Ehime-hadaka No. 1, were enhanced in the absence ofgibberellic acid (GA₃), while the enzyme secretion was absolutelydependent upon its presence. GA₃ was required for both inductionand secretion of a-amylase. The longer the preincubation ofthe tissue without GA₃, the longer was the lag period beforesecretion of both a group of phosphatases and a-amylase. Changesin the fine structure of aleurone cells were also investigated.Characteristics of the phase transition from enzyme accumulationto enzyme secretion seemed to be a development of a bundledtype of endoplasmic reticulum. ¹Present address: Institute of Biological Sciences, The Universityof Tsukuba, Ibaraki 300-31, Japan. (Received August 25, 1975; )     

Observations on the Germ Aleurone of Barley. Morphology and Histochemistry

The germ aleurone over the embryonic axis of barley was examinedin strips of tissue peeled off harvest-ripe grains. The germaleurone is only one cell thick but resembles 'normal' aleuronein being composed of living cells with dense, lipid-rich cytoplasmand thick walls containing phenolic material. In contrast tothe cells of the 'normal' aleurone, germ aleurone cells containvery few phytin or protein deposits. When the germ aleuroneis ruptured during germination, the walls at the torn edge becomethickened with shiny golden-brown material, and 'sealed' tothe testa. Two days after germination, lignin can be detectedin the walls of a single row of germ aleurone cells adjoiningthe scutellum. The role of the germ aleurone in defence againstmicroorganisms is discussed. It is suggested that the metabolicactivities in the germ aleurone in imbibed grains compete withthe embryo for oxygen, and thus may be one of the factors whichdetermine whether a grain germinates or remains dormant.Copyright1994, 1999 Academic Press Barley, Hordeum vulgare L., germ aleurone, histochemistry, defence mechanism, lignin, dormancy, microorganisms, pre-mature germination     

Rates of Cell Division in Developing Barley Endosperms

Counts of nuclei in enzyme digested endosperms of barley cultivarBomi show that the final number of cells, 170000, is reachedbetween 18 and 21d after anthesis. Based on the number of cellprofiles in transverse mid-grain sections, starchy endospermcells divide up to day 14. Thereafter, cell proliferation isrestricted to the aleurone layer. Hordeum vulgare, starchy endosperm, aleurone, mitotic activity, light microscopy     

Observations on the Germ Aleurone of Barley. Phenol Oxidase and Peroxidase Activity

Strips of tissue containing the germ aleurone layer were removedfrom dry, harvest-ripe grains of barley (Hordeum vulgare L.)and incubated in buffered solutions of phenolic compounds, withand without the addition of hydrogen peroxide. Peroxidase ando-diphenol oxidase activity were found in the material releasedinto the incubation medium, and in the cytoplasm of the germaleurone cells. Peroxidase activity was located in the cellwalls and appeared to be high in the region where the germ aleuronecovering the embryonic axis merges into that which adheres tothe scutellum i.e. the region in which a row of germ aleuronecells becomes lignified following germination. Monophenol oxidaseactivity was not detected in the released enzymes or in theintact tissue. Although hydroquinone was oxidized in the cytoplasmof the germ aleurone tissue, unequivocal evidence of the presenceof laccase was not obtained. The oxidation of endogenous phenolicsubstances by phenol oxidases and peroxidases is discussed inrelation to anti-microbial defence mechanisms which appear tooperate in the germ aleurone during germination.Copyright 1994,1999 Academic Press Barley, Hordeum vulgare L., germ aleurone, catechol oxidase, laccase, peroxidase, defence mechanisms, germination     

Gibberellic Acid Controls the Secretion of Acid Phosphatase in Aleurone Layers and Isolated Aleurone Protoplasts of Avena fatua

The role of gibberellic acid (GA₃) in controlling the secretion(across the plasma membrane) and release (through the cell wall)of acid phosphatase (E.C. 3.1.3.2 [EC] .) from Avena aleurone layershas been investigated. Evidence from this comparative studywith intact aleurone layers and isolated aleurone protoplastsreveals that the secretion of acid phosphatase is under GA₃control. The mechanism underlying secretion and release of theenzyme from aleurone cells is discussed. Key words: Avena fatua, Acid phosphatase, Aleurone protoplasts, Gibberellic acid, Secretion     

Cell death of barley aleurone protoplasts is mediated by reactive oxygen species

The barley aleurone layer is a terminally differentiated secretory tissue whose activity is hormonally controlled. The plant hormone gibberellic acid (GA) stimulates the secretion of hydrolytic enzymes and triggers the onset of programmed cell death (PCD). Abscisic acid (ABA) antagonizes the effects of GA and inhibits enzyme secretion and PCD. Reactive oxygen species (ROS) are key players in many types of PCD, and data presented here implicate ROS in hormonally regulated death of barley aleurone cells. Incubation of aleurone layers or protoplasts in H(2)O(2)-containing media results in death of GA-treated but not ABA-treated aleurone cells. Cells that are programmed to die are therefore less able to withstand ROS than cells that are programmed to remain alive. Illumination of barley aleurone protoplasts with blue or UV-A light results in a rapid increase in intracellular H(2)O(2) production. GA-treated protoplasts die rapidly in response to this increase in intracellular H(2)O(2) production, but ABA-treated protoplasts do not die. The rate of light-induced death could be slowed by antioxidants, and incubating protoplasts in the dark with the antioxidant butylated hydroxy toluene reduces the rate of hormonally induced death. Taken together, these data demonstrate that GA-treated aleurone protoplasts are less able than ABA-treated protoplasts to tolerate internally generated or exogenously applied H(2)O(2), and strongly suggest that ROS are components of the hormonally regulated cell death pathway in barley aleurone cells.     

Effects of gibberellic acid and abscisic acid on α-amylase activity and ultrastructure of aleurone cells ofAvena sativa L.

The effects of GA₃ and/or ABA on the α-amylase activity and the ultrastructure of aleurone cells in halves of seeds without embryos (embryo-less half seeds) of oats (Avena sativa L.) were studied. α-Amylase activity was detected by the starch-agar gel method in the aleurone layers of embryo-less half seeds soaked in 1 μM GA₃ solution or 100 μM GA₃+10 μM ABA solution but not in those of seeds soaked in distilled water, 10 μM ABA solution, or 1 μM GA₃+10 μM ABA solution. Ultrastructural examinations of aleurone cells with α-amylase activity showed a decrease in the number of sphaerosomes, the appearance of flattened saccules pressed to the surface of aleurone grains, and the development and transformations of the rER from a slender form to the one with wide inner spaces. In the aleurone cells in which the enzyme activity was not detected, components of the rER showed only slender profiles. The number of sphaerosomes did not decrease, and no flattened saccules appeared in the aleurone cells treated with 10 μM ABA or 1 μM GA₃+10 μM ABA.     

Heat shock proteins do not provide thermoprotection to normal cellular protein synthesis, α-amylase mRNA and endoplasmic reticulum lamellae in barley aleurone layers

Heat shock in barley ( Hordeum vulgare L. cv. Himalaya) aleurone layers induces the synthesis of heat shock proteins (hsps) and suppresses the synthesis and secretion of α-amylase, the principal secretory protein. This is accompanied by the destabilization of α-amylase mRNA and a concomitant dissociation of ER lamellae. In the absence of heat shock α-amylase mRNA is extremely stable (Belanger et al. 1986. Proc. Natl. Acad. Sci. USA 83: 1354–1358). In most organisms there is a direct correlation between the synthesis of hsps and thermotolerance. The ability of hsps to provide thermoprotection to secretory protein synthesis, α-amylase mRNA and ER lamellae was analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of pulse-chased, [³⁵S]-methionine-labeled proteins revealed that the half-life of hsps in barley aleurone cells recovering from heat shock was approximately 12 h. Within approximately 6 h, there was a recovery of α-amylase mRNA and a reformation of ER lamellae. Heat shock protein synthesis was induced by either heat shock (40°C) or arsenite, the cells were allowed to recover for 8 h, then were re-exposed to heat shock. Results from SDS-PAGE showed that, despite the presence of hsps, α-amylase synthesis was suppressed. Northern blot hybridizations showed that α-amylase mRNA levels were reduced in heat-shocked tissues. Transmission electron microscopy demonstrated that ER lamellar structures were dissociated. The synthesis of hsps did not enable barley aleurone cells to sustain the synthesis of any proteins at lethal temperature. In contrast, similar conditions established thermotolerance and provided thermoprotection to protein synthesis in germinating barley embryos. Our findings suggest that the aleurone layer does not become thermotolerant following the induction of hsp synthesis.     

Expression of the 30 kD cysteine endoprotease B in germinating barley seeds

The expression of a 30 kD cysteine endoprotease (EP-B) was studied by in situ hybridization and immunomicroscopy to clarify its role in germinating barley grains. At the beginning of germination, EP-B mRNA was expressed in the scutellar epithelium and aleurone cells next to the embryo. Later, mRNA levels were highest in the aleurone layer proceeding to the distal end of the grain. During the first day of germination, EP-B protein was strongly localized to the germ aleurone and scutellar epithelium from where the secretion into the starchy endosperm began. Secretion was also observed to proceed along the aleurone layer to the distal end. These results show that EP-B is differentially localized during germination, and both scutellum and aleurone layer are able to synthesize and secrete EP-B protein.     

High pressure freezing and freeze substitution reveal new aspects of fine structure and maintain protein antigenicity in barley aleurone cells

High pressure freezing and freeze substitution (HPF-FS) were used to prepare barley ( Hordeum vulgare L. cv Himalaya) aleurone protoplasts for transmission electron microscopy (TEM). We show that HPF-FS is superior to conventional chemical fixation and dehydration techniques for the preservation of cellular fine structure and antigenicity of proteins in barley aleurone protoplasts. HPF-FS extracted fewer proteins from the cytosol and organelles of aleurone protoplasts and maintained the details of cellular structure. The cortical cytoskeleton, made up of microtubules, was observed for the first time by TEM in barley aleurone protoplasts prepared by HPF-FS. Organelles such as protein storage vacuoles retained their proteinaceous contents, and other cellular organelles (including the Golgi apparatus, the nucleus and mitochondria) were also well preserved in protoplasts fixed by HPF-FS. Antibodies to the vacuolar enzyme nuclease I, the tonoplast aquaporin α-TIP and the glyoxysomal enzyme malate synthase showed that the antigenicity of organellar enzymes and membrane proteins was preserved in cells prepared by HPF-FS. We conclude that HPF-FS is superior to chemical fixation for the preparation of plant protoplasts for TEM and is the method of choice for the preservation of aleurone protoplasts for structural and immunochemical studies.     

Evidence of two isomers of phosphatidylinositol in plant tissue

The structure of phosphatidylinositol in barley (Hordeum vulgare) aleurone layers was investigated by chemical degradation. In vivo myo-[2-³H]inositol-labeled phosphatidylinositol was first converted to glycerophosphoinositol and, subsequently, after removal of the glycerol moiety, to inositol monophosphate. Here, we present data that show that, in addition to the commonly occurring 1,2-diacylglycero-3-(d-myo-inositol-1-phosphate), barley aleurone cells contain a novel second isomer of phosphatidylinositol that differs in structure of the head group.     

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.     

Effect of gibberellic Acid and actinomycin d on the formation and distribution of rough endoplasmic reticulum in barley aleurone cells

Analysis of structural changes in barley aleurone cells during germination or following incubation of isolated layers in gibberellic acid with or without actinomycin D revealed extensive development of rough endoplasmic reticulum. Following the assembly of stacked rough endoplasmic reticulum, vesiculation occurred mainly in basal regions of the cell, resulting in a polar distribution of rough endoplasmic reticulum vesicles. It is postulated that these vesicles are involved in protein secretion, because smooth vesicles, derived from the rough endoplasmic reticulum, apparently become appressed to the plasma membrane. The increased α-amylase in the ambient medium and in cell homogenates correlated directly with formation and subsequent vesiculation of the rough endoplasmic reticulum. Furthermore, when cells were treated with actinomycin D and gibberellic acid, α-amylase synthesis was inhibited by 45% and secretion by 63%. These cells were characterized cytologically by large areas of disarrayed segments of fragmented rough endoplasmic reticulum, corresponding to a high intracellular level of α-amylase. In addition, small lipid bodies common to the segmented regions of rough endoplasmic reticulum were surrounded by fine fibrous material, short segments of rough endoplasmic reticulum, and free ribosomes, suggesting that actinomycin D had interfered with development and organization of rough endoplasmic reticulum.     

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.     

Heat shock temperature acclimation of normal secretory protein synthesis in barley aleurone cells

Exposure of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers to 40°C for a period of 3 h results in the selective suppression of the synthesis and secretion of hydrolytic enzymes; other normal cellular protein synthesis continues during heat shock. This suppression is correlated with secretory protein mRNA destabilization and the dissociation of stacked ER lamellae during heat shock (Belanger et al. 1986, Proceedings of the National Academy of Sciences USA 83, pp. 1354–1358). In this report we examined the effect of exposure to extended periods of heat shock. If exposure to 40°C was continued for a period of 18 h, the synthesis of α-amylase, the predominant secreted hydrolase, resumed. This was accompanied by increased α-amylase mRNA levels and the reformation of ER lamellae. Though initial exposure (3 h) to 40°C reduced protein secretion to ～10% of that observed in aleurone cells maintained at 25°C, exposure for prolonged periods (16–20 h) permitted the resumption of protein secretion to ～66% of non-heat-shocked control levels. The resumption of normal secretory protein synthesis during prolonged exposure to 40°C was correlated with an increase in the incorporation of [¹⁴C]glycerol into phosphatidylcholine and an increase in the ratio of saturated to unsaturated fatty acids in lipids isolated from ER membrane preparations. Increased fatty acid saturation has been demonstrated to enhance thermostability in biological membranes, and such changes in membrane composition may be important to the recovery of secretory protein synthesis at the ER.     

Premature Drying Increases the GA-Responsiveness of Developing Aleurone Layers of Barley (Hordeum vulgare L.) Grain

The effect of premature drying on the sensitivity of aleuronelayer cells of developing barley (Hordeum vulgare L.) grainto gibberellic acid (GA₃) was investigated. The capacity ofbarley aleurone layer cells to respond to GA₃, as indicatedby -amylase synthesis and secretion by de-embryonated grain,increased during the later stages of development. Aleurone layersof immature grain (younger than 30 d after anthesis; DAA) werenot capable of producing amylase in response to GA₃; however,premature drying at this stage promoted GA-responsiveness resultingin the induction of mRNA for -amylase and increased -amylasesynthesis and secretion. Preincubation of the immature grainor its maintenance at 100% relative humidity prior to exposureof the de-embryonated grain to GA₃ also led to an enhanced capacityof the aleurone layer to produce amylase and its mRNA as comparedto the fresh, untreated grain. However, the amount of mRNA andenzyme produced was much lower than that induced by prematuredrying. Moreover, following these nondrying treatments, thealeurone layer cells remained unresponsive to exogenous GA₃;the same amount of enzyme was produced in the absence of appliedGA₃. Transient expression of chimeric gene constructs in aleuronelayer cells of de-embryonated grain suggest that drying up-regulatesthe -amylase gene promoter in response to GA₃. We conclude thatdesiccation is required for barley aleurone layer cells to becomeresponsive to GA₃ and hence express their full potential foramylase synthesis and secretion. ³Present address: Department of Biochemistry, University ofMissouri, 117 Schweitzer Hall, Columbia MO 65211, U.S.A.     

Heat shock response of warm-incubated barley aleurone layers

Heat shock suppresses secretory protein synthesis in GA(3)-stimulated barley (Hordeum vulgare cv. Himalaya) aleurone layers by selectively destabilizing their mRNAs and dissociating the stacked rough endoplasmic reticulum (ER) lamellae upon which they are translated. Heat shock also increases phosphatidylcholine (PC) synthesis, and these PC molecules have increased levels of fatty acid saturation. This appears to be adaptive, for aleurone layers maintained at heat shock temperatures for 18 h resynthesize secretory protein mRNAs, rebuild stacked ER lamellae, and resume secretory protein synthesis. In the present study aleurone layers were incubated at warmer than normal pre-heat shock temperatures to determine whether this would favor the formation of heat-resistant ER lamellae that could continue secretory protein synthesis during heat shock. Western blot and SDS-PAGE analyses showed that such treatment did not induce heat shock protein (HSP) synthesis, but it preserved significant secretory protein synthesis during heat shock. Northern hybridizations revealed that levels of mRNAs encoding secretory proteins were several-fold elevated as compared to 25°C preincubated controls, and transmission electron microscopic observations revealed stacked ER lamellae. Thin layer and gas chromatography showed that PC molecules in warm-incubated barley aleurone layers had more fatty acid saturation than did controls. These observations indicate that previous incubation temperature influences both the induction of HSP synthesis and the suppression of normal protein synthesis in the heat shock response. However, we found that it does not affect the temperature at which heat shock becomes lethal.